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https://openalex.org/W2809072644	https://www.rrh.org.au/journal/download/pdf/89/	English	null	Factors influencing student nurses in their choice of a rural clinical placement site	Rural and remote health	2,001	cc-by	4,143	O R I G I N A L R E S E A R C H Factors influencing student nurses in their choice of a rural clinical placement site S Smith, H Edwards, M Courtney, K Finlayson S Smith, H Edwards, M Courtney, K Finlayson Centre for Nursing Research, Queensland University of Technology, Austral Centre for Nursing Research, Queensland University of Technology, Australia Smith S, Edwards H, Courtney M, Finlayson K Factors influencing student nurses in their choice of a rural clinical placement site Rural and Remote Health 1 (online), 2001: no 89 Available from: http://rrh.deakin.edu.au A B S T R A C T Rural health and government bodies have identified the need for greater numbers of health professionals in rural and remote health care settings. Providing students with the opportunity to experience a rural clinical placement has been suggested as one strategy for future health professionals to gain familiarity with the rural workplace and an awareness of the employment opportunities available in these areas. Although substantial numbers of student nurses have participated in a rural undergraduate clinical placement program at Queensland University of Technology since 1996, available places remain unfilled. This study aimed to investigate the factors influencing student nurses in their choice of a rural or metropolitan clinical placement. This study utilised a descriptive survey design. All final year Bachelor of Nursing students in 2000 (n = 212) were included in the sample, with a 65% response rate for the pre-test (n = 137). Findings demonstrated that possession of a rural background, previous work experience in a rural community and family, financial and/or employment commitments all influenced students’ choice of undertaking a rural clinical placement. settings. This is despite a need for greater numbers of health professionals in rural and remote areas being repeatedly identified by rural health and government peak bodies, the National Rural Health Alliance and the Australian Health © S Smith, H Edwards, M Courtney, K Finlayson, 2001. A licence to publish this material has been given to Deakin University http://rrh deakin edu au/ 1 © S Smith, H Edwards, M Courtney, K Finlayson, 2001. A licence to publish this material has been given to Deakin University http://rrh.deakin.edu.au/ 1 Department Healthy Horizons Policy1. Previous studies suggest that familiarity with the workplace is a positive influence on future work intentions2,3. However, a recent national survey of Australian rural nurses found only 8% had experienced any fieldwork in rural practice during their training4. This study is part of a larger project which aimed to provide evidence that student nurses who experience rural and remote health care settings during their studies are more likely to be recruited in their graduate year to rural settings. isolation, cultural differences, communication and information and documentation regarding the clinical venues. Students could access the information database on any of the clinical venues which provided them with details about travel options to the venue and within the town, accommodation availability, sporting events and other activities as well as tourist information. Collaboration with the clinical venue staff encouraged appropriate support for the students and preceptors through discussion of clinical site needs and the provision of guidelines for facilitation. A number of difficulties associated with rural clinical placements have been identified in the literature. Nominated restraints include family or carer responsibilities, the commitments associated with part-time employment, the financial burden and transport worries5,6. Concerns have been raised about the type of clinical experience available and distance from professional resources5,6. A study of rural placements in Georgia, USA, found that while some students believed they wouldn't obtain quality work experience, others worried about the level of independence or self- direction needed in a rural setting5. Social isolation and a lack of recreation facilities have also been reported as barriers to rural clinical placements6,7. Anecdotal evidence indicates rural placements have a positive effect in raising awareness of the advantages of rural practice as a registered nurse. Although substantial numbers of student nurses have successfully undertaken a rural placement, available places go unfilled. To gain an understanding of the characteristics of students who select a rural placement, it was important to obtain information on factors influencing their choice of a rural or metropolitan clinical placement setting. It is hoped this information will lead to the development of successful recruitment strategies for graduate nurses in rural and remote areas. Introduction and Background There is a paucity of literature in the area of recruitment strategies for graduate nurses in rural and remote health care © S Smith, H Edwards, M Courtney, K Finlayson, 2001. A licence to publish this material has been given to Deakin University http://rrh.deakin.edu.au/ 1 1 © S Smith, H Edwards, M Courtney, K Finlayson, 2001. A licence to publish this material has been given to Deakin University http://rrh.deakin.edu.au/ 2 Data analysis 4. Did the choice of a region of placement have any relationship with other demographic factors? Quantitative data were analysed using an SPSS database. Frequency distributions, Chi-square analysis (for nominal data) and ANOVA (for interval data) were used to describe differences between the characteristics of students choosing a rural or remote placement and students choosing a metropolitan placement. Design Characteristics of students who chose a rural placement Thirty (22%) of the 137 students who responded chose a rural setting for their clinical placement. This study utilised a descriptive survey design to collect pre- test data. A quasi-experimental pre-post test design was used for the larger project. • Previous experience of a rural lifestyle • Previous experience of a rural lifestyle Of the 58 students who had lived previously in a rural community, 19 (33%) chose a rural clinical placement and 39 (67%) chose a metropolitan setting. However, of the group of 30 students who chose a rural clinical placement, 19 (63%) had lived previously in a rural area. The proportion of rural clinical placement students who had lived previously in a rural area (63%) was significantly higher than the proportion of metropolitan clinical placement students who had lived previously in a rural area (37%), (c2 = 6.877, df = 2, p = 0.028). 2. Did the number of years lived in a rural area have any impact on the choice of region of placement? commencement of clinical placements. Participants completed the questionnaire during the session and returned it anonymously to the researcher. 3. Did the choice of a region of placement have any relationship with having worked previously in a rural area? Ethical approval for the study was obtained from QUT Human Research Ethics Committee. Instrument A questionnaire consisting of 24 items was developed by the researchers and contained a combination of open7 and closed8 questions. The questions related to demographics, previous experience of a rural lifestyle, previous work experience and issues of importance regarding the clinical placement experience. Piloting of the questionnaire was conducted prior to commencement of the study. • Length of time lived in a rural community • Length of time lived in a rural community Of the students who had lived previously in a rural area and chose a rural clinical placement: 17% (n = 3) had lived in a rural community for less than 5 years, 39% (n = 7) had lived in a rural community for 6 - 10 years, Aims All final year nursing students at Queensland University of Technology (QUT) are encouraged to select a placement of interest in first semester of their final year. The motivation of students to try something different is encouraged after two years of clinical placement in metropolitan clinical venues. Some students select to go overseas, others rural or remote and many stay within the usual confines of the metropolitan setting for a number of reasons. This study was part of a larger research project, of which the aims were to: • Evaluate the effectiveness of the Clinical Placement Support Scheme for final year Bachelor of Nursing students as a recruitment strategy for rural and remote health care services; and • Develop an increasing awareness in final year nursing students of the employment opportunities available for new graduates in rural and remote areas. Since 1996, a rural undergraduate clinical placement program has been available to student nurses at QUT. The Rural Clinical Support Scheme was comprised of a rural placement grant that assisted students with the cost of travel. The granting model was based on the number of kilometres from Brisbane, the State's capital city, and the funding ranged from $100 to $200 per student. Students were provided with a pre-briefing session prior to placement which addressed a number of important issues such as The following research questions were explored in this study: The following research questions were explored in this study: 1. Did previous experience of living in a rural community have any impact on the choice of region of placement? 2 Population and sample The population under study consisted of all final year Bachelor of Nursing students at Queensland University of Technology who undertook a clinical placement in 2000 (n = 212). One hundred and thirty-seven questionnaires were returned in the pre-test (response rate 65%). © S Smith, H Edwards, M Courtney, K Finlayson, 2001. A licence to publish this material has been given to Deakin University http://rrh.deakin.edu.au/ 3 • Financial/employment considerations • Financial/employment considerations • Financial/employment considerations Thirty- one students (28%) from the total student sample had applied for a rural scholarship, either from Queensland University of Technology, Royal College of Nursing Australia or Queensland Health. Major issues influencing application for a scholarship were financial reasons (42%) and the opportunity to gain diversity in clinical experience (33%). Reasons identified for not applying for a scholarship included financial/employment obligations in the local area (31%) and a lack of interest in working in a rural area (33%). • Financial/employment considerations Thirty- one students (28%) from the total student sample had applied for a rural scholarship, either from Queensland University of Technology, Royal College of Nursing Australia or Queensland Health. Major issues influencing application for a scholarship were financial reasons (42%) and the opportunity to gain diversity in clinical experience (33%). Reasons identified for not applying for a scholarship included financial/employment obligations in the local area (31%) and a lack of interest in working in a rural area (33%). Procedure 44% (n = 8) had lived in a rural community for more than 10 years. Pre-test questionnaires were distributed to participants at a prebriefing session held at the university one week prior to (One student did not respond to this question.) © S Smith, H Edwards, M Courtney, K Finlayson, 2001. A licence to publish this material has been given to Deakin University http://rrh.deakin.edu.au/ 3 3 © S Smith, H Edwards, M Courtney, K Finlayson, 2001. A licence to publish this material has been given to Deakin University http://rrh.deakin.edu.au/ 4 In comparison, of the students who had lived previously in a rural area but chose a metropolitan placement: higher than students undertaking a rural placement (Rural students: M = 3.45, SD = 1.27; Metropolitan students: M = 4.03, SD = 1.12; F = 5.77, df = 1, p=0.018). 41% (n = 16) had lived in a rural community for less than 5 years, 31% (n = 12) had lived in a rural community for 6 – 10 years, • Home and family considerations • Home and family considerations Family issues were nominated by 57% of students as a reason for not considering future employment in a rural setting and by 24% of students as a reason for not applying for a scholarship to undertake a rural clinical placement. Students who had chosen a metropolitan placement rated the importance of family responsibilities significantly higher than students who selected a rural placement (Rural students: M = 3.55, SD = 1.38; Metropolitan students: M = 4.26, SD = 1.03; F = 9.292, df = 1, p = 0.003). Rural placement students also rated the provision of appropriate living conditions significantly higher than metropolitan placement students (Rural students: M = 3.88, SD = 1.03; Metropolitan students: M = 3.03, SD = 1.18; F = 11.02, df = 1, p = 0.001). 28% (n = 11) had lived in a rural community for more than 10 years. • Worked previously in a rural community Of the 32 students who had worked previously in a rural community, 11 (34%) chose a rural clinical placement, while 21 (66%) chose a metropolitan placement. However, 37% of the students who chose a rural clinical placement had worked previously in a rural community, compared to 20% of the students who chose a metropolitan setting, a significantly higher difference (c2 = 5.873, df = 2, p = 0.048). • Worked previously in a rural community Of the 32 students who had worked previously in a rural community, 11 (34%) chose a rural clinical placement, while 21 (66%) chose a metropolitan placement. while 21 (66%) chose a metropolitan placement. However, 37% of the students who chose a rural clinical placement had worked previously in a rural community, compared to 20% of the students who chose a metropolitan setting, a significantly higher difference (c2 = 5.873, df = 2, p = 0.048). licence to publish this material has been given to Deakin University © S Smith, H Edwards, M Courtney, K Finlayson, 2001. A licence to publish this material has been given to Deakin University http://rrh deakin edu au/ 4 Characteristics of students who chose a rural placement © S Smith, H Edwards, M Courtney, K Finlayson, 2001. A licence to publish this material has been given to Deakin University http://rrh.deakin.edu.au/ 5 Characteristics of students who chose a rural placement • Rural background Possession of a rural background has been identified in previous studies as being positively associated with future employment in country areas9-13. It was interesting that of the 58 students who had lived previously in a rural community, only 19 (33%) chose a rural clinical placement setting. However, the proportion of rural clinical placement students who had lived previously in a rural area (63%) was still significantly higher than the proportion of metropolitan clinical placement students who had lived previously in a rural area (37%). A survey of Victorian rural nurses supports these findings, with 67% of the nurses reporting they had spent most of their childhood in a rural area11. These participants indicated that rural background and y g p when considering recruitment to a rural area. It has been suggested that universities need to recruit greater numbers of students with rural backgrounds to the health care professions in order to take advantage of this trend14, and recent government initiatives aim to encourage this idea15. • Length of time in a rural community When examining the number of years lived previously in a rural community, the data suggested that the longer a student had lived previously in a rural community, the more likely they were to choose a rural placement. Forty-four percent of the students who had lived in a rural community and chose a rural placement had lived in a rural area for over ten years. In contrast, 41% of students who had lived previously in a rural area and chose a metropolitan placement had lived in a rural area • Rural background Possession of a rural background has been identified in previous studies as being positively associated with future employment in country areas9-13. It was interesting that of the 58 students who had lived previously in a rural community, only 19 (33%) chose a rural clinical placement setting. However, the proportion of rural clinical placement students who had lived previously in a rural area (63%) was still significantly higher than the proportion of metropolitan clinical placement students who had lived previously in a rural area (37%). Table 1: Demographic characteristics of final year Bachelor of Nursing student lifestyle issues were among the most important factors when considering recruitment to a rural area. It has been suggested that universities need to recruit greater numbers of students with rural backgrounds to the health care professions in order to take advantage of this trend14, and recent government initiatives aim to encourage this idea15. lifestyle issues were among the most important factors when considering recruitment to a rural area. It has been suggested that universities need to recruit greater numbers of students with rural backgrounds to the health care professions in order to take advantage of this trend14, and recent government initiatives aim to encourage this idea15. © S Smith, H Edwards, M Courtney, K Finlayson, 2001. A licence to publish this material has been given to Deakin University http://rrh.deakin.edu.au/ 5 Discussion The demographic information obtained in this study had implications on the students' choice of clinical practice site. Over half of the student sample were aged >24yrs, with 31% aged 24 - 34 years and 20% aged >35 years. In fact, only around one-third of nursing students currently enrolled at QUT are recent school leavers, while approximately two thirds are mature age students. The high proportion of mature age students at QUT is not an isolated case, as Queensland Tertiary Admissions Centre's figures show, many South-East Queensland universities have a similar proportion of mature age nursing students. The high level of family, carer, financial and employment commitments identified in this study reflects the mature age of the group and the strong ties many students have to the local area. Respondents were asked to rate the importance of issues regarding their clinical placement experience on a scale from 1 to 5, from 'not at all important' to 'extremely important'. Analysis of results (using ANOVA) found rural students rated the importance of support for costs associated with placement significantly higher than students undertaking a metropolitan placement (Rural students: M = 3.97, SD = 1.02; Metropolitan students: M = 3.25, SD = 1.44; F = 6.125, df = 1, p= 0.015), while metropolitan students rated the importance of maintaining work commitments significantly 4 © S Smith, H Edwards, M Courtney, K Finlayson, 2001. A licence to publish this material has been given to Deakin University http://rrh deakin edu au/ 5 • Worked previously in a rural community There was a significantly higher proportion of students who had worked previously in a rural community and chose a rural clinical placement than those who had worked previously in a rural community and chose a metropolitan clinical placement. This trend could be linked to the positive association between familiarity with the work environment and subsequent employment noted by Glover, Clare, Longston and De Bellis3 and Collins, Hilde and Shriver2. Providing students with some work experience in a rural area may encourage greater familiarity with possible employment settings. The nursing graduates surveyed by Collins et al.2 felt their site of clinical practice was an important influence on their choice of employment, with 28% beginning their careers in the same workplace. Acknowledgements Funding for this research project was received from a Queensland University of Technology Scholarship in the Professions Grant. We would like to thank Ms Rekha Sharma for her assistance in the analysis of data for this project, and also the members of the Reference Committee for their valuable contribution. Conclusion This study supports earlier research demonstrating that students who have previously lived and/or worked in a rural area are more likely to choose a rural setting for clinical placements or postgraduate employment. However, considering nearly half of the students who choose a rural placement had no previous experience of a rural lifestyle, the value of rural clinical placements as a method of increasing awareness of employment opportunities in the rural setting is considerable. Family, financial and employment commitments were also found to influence students’ choice of undertaking a rural clinical placement. These factors should be considered in the development of recruitment and retention strategies for health professionals to rural areas. • Financial and family considerations A considerable number of students indicated family (24%), financial or employment (31%) commitments influenced their choice of a clinical placement site. Students who chose a metropolitan clinical placement rated the importance of maintaining work commitments and family responsibilities significantly higher than students selecting a rural area for their placement, while provision of financial support for costs associated with the placement and appropriate accommodation was significantly more important to the rural clinical placement students. for less than five years (28% for more than ten years). However, the numbers involved were too small to detect any statistically significant differences. recruitment to a rural location. Providing infrastructure for the needs of nursing students' family obligations, such as the availability of employment opportunities, education facilities and social support for carers, are thus of vital importance when considering recruitment strategies for health professionals to rural areas. Characteristics of students who chose a rural placement A survey of Victorian rural nurses supports these findings, with 67% of the nurses reporting they had spent most of their childhood in a rural area11. These participants indicated that rural background and • Length of time in a rural community • Length of time in a rural community When examining the number of years lived previously in a rural community, the data suggested that the longer a student had lived previously in a rural community, the more likely they were to choose a rural placement. Forty-four percent of the students who had lived in a rural community and chose a rural placement had lived in a rural area for over ten years. In contrast, 41% of students who had lived previously in a rural area and chose a metropolitan placement had lived in a rural area 5 © S Smith, H Edwards, M Courtney, K Finlayson, 2001. A licence to publish this material has been given to Deakin University http://rrh.deakin.edu.au/ 6 2. Collins P, Hilde E, Shriver C. Recruiting factors: Rural and urban settings. Nursing Management 1993; 24(4): 97-8. © S Smith, H Edwards, M Courtney, K Finlayson, 2001. A licence to publish this material has been given to Deakin University http://rrh deakin edu au/ 6 References Similar concerns have been identified in recruitment literature. In 1999, a national survey of rural Australian nurses reported that 'self, family or friends live in the area' and 'marriage commitments or family' were listed in the top four most frequent responses to essential factors influencing original recruitment to rural nursing4. Duffy, Siegloff and McGrail11 reported Victorian rural nurses indicated their partner's employment was the most important factor in 1. Healthy Horizons. Healthy Horizons: A framework for improving the health of rural, regional and remote Australians: National Rural Health Policy Forum and the National Rural Health Alliance, 1999. 2. Collins P, Hilde E, Shriver C. Recruiting factors: Rural and urban settings. Nursing Management 1993; 24(4): 97-8. © S Smith, H Edwards, M Courtney, K Finlayson, 2001. A licence to publish this material has been given to Deakin University http://rrh.deakin.edu.au/ 6 6 © S Smith, H Edwards, M Courtney, K Finlayson, 2001. A licence to publish this material has been given to Deakin University http://rrh.deakin.edu.au/ 7 10. Huntley B. Factors influencing recruitment and retention: Why RNs work in rural and remote area hospitals. The Australian Journal of Advanced Nursing 1995; 12(2): 14-19. 10. Huntley B. Factors influencing recruitment and retention: Why RNs work in rural and remote area hospitals. The Australian Journal of Advanced Nursing 1995; 12(2): 14-19. 3. Glover P, Clare J, Longston D, De Bellis A. Should I take my first offer? A graduate survey. Australian Journal of Advanced Nursing 1998; 15(2): 17-25. 4. Stephenson J, Blue I, Petkov J. A National Survey of Australian Rural Nurses. Whyalla Norrie, South Australia: The Association for Australian Rural Nurses Inc., 1999. 11. Duffy E, Siegloff LH, Siegloff LMF, McGrail M. Victorian Rural Nurse Project: Workforce Database Final Report - December 1999. Traralgon, Victoria: Monash University Centre for Rural Health, 2000. 5. McDonough JE, Lambert VA, Billue JS. A rural nursing practicum: Making it work. Nurse Educator 1992; 17(4): 30-34. 12. Rabinowitz HK, Diamond JJ, Hojat M, Hazelwood CE. Demographic, educational and economic factors related to recruitment and retention of physicians in rural Pennsylvania.' Journal of Rural Health 1999; 15(2): 210-1. 6. Barney T, Russell M, Clark M. Evaluation of the provision of fieldwork training through a rural student unit. Australian Journal of Rural Health 1998; 6: 202-07. 13. Wood D. Effects of educational focus on a graduate nurse's initial choice of practice area. Journal of Professional Nursing 1998; 14(4): 214-9. 7. Mitchell R. Perceived inhibitors to rural practice among physiotherapy students. Australian Journal of Physiotherapy 1996; 42(1): 47-52. 14. Hays RB, Acklin F, Chan P. et al. The University of Sydney Rural Careers Project. The Australian Journal of Rural Health 1993; 1(3): 23-25. 8. Queensland Tertiary Admissions Centre 2001. Queensland. 9. Easterbrook M, Godwin M, Wilson R. et al. Rural background and clinical rural rotations during medical training: Effect on practice location. Canadian Medical Association Journal 1999; 160(8): 1159-64. 15. National Rural Health Alliance. 8 June, 'Budget 2001' (Media Release, 22 May 2001). National Rural Health Alliance e-forum, Retrieved from http://www.ruralhealth.org.au/22501.htm 7
https://openalex.org/W4386436837	https://zenodo.org/record/8317268/files/AJUV_Dossier_BERTOLOTTI.pdf	es		De la prueba incorporada a la causa, tenemos que estamos en presencia de una persona transexual. Un análisis del modelo patologizador para el reconocimiento de la identidad de género en la jurisprudencia de Córdoba	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	11,143	Revista Argumentos (ISSN: 2525-0469) Núm. 16 2023, pp. 155-176 Sección: Dossier Centro de Perfeccionamiento Ricardo C. Núñez [En Línea] http://revistaargumentos.justiciacordoba.gob.ar/ _____________________________________________________________________ De la prueba incorporada a la causa, tenemos que estamos en presencia de una persona transexual. Un análisis del modelo patologizador para el reconocimiento de la identidad de género en la jurisprudencia de Córdoba Martín D. E. Bertolotti* Resumen: En 2009, MV demandó judicialmente el reconocimiento de su identidad de género autopercibida. En 2011 un tribunal de Córdoba, luego de testimonios y una serie de pericias de todo tipo, hizo lugar a su derecho. Esto fue solo el comienzo de un largo proceso, que llevaría hasta 2018, para lograr el acceso de MV a derechos básicos como la justicia, la salud o la identidad, aun con una sentencia favorable y con una nueva ley de identidad de género vigente. El presente trabajo aborda en una primera parte los diferentes paradigmas o modelos de gestión legal del reconocimiento de la identidad de género: modelo patologizador, modelo de derechos humanos y modelo mixto. Luego, se enfoca en el marco normativo argentino posterior a la sanción de la Ley de Identidad de Género que receptó el modelo de derechos humanos. En la segunda parte, se analiza el caso de MV resuelto en 2011, antes de la Ley de Identidad de Género, y que constituye un ejemplo del paradigma patologizador. Asimismo, se analiza la ejecución de sentencia de ese fallo donde se evidencian prácticas del modelo patologizador aun cuando la nueva ley ya estaba vigente. Palabras claves: Identidad de género, Modelo patologizador, Modelo de derechos humanos, Ley 26.743, Prácticas institucionales Abstract: In 2009, MV sued in court for recognition of his self-perceived gender identity. In 2011, a court in Córdoba, after testimonies and a series of expert opinions of all kinds, made room for his right. This was just the beginning of a long process that would take until 2018, to achieve MV access to basic rights such as justice, health or identity, even with a favorable sentence and with a new gender identity law in force. This paper addresses in a first part the different paradigms or models of legal management of the recognition of gender identity: pathologizing model, human rights model and mixed model. It focuses on the Argentine regulatory framework after the enactment of the gender identity law that receives the human rights model. In the second part, the case of MV resolved in 2011, before the gender identity law, is analyzed. This case is an example of the pathologizing paradigm. Likewise, the execution of the sentence of that ruling is analyzed, which shows that the pathologizing practices prevailed over the enactment of the new regulation. Keywords: Gender identity, Pathologizing model, Human rights model, 26,743 Act, Institutional practices _______________________ * El autor agradece a María Alejandra Reinatto, Marisa Fassi, Wilfrido Pérez y a todo el equipo de la Oficina de Derechos Humanos y Justicia del Poder Judicial de la Provincia de Córdoba por su apoyo y colaboración en la producción de este trabajo. Doctorando en Ciencia Política (Université du Québec à Montréal), Magister en Relaciones Internacionales (Centro de Estudios Avanzados. Universidad Nacional de Córdoba [UNC]). Abogado (UNC). Adscripto a la cátedra C Derecho Internacional Público (UNC). Investigador Junior en Équipe de recherche sur l’inclusion et la gouvernance en Amérique Latine ERIGAL. Pasante de Investigación Chaire de recherche sur la diversité sexuelle et la pluralité des genres (Université du Québec à Montréal). Auxiliar en la Oficina de Derechos Humanos y Justicia del Poder Judicial de Córdoba. Integrante del equipo de investigación AJuV (Poder Judicial de la Provincia de Córdoba). Correo: mbertolotti@justiciacordoba.gob.ar bertolotti.martin@courrier.uqam.ca ORCID 0009-0001-2738-0424 155 Es de esperar que, con el tiempo y el avance de los conocimientos científicos, más la humanización de la ciencia y de la sociedad, no se llegue a estos extremos, que una persona tenga que soportar tanto y tantos años para lograr una reivindicación tan justa y humana. Extracto de la pericia médica (Sentencia 482, 2011, p. 28) Introducción La frase que titula este artículo se encuentra en la página 30 de la Sentencia 482 del año 2011 en autos “MDCV - Ordinario - Otros - Expte.: 1679035/36”, dictada luego de la producción de informes psicológicos, testimoniales, una pericia médica, una pericia psiquiátrica y una pericia psicológica entre otros medios de prueba. Esta fue la realidad que tuvo que enfrentar MV para lograr el reconocimiento y rectificación de su identidad. El caso que se analiza en este artículo es revelador por varias razones, pero principalmente por su ubicación temporal. El procedimiento ante la justicia civil y comercial iniciado en mayo de 2009 obtuvo sentencia en septiembre de 2011, es decir antes de la sanción de la Ley de Identidad de Género 26.743 (9 de mayo de 2012), la que, como se abordará, hubiera evitado la judicialización de la situación. Por otra parte, la ejecución de la sentencia obtenida se extendió hasta 2018, es decir con la Ley de Identidad de Género ya vigente. No obstante, la sanción de la ley no evitó una multitud de prácticas patologizantes que MV debió enfrentar para tener un efectivo acceso a justicia y a otros derechos básicos. Los objetivos de este trabajo son, por un lado, explicar los diferentes paradigmas o modelos de gestión legal para el reconocimiento del derecho a la identidad de género, y en particular el modelo seguido por la legislación argentina. Asimismo, mediante el análisis del caso mencionado previamente, se buscará mostrar la aplicación del paradigma patologizador por parte de la justicia de Córdoba antes de la sanción de la Ley de Identidad de Género 26.743, y demostrar cómo las prácticas institucionales patologizadoras perpetuaron este modelo aún después de la sanción de la ley, restringiendo el acceso a justicia y a otros derechos de las personas trans, como un ejemplo de lo que Georgina Waylen (2014) llama el aspecto informal de las instituciones. Para la elaboración de este trabajo, en primer lugar, se realizó una revisión de literatura y un análisis de la normativa nacional y comparada. Posteriormente se tomó el fallo que resolvió en 2011 el caso de MV y se extrajeron los argumentos que integraron el razonamiento del tribunal, los que fueron analizados en base a los diferentes paradigmas o modelos de gestión legal del reconocimiento de la identidad de género previamente identificados. Finalmente, para poder analizar las prácticas institucionales posteriores a la resolución judicial se realizó un análisis documental del expediente llevado adelante por la Oficina de Derechos Humanos y Justicia del Poder Judicial de Córdoba para la ejecución de sentencia, y asimismo se realizó una entrevista semidirigida a la encargada de este caso en la oficina mencionada. El artículo se divide en dos partes para su mejor comprensión. La primera está destinada a la explicación de los modelos de gestión legal para el reconocimiento del 156 derecho a la identidad de género y el marco normativo argentino. La segunda parte aborda el caso MDCV – Ordinario – Otros – Expte.: 1679035/36 divido en dos momentos. Primero el procedimiento judicial hasta la sentencia y luego el proceso de ejecución de sentencia o de efectivización de derechos. PRIMERA PARTE A. Paradigmas o modelos para el reconocimiento de la identidad de género Como bien señala Emiliano Litardo (2018), a nivel internacional existen al menos tres modelos de gestión legal para el reconocimiento del derecho a la identidad de género: el modelo patologizador, el modelo de derechos humanos y el modelo intermedio o mixto. El modelo patologizador se sostiene en una concepción binaria del género (varón-mujer) en la cual existen cuerpos sexuados, cuyo sexo se asigna al momento del nacimiento en base a criterios médicos principalmente sobre los genitales externos. En este sistema determinista, a una persona con una cierta genitalidad se le asigna un sexo y se espera que se identifique y comporte en base a normas sociales de género esperadas para ese sexo. Por lo tanto, todas aquellas identidades y corporalidades que no se encuadren en esta predeterminación serán consideradas patológicas. Así, serán, en primer lugar, sometidas a pruebas médicas, psicológicas y psiquiátricas para demostrar su existencia y, en segundo lugar, sometidas a la tutela del sistema judicial que autorizará el “reajuste” o la “corrección” de la identidad legalmente reconocida de la persona o de su corporalidad. Este modelo puede estar positivizado en una ley, como por ejemplo la ley española de 2007.1 Dicha ley, en su artículo 4, establecía como requisitos el diagnostico de disforia de género mediante informe de médico o psicólogo clínico y un tratamiento médico durante al menos dos años para acomodar las características físicas de la persona a las correspondientes al sexo reclamado. Asimismo, la ausencia de legislación lleva generalmente a la aplicación de este modelo, como era la situación en Argentina hasta el año 2012, lo que se ilustrará más adelante mediante el caso de MV. El modelo de derechos humanos, en cambio, se funda en la autopercepción de la identidad de género, la autonomía de la voluntad y el derecho al libre desarrollo de la personalidad. En este modelo, el género es una cuestión declarativa y no es un atributo de un cuerpo sexuado. El libre desarrollo de la personalidad nos habla de la posibilidad de cada persona de elegir su propio plan de vida y de la ausencia de injerencia externa en este. Este modelo reconoce por lo tanto que existe una amplia diversidad de géneros y que ninguna identidad es patológica. Por todo esto, rechaza el sometimiento de la persona a toda prueba psicológica, psiquiátrica o médica de cualquier tipo. Es importante remarcar que este modelo no solo no requiere de las pruebas mencionadas, sino que tampoco obliga a la persona a someterse a cirugías o 1 Esta ley de 2007 fue remplazada por la Ley 4/2023, del 28 de febrero de 2023, para la igualdad real y efectiva de las personas trans y para la garantía de los derechos de las personas LGTBI. Con la nueva ley, España adoptó un modelo de derechos humanos de gestión legal para el reconocimiento del derecho a la identidad de género. 157 tratamientos médicos para el reconocimiento de su identidad de género. Así, este modelo armoniza el derecho a la identidad de la persona con el goce de otros derechos como la vida privada y la integridad. Así lo ha entendido la Corte Interamericana de Derechos Humanos en la Opinión Consultiva 24/17: el procedimiento de solicitud de cambio de nombre, adecuación de la imagen y rectificación de la referencia al sexo o género, en los registros y documentos de identidad, no podrá requerir que se lleven a cabo intervenciones quirúrgicas totales o parciales ni terapias hormonales, esterilizaciones o modificaciones corporales para sustentar el requerimiento, para otorgar lo solicitado o para probar la identidad de género que motiva dicho procedimiento, por cuanto podría ser contrario al derecho a la integridad personal contenido en el artículo 5.1 y 5.2 de la Convención Americana. En efecto, someter el reconocimiento de la identidad de género de una persona trans a una operación quirúrgica o a un tratamiento de esterilización que no desea, implicaría condicionar el pleno ejercicio de varios derechos, entre ellos, a la vida privada (artículo 11.2 de la Convención), a escoger libremente las opciones y circunstancias que le dan sentido a su existencia (artículo 7 de la Convención), y conllevaría a la renuncia del goce pleno y efectivo de su derecho a la integridad personal. (OC 24/17 Corte IDH, párrafo 146) Un tercer modelo es el llamado modelo mixto. La legislación de Ecuador (Ley orgánica de gestión de la identidad y datos civiles de 2015), de Uruguay (Ley 18.620 “Regulación del derecho a la identidad de género, cambio de nombre y sexo registral” de 2009) y de Chile (Ley 21.120 “Reconoce y da protección al derecho a la identidad de género” de 2018) son ejemplos de este modelo. Las legislaciones mencionadas de Uruguay y Ecuador reconocen la autopercepción del género, pero agravan los requisitos estableciendo procedimientos judiciales, exigiendo la permanencia y estabilidad de dos años en la identidad autopercibida y pruebas testimoniales de tal vivencia (Litardo, 2018). En 2018, Uruguay sancionó la Ley Integral para Personas Trans 19.684, mediante la cual se optó –entre otras medidas favorables– por un procedimiento administrativo, y se dejó de exigir un informe técnico de un equipo multidisciplinario y especializado en identidad de género y diversidad, así como también se eliminó el requisito de presentar testigos. Con esta modificación a la norma el país se encuadra en el modelo de derechos humanos. La ley chilena de 2018, si bien reconoce la autodeterminación del género, restringe el concepto de identidad de género a “la convicción personal e interna de ser hombre o mujer, tal como la persona se percibe a sí misma, la cual puede corresponder o no con el sexo y nombre verificados en el acta de inscripción del nacimiento (cursivas agregadas)” (Ley 21.120, art. 1, párr. 2). Establece que nunca podrán exigirse tratamientos médicos o cirugías, pero solicita la declaración de dos testigos. Para el caso de personas menores de edad establece un procedimiento judicial en el cual está prevista expresamente una serie de informes psicológicos y psicosociales. La ley 158 establecía que las personas casadas debían realizar el trámite de manera judicial y su aprobación disolvía el vínculo matrimonial. Esta diferenciación para las personas casadas fue modificada en 2021 con la sanción de la Ley 21.400 de matrimonio entre personas del mismo sexo. B. La Ley 26.743 de identidad de género Hasta 2012 no existía en Argentina ninguna disposición legal que permitiera la modificación del género en los registros o en la documentación personal, ni el acceso a tratamientos médicos de ningún tipo para la adecuación del cuerpo a la identidad autopercibida. El modelo patologizador se desarrollaba mediante la jurisprudencia de los tribunales, mientras que las disposiciones normativas existentes eran criminalizadoras, como los códigos de faltas,2 o prohibitivas, como la ley de ejercicio de la medicina. La Ley 17.132 de ejercicio de la medicina, en su art. 19 inc. 4, expresamente prohibía “llevar a cabo intervenciones quirúrgicas que modifiquen el sexo del enfermo, salvo que sean efectuadas con posterioridad a una autorización judicial (cursivas agregadas)”. Como lo señala Mauro Cabral (2003), la dificultad para obtener autorizaciones judiciales para realizarse cirugías destinadas a modificar los genitales y la ausencia de servicios especializados en cirugías de reconstrucción genital en Argentina llevaban a muchas personas trans a migrar a Chile para acceder a estas. Anahí Farji Neer (2015) señala que a partir de 1995 se presentaron diferentes proyectos de ley para regular los procesos de cambio de nombre y sexo legal, pero que ninguno fue discutido hasta 2011. La autora atribuye el tratamiento de la ley a una reconfiguración en el vínculo entre el Estado y los colectivos sexo-políticos locales y la sanción previa de la Ley 26.618 de “matrimonio igualitario” en 2010. La Ley de Identidad de Género 26.743, sancionada el 9 de mayo de 2012, reconoce que toda persona tiene derecho al reconocimiento de su identidad de género, al libre desarrollo de su personalidad conforme a su identidad de género y a ser tratada de acuerdo con su identidad de género y, en particular, a ser identificada de ese modo en los instrumentos que acreditan su identidad (nombre de pila, imagen y sexo registrado). La propia ley brinda un concepto amplio de identidad de género, la que se entiende como la vivencia interna e individual del género tal como cada persona la siente, que puede corresponder o no con el sexo asignado al momento del nacimiento, incluyendo la vivencia personal del cuerpo (Ley 26.743, art. 2). La identidad de género, a diferencia de lo que ocurre, por ejemplo, en la ley chilena ya mencionada, no se circunscribe al binomio varón-mujer. Asimismo, la ley establece un procedimiento administrativo ante el Registro Nacional de las Personas basado en la mera declaración de la persona interesada y expresamente establece que en ningún caso será requisito 2 Los códigos de faltas provinciales persiguieron a las personas trans de manera explícita hasta bien entrados los años 2000. Asimismo, según la Federación Argentina LGBT hay también artículos de códigos contravencionales “menos explícitos” que realizan alusiones indirectas o contienen figuras que son utilizadas para perseguir y detener a gays, lesbianas, bisexuales y personas trans (Federación Argentina LGBT, 2008). En Córdoba, la discriminación más flagrante de los códigos de faltas todavía acarrea problemas para las personas trans, como fue el caso de detención de la activista trans Ivanna Aguilera en abril de 2021 por parte de la Policía de Córdoba por una orden de captura del año 1988 en virtud del art 19 del entonces código de faltas por “no llevar vestimenta acorde al género”. Si bien ese artículo fue derogado en 1994, la orden de captura seguía vigente (Pizzarro, 2021). 159 acreditar intervención quirúrgica por reasignación genital total o parcial, ni acreditar terapias hormonales u otro tratamiento psicológico o médico (art. 4). Es decir, la ley se enmarca expresamente en el modelo de derechos humanos. Eleonora Lamm se refiere de la siguiente manera: Esta ley significó el paso del paradigma médico psiquiátrico al de los derechos humanos, de modo que esta ley, la más liberal del globo y ejemplo a nivel mundial, se funda en dos decisiones de política legislativa centrales: 1) se permite el cambio de género en el registro civil sin necesidad de acreditar ningún requisito, expresamente se manifiesta la no necesidad previa de modificación corporal alguna ni certificación de ningún tipo y 2) la autoridad pública que interviene es administrativa (registro civil) y no judicial. Se trata de dos consideraciones claves para poner fin a la patologización y estigmatización. (Lamm, 2018, p. 238) La ley establece que el registro civil de la jurisdicción donde fue asentada el acta de nacimiento deberá emitir una nueva partida con los cambios realizados. Para garantizar la confidencialidad del trámite, y evitar eventuales tratos discriminatorios, en la nueva partida no puede haber ninguna alusión a que hubo un cambio en virtud de la identidad de género y el acta original solo será accesible para la persona titular y quienes tengan su expresa autorización de esta o de autoridad judicial. El nuevo DNI mantendrá el número original. La ley garantiza también el acceso a intervenciones quirúrgicas totales y parciales y/o tratamientos integrales hormonales de adecuación corporal, sin necesidad de requerir autorización judicial o administrativa, y expresamente deroga el ya mencionado art. 19 inc. 4 de la ley de ejercicio de la medicina. Para el acceso a los tratamientos integrales hormonales, no será necesario acreditar la voluntad en la intervención quirúrgica de reasignación genital total o parcial, bastando el consentimiento informado. La ley también trajo una solución al debate sobre el acceso a los tratamientos y cirugías en base a criterios diagnósticos. La American Psychiatric Association en las diferentes versiones de su Manual diagnóstico y estadístico de los trastornos mentales (conocido como DSM, por su sigla en inglés) fue modificando la manera de abordar la situación de las personas trans. En su versión más primitiva el manual hablaba de “transexualismo” como un “trastorno de la identidad de género”. En la versión actual del manual (quinta edición, 2014) se aborda como “disforia de género”, poniendo el foco no en la identidad sino en la angustia que la persona puede sentir cuando su identidad no corresponde con el sexo asignado al nacer. Por su parte, la Clasificación Internacional de Enfermedades de la Organización Mundial de la Salud en su onceava edición ha eliminado de la lista de trastornos mentales a la transexualidad y al travestismo, aunque ha creado el diagnóstico de discordancia de género dentro de la categoría “condiciones relacionadas con la salud sexual”. Mantener estos diagnósticos continúa siendo patologizar la identidad de género y existen hoy diferentes campañas 160 que buscan erradicarlos finalmente.3 Uno de los argumentos utilizados para mantener estas figuras es que ello permite que cirugías y tratamientos sean cubiertos por el sistema de salud. La técnica legislativa de la Ley 26.743 resuelve este debate al incluir estas prestaciones expresamente en el Plan Médico Obligatorio (Ley 26.743, art. 11), no solo sobre la base del derecho a la salud (entendido de manera integral), sino también sobre la base del libre desarrollo de la personalidad. Asimismo, la ley establece que el nombre de pila y el género autopercibido deben ser utilizados al solo requerimiento de la persona interesada, aun cuando el cambio registral no se haya realizado formalmente; mostrando que la rectificación registral no tiene un carácter constitutivo. C. Reconocimiento de la identidad de género de niñas, niños y adolescentes La Ley de Identidad de Género establece que la solicitud de cambio registral, de nombre y de imagen debe ser realizada a través de los representantes legales de la persona menor de edad y con el expreso consentimiento de esta. Se deberán tener en cuenta los principios de capacidad progresiva e interés superior. Asimismo, establece que el trámite será siempre administrativo, salvo cuando: • Se niegue o sea imposible obtener el consentimiento de representantes legales. En cuyo caso se debe recurrir vía sumarísima a la justicia (art. 5). • En los casos de intervenciones quirúrgicas a personas menores de edad se necesita autorización judicial, esta deberá expedirse en un plazo no mayor a 60 días desde la solicitud (art. 11). Sin embargo, tres años después de la sanción de la Ley 26.743, Argentina sancionó un nuevo Código Civil y Comercial (CCyC) lo que hace necesario armonizar ambas normas. El art. 26 del CCyC establece que se presume que adolescentes entre 13 y 16 años tienen aptitud para decidir por sí respecto de aquellos tratamientos que no resultan invasivos, ni comprometen su estado de salud o provoquen un riesgo grave en su vida o integridad física. Cuando esos tratamientos no tienen estas características deben prestar el consentimiento informado con la asistencia de sus progenitores. A partir de los 16 años las personas adolescentes son consideradas como adultas para las decisiones atinentes al cuidado de su cuerpo. En el año 2015, la Secretaría de Salud Comunitaria del Ministerio de Salud emitió la Res. 65/2015 justamente sobre esta armonización. En primer lugar, la resolución establece que todas las prácticas previstas en la Ley de Identidad de Género deben entenderse como constitutivas del cuidado del propio cuerpo. De la resolución puede extraerse que, para las personas menores de 13 años de edad, debe seguirse lo dispuesto por la Ley de Identidad de Género, es decir toda solicitud debe ser realizada a través de representantes legales y con el expreso consentimiento de la persona menor de edad. El trámite será siempre administrativo, salvo cuando se niegue o sea imposible obtener el consentimiento de representantes legales o cuando se trate de intervenciones quirúrgicas. 3 Como por ejemplo de la campaña Stop Trans Pathologization creada en España en el año 2007 (Farji Neer, 2015, p. 363). 161 Para las personas de entre 13 y 16 años de edad, la regla varía según el tipo de intervención. Las prácticas no invasivas podrán solicitarse por sí mismas, mientras las prácticas como intervenciones quirúrgicas deberán solicitarse únicamente con la asistencia de personas que ejerzan el rol de cuidado y acompañen el proceso. Las personas a partir de los 16 años son consideradas como adultas (art. 26 CCyC), por lo cual para la realización de trámites y prácticas (tratamientos y cirugías) relativas a la identidad de género deben cumplir solamente los requisitos previstos para las personas adultas. D. Ampliación del reconocimiento de la identidad de género después de la Ley 26.743 Según datos del Registro Nacional de las Personas (RENAPER), hasta el 31 de marzo de 2022 unas 12.655 personas habían rectificado su información registral en base a su identidad de género (RENAPER, 2022). Si bien, como se mencionó anteriormente, la Ley 26.743 no se circunscribe al binomio varón-mujer. Hasta julio de 2021, en la práctica el sistema siguió siendo binario. Una persona podía corregir sus documentos y rectificar su partida de nacimiento conforme a su autopercepción, pero el RENAPER reconocía como únicas opciones los marcadores de sexo masculino y femenino, lo que distaba de ser la vivencia del género tal como cada persona la siente. En 2018, la Dirección de Registro Civil de la Provincia de Mendoza mediante la Resolución 420/2018 en base al artículo 2 de la Ley 26.743 hizo lugar, luego de un dictamen de la Subdirección de Derechos Humanos de la Corte Suprema de Justicia de Mendoza, a la solicitud de dos personas que requirieron el reconocimiento legal de su identidad de género autopercibida y la correspondiente rectificación registral, y solicitaron de manera expresa que en su partida de nacimiento no se consigne sexo alguno (Gobierno de Mendoza, 2018; De la Rosa, 2021). En 2019, un fallo del Juzgado de Familia y Minoridad de Tierra del Fuego, a cargo del juez Alejandro Ferreto, resolvió el amparo de S.L.S.B. reconociendo su identidad “no binario-igualitario”. La medida se extendió también al hijo de S.L.S.B. ya que el juez ordenó rectificar el acta de nacimiento de este para que en el campo destinado a sus progenitores se reemplace por el nombre actual de S.L.S.B. (Arias, 2019). En 2020, por vía administrativa en la provincia de Misiones, L.L. logró el reconocimiento de su identidad no binaria y el otorgamiento de una nueva partida de nacimiento rectificada. Según la Asociación Todes con DNI, avances como estos se dieron en diferentes provincias de Argentina (Ojeda, 2020). En 2019, el Juzgado Nacional en lo Civil N.º 7 a cargo de la jueza Myriam Cataldi ordenó la rectificación registral de Lara Bertolini reconociendo su identidad de género como “feminidad travesti”. Sin embargo, el juzgamiento fue revertido por la Sala G de la Cámara Nacional de Apelaciones (Agencia Presentes, 2019). El fallo de primera instancia no solo reconoció el avance de derechos en la materia sino también el dinamismo conceptual: Ahora bien, no podemos dejar de resaltar que la Ley 26.743 se sancionó en el año 2012, es decir con anterioridad a la entrada en vigencia del Código Civil y Comercial de la Nación 162 (CCyC) en el 2015, y desde entonces, afortunadamente, mucha agua ha pasado bajo el puente y mucho se ha avanzado en el reconocimiento de derechos. El camino en este campo se fue abriendo progresivamente, reflejándose en la evolución de ciertos conceptos, -como veremos más adelante- los que fueron interpretados ampliamente en el reciente caso del Registro Provincial de Mendoza, en su Resolución Nro. 420, que marcó, sin dudas, la ruptura de la concepción binaria del género. (Bertolini, Lara María c/ Ministerio del Interior OP y V s/Información Sumaria, 2019, p. 4) Algunos organismos también reconocieron mediante resoluciones administrativas designaciones no binarias en términos de sexo/género, como por ejemplo la Resolución General Conjunta 5007/2021 AFIP y ANSES que establece que para los numero de CUIL y otros se utilizara 20, 23, 24 o 27 de forma aleatoria, genérica y no binaria en términos de sexo/género. Este camino condujo a que finalmente en julio de 2021 a partir del Decreto Presidencial N.º 476/21 se permitiera al RENAPER a emitir documentos con el marcador de sexo: X. Artículo 2º.- Determínase que las nomenclaturas a utilizarse en los Documentos Nacionales de Identidad y en los Pasaportes Ordinarios para argentinos en el campo referido al “sexo” podrán ser “F” -Femenino-, “M” - Masculino- o “X”. Esta última se consignará, de conformidad con lo establecido en el artículo 4° del presente decreto, en aquellos supuestos de personas nacionales cuyas partidas de nacimiento sean rectificadas en el marco de la Ley N° 26.743, cualquiera sea la opción consignada en la categoría “sexo”, siempre que no sea “F” -Femenino- o “M” -Masculino-, o bien si el “sexo” no se hubiere consignado (Dto. 476/21, art. 2) Artículo 4º.- A los fines del presente decreto, la nomenclatura “X” en el campo “sexo” comprenderá las siguientes acepciones: no binaria, indeterminada, no especificada, indefinida, no informada, autopercibida, no consignada; u otra acepción con la que pudiera identificarse la persona que no se sienta comprendida en el binomio masculino/femenino (Dto. 476/21, art. 4) El Decreto 476/21 integra y completa a la Ley de Identidad de Género 26.743. Sin embargo, existen críticas sobre la utilización de la X en DNI y pasaportes, atento a que esta no representa acabadamente el abanico de posibilidades identitarias y sigue jerarquizando el binomio masculino (M) femenino (F). El decreto reconoce la posibilidad de optar en los documentos de identificación personal por “F” –Femenino–, “M” –Masculino– o “X”. De esta manera, crea estas categorías rígidas en la que la X abarca diferentes identidades que no quedan representadas en igualdad de condiciones que F y M. Algunas organizaciones 4 4 Como por ejemplo la organización Todes con DNI: todescondni.com 163 propusieron formas más igualitarias para reconocer del abanico de identidades, como por ejemplo colocar un campo abierto a completar con la palabra más acorde a la identidad propia. Asimismo, el propio decreto señala que la X es para las personas que “no se sienta[n]comprendida[s] en el binomio masculino/femenino” por lo cual es una categoría residual a este binomio. Finalmente, cabe decir que tanto la Ley 26.743 como el Decreto 476/21 reconocen la posibilidad del cambio de las partidas de nacimiento y de los documentos de identidad, pero no reconocen la posibilidad de que las personas recién nacidas sean inscriptas con una opción diferente al femenino o masculino, por lo cual en la práctica “M o F” seguirá siendo la regla y “X” la excepción. SEGUNDA PARTE A. Caso MDCV: el proceso judicial 2009-2011 El 26 de mayo de 2009, MV, una persona de sesenta y dos años a la que se le había asignado el sexo femenino al momento de nacer, interpuso una demanda ante un juzgado de primera instancia con competencia en lo civil y comercial de la ciudad de Córdoba. El objeto de la demanda según surge de la relación de la causa era “que se autorice la intervención quirúrgica para adecuar órganos genitales; se modifique la partida de nacimiento con cambio de nombre de pila y sexo; se expida un nuevo DNI que acredite su condición de varón” (Sentencia 482, 2011, p. 2). El relato de MV está cargado de diferentes procesos de exclusión. Entre ellos destacan problemas de conducta al inicio de la etapa escolar fundados en su rechazo a la vestimenta y normas de comportamiento pensadas para un género que no era el propio, el abandono de la escuela secundaria “porque no soportaba más la situación de vestirse como no lo sentía” y la exclusión laboral por no poder tener nunca un trabajo formal al no poder presentar documentación con su nombre y género autopercibido. En la relación de los hechos, el actor manifestó haber visitado desde niño a diferentes profesionales médicos y psicólogos e incluso que a la edad de 14 años uno de estos le recomendó operarse y solicitar el cambio de DNI. La postura tomada por el tribunal fue que el actor debía demostrar que era una persona trans. Su declaración no era suficiente. El relato de su vivencia no era suficiente. Incluso su apariencia, la que parecía suficiente para los peritos médicos, tampoco bastaba. Por lo tanto, el tribunal se valió de una serie de medios probatorios. En primer lugar, un informe aportado por el actor de un psicoterapeuta especializado en sexología clínica. Luego, tres informes periciales oficiales: una pericia médica, una pericia psiquiátrica y una pericia psicológica. Finalmente, declaraciones testimoniales. Las pruebas médicas apuntaban a confirmar un diagnóstico patológico. Por su parte, la prueba testimonial buscaba demostrar una cierta permanencia en el tiempo, confirmar una idea de perdurabilidad e irreversibilidad. La importancia de la prueba era categórica para el tribunal, el que escribe “de la prueba incorporada a la causa, tenemos que estamos en presencia de una persona transexual” (Sentencia 482, 2011, p. 30) 164 Entre los razonamientos expuestos por el tribunal sobresale el determinismo biológico, la diagnosis, la irreversibilidad, el sufrimiento y el derecho a la vida y la salud. Determinismo biológico: el tribunal considera necesario en primer lugar definir “al transexual”. Para ello, toma una definición de Carlos Fernández Sessarego según la cual una persona trans es aquella que “no obstante pertenecer biológicamente y desde su nacimiento a un sexo, con el que aparece inscripto en el registro civil, siente y vive como si fuera del sexo opuesto” (Sentencia 482, 2011, p. 30). De la definición surge, en primer lugar, el determinismo biológico. La persona pertenece a un determinado sexo como una realidad incuestionable. No se menciona que es una mirada externa (médica) basada en la genitalidad la que asigna un cierto sexo a la persona. En segundo lugar, la persona “siente y vive como si fuera del “sexo opuesto”. Lo que da la idea de que no solo existe un determinismo biológico, sino que ese determinismo es binario, un sexo opuesto al otro, en este caso mujer-varón. Diagnosis: “de los informes periciales, surge que el diagnóstico del solicitante es transexual. Dicho diagnóstico es categórico” (Sentencia 482, 2011, p. 31). Es decir, según el tribunal la persona tiene una condición patológica, la que una vez diagnosticada permitirá de alguna manera curarla. Este análisis deductivo queda explicitado en otro pasaje de la sentencia: “además, sin duda que la intervención quirúrgica permitirá al peticionante lograr un equilibrio entre soma y psiquis, permitiéndole, gozar de una vida sexual y de relación, lo más normal posible” (Sentencia 482, 2011, p. 32). Así, el tribunal autoriza la intervención quirúrgica para curar esta patología y permitirle al peticionante una “vida normal”. Como se mencionó en la primera parte de este artículo, el modelo patologizador conjuga el discurso médico (diagnóstico) con el discurso jurídico (autorización judicial). Algunos de los diagnósticos receptados en la sentencia, provenientes de las múltiples pruebas médicas son: • Informe de especialista en sexología clínica: “De dicho informe surge que, [el especialista] ha evaluado a MDCV, luego de varias entrevistas, determinando el diagnóstico del mismo en disforia de género. Asimismo, realiza el marco conceptual de disforia de género, estableciendo que son sinónimos de transexualismo, transgenerismo, síndrome Harry Benjamín” (Sentencia 482, 2011, pp. 23-24). • Pericia médica: “La impresión clínica que se recoge durante toda la entrevista y en el examen pericial, es de que los genitales externos que presenta el actor no coinciden con su condición antropológica masculina” (Sentencia 482, 2011, p. 27). • Pericia psiquiátrica “El examinado padece de un síndrome transexual, pertenece a un género o identidad sexual masculino” (Sentencia 482, 2011, p. 28). Irreversibilidad: en el informe emitido por el psicoterapeuta del actor puede leerse “la transexualidad es el sentimiento irreversible de pertenecer al sexo contrario al legalmente establecido” (Sentencia 482, 2011, p. 24). Asimismo, en la causa se receptaron dos testimonios. El primero de una familiar del actor que dijo conocerlo desde que nació y el segundo del psicoterapeuta que trató al actor previamente al inicio del procedimiento judicial. El primer testimonio se orientó a demostrar que el actor desde niño siempre se identificó con todo lo asociado al género masculino (ropa, 165 juegos, etc.). En la transcripción del segundo testimonio se advierte una pregunta del tribunal sobre la posibilidad de reversión de la identidad del actor, a lo que el testigo especialista respondió “habiéndose adquirido la constancia de identidad genérica no hay hasta el momento ninguna posibilidad de que pueda revertirse”. Previamente el testigo había afirmado que la identidad de género se comienza a construir alrededor del año y medio de edad y que una vez construido “se cierra a cal y canto” (Sentencia 482, 2011, p. 26). El perito psiquiatra luego de diagnosticar “síndrome transexual” refirió que las características psíquicas asociadas a este son irreversibles y que no existe la posibilidad de que el actor pueda vivir de otro modo y que su decisión no responde a un impulso sino a algo “sostenido y anhelado desde hace muchos años” (Sentencia 482, 2011, p. 29). Sufrimiento: un razonamiento que surge en diferentes oportunidades a lo largo del fallo es que las personas trans sufren por estar “atrapadas en un cuerpo equivocado”. No solamente este razonamiento va de la mano con los ya nombrados, en cuanto la consideración de tratamientos médicos y cirugías como una forma de “corregir” el cuerpo o “curar” una patología. Sino que, además, se asocia de una manera determinante la identidad con la corporalidad. Al margen de que en el caso concreto el actor solicitaba la realización de intervenciones quirúrgicas, el marco conceptual general elimina cualquier posibilidad de que una persona pueda tener una identidad masculina sin modificar su corporalidad. Las cirugías son necesarias para “liberar” a la persona de ese cuerpo que le es ajeno. Así, nuevamente se vuelve al determinismo: identidad masculina = pene y no mamas (necesita hormonas, faloplastia, mastectomía); identidad femenina = vagina y mamas (necesita hormonas, vaginoplastia, mamoplastia). Esto se ejemplifica con el siguiente extracto: La transexualidad, ha sido descripta por Matilde Zavala de González, como aquella en la que se perciben atrapadas en un cuerpo que no sienten como propio […] Estamos en definitiva ante una persona que se encuentra atrapada en un cuerpo que tiene un sexo distinto al que siente, el cual rechaza (Sentencia 482, 2011, p. 31). Derecho a la vida y a la salud: tratar a la persona trans como alguien que sufre, que está atrapada en cuerpo extraño, que su identidad es por lo tanto patologizada y que necesita del sistema judicial y del sistema médico para poder corregir esta situación y lograr una vida “lo más normal posible” lleva a que el fundamento central del fallo descanse sobre el derecho a la salud, el cual relaciona íntimamente con el derecho a la vida. El derecho a la preservación a la salud, comprendido, como se dijo dentro del derecho a la vida, debe ser garantizado en forma impostergable por la autoridad pública con acciones positivas, sin perjuicio de las obligaciones que deban asumir en su cumplimiento las jurisdicciones locales, las obras sociales o las entidades de la llamada medicina prepaga. (Sentencia 482, 2011, p. 20). 166 El tribunal al abordar el derecho a la salud como fundamento del fallo entendió que este se completa con otras garantías como la intimidad personal, la propia imagen y la dignidad personal. Asimismo, aun fundándose en criterios médicos ya mencionados, reconoce que “el derecho a la identidad de género y orientación sexual” involucra una serie de derechos fundamentales. Además, existen otras garantías constitucionales que coadyuvan y completan el derecho a la salud en su más amplia significación como el art. 33 CN que protege el derecho a la intimidad personal y a la propia imagen (art. 19 CN), así como a la dignidad personal (ar. 33 CN). Si bien como hemos analizado no existe una normativa precisa para el caso, hemos visto que el derecho a la identidad de género y orientación sexual, por su contenido personalísimo, involucra toda una serie de derechos fundamentales como se dijo, el derecho a la dignidad personal, a la libertad, a la personalidad, a la no discriminación, a la vida privada, a la salud, a trabajar, al proyecto de vida, a una adecuada calidad de vida etc. (Sentencia 482, 2011, p. 22). El procedimiento judicial finalizó mediante la Sentencia 482 de fecha 16 de septiembre de 2011. El objeto de este análisis no es hacer un juicio de valor del fallo del Tribunal Civil y Comercial, sino exponer la materialización del modelo patologizador en la jurisprudencia. Lo resuelto por el fallo en el caso concreto fue favorable al actor e hizo lugar a todas sus legítimas pretensiones. Al momento de fallar el tribunal hizo una revisión de los diferentes procesos de exclusión (escolar, laboral, familiar, social) vividos por el actor, y en una suerte de reivindicación de la voluntad de este, agrega que “el peticionante es consciente de los efectos que la cirugía le provocará y ha elegido libremente este camino. Por lo tanto, en base a la decisión adoptada por el actor, el derecho no puede dejar de darle una respuesta favorable a su pretensión” (Sentencia 482, 2011, p. 34). El tribunal autorizó la intervención quirúrgica. Ordenó inmovilizar el acta de nacimiento original y emitir una nueva partida y un nuevo DNI. Si bien ordenó al registro civil abstenerse de expedir el acta inmovilizada, hizo una excepción para casos de matrimonio y adopción. En esos casos, el Registro debía informar fehacientemente a la futura persona contrayente de la situación del actor –lo que recuerda a lo mencionado previamente sobre la ley de identidad de género chilena– y al juez o autoridad competente en casos de adopción. La Ley de Identidad de Género 26.743 expresamente recepta la confidencialidad sobre la rectificación registral (art. 6 y 9), lo mismo ha receptado, por ejemplo, la nueva ley española de 2023. La Corte Interamericana de Derechos Humanos ha seguido la misma tesitura en la OC-24/17 sosteniendo que la publicidad no deseada sobre un cambio de identidad de género puede poner a la persona en una situación de mayor vulnerabilidad a diversos actos de discriminación en su contra, en su honor o en su reputación y puede ser un obstáculo para el ejercicio de otros derechos humanos (párr. 135). Los tres ejemplos forman parte del modelo o paradigma de derechos humanos. 167 Ahora bien, en el fallo que estudiamos vemos que se adopta un modelo mixto. Por regla general, el tribunal resolvió que el registro civil debe abstenerse de expedir el acta de nacimiento original, pero ha hecho dos excepciones: matrimonios y adopciones donde el mismo registro civil (no dice a pedido de parte) deberá informar fehacientemente a la persona contrayente o al juez competente. ¿Cómo se explican estas excepciones? Son ejemplos del modelo patologizador. Este modelo entiende que la desjudicialización y la posibilidad de modificar los registros van en contra del orden público. Este enfoque critica que sea la “voluntad no comprobada” de la persona, es decir, no comprobada por una autoridad externa médica o judicial la que prime a la hora de definir su identidad y su plan de vida. Esta situación, en el caso del matrimonio, induciría categóricamente al error, como vicio del consentimiento, de la otra persona contrayente (García de Solavagione et al., 2013). Este enfoque no esconde su paternalismo al referirse a la “protección del contrayente de buena fe” y somete a la persona no solo a la injerencia externa sobre su identidad, sino también a la elección de su propio plan de vida a un supuesto “bien social” protegido por el orden público. Esta postura privilegia un modelo de familia formado por personas cis-género (no trans), descrinando los demás modelos de familia (a los que patologiza) presuponiendo que la persona que se case con una persona trans estaría incurriendo, salvo prueba en contrario, en el error como vicio del consentimiento. Y, asimismo, que las personas adoptantes trans deben quedar en una categoría residual frente a las adoptantes cis-género. B. Caso MDCV: La ejecución de sentencia 2012-2018 La obtención de una sentencia favorable a las pretensiones del actor, aun luego de todo el sometimiento a pruebas médicas patologizantes, fue el primer paso para la obtención del reconocimiento de su identidad por parte del Estado y de la materialización de sus derechos. El primer paso porque, como se expondrá a continuación, lograr la ejecución de la sentencia no estuvo libre de desafíos y llevó más tiempo que el trámite judicial. En esta oportunidad el actor recurrió nuevamente al poder judicial, a través de la Oficina de Derechos Humanos y Justicia, no ya para obtener una autorización, sino para conseguir un acompañamiento que permitiría llevar a la práctica la sentencia obtenida en 2011. Cabe mencionar que casi la totalidad del proceso para lograr la ejecución de la sentencia se desarrolló con la Ley de Identidad de Género 26.743 ya vigente. Esta situación nos lleva a cuestionarnos también el cambio institucional que implicó esta ley en la práctica. Georgina Waylen (2014) aborda el cambio institucional en materia de género, haciendo hincapié tanto en los aspectos formales de las instituciones (por ejemplo, las leyes) como en el aspecto informal de estas (por ejemplo, las prácticas). La autora reconoce que el aspecto informal es el más difícil de visualizar y que es justamente este aspecto el que puede facilitar o subvertir el cambio institucional impulsado mediante modificaciones formales. Desde el punto de vista formal, autores como Mahoney y Thelen (2010) identifican diferentes mecanismos de cambio institucional: desplazamiento, 168 superposición, deriva y conversión. Se trata de desplazamiento cuando se crea una nueva institución que reemplaza a las anteriores y es producto de una exitosa presión por parte de los actores que impulsaron la nueva institución. Es este mecanismo el que nos interesa en nuestro caso. La Ley de Identidad de Género 26.743 fue una nueva institución que implantó un nuevo modelo de derechos humanos y desplazó al anterior modelo patologizador. Ahora bien, la ley fue sancionada en 2012 pero a MV le llevó hasta 2018 acceder realmente a sus derechos, con innumerables prácticas patologizantes en el transcurso, aun teniendo una sentencia favorable y el acompañamiento de la Oficina de Derechos Humanos y Justicia del Poder Judicial. Waylen explica esta situación desde el institucionalismo histórico, según el cual las instituciones no son ni neutrales, ni cohesivas, ni tampoco coherentes; sino que son el resultado de una lucha de poder. Una lucha en la cual los diseñadores del cambio institucional (activistas y legisladores que impulsaron y votaron la Ley 26.743) no son sus implementadores (en nuestro caso, principalmente el sistema de salud). Este aspecto informal, estas prácticas institucionales subvirtieron –y subvierten– el cambio institucional que trajo la ley permitiendo que los criterios médicos patologizantes –como ejercicio del poder de la autoridad médica– se perpetuaran en el tiempo, generando que el modelo de derechos humanos en la práctica no fuera tal. Según Waylen (2014) este aspecto informal es muy difícil de descubrir y requiere de métodos específicos. Para intentar descubrirlo, en nuestro caso particular, nos hemos valido del análisis documental del expediente llevado por la Oficina de Derechos Humanos y Justicia, donde se describen las diferentes intervenciones y prácticas realizadas, al mismo tiempo que se adjuntan copias y transcripciones de las comunicaciones entre los diferentes actores. Asimismo, se entrevistó a la persona encargada del caso –quien acompañó muy de cerca a MV– para dilucidar aún más estos aspectos informales. El primer acercamiento de MV con la Oficina de Derechos Humanos fue el 9 de abril de 2012, un mes antes de la sanción de la Ley de Identidad de Género. El primer motivo fue lograr la ejecución de la primera parte de la sentencia de 2011: la obtención de un DNI con la adecuación de su nombre y su marcador de sexo. La actuación de la Oficina logró que en poco más de un mes el actor tuviera un nuevo DNI en ese momento con nuevo número, a diferencia de lo que hoy prescribe la ley. Este primer logro no parece significativo teniendo en cuenta que MV contaba con una sentencia firme a su favor, sin embargo, para la encargada de su caso fue el primer paso para la construcción de una relación de confianza que se mantuvo durante los siguientes seis años: “eso [obtención del DNI] a él lo habilita y le da cierto nivel de confianza como para seguir adelante con alguna otra transformación” (Entrevista M.A.R., 2022). Las acciones desarrolladas desde la Oficina de Derechos Humanos y Justicia se orientaron no solamente a lograr la plena ejecución de la sentencia del Juzgado Civil y Comercial, sino también a lograr una mejora integral de la calidad de vida de MV quien estaba atravesado por múltiples vulnerabilidades. Así, las acciones se orientaron a la obtención de un nuevo DNI, a la adecuación quirúrgica de sus genitales (mastectomía y faloplastia) pero también a la obtención de una jubilación, una obra social y a fortalecer sus vínculos sociales. Cada uno de estos logros no estuvo desprovisto de dificultades en un periodo que se extendió entre abril de 2012 y mayo de 2018. 169 Luego del DNI, el siguiente paso fue la realización de la mastectomía o extirpación quirúrgica de las mamas. El 18 de abril de 2012 se envió una nota para “realizar una intervención quirúrgica de reasignación de órganos” a un reconocido centro público de salud de la ciudad de Córdoba con amplia trayectoria en cirugías plásticas y estéticas. Luego de la primera entrevista, en foja 5 del expediente de la Oficina de Derechos Humanos y Justicia, puede leerse que “mediando a) autorización del jefe de servicio, b) la realización de estudios prequirúrgicos -incluido examen psiquiátrico- y que todo se encuentre en condiciones, c) que el servicio no se encuentre sobrepasado de pacientes, allí podría llevarse a cabo solo la mastectomía (Oficina de Derechos Humanos y Justicia, 2012-2018, p. 5). Vemos ya desde el inicio los aspectos informales señalados por Waylen (2014). Aunque la nueva ley adopta un enfoque basado en la autonomía de la voluntad y expresamente rechaza las pruebas psiquiátricas, el derecho de MV se subordina al poder médico que exige la prueba como condición prequirúrgica. No bastando con ello, somete el derecho de MV lisa y llanamente a la “autorización del jefe del servicio” y como para que no queden dudas de que el reclamo del actor es de segunda categoría, lo condicionan a “que el servicio no se encuentre sobrepasado de pacientes”. Cabe decir que en la nota y en la entrevista expresamente se había aclarado que la intervención quirúrgica era ordenada por una sentencia firme, para la cual ya se habían realizado las pericias enunciadas anteriormente. Sin embargo, esta no fue la única conducta patologizante por parte del centro de salud: Yo lo acompaño, entramos a una sala donde había de siete a diez personas médicas y médicos, MV y yo, donde a MV se lo intenta someter a una serie de preguntas […] ¿Y usted en qué grado se siente hombre? ¿Y usted qué órganos tiene que se está tapando? Bueno, esa fue la última pregunta, yo dije: listo. Porque aparte preguntaba uno y antes que MV contestara preguntaba otro y aparte el lenguaje gestual que yo advertía se miraban entre ellos y lo miraban a MV una invasión a nivel de lenguaje gestual horrorosa […] ahí fue donde yo dije discúlpenme yo sé que ustedes tienen que tener en su poder los estudios y demás, esto se trata de un primer paso y ustedes tienen todos los elementos por escrito y los estudios por escrito. En ese momento ocurre algo que a mí me paralizó y paralizó a todos los médicos que fue que MV se paró y llorando se abrió la camisa. Yo ahí me le abalancé, le cerré la camisa. Les dije señores, esta charla la seguimos en otro momento, esta persona no se merece esto. (Entrevista M.A.R., 2022) La fecha original de la mastectomía fue reprogramada para que MV presente unos exámenes prequirúrgicos actualizados. Con fecha 29 de agosto de 2012 –ya en vigencia la Ley de Identidad de Género– expresamente le exigieron nuevos aptos psiquiátricos y psicológicos, pese a la reiteración por parte de la Oficina de Derechos Humanos y Justicia de que se trataba de una sentencia firme y que ya se habían realizado ese tipo de pericias. La mastectomía se realizó finalmente en diciembre de 170 2012, es decir, aproximadamente ocho meses después de la primera entrevista con el centro de salud. Luego de su recuperación, MV reunió los documentos necesarios para iniciar sus trámites jubilatorios. Como se expuso previamente, MV nunca tuvo un trabajo registrado debido a la imposibilidad de presentar documentos que representaran su verdadera identidad. A las necesidades de cualquier persona de su edad se sumaba que las cirugías previstas implican largos postoperatorios durante los cuales se vería imposibilitado de trabajar y así obtener los ingresos necesarios para su subsistencia. Con la intervención de la Oficina de Derechos Humanos y Justicia se logró que la ANSES le otorgara el beneficio jubilatorio mediante el sistema de moratorias para trabajadores informales o planes de inclusión previsional (PIP). Una iniciativa que permitió que muchos trabajadores y trabajadoras que, como MV, habían trabajado sin registración accedan a la jubilación (Corsiglia, 2018). MV obtuvo el beneficio el 6 de mayo de 2014 a poco más de un año de haber iniciado el trámite. El recorrido de MV para lograr la ejecución de la sentencia de 2011 fue arduo. La falta de confianza del actor en las instituciones –las que históricamente lo habían excluido como la escuela, el mercado formal de trabajo, los servicios de salud, el Estado en general– se vio reforzada por actitudes como la del equipo médico relatada anteriormente, sumado a resultados físicos desalentadores –la mastectomía implicó otras cirugías posteriores de reconstrucción–, y el desgaste producido por una serie de estudios médicos y psicológicos que lo acompañaron durante todo el proceso. Esto implicó la necesidad de un apoyo emocional. De todos los objetivos planteados, la faloplastia, a cargo de otro centro de salud, el Hospital de Clínicas, fue el que produjo una mayor patologización del actor y registró las mayores dilaciones. Según el expediente, el trámite que oficialmente dio inicio al recorrido para la realización de esta cirugía fue realizado el 3 de diciembre de 2013. El proceso estuvo cargado de dilaciones, estudios médicos que excedían los estudios prequirúrgicos rutinarios y la imposición de una terapia psicológica a cargo de una profesional asociada al equipo que realizaría la cirugía. Estudios que muchas veces fueron realizados ad honorem por la intermediación de la Oficina de Derechos Humanos y Justicia atento a la vulnerabilidad económica del actor y la ausencia de obra social al inicio del trámite. Del expediente surgen también las constantes reprogramaciones de la fecha de la cirugía que aparejaban la nueva realización de estos estudios. Durante los tres últimos años antes de la cirugía el médico le hacía hacer todos los estudios, le hacía tomar hormonas que no le hacían falta porque MV era un hombre, le hacían los chequeos y todo porque ya llegaba la cirugía en noviembre y en octubre le decía no, vamos a tener que pasarlo. Entonces volvíamos a empezar al año siguiente con todo lo que significaba para él volver a la psicóloga volver a hacer los estudios. Bueno, va a ser en diciembre y en diciembre decían, no. Ahí es donde yo intervengo y pido una entrevista con [el médico] y le planteo ¿doctor por qué usted está haciendo esto con este ser humano? llevamos dos años y le llevé la carpeta […] usted lo manda al psicólogo, pero no está 171 viendo lo que usted y su equipo le están ocasionando. (Entrevista M.A.R., 2022) Finalmente, luego de aproximadamente tres años de gestiones en el Hospital de Clínicas, le informaron que la faloplastia no podía realizarse por cuestiones internas del centro de salud y la disolución del equipo encargado de hacerlo. Cabe aclarar que, por un lado, se han encontrado grandes resistencias por parte de profesionales de la salud para llevar a cabo el efectivo cumplimiento de lo dispuesto por el tribunal. Esto ha ocasionado -y de hecho sigue haciendo-, interminables recorridos y esperas en las instituciones, sin que fueran comprendidas las delicadas circunstancias psicofísicas y emocionales del Sr. MV sobre el cual -agravando su condición de vulnerabilidad ya descripta- se suma su edad, corriendo el riesgo de que cuando por fin obtenga las respuestas institucionales, estas no sean ya las adecuadas o lo que es peor las posibles. Por otro costado, también ha intervenido en el retraso de las intervenciones, los inconvenientes que devienen de la carencia de profesionales especializados en este tipo de cirugías, sumados a aquellos que derivan de la falta de insumos -de todo tipo- o de turnos disponibles en los Hospitales. (Oficina de Derechos Humanos y Justicia, 2012-2018, pp. 23-24) Resistencia por parte de profesionales de la salud, el paso del tiempo y la suspensión del goce de derechos fundamentales como lo son el acceso a la salud y el reconocimiento de la identidad, así como la falta de profesionales idóneos e insumos son apenas los puntos más sobresalientes de esta realidad. Así, ante la imposibilidad de realizar la cirugía en Córdoba se comenzaron las gestiones en el único centro de salud público del país en aquel momento para dar respuesta a estas situaciones ubicado en La Plata. La recepción por parte del equipo médico de La Plata fue calificada como buena por parte del personal judicial que acompañaba a MV. Sin embargo, a los desafíos ya conocidos se planteaban nuevos. Entre los primeros estaba la carencia de profesionales formados para realizar este tipo de cirugías lo que se traducía en un tiempo de espera elevado. Entre los nuevos desafíos destacaban el costo del traslado hasta La Plata y el acompañamiento de MV en su postoperatorio. Este último punto fue de especial atención para la Oficina de Derechos Humanos y Justicia quien en reiteradas oportunidades señaló entre las vulnerabilidades del actor la ausencia de redes de apoyo y vínculos sólidos. El rechazo por parte de la familia de origen, así como la marginación educativa y laboral son realidades que afectan a muchas personas trans (Berkins y Fernández, 2005; Ministerio Público de la Defensa de la Ciudad Autónoma de Buenos Aires, 2017). Para lograr conseguir a alguien que acompañe a MV en su estancia en La Plata, la Oficina realizó un trabajo conjunto con la entonces Coordinación Nacional de Diversidad Sexual. Así, finalmente MV logró la realización de la cirugía en mayo de 2018 en La Plata, aproximadamente cuatro años y medio después de la primera entrevista con ese 172 objetivo en el Hospital de Clínicas y nueve años después de que inició el proceso ante el Juzgado Civil y Comercial. Esto es solo un resumen de los puntos más sobresalientes del camino recorrido por MV para lograr ejercer sus derechos, incluso cuando estos habían sido reconocidos por una sentencia firme y cuando en el camino una ley nacional había cambiado el paradigma sobre el reconocimiento de la identidad de género. Antes de finalizar es importante también tener en cuenta las interseccionalidades (Crenshaw, 2017) de MV, las cuales se retroalimentan y quedan muy patentes en el expediente. Solo a modo de ejemplo surge del caso la vulnerabilidad económica del actor (a causa de las lógicas de exclusión ya mencionadas) lo que sumado al desamparo familiar y afectivo lo llevaban a vivir en una pensión donde había sufrido varios robos. MV no tenía donde dejar las prótesis, fíjate el nivel de vulnerabilidad, él vivía en una pensión donde cada dos por tres entraban y le robaban cosas. Entonces yo me llevé las prótesis a mi escritorio… él me entregó toda la documentación que él tenía de su infancia y demás porque no tenía donde guardarla… un entrecruzamiento de vulnerabilidades donde esta gran batalla fue la batalla de su vida. (Entrevista M.A.R., 2022) Entre las múltiples vulnerabilidades de MV se encontraba su edad. Él tenía 62 años al momento de entablar la demanda y 71 años cuando le practicaron la última cirugía. A lo largo del expediente puede verse como la cuestión de la edad era utilizada para cuestionar la validez de su reclamo. porque [el médico] aparte me decía tengo gente muy joven, que lo requiere más. Entonces yo ahí le decía ¿cuál es la medida de la dignidad? Porque una persona no logra constituirse en su dignidad por las razones que sea cuál es la medida que usted pone para la dignidad ¿una cuestión cronológica? […] No importa si tiene sesenta, setenta u ochenta años, está en condiciones físicas, psicológicas y psíquicas ¡vamos para adelante! no es usted quien tiene que establecer ese límite porque acá se trata de la dignidad de un ser humano ¿usted va a poner la medida de la dignidad? (Entrevista M.A.R., 2022) Cabe mencionar que como señala el Protocolo de acceso a justicia para personas mayores del Poder Judicial de Córdoba, la edad cronológica no es una pauta objetiva para adoptar medidas de protección o para restringir derechos (2020, p. 15). Conclusión La historia relatada en estas páginas es una historia de transición. Una transición desde el olvido y la persecución estatal hacia el reconocimiento de la dignidad humana. Una transición desde un modelo patologizador a un modelo de derechos humanos. Una transición hacia una sociedad más justa que respete el plan de vida de 173 cada persona y que valore las vivencias de cada una por sobre la injerencia de voluntades externas. Para contar esta historia se ha optado, en una primera parte, por dar un panorama normativo, en el que se ha desarrollado el marco legal argentino, su evolución y se han explicado algunos ejemplos a nivel de derecho comparado. La diferencia entre el paradigma patologizador y el paradigma de derechos humanos ha explicado cada ejemplo. En una segunda parte, nos hemos adentrado en el caso de MV, por un lado, para mostrar cómo era el razonamiento judicial anterior a la sanción de la Ley 26.743 y del cambio de paradigma. El fallo del Juzgado Civil y Comercial se apoya en testimonios y diferentes pruebas médicas y psicológicas. El razonamiento del tribunal construye un “sujeto transexual” al que entiende como un sujeto que sufre, que está “atrapado” en un cuerpo que no siente propio, que ese cuerpo es del “sexo opuesto” (binario) y que esta es una condición irreversible. Por tal, la finalidad del fallo es liberar y sanar a esta persona para que lleve “una vida lo más normal posible”. En 2012, Argentina sancionó la Ley de Identidad de Género 26.743, que adoptó un modelo completamente opuesto al reflejado en el fallo. La identidad de género es entendida como la vivencia del género tal como cada persona la siente, lo que implica que el abanico de posibilidades es enorme. Se reconoce el derecho al libre desarrollo de la personalidad y la autonomía para elegir el propio plan de vida sin injerencias externas y se prohíben las pruebas médicas y psicológicas para el reconocimiento de la identidad de género. Este cambio de modelo o paradigma, sin embargo, tiene algunos matices. El caso de MV muestra como las prácticas patologizantes se extendieron ya con la Ley de Identidad de Género vigente y como eran ejercidas sin miramientos por diferentes –no todos– actores médicos. Es lo que Waylen (2014) llama del aspecto informal de las instituciones que subvierte el cambio institucional que en nuestro caso trajo la Ley 26.743. El ejercicio del poder dentro de las instituciones siguió –y sigue– en la práctica subordinando la identidad de las personas a injerencias externas. Tener en cuenta este aspecto informal es sumamente relevante en Estados como Argentina, donde a simple vista el marco normativo parece sumamente abierto y receptivo. Por un lado, tenemos una avanzada legislación en materia de identidad de género (Ley 26.743), la posibilidad de tener marcadores de género fuera del binomio M/F en los documentos de identidad (Dto. 476/21) una ley de inclusión laboral trans (27.636) y, sin embargo, acceder a prácticas médicas, a la salud integral, al trabajo formal o a una vida libre de violencia está lejos de ser una realidad de las personas trans en Argentina. Es en este punto que debe ponerse atención ahora para garantizar el acceso a justicia de las personas trans en particular, y LGBTIQ+ en general. Se hace necesario advertir cuáles son las prácticas institucionales, cotidianas, no escritas que subvierten la legislación vigente y restringen el acceso a justicia y a otros derechos básicos. Esto marca la necesidad de adoptar otras estrategias de control, de capacitación y sensibilización, así como protocolos de actuación institucionales que acompañen a las reformas legislativas para que estas no se conviertan en letra muerta. 174 Bibliografía Agencia Presentes. (2019, 1 de marzo). Por primera vez, un documento de identidad dirá «femineidad travesti». Presentes. Periodismo de Género. https://agenciapresentes.org/2019/03/01/porprimera-vez-un-documento-de-identidad-dira-femeinidad-travesti/ American Psychiatric Association. (2014). DSM-5: Manual diagnóstico y estadístico de los trastornos mentales. Editorial Médica Panamericana. Arias, M. (2019, 17 de diciembre). Ushuaia: la Justicia ordenó que se confeccione un DNI "no-binarioigualitario". La Nación. https://www.lanacion.com.ar/sociedad/ushuaia-justicia-ordeno-seconfeccione-dni-no-binario-igualitario-nid2316665/ Berkins, L. y Fernández, J. (2005). La gesta del nombre propio. Informe sobre la situación de la comunidad travesti en la Argentina. Asociación Madres de Plaza de Mayo. Bertolini, Lara María c/ Ministerio del Interior OP y V s/Información Sumaria, 48756/2018 (Juzgado Nacional en lo Civil N.º 7 01 de marzo de 2019). Cabral, M. (2003). Ciudadanía (trans) sexual. Proyecto sexualidades, salud y derechos humanos en América Latina. Corsiglia, L. (2018). Las mujeres, el derecho a la jubilación y las disputas en torno a los sentidos de los legítimo. Revista Escenarios, 28, 1-14. Crenshaw, K. (2017). On intersectionality: Essential writings. The New Press. De la Rosa, I. (2021, 21 de julio). La historia de Gero Caro, la primera persona de Mendoza en recibir su DNI no binario. Los Andes. https://www.losandes.com.ar/sociedad/la-historia-de-gero-caro-laprimera-persona-de-mendoza-en-recibir-su-dni-no-binario/ Farji Neer, A. (2015). Cuerpo, derechos y salud integral: Análisis de los debates parlamentarios de las leyes de Identidad de Género y Fertilización Asistida (Argentina, 2011-2013). Salud Colectiva, 11, 351-365. Federación Argentina LGBT. (2008). Informe sobre códigos contravencionales y de faltas de las provincias de la República Argentina y la Ciudad Autónoma de Buenos Aires en relación con la discriminación y la represión a gays, lesbianas, bisexuales y trans. García de Solavagione, A., Moisset de Espanés, L., Palmero, J. C., Méndez Costa, M. J. y Trigo Represas, F. (2013). El estatuto de los vicios del consentimiento matrimonial y la contradicción con las leyes relativas a la identidad de género. Gobierno de Mendoza. (2018, 2 de noviembre). Solicitud de partida de nacimiento sin consignación de sexo dentro del marco de la Ley de Identidad de Género 26743. Prensa del Gobierno de Mendoza. https://www.mendoza.gov.ar/prensa/solicitud-de-partida-de-nacimiento-sin-consignacion-desexo-dentro-del-marco-de-la-ley-de-identidad-de-genero-lig-26-743/ Lamm, E. (2018). Identidad de género. Sobre la incoherencia legal de exigir el sexo como categoría jurídica. Actualidad Jurídica Iberoamericana, 8, 230-278. Litardo, E. (2018). La identidad de género en América: aproximaciones para una teoría democrática del género. Sistemas judiciales: una perspectiva sobre la administración de justicia, 18(22), 45-66. Mahoney, J. y Thelen, K. (Eds.) (2010). Explaining institutional change: Ambiguity, agency, and power. Cambridge University Press. Ministerio Público de la Defensa de la Ciudad Autónoma de Buenos Aires. (2017). La revolución de las mariposas. A diez años de La Gesta del Nombre Propio. https://www.mpdefensa.gob.ar/sites/default/files/la_revolucion_de_las_mariposas.pdf Oficina de Derechos Humanos y Justicia 2012-2018. Expediente Oficina de Derechos Humanos y Justicia, MV 04-2012. Ojeda, C. (2020, 26 de febrero). Es no binarie, cambió su partida y lucha por su DNI. Presentes. Periodismo de Género. https://agenciapresentes.org/2020/02/26/es-no-binarie-cambio-supartida-y-lucha-por-su-dni/ 175 Pizzarro, O. (2021, 14 de abril). Abuso policial. Detención discriminatoria por parte de la policía de Córdoba. La Izquierda Diario. https://www.laizquierdadiario.com/Detencion-discriminatoria-porparte-de-la-policia-de-Cordoba Poder Judicial de la Provincia de Córdoba. (2020). Protocolo de actuación para el acceso a justicia para personas mayores. Córdoba. Registro Nacional de las Personas. (2022). A 10 años de la Ley de Identidad de Género, más de 12 mil personas cambiaron su identidad autopercibida en el DNI. https://www.argentina.gob.ar/noticias/10-anos-de-la-ley-de-identidad-de-genero-mas-de-12mil-personas-cambiaron-su-identidad Sentencia 482, 5094686 - MDCV - ORDINARIO - OTROS (JUZG 1A INST CIV COM 6A NOM 16 de septiembre de 2011). Waylen, G. (2014). Informal institutions, institutional change, and gender equality. Political Research Quarterly, 67(1), 212-223. DOI: 10.5281/zenodo.8317268 176
https://openalex.org/W2804478446	https://digital.csic.es/bitstream/10261/189042/1/document.pdf	English	null	Effect of copper-enriched layers on localized corrosion of aluminium-copper alloys	Revista Facultad de Ingeniería/Revista Facultad de Ingeniería	2,018	cc-by	5,021	Abstract Copper-enriched layers were developed onto aluminum-copper alloys using alkaline etching in sodium hydroxide, for both, sputter deposited and bulk conditions. Enriched alloys were evaluated by potentiodynamic polarization in sodium chloride solution in order to determine the effect of the enriched layers on the pitting potential of the alloys. Rutherford backscattering spectroscopy was employed to quantify the enrichments and their locations just beneath the alumina-based oxides remaining from the etching. For the sputter deposited aluminum-copper alloys, the results show some scattering of the pitting potential data, and no correlation between pitting potential and the alloy enriched layer. In the case of bulk Al-2wt.%Cu alloy, with the copper in solid-solution, the pitting potential increased for the enriched specimens, indicating also a different pit morphology, with respect to the non-enriched alloy. Keywords: aluminium-copper alloys; anodizing; copper enrichment; pitting. Efeito das camadas de enriquecimento de cobre sobre corrosão localizada em ligas alumínio-cobre Efeito das camadas de enriquecimento de cobre sobre corrosão localizada em ligas alumínio-cobre María Ángeles Arenas-Vara* Peter Skeldon Sandra Judith García-Vergara* Fecha de recepción: 17 de enero de 2018 Fecha de aprobación: 6 de abril de 2018 * Ph.D. Centro Nacional de Investigaciones Metalúrgicas – CENIM (Madrid, España). geles@cenim.csic.es. Ph.D. University of Manchester (Manchester, Reino Unido). peter.skeldon@manchester.ac.uk. * Ph.D. Universidad Industrial de Santander (Bucaramanga-Santander, Colombia). sagarver@uis.edu.co. ORCID: 0000-0002-4480-6255. Resumen Fueron desarrolladas capas de enriquecimiento de cobre mediante ataque alcalino en hidróxido de sodio sobre aleaciones aluminio-cobre. Se usaron aleaciones tanto depositadas por pulverización catódica, como vaciadas convencionalmente. Las aleaciones con las capas de enriquecimiento de cobre fueron estudiadas mediante polarización potenciodinámica en una solución de cloruro de sodio, para poder determinar si existe alguna correlación entre las capas de enriquecimiento de cobre y el potencial de picadura de las aleaciones. Los resultados no son concluyentes en el caso de las aleaciones depositadas por pulverización catódica; sin embargo, en el caso de la aleación en condición de vaciado, con el cobre en solución sólida, el potencial de picadura aumenta por la presencia de la capa de enriquecimiento, mostrando además una morfología diferente. Palabras clave: aleaciones aluminio-cobre; anodizado; enriquecimiento de cobre; potencial de * Ph.D. Centro Nacional de Investigaciones Metalúrgicas – CENIM (Madrid, España). geles@cenim.csic.es. Ph.D. University of Manchester (Manchester, Reino Unido). peter.skeldon@manchester.ac.uk. * Ph.D. Universidad Industrial de Santander (Bucaramanga-Santander, Colombia). sagarver@uis.edu.co. ORCID: 0000-0002-4480-6255. Revista Facultad de Ingeniería (Rev. Fac. Ing.) Vol. 27 (48), pp. 7-15. Mayo-Agosto, 2018. Tunja-Boyacá, Colombia ISSN Impreso 0121-1129, ISSN Online 2357-5328, DOI: http://doi.org/10.19053/01211129.v27.n48.2018.8016 7 Effect of copper-enriched layers on localized corrosion of aluminium-copper alloys Revista Facultad de Ingeniería (Rev. Fac. Ing.) Vol. 27 (48), pp. 7-15. Mayo-Agosto, 2018. Tunja-Boyacá, Colombia. Para citar este artículo: M. A. Arenas-Vara, P. Skeldon, and S. J. García-Vergara, “Effect of copper-enriched layers on localized corrosion of aluminium-copper alloys,” Revista Facultad de Ingeniería, vol. 27 (48), pp. 7-15, may. 2018. A. Sample preparation and characterization Layers of Al-0.95at.%Cu alloy were prepared using an Atom Tech Ltd magnetron sputtering system with separate 99.999% aluminium and 99.95% copper targets. The alloy was deposited onto electropolished high purity aluminium substrates. The chamber was firstly evacuated to 6×10-7 mbar, and then deposition was carried out in 99.998% argon at 5.5 ×10-3 mbar. The substrates were attached to a large copper disk, whose temperature increased to 305 K during deposition of the alloys. Another set of experiments was carried out using heat-treated Al-2wt.%Cu alloy, with the copper in solid solution. Specimens of the alloy were heated to 550 °C for 3 h and then quenched in water. Rectangular pieces of the alloy were mounted in standard 00 size BEEM capsules, which were then filled with an epoxy resin. The filled capsule was cured in an oven at 60 °C for 48 h. Following the procedure used to prepare the samples for TEM by ultramicrotomy, the hardened resin blocks containing the specimens were trimmed using a glass knife in a Leica Ultracut UCT ultramicrotome. The final area was about 0.20 cm2. This procedure was used to avoid inclusions or contamination by the normal polishing procedures. A high purity aluminum specimen was also prepared using the previous procedure. In almost all applications of aluminum alloys, the fabricated alloys are pretreated and surface finished, thereby providing a functionalized surface. Etching, chemical polishing, acid picking, electropolished and anodized pretreatments of copper containing aluminum alloys normally results in the formation of a thin layer of alloy, about 2 to 4 nm thick and highly enriched in copper, immediately beneath the residual or anodic alumina film due to the less negative Gibbs free energy per equivalent for formation of copper oxide compared with that of alumina [3-6]. Further, for anodic oxidation of the alloys, once a steady enrichment of about 40 at% Cu, equivalent to about 5.4×1019 Cu atoms m-2, is achieved, oxidation of copper proceeds and copper species are incorporated into the film at the alloy/film interface, in the alloy proportions [6]. The presence of copper in the anodic film leads to the generation of oxygen gas within the anodic films, resulting in oxygen gas containing bubbles at high pressure, consequently, introducing lateral porosity to the porous anodic films formed on copper-containing aluminum alloys [7]. Resumo Foram desenvolvidas camadas de enriquecimento de cobre mediante ataque alcalino em hidróxido de sódio sobre ligas alumínio-cobre. Usaram-se ligas tanto depositadas por pulverização catódica, como esvaziadas convencionalmente. As ligas com as camadas de enriquecimento de cobre foram estudadas mediante polarização potenciodinâmica em uma solução de cloreto de sódio, para poder determinar se existe alguma correlação entre as camadas de enriquecimento de cobre e o potencial de picadura das ligas. Os resultados não são concluintes no caso das ligas depositadas por pulverização catódica; porém, no caso da liga em condição de esvaziado, com o cobre em solução sólida, o potencial de picadura aumenta pela presença da camada de enriquecimento, mostrando além disso, uma morfologia diferente. Palavras chave: ligas alumínio-cobre; anodizado; enriquecimento de cobre; potencial de picadura. Para citar este artículo: Para citar este artículo: M. A. Arenas-Vara, P. Skeldon, and S. J. García-Vergara, “Effect of copper-enriched layers on localized corrosion of aluminium-copper alloys,” Revista Facultad de Ingeniería, vol. 27 (48), pp. 7-15, may. 2018. 8 María Ángeles Arenas-Vara - Peter Skeldon - Sandra Judith García-Vergara I. Introduction correlation between the enriched alloy layer and the pitting potential of aluminium copper alloys. Aluminum-copper alloys are widely used in aerospace and automotive industries due to their high strength to weight ratio and damage tolerance. These properties result from alloying with copper as the principal alloying element and appropriate thermomechanical processing. However, copper also has significant influence on the corrosion behaviour of the alloys. Copper-containing intermetallic particles often form microgalvanic couples with the surrounding alloy, consequently, increasing the susceptibility of the alloys to localized attack [1, 2]. A. Enrichment of copper developed by alkaline etching Specimens of freshly deposited and anodic etched alloy were sectioned to a nominal thickness of ~10 nm by ultramicrotomy and examined by Transmission electron microscopy TEM using a JEOL FX 200 II instrument. Table 1 lists the enrichments of copper from RBS and the expected amount of copper in the enriched layer generated in the Al-0.95at.%Cu alloy by alkaline etching in sodium hydroxide for 12 to 75 s. The average compositions of the copper enriched layer are also given assuming a layer of 2 nm in thickness with a weighted average of the atomic densities of aluminium and copper. Only for the longest time does copper oxidize during etching. The atomic density of the outer layer film material used in the simulations was 0.92 ´ 1023 atoms cm-3, corresponding to that of anodic alumina of density 3.1 g cm-3. The RBS analysis shows that the copper enrichment increases as the time of anodic etching increases. A. Sample preparation and characterization The copper-enriched layer were generated, for both type of samples, by anodic etching at a constant current density of 5 mA cm-2 in 0.1 M sodium hydroxide solution at 293 K by different times. Individual specimens were then connected, as the anode, to the power supply (METRONIX 6911 DC) and a stainless- steel sheet was connected as a cathode. The voltage- time responses of the anodic etching were recorded on a PC, with a PICOLOG recorder program. After the anodic etching, the specimens were quickly rinsed with deionized water and dried in cool air stream. The specimens were then immersed for 30 s in 15% nitric acid, to remove any smut layer, rinsed with deionized water and dried in a cool air stream. Many studies have been devoted to the electrochemical behaviour of aluminium alloys in sodium chloride solutions [1-3]. All these studies reveal that the exposure of aluminium alloys to chloride ions results in localized corrosion (pitting corrosion). Pitting is the most common form of corrosion for aluminium and its alloys. Of the common electrochemical testing methods, potentiodynamic polarisation allows determining pitting susceptibility by revealing the pitting potential (Ep). Features like, inclusions, intermetallic particles (coarse or fine), surface roughness and electrochemical potential are factors that influence the nucleation and growth of metastable pits [8]. The aim of this paper is to study if there is a The composition of the sputter deposited alloy and the specimens with the copper enriched layers were determined by Rutherford backscattering Revista Facultad de Ingeniería (Rev. Fac. Ing.) Vol. 27 (48), pp. 7-15. Mayo-Agosto, 2018. Tunja-Boyacá, Colombia. 9 Effect of copper-enriched layers on localized corrosion of aluminium-copper alloys saturated calomel reference electrode (SCE). The data were recorded using a potentiostat-galvanostat EG&G 264, Solartron connected to a computer, using the Corr software. spectroscopy (RBS) [9], using 2.00-2.5 MeV He+ ions produced by the 2.5 MeV Van de Graaff accelerator of the University of Paris. The beam current and the bean diameter were 60 nA and 0.5 mm, respectively, with the beam incident normal to the specimen surface. The scattered particles were detected at 165° to the direction of the incident bean. The data were interpreted using the RUMP program [10]. III. Results B. Corrosion test Further, the curves suggest an slightly in- creased corrosion current density (icorr) of 3.5 ´ 10-7 A cm-2 following 43 s of etching, when the enrichment is about 4.5 ´ 1015 Cu atoms cm-2 compare with the non-enriched alloy. The current density subsequently increases due to pitting. However, identification of the pitting potential (Ep) was hindered by absence of a clearly defined change in behaviour, which may relate to the use of thin alloy layers. Ep is here considered as the potential at which the current starts to increase rapidly above the background passive current [11]. Generally, the Al-Cu alloy appears to disclose an in- crease in the pitting potential in comparison with that of aluminium. This behaviour is in general agreement with previous work [12]. -1065 mV for the as-deposited alloy, -972 mV and -896 mV following 12 and 43 s of alkaline etching, respectively. Further, the curves suggest an slightly in- creased corrosion current density (icorr) of 3.5 ´ 10-7 A cm-2 following 43 s of etching, when the enrichment is about 4.5 ´ 1015 Cu atoms cm-2 compare with the non-enriched alloy. The current density subsequently increases due to pitting. However, identification of the pitting potential (Ep) was hindered by absence of a clearly defined change in behaviour, which may relate to the use of thin alloy layers. Ep is here considered as the potential at which the current starts to increase rapidly above the background passive current [11]. Generally, the Al-Cu alloy appears to disclose an in- crease in the pitting potential in comparison with that of aluminium. This behaviour is in general agreement with previous work [12]. Revista Facultad de Ingeniería (Rev. Fac. Ing.) Vol. 27 (48), pp. 7-15. Mayo-Agosto, 2018. Tunja-Boyacá, Colombia. B. Corrosion test Potentiodynamic polarization curves of the aluminium- copper alloys, enriched aluminium-copper alloys and high purity aluminium were recorded in de-aerated 0.1 M sodium chloride solution at 293 K at a scan rate of 0.2 mV s-1. The working area, defined with Lacomite, was about 0.2-1.0 cm2, for the sputter deposited and bulk alloys, respectively. A three-electrode cell was employed, with a platinum counter electrode and a Table 1 Results of RBS analyses of Al-0.95at.% Cu alloy after etching at 5 mA cm-2 in 0.1 M sodium hydroxide solution at 293 K Time of etching (s) Enrichment in alloy (Cu atoms cm-2) Average composition of enriched layer (at.%Cu) RBS Expected 12 1.2 x 1015 1.2 x 1015 10 22 2.1 x 1015 2.1 x 1015 18 43 4.5 x 1015 4.6 x 1015 37 75 6.5 x 1015 7.9 x 1015 55 Table 1 Results of RBS analyses of Al-0.95at.% Cu alloy after etching sodium hydroxide solution at 293 K to within ~ 8 nm, which are usual for the sputtering conditions. In the presented example, etching for 75 s reduced the alloy thickness by 148 nm, in reasonable agreement with the calculated reduction of 132 nm from the time of etching. to within ~ 8 nm, which are usual for the sputtering conditions. In the presented example, etching for 75 s reduced the alloy thickness by 148 nm, in reasonable agreement with the calculated reduction of 132 nm from the time of etching. Figure 1 shows typical transmission electron micro- graphs of the Al-0.95at.%Cu alloy following deposi- tion and after 75 s of etching in sodium hydroxide. The alloy is columnar-grained, with a typical grain width of ~ 40 nm, and the selected surface was locally flat Revista Facultad de Ingeniería (Rev. Fac. Ing.) Vol. 27 (48), pp. 7-15. Mayo-Agosto, 2018. Tunja-Boyacá, Colombia. 10 María Ángeles Arenas-Vara - Peter Skeldon - Sandra Judith García-Vergara Fig. 1. Transmission electron micrographs of the Al-0.95at.%Cu alloy following (a) deposition and (b) etching for 75 s at 5 mA cm-2 in 0.1 M sodium hydroxide solution at 293 K. Fig. 1. Transmission electron micrographs of the Al-0.95at.%Cu alloy following (a) deposition and (b) etching for 75 s at 5 mA cm-2 in 0.1 M sodium hydroxide solution at 293 K. -1065 mV for the as-deposited alloy, -972 mV and -896 mV following 12 and 43 s of alkaline etching, respectively. B. Potentiodynamic polarization behaviour in sodium chloride solution 1) Sputtering-deposited Al-0.95at.%Cu alloy. Figure 2 shows the potentiodynamic polarization curves of Al-0.95at.%Cu alloy in de-aerated sodium chloride solution at 298 K, following alkaline etching. Indi- vidual specimens were immersed for 120 min prior to polarization. The potential was scanned at 0.2 mV s-1 starting at about 200 mV below the corrosion potential. The cathodic current density, due to decomposition of the water, does not show significant changes with the alloy enrichment, exhibiting values of 0.01–0.02 mA cm-2. The corrosion potential increases as the etching time increases, due to the presence of the copper-en- riched alloy layer, being the corrosion values about 11 Effect of copper-enriched layers on localized corrosion of aluminium-copper alloys Fig. 2. Potentiodynamic polarization curves, measured at a scan rate of 0.2 mV s-1, for Al-0.95at.%Cu alloy in de- aerated 0.1 M sodium chloride solution at 293 K. The curves were measured for the as-deposited alloy, and following etching of the alloy for 12 and 43 s at 5 mA cm-2 in 0.1 M sodium hydroxide solution at 293 K. Fig. 2. Potentiodynamic polarization curves, measured at a scan rate of 0.2 mV s-1, for Al-0.95at.%Cu alloy in de- aerated 0.1 M sodium chloride solution at 293 K. The curves were measured for the as-deposited alloy, and following etching of the alloy for 12 and 43 s at 5 mA cm-2 in 0.1 M sodium hydroxide solution at 293 K. Then, considering the results for the sputtering-depos- ited Al-0.95at.%Cu alloy in which copper does not oxidize, there is an increase in the corrosion potential by about 160 mV vs. SCE with enrichment by 4.5 ´ 1015 Cu atoms cm-2. The influence of the enriched al- loy layer on the free corrosion potential of aluminium alloys has been reported previously [13]. The present results confirm the key role of the copper enrichment in determining the corrosion potential of aluminium alloys. Further, the cathodic current density increases in the presence of the enriched alloy layer, suggest- ing changes in the cathodic kinetics. The pitting po- tential, however, is not well defined, probably due to the relatively low thickness of the initial alloy layers, which is further reduced by the anodic etching in sodi- um hydroxide solution. B. Potentiodynamic polarization behaviour in sodium chloride solution Although the thin layers have the advantage that surface preparation is not required prior to anodic etching, unlike bulk alloys that require mechanical treatment to generate a non-enriched ini- tial surface, this is offer by their low thickness. Figure 3. The polarization curve of high pure alumin- ium is also included for comparison. The curves dis- play a positive shift of the corrosion potential, as the enrichment of copper increases; however, the magni- tude is smaller compared with that observed in Figure 2, for sputtering-deposited aluminium-copper alloy. The anodic region of Al-Cu specimens presents pas- sive current densities in the range typically between 0.3–6 mA cm2, with pitting subsequently commencing at potentials of about -600 mV vs. SCE. Pure alumini- um exhibits a passive current density of about 0.4 mA cm-2 and a pitting potential of about -689 mV vs. SCE. After this point, the current density increases rapidly due to the pitting corrosion. Compared with the pre- vious results for the sputtering-deposited alloy, the pitting potential is clearly distinguished for the bulk alloy. Further, measurements performed in triplicate for each specimen, show a very good reproducibility. The enrichment measured by RBS reveals a level of about 4.0 ´ 1015 Cu atoms cm-2 after 41 s of etching. However, no particular difference in the pitting poten- tial is displayed between specimens in the as-received and alkaline-etched conditions, except for the possibly formation of metastable pits on the specimen in the as-received condition. Figure 3. The polarization curve of high pure alumin- ium is also included for comparison. The curves dis- play a positive shift of the corrosion potential, as the enrichment of copper increases; however, the magni- tude is smaller compared with that observed in Figure 2, for sputtering-deposited aluminium-copper alloy. The anodic region of Al-Cu specimens presents pas- sive current densities in the range typically between 0.3–6 mA cm2, with pitting subsequently commencing at potentials of about -600 mV vs. SCE. Pure alumini- um exhibits a passive current density of about 0.4 mA cm-2 and a pitting potential of about -689 mV vs. SCE. After this point, the current density increases rapidly due to the pitting corrosion. Compared with the pre- vious results for the sputtering-deposited alloy, the pitting potential is clearly distinguished for the bulk alloy. Further, measurements performed in triplicate for each specimen, show a very good reproducibility. B. Potentiodynamic polarization behaviour in sodium chloride solution The enrichment measured by RBS reveals a level of about 4.0 ´ 1015 Cu atoms cm-2 after 41 s of etching. However, no particular difference in the pitting poten- tial is displayed between specimens in the as-received and alkaline-etched conditions, except for the possibly formation of metastable pits on the specimen in the as-received condition. 2) Bulk aluminium-copper alloy. The potentiodynam- ic polarization curves of the bulk Al-2wt.%Cu alloy, in both the as-received and alkaline-etched conditions, in de-aerated sodium chloride solution is presented in Revista Facultad de Ingeniería (Rev. Fac. Ing.) Vol. 27 (48), pp. 7-15. Mayo-Agosto, 2018. Tunja-Boyacá, Colombia. 12 María Ángeles Arenas-Vara - Peter Skeldon - Sandra Judith García-Vergara Fig. 3. Potentiodynamic polarization curves, measured at a scan rate of 0.2 mV s-1, for Al-2wt.%Cu alloy in de- aerated 0.1 M sodium chloride solution at 293 K. The curves were measured for high pure aluminium, for as-received Al-2wt.%Cu alloy, and following etching of the alloy for 41 s at 5 mA cm-2 in 0.1 M sodium hydroxide solution at 293 K. Fig. 3. Potentiodynamic polarization curves, measured at a scan rate of 0.2 mV s-1, for Al-2wt.%Cu alloy in de- aerated 0.1 M sodium chloride solution at 293 K. The curves were measured for high pure aluminium, for as-received Al-2wt.%Cu alloy, and following etching of the alloy for 41 s at 5 mA cm-2 in 0.1 M sodium hydroxide solution at 293 K. Pitting corrosion is considered to be one of the princi- pal mechanisms for damage of high-strength alumin- ium alloys. Pitting corrosion occurs in the presence of aggressive anionic species, and chloride ions are usually, although not always, the cause. Chloride is an anion of a strong acid, and many metal cations exhib- it considerable solubility in chloride solutions [12]. A critical factor in pitting corrosion is that the phenom- enon is considered to be autocatalytic in nature; once a pit starts to grow, the conditions developed are such that further pit growth is promoted. The resistance to pitting for pure metals is usually measured in terms of the pitting potential, Ep, which characterizes the elec- trochemical stability of the passive film [14]. pitting potential was increased by about 200 mV as the concentration of copper increased to 5 wt.%, with respect to aluminium. B. Potentiodynamic polarization behaviour in sodium chloride solution Enrichment of the alloying ele- ments at the alloy/alumina film interface in aluminium alloys has been observed as a consequence of several surface treatments, including electropolishing [17]. Accordingly, the enrichment of copper was present in the specimens examined by Galvele et al., and the observation regarding an improved resistance to pit- ting corrosion could reflect on the influence of this en- riched alloy layer. Following their observations, a pos- itive effect of the enriched alloy layer was expected in the present study. The pitting potential of the bulk alu- minium-copper alloy increases in about 100 mV com- pared with aluminium. However, the potentiodynam- ic polarization curves show that the pitting potential for the specimens in as-received and alkaline etched conditions, exhibits approximately the same value around -600 mV vs. SCE. The thickness of enriched alloy layer is about 2–2.5 nm and the layer is probably consisted of copper-rich clusters, which are of size of the order of nanometres. Pits can penetrate the alloys in a fraction of seconds, and the effect of the enriched alloy layer cannot be detected. From the present obser- vations, it is possible to conclude that the enrichment of copper in the alloy is not a key factor in the pitting corrosion of aluminium alloys. On the other hand, pits A better approach to understanding the effect of the alloy enrichment on the pitting potential was achieved using a bulk Al-2wt.%Cu alloy. Since the aluminium is a reactive metal, the homogeneous addition of al- most any metal (except magnesium and zinc) to al- uminium results in an increase in pitting potential, provided that the structure remains single phase [15, 16]. Galvele et. al. reported that the pitting potential of aluminium-copper alloys increased with copper con- centration as long as copper was in solid solution [12]. They examined electropolished aluminium-copper al- loys with copper content between 1 to 33 wt.%. The Revista Facultad de Ingeniería (Rev. Fac. Ing.) Vol. 27 (48), pp. 7-15. Mayo-Agosto, 2018. Tunja-Boyacá, Colombia. 13 Effect of copper-enriched layers on localized corrosion of aluminium-copper alloys in aluminium alloys are typically associated with in- termetallic particles [18, 19]. For aluminium-copper alloys, the presence of the intermetallic q-phase (Al- 2Cu) reduces the resistance to pitting at levels similar to those achieved in aluminium alloyed with little cop- per [20]. B. Potentiodynamic polarization behaviour in sodium chloride solution In the commercial alloys, pitting corrosion is normally associated with the presence of two constitu- ent particles, Al-Cu-Mn-Fe and Al-Cu-Mg containing particles [21]. Recently it was found that intergranular corrosion occurs first and crystallographic pitting ini- tiates from the crevice wall behind the intergranular corrosion front of aluminium-copper alloys exposed to chloride solutions. Due to copper enrichment along the pit walls, the crystallographic pits in the AA2024 alloy are characterized with relatively irregular pit walls on a much finer dimension compared to the well-defined half-cube shape crystallographic pits in pure aluminium [22]. The present results confirm the influence of the enrichment of the alloying elements in the corrosion behaviour of surface-treated aluminium alloys. Also, they suggested that further investigation is necessary to clarify the behaviour of alloy enriched aluminium alloys in chloride solutions. Electrochem. Soc., vol. 161, pp. C535-C543, Dec. 2014. DOI: https://doi.org/10.1149/2.0361412jes. [2] [2] N. Birbilis, and R. G. Buchheit, “Electrochemical Characteristics of Intermetallic Phases in Aluminum Alloys,” J. Electrochem. Soc., vol. 152, pp. B140-B151, Apr. 2015. DOI: https://doi. org/10.1149/1.1869984. [3] [3] H. H. Strehblow, and C. J. Doherty, “Examination of aluminum copper films during anodic oxidation,” J. Electrochem. Soc., vol. 125, pp. 30–33, Jan. 1978. DOI h //d i /10 1149/1 2131392 DOI: https://doi.org/10.1149/1.2131392. [4] [4] M. A. Paez, T. M. Foong, C.T. Ni, G.E. Thompson, K. Shimizu, H. Habazaki, P. Skeldon, and G.C. Wood, “Barrier-type anodic film formation on an Al- 3.5 wt% Cu alloy,” Corros. Sci., vol. 38 (1), pp. 59- 72, Jan.1996. [5] [5] X. Zhou, H. Habazaki, K. Shimizu, P. Skeldon, G.E. Thompson, and G.C. Wood, “Copper enrichment in Al–Cu alloys due to electropolishing and anodic oxidation,” Thin Solid Films, vol. 293 (1-2), pp. 327– 332, Jan. 1997. DOI: https://doi.org/10.1016/S0040- 6090(96)09117-1. [6] Y. Liu, E. A. Sultan, E. V. Koroleva, P. Skeldon, G. E. Thompson, X. Zhou, K. Shimizu, and H. Habazaki, “Grain orientation effects on copper enrichment and oxygen generation during anodizing of an Al-1 at.% Cu alloy,” Corros. Sci., vol 45(4), pp. 789–797, Apr. 2003. DOI: https://doi.org/10.1016/S0010- 938X(02)00126-9. IV. Conclusions [7] Y. Ma, X. Zhou, G. E. Thompson, M. Curioni, T. Hashimoto, P. Skeldon, P. Thomson, and M. Fowles, “Anodic film formation on AA 2099-T8 aluminium alloy in tartaric-sulphuric acid,” J. Electrochem. Soc., vol. 158 (2), pp. C17–C22, Feb. 2011. DOI: https:// doi.org/10.1149/1.3523262. The corrosion potential of an alkaline-etched, sol- id-solution Al-0.95at.%Cu alloy or bulk Al-2wt.%Cu, in de-aerated sodium chloride solution moves to more noble values as the level of copper enrichment in the alloy increases. The latter is determined by the extent of prior etching. The potential shift is more relevant for Al-0.95at.%Cu alloy after etching, about 160 mV as the enrichment rises to 4.5 ´ 1015 copper atoms cm-2. Additionally, the absence of a clearly defined pitting potential of the sputtering deposited aluminium-cop- per alloys is related to the thickness of the alloy lay- ers, suggesting that pit penetration of the alloy layers is very fast, and that the influences of the aluminium substrate cannot be avoided. Finally, the copper-en- riched layer did not show a significant effect on the pitting potential, either in the as-deposited alloy or in the bulk alloy, suggesting that it is not a critical factor determining the resistance to pitting. [8] [8] R. K. Gupta, N. L. Sukiman, M. K. Cavanaugh, B. R. W. Hinton, C. R. Hutchinson, and N. Birbilis, “Metastable pitting characteristics of aluminium alloys measured using current transients during potentiostatic polarization,” Electrochim. Acta, vol. 66, pp. 245–254. Apr. 2012. DOI: https://doi. org/10.1016/j.electacta.2012.01.090. [9] [9] J. Pierrière, “Rutherford backscattering spectrometry,” Vacuum, vol. 37 (5-6), pp. 429-432, May. 1987. DOI: https://doi.org/10.1016/0042- 207X(87)90327-7. [10] L. R. Doolittle, “Algorithms for the rapid simulation of Rutherford backscattering spectra,” Nucl. Instrum. Meth.B, vol. 9(1), pp. 344-351, Jun. 1985. DOI: https://doi.org/10.1016/0168-583X(85)90762-1. p g ( ) [11] G. S. Frankel, J. R. Scully, and C. V. Jahnes, “Repassivation of Pits in Aluminum Thin Films,” J. Electrochem. Soc., vol. 143 (6), pp. 1834-1840, Jun. 1996. DOI: https://doi.org/10.1149/1.1836912. References [1] M. K. Cavanaugha, J.-C. Lia, N. Birbilis, and R. G. Buchheit, “Electrochemical Characterization of Intermetallic Phases Common to Aluminum Alloys as a Function of Solution Temperature,” J. [12] J. R. Galvele, “Transport processes in passivity breakdown—II. Full hydrolysis of the metal ions,” Revista Facultad de Ingeniería (Rev. Fac. Ing.) Vol. 27 (48), pp. 7-15. Mayo-Agosto, 2018. Tunja-Boyacá, Colombia. 14 María Ángeles Arenas-Vara - Peter Skeldon - Sandra Judith García-Vergara [18] G. D. Davis, B. A. Shaw, B. J. Rees, and C. A. Pecile, “Electrochemical behavior and surface chemistry of non-equilibrium aluminum–tantalum alloys: Solute- rich interphase mechanisms,” Surf. Int. Anal., vol. 23 (9), pp. 609-617, Aug. 1995. DOI: https://doi. org/10.1002/sia.740230906. Corros. Sci., vol. 21 (8), pp. 551-579, Aug. 1981. DOI: https://doi.org/10.1016/0010-938X(81)90009-3. Corros. Sci., vol. 21 (8), pp. 551-579, Aug. 1981. DOI: https://doi.org/10.1016/0010-938X(81)90009-3. [13] [13] S. Garcia-Vergara, F. Colin, P. Skeldon, G. E. Thompson, P. Bailey, T. C. Q. Noakes, H. Habazaki, and K. Shimizu, “Effect of Copper Enrichment on the Electrochemical Potential of Binary Al-Cu Alloys,” J. Electrochem. Soc., vol. 151 (1), pp. B16-B21, Jan. 2004. DOI: https://doi.org/10.1149/1.1627343. [19] [19] G. S. Frankel, R. C. Newman, C. V. Jahnes, and M. A. Russak, “On the Pitting Resistance of Sputter- Deposited Aluminum Alloys,” J. Electrochem. Soc., vol. 140 (8), pp. 2192-2197, Aug. 1993. DOI: https:// doi.org/10.1149/1.2220794. [14] A. Broli, and H. Holtan, “Use of potentiokinetic methods for the determination of characteristic potentials for pitting corrosion of aluminium in a deaerated solution of 3%NaCl,” Corros. Sci., vol. 13(4), pp. 237-246, Apr. 1973. DOI: https://doi. org/10.1016/0010-938X(73)90002-4.i g [20] M. M. Lohrengel, “Thin anodic oxide layers on aluminium and other valve metals: high field regime,” Mat. Sci. Eng. R, vol. 11(6), pp. 243-294, Dec. 1993. DOI: https://doi.org/10.1016/0927- 796X(93)90005-N. g [15] G. S. Frankel, “The growth of 2-D pits in thin film aluminium,” Corros. Sci., vol. 30 (12), pp. 1203- 1218, Dec. 1990. DOI: https://doi.org/10.1016/0010- 938X(90)90199-F. [21] X. Zhou, C. Luo, T. Hashimoto, A.E. Hughes, and G.E. Thompson, “Study of localized corrosion in AA2024 aluminium alloy using electron tomography,” Corros. Sci., vol. 58, pp. 299-306, May. 2012. DOI: https://doi.org/10.1016/j.corsci.2012.02.001. [16] G. S. Frankel, M. A. Russak, C. V. Jahnes, M. Mirzamaani, and V. A. Brusic, “Pitting of Sputtered Aluminum Alloy Thin Films,” J. Electrochem. Soc., vol. 136(4), pp.1243-1244, Apr. 1989. DOI: https:// doi.org/10.1149/1.2096864. [22] X. Zhang, X. Zhou, T. Hashimoto, and B. Revista Facultad de Ingeniería (Rev. Fac. Ing.) Vol. 27 (48), pp. 7-15. Mayo-Agosto, 2018. Tunja-Boyacá, Colombia. References Liu, “Localized corrosion in AA2024-T351 aluminium alloy: Transition from intergranular corrosion to crystallographic pitting,” Mat. Characterization, vol. 130, pp. 230-236, Aug. 2017. DOI: https://doi. org/10.1016/j.matchar.2017.06.022. g [17] X. Zhou, H. Habazaki, K. Shimizu, P. Skeldon, G. E. Thompson, and G. C. Wood, “Enrichment-dependent anodic oxidation of Zinc in Al-Zn Alloys,” Corros. Sci., vol. 38 (9), pp. 1563-1577, Sep. 1996. 15
https://openalex.org/W2262219816	https://journals.iucr.org/e/issues/2010/04/00/hb5376/hb5376.pdf	English	null	<i>tert</i>-Butyl<i>N</i>-[(<i>S</i>)-1-hydrazinecarbonyl-2-hydroxyethyl]carbamate	Acta crystallographica. Section E	2,010	cc-by	3,480	Alessandra C. Pinheiro,a Marcus V. N. de Souza,b Solange M. S. V. Wardell,c James L. Wardelld‡ and Edward R. T. Tiekinke* aInstituto de Tecnologia em Farmacos, Fundac¸a˜o Oswaldo Cruz (FIOCRUZ), FarManguinhos, Rua Sizenando Nabuco, 100, Manguinhos, 21041-250 Rio de Janeiro, RJ, Brazil, bInstituto de Tecnologia em Farmacos, Fundac¸a˜o Oswaldo Cruz (FIOCRUZ), FarManguinhos, Rua Sizenando Nabuco, 100, Manguinhos, 21041-250 Rio de Janeiro, RJ, Brazil, cCHEMSOL, 1 Harcourt Road, Aberdeen AB15 5NY, Scotland, dCentro de Desenvolvimento Tecnolo´gico em Sau´de (CDTS), Fundac¸a˜o Oswaldo Cruz (FIOCRUZ), Casa Amarela, Campus de Manguinhos, Av. Brasil 4365, 21040-900 Rio de Janeiro, RJ, Brazil, and eDepartment of Chemistry, University of Malaya, 50603 Kuala Lumpur, Malaysia Table 1 Hydrogen-bond geometry (A˚ , ). D—H A D—H H A D A D—H A O2—H1o N1i 0.84 (3) 1.94 (3) 2.776 (4) 174 (5) N1—H1n O2i 0.91 (3) 2.24 (3) 3.121 (4) 162 (4) N1—H2n O1ii 0.91 (1) 2.29 (2) 3.070 (4) 144 (3) N2—H3n O1iii 0.88 (2) 2.18 (2) 2.985 (4) 152 (3) N3—H4n O3iv 0.88 (1) 2.02 (1) 2.892 (4) 172 (3) Symmetry codes: (i) x; y þ 1 2; z þ 1; (ii) x þ 1; y 1 2; z þ 1; (iii) x; y 1; z; (iv) x; y þ 1; z. Correspondence e-mail: edward.tiekink@gmail.com Received 25 March 2010; accepted 25 March 2010 Key indicators: single-crystal X-ray study; T = 120 K; mean (C–C) = 0.004 A˚; R factor = 0.046; wR factor = 0.149; data-to-parameter ratio = 9.3. In the title compound, C8H17N3O4, the dihedral angle between the hydrazinecarbonyl and carbamate groups is 44.94 (12), and the carbonyl groups are anti to each other. In the crystal, the hydroxy group forms an O—H Na (a = amine) hydrogen bond and each of the four N—H atoms forms an N—H O hydrogen bond; the hydrazinecarbonyl O atom accepts two such bonds. This results in two-dimensional arrays in the ab plane, mediated by the hydrogen bonding, sandwiched by tert- butyl groups. Data collection: COLLECT (Hooft, 1998); cell reﬁnement: DENZO (Otwinowski & Minor, 1997) and COLLECT; data reduc- tion: DENZO and COLLECT; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to reﬁne structure: SHELXL97 (Sheldrick, 2008); molecular graphics: ORTEP-3 (Farrugia, 1997) and DIAMOND (Brandenburg, 2006); software used to prepare material for publication: publCIF (Westrip, 2010). The use of the EPSRC X-ray crystallographic service at the University of Southampton, England, and the valuable assis- tance of the staff there is gratefully acknowledged. organic compounds b = 5.0074 (4) A˚ c = 16.2388 (15) A˚ = 94.483 (5) V = 561.57 (8) A˚ 3 Z = 2 Mo K radiation = 0.10 mm1 T = 120 K 0.26 0.14 0.03 mm Data collection Nonius KappaCCD diffractometer Absorption correction: multi-scan (SADABS; Sheldrick, 2007) Tmin = 0.616, Tmax = 0.746 6687 measured reﬂections 1428 independent reﬂections 1168 reﬂections with I > 2(I) Rint = 0.062 Reﬁnement R[F 2 > 2(F 2)] = 0.046 wR(F 2) = 0.149 S = 1.23 1428 reﬂections 154 parameters 6 restraints H atoms treated by a mixture of independent and constrained reﬁnement max = 0.30 e A˚ 3 min = 0.34 e A˚ 3 Table 1 Hydrogen-bond geometry (A˚ , ). D—H A D—H H A D A D—H A O2—H1o N1i 0.84 (3) 1.94 (3) 2.776 (4) 174 (5) N1—H1n O2i 0.91 (3) 2.24 (3) 3.121 (4) 162 (4) N1—H2n O1ii 0.91 (1) 2.29 (2) 3.070 (4) 144 (3) N2—H3n O1iii 0.88 (2) 2.18 (2) 2.985 (4) 152 (3) N3—H4n O3iv 0.88 (1) 2.02 (1) 2.892 (4) 172 (3) Symmetry codes: (i) x; y þ 1 2; z þ 1; (ii) x þ 1; y 1 2; z þ 1; (iii) x; y 1; z; (iv) x; y þ 1; z. Data collection: COLLECT (Hooft, 1998); cell reﬁnement: DENZO (Otwinowski & Minor, 1997) and COLLECT; data reduc- Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 Mo K radiation = 0.10 mm1 T = 120 K 0.26 0.14 0.03 mm = 94.483 (5) V = 561.57 (8) A˚ 3 Z = 2 T = 120 K 0.26 0.14 0.03 mm Data collection Nonius KappaCCD diffractometer Absorption correction: multi-scan (SADABS; Sheldrick, 2007) Tmin = 0.616, Tmax = 0.746 6687 measured reﬂections 1428 independent reﬂections 1168 reﬂections with I > 2(I) Rint = 0.062 Reﬁnement R[F 2 > 2(F 2)] = 0.046 wR(F 2) = 0.149 S = 1.23 1428 reﬂections 154 parameters 6 restraints H atoms treated by a mixture of independent and constrained reﬁnement max = 0.30 e A˚ 3 min = 0.34 e A˚ 3 Alessandra C. Pinheiro,a Marcus V. N. de Souza,b Solange M. S. V. Wardell,c James L. Wardelld‡ and Edward R. T. Tiekinke* JLW acknowledges support from CAPES and FAPEMIG (Brazil). Related literature For background to the use of serinyl compounds as potential anti-tuberculosis agents, see: Pinheiro et al. (2007). Experimental Crystal data C8H17N3O4 Mr = 219.25 Monoclinic, P21 a = 6.9274 (5) A˚ Brandenburg, K. (2006). DIAMOND. Crystal Impact GbR, Bonn, Germany. Farrugia, L. J. (1997). J. Appl. Cryst. 30, 565. Hooft, R. W. W. (1998). COLLECT. Nonius BV, Delft, The Netherlands. Otwinowski, Z. & Minor, W. (1997). Methods in Enzymology, Vol. 276, Macromolecular Crystallography, Part A, edited by C. W. Carter Jr & R. M. Sweet, pp. 307–326. New York: Academic Press. Pinheiro, A. C., Kaiser, C. R., Lourenc¸o, M. C. S., de Souza, M. V. N., Wardell, S. M. S. V. & Wardell, J. L. (2007). J. Chem. Res. pp. 180–184. Sheldrick, G. M. (2007). SADABS. Bruker AXS Inc., Madison, Wisconsin, USA. Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. Supplementary data and ﬁgures for this paper are available from the IUCr electronic archives (Reference: HB5376). tert-Butyl N-[(S)-1-hydrazinecarbonyl-2- hydroxyethyl]carbamate Alessandra C. Pinheiro,a Marcus V. N. de Souza,b Solange M. S. V. Wardell,c James L. Wardelld‡ and Edward R. T. Tiekinke* S1. Comment Continuing our interests in serinyl compounds as potential anti-tuberculosis agents (Pinheiro et al., 2007), we have prepared the title compound, tert-butyl N-[1(S)-1-(hydrazinecarbonyl)-2-hydroxyethyl]carbamate (I) from L-serine methyl ester hydrochloride, as a precursor of a series of tert-butyl N-(2-hydroxy-1-(S)-{N′-[(1E)-(2-aryl)methylidene]- hydrazinecarbonyl}ethyl)carbamates. We now report the syntheses and structure of (I). The molecular structure of (I), Fig. 1, is twisted with the dihedral angle formed between the least-squares planes through the hydrazinecarbonyl (r.m.s. deviation = 0.0045 Å) and carbamate (r.m.s. deviation = 0.021 Å) residues being 44.94 (12) °. The carbonyl-O1 and O3 atoms lie to opposite sides of the molecule as seen in the pseudo O1–C1···C4–O3 torsion angle of -176.7 (3) °. Finally, each of the N–H groups is anti to the adjacent carbonyl so that the N–H groups, too, lie to opposite sides of the molecule. Although the absolute structure could not be determined experimentally, the assignment of the S-configuration at the C2 atom is based on the starting reagents. There are five acidic H atoms in the structure, and each of these forms a significant hydrogen bonding interaction, Table 1. The hydroxyl-O2–H forms an O– H···N bond with the amino-N1 atom. The carbonyl-O1 atom accepts two N–H hydrogen bonds, one from the amino-N1 atom and the other from the hydrazine-N2. The second amino-N1–H atom forms a hydrogen bond with the hydroxyl-O2 atom, and, finally, the carbamate-N3–H interacts with the O3-carbonyl atom. The hydrogen bonds cooperate with each other to form a 2-D array in the ab plane, Fig. 2, and these stack along the c axis being sandwiched by the t-butyl groups, Fig. 3. Fig. 3. Fig. 3. tert-Butyl N-[(S)-1-hydrazinecarbonyl-2-hydroxyethyl]carbamate Alessandra C. Pinheiro, Marcus V. N. de Souza, Solange M. S. V. Wardell, James L. Wardell and Edward R. T. Tiekink S2. Experimental To a stirred ethanol solution (10 ml) of methyl (2S)-2-[(tert-butoxycarbonyl)amino]-3-hydroxypropanoate (0.3 g, 1.37 mmol), obtained from L-serine methyl ester hydrochloride and (BOC)2O, at room temperature was added N2H4.H2O (80%, 5.5 mmol). The reaction mixture was stirred for 24 hours at room temperature and concentrated under reduced pressure. The residue was columned chromatographed on silica gel using a gradient of 0 to 5% chloroform in methanol, affording the title compound as a white solid in 70% yield. The crystals used in the structural study were grown from EtOH solution, m. pt. 403–404 K. 1H NMR (500 MHz, DMSO-d6) δ (ppm): 9.02 (1H, s, NHNH2), 6.58 (1H, d, J = 8.2, NHCH), 4.81 (1H, t, J = 5.6, OH), 4.19 (2H, s, NHNH2), 3.93 (1H, m, CH), 3.60–3.40 (2H, m, CH2OH), 1.37 (9H, s, (CH3)3C). 13C NMR (125 MHz, DMSO-d6) δ (ppm): 169.7 (COCH), 155.1 (COO), 78.1 ((CH3)3C), 61.9 (CH2OH), 55.5 (CH), 28.2 ((CH3)3C). IR (cm-1, KBr): 3281 (O—H), 1699 (COCH), 1668 (COO). EM/ESI (m/z [M—H]-): 218.1. Experimental Experimental Monoclinic, P21 a = 6.9274 (5) A˚ Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. Westrip, S. P. (2010). publCIF. In preparation. ‡ Additional correspondence author, e-mail: j.wardell@abdn.ac.uk. o994 Pinheiro et al. Acta Cryst. (2010). E66, o994 doi:10.1107/S1600536810011438 supporting information Acta Cryst. (2010). E66, o994 [doi:10.1107/S1600536810011438] Acta Cryst. (2010). E66, o994 [doi:10.1107/S1600536810011438] Acta Cryst. (2010). E66, o994 [doi:10.1107/S1600536810011438] Acta Cryst. (2010). E66, o994 [doi:10.1107/S1600536810011438] supporting information Uiso(H) = 1.5Ueq(O). The N-bound H atoms were treated similarly with N–H = 0.880±0.001 and 0.910±0.001 Å, and with Uiso(H) = 1.2Ueq(N). In the absence of significant anomalous scattering effects, 1067 Friedel pairs were averaged in the final refinement. Figure 1 The molecular structure of (I) showing displacement ellipsoids at the 50% probability level. S3. Refinement The C-bound H atoms were geometrically placed (C–H = 0.98–1.00 Å) and refined as riding with Uiso(H) = 1.2-1.5Ueq(parent atom). The O-bound H atom was refined with the distance restraint O–H = 0.840±0.001, and with sup-1 Acta Cryst. (2010). E66, o994 supporting information Figure 1 Figure 1 The molecular structure of (I) showing displacement ellipsoids at the 50% probability level. g The molecular structure of (I) showing displacement ellipsoids at the 50% probability level. g The molecular structure of (I) showing displacement ellipsoids at the 50% probability level. sup-2 Acta Cryst. (2010). E66, o994 supporting information Figure 2 A view of a supramolecular array in (I) in the ab plane. The O–H···N and N–H···O hydrogen bonding interactions are shown as orange and blue dashed lines, respectively. Colour code: O, red; N, blue; C, grey; and H, green. Figure 2 Figure 2 g A view of a supramolecular array in (I) in the ab plane. The O–H···N and N–H···O hydrogen bonding interactions are shown as orange and blue dashed lines, respectively. Colour code: O, red; N, blue; C, grey; and H, green. g A view of a supramolecular array in (I) in the ab plane. The O–H···N and N–H···O hydrogen bonding interactions are shown as orange and blue dashed lines, respectively. Colour code: O, red; N, blue; C, grey; and H, green. sup-3 Acta Cryst. (2010). E66, o994 supporting information e 3 w of the crystal packing in (I) in projection down the b axis, showing the stacking of layers. The O–H···N and N– Figure 3 g A view of the crystal packing in (I) in projection down the b axis, showing the stacking of layers. The O–H···N and N– H···O hydrogen bonding interactions are shown as orange and blue dashed lines, respectively. Colour code: O, red; N, blue; C, grey; and H, green. tert-Butyl N-[(S)-1-hydrazinecarbonyl-2-hydroxyethyl]carbamate sup 4 A t C t (2010) E66 994 Crystal data C8H17N3O4 Mr = 219.25 Monoclinic, P21 Hall symbol: P 2yb a = 6.9274 (5) Å b = 5.0074 (4) Å c = 16.2388 (15) Å β = 94.483 (5)° V = 561.57 (8) Å3 Z = 2 F(000) = 236 Dx = 1.297 Mg m−3 Mo Kα radiation, λ = 0.71073 Å Cell parameters from 10838 reflections Crystal data C8H17N3O4 Mr = 219.25 Monoclinic, P21 Hall symbol: P 2yb a = 6.9274 (5) Å b = 5.0074 (4) Å c = 16.2388 (15) Å β = 94.483 (5)° V = 561.57 (8) Å3 Z = 2 F(000) = 236 Dx = 1.297 Mg m−3 Mo Kα radiation, λ = 0.71073 Å Cell parameters from 10838 reflections sup-4 Acta Cryst. (2010). E66, o994 supporting information θ = 2.9–27.5° µ = 0.10 mm−1 T = 120 K Data collection Nonius KappaCCD diffractometer Radiation source: Enraf–Nonius FR591 rotating anode 10 cm confocal mirrors monochromator Detector resolution: 9.091 pixels mm-1 φ and ω scans Absorption correction: multi-scan (SADABS; Sheldrick, 2007) Refinement Refinement on F2 Least-squares matrix: full R[F2 > 2σ(F2)] = 0.046 wR(F2) = 0.149 S = 1.23 1428 reflections 154 parameters 6 restraints Primary atom site location: structure-invariant direct methods Plate, colourless 0.26 × 0.14 × 0.03 mm Plate, colourless 0.26 × 0.14 × 0.03 mm Tmin = 0.616, Tmax = 0.746 6687 measured reflections 1428 independent reflections 1168 reflections with I > 2σ(I) Rint = 0.062 θmax = 27.5°, θmin = 3.0° h = −8→8 k = −6→6 l = −21→18 Secondary atom site location: difference Fourier map Hydrogen site location: inferred from neighbouring sites H atoms treated by a mixture of independent and constrained refinement w = 1/[σ2(Fo2) + (0.0838P)2] where P = (Fo2 + 2Fc2)/3 (Δ/σ)max = 0.001 Δρmax = 0.30 e Å−3 Δρmin = −0.34 e Å−3 Absolute structure: nd H atoms treated by a mixture of independent and constrained refinement w = 1/[σ2(Fo2) + (0.0838P)2] where P = (Fo2 + 2Fc2)/3 (Δ/σ)max = 0.001 Δρmax = 0.30 e Å−3 Δρmin = −0.34 e Å−3 Absolute structure: nd Acta Cryst. (2010). E66, o994 Special details Geometry. All s.u.'s (except the s.u. in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell s.u.'s are taken into account individually in the estimation of s.u.'s in distances, angles and torsion angles; correlations between s.u.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell s.u.'s is used for estimating s.u.'s involving l.s. planes. Refinement. Refinement of F2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F2, conventional R-factors R are based on F, with F set to zero for negative F2. The threshold expression of F2 > 2σ(F2) is used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F2 are statistically about twice as large as those based on F, and R- factors based on ALL data will be even larger. Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) x y z Uiso/Ueq O1 0.2906 (3) 0.6236 (5) 0.55336 (15) 0.0226 (6) O2 −0.0983 (3) 0.3069 (5) 0.64307 (16) 0.0245 (6) H1O −0.149 (6) 0.452 (5) 0.627 (3) 0.037 O3 0.4633 (4) −0.1207 (6) 0.74118 (17) 0.0298 (7) O4 0.6317 (3) 0.2100 (5) 0.81155 (14) 0.0223 (6) N1 0.2779 (4) 0.2663 (6) 0.42051 (18) 0.0223 (7) H1N 0.205 (5) 0.414 (5) 0.409 (3) 0.027* H2N 0.4053 (15) 0.288 (9) 0.412 (2) 0.027* N2 0.2663 (4) 0.1984 (5) 0.50498 (18) 0.0198 (6) H3N 0.230 (5) 0.033 (3) 0.513 (2) 0.024* N3 0.4237 (4) 0.3155 (5) 0.70510 (18) 0.0207 (6) l atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) x y z Uiso/Ueq sup-5 Acta Cryst. (2010). E66, o994 supporting information supporting information supporting information H4N 0.429 (6) 0.485 (2) 0.720 (2) 0.025 C1 0.2715 (4) 0.3813 (8) 0.5650 (2) 0.0181 (7) C2 0.2480 (4) 0.2659 (8) 0.65093 (19) 0.0182 (7) H2 0.2267 0.0688 0.6460 0.022* C3 0.0743 (2) 0.3924 (5) 0.68777 (11) 0.0213 (7) H3A 0.0723 0.3398 0.7465 0.026* H3B 0.0842 0.5894 0.6852 0.026* C4 0.5023 (2) 0.1141 (5) 0.75191 (11) 0.0193 (7) C5 0.7406 (2) 0.0190 (5) 0.86737 (11) 0.0218 (7) C6 0.8669 (2) 0.2032 (5) 0.92338 (11) 0.0317 (9) H6A 0.9567 0.3001 0.8904 0.047* H6B 0.9406 0.0969 0.9657 0.047* H6C 0.7847 0.3310 0.9501 0.047* C7 0.6005 (6) −0.1336 (9) 0.9179 (2) 0.0300 (8) H7A 0.5109 −0.0078 0.9411 0.045* H7B 0.6735 −0.2291 0.9629 0.045* H7C 0.5272 −0.2620 0.8823 0.045* C8 0.8656 (5) −0.1602 (7) 0.8176 (2) 0.0246 (8) H8A 0.7860 −0.3046 0.7922 0.037* H8B 0.9705 −0.2366 0.8542 0.037* H8C 0.9207 −0.0549 0.7743 0.037* Atomic displacement parameters (Å2) U11 U22 U33 U12 U13 U23 O1 0.0269 (12) 0.0135 (13) 0.0271 (14) −0.0025 (11) −0.0007 (10) 0.0009 (10) O2 0.0192 (12) 0.0197 (14) 0.0342 (14) 0.0009 (10) −0.0008 (10) 0.0023 (11) O3 0.0372 (13) 0.0126 (13) 0.0370 (15) −0.0006 (12) −0.0138 (11) 0.0009 (11) O4 0.0314 (12) 0.0113 (12) 0.0230 (12) 0.0012 (10) −0.0066 (10) 0.0013 (10) N1 0.0214 (14) 0.0213 (17) 0.0241 (15) −0.0002 (12) 0.0013 (12) −0.0001 (13) N2 0.0235 (14) 0.0133 (13) 0.0225 (15) −0.0017 (12) 0.0015 (11) 0.0000 (13) N3 0.0231 (13) 0.0110 (14) 0.0272 (15) −0.0018 (12) −0.0031 (12) −0.0014 (13) C1 0.0143 (13) 0.0129 (16) 0.0266 (18) 0.0004 (13) −0.0010 (12) 0.0017 (15) C2 0.0182 (15) 0.0140 (17) 0.0215 (17) −0.0020 (13) −0.0032 (13) −0.0013 (13) C3 0.0225 (15) 0.0165 (16) 0.0252 (17) −0.0019 (15) 0.0030 (13) −0.0005 (14) C4 0.0212 (15) 0.0121 (17) 0.0243 (18) −0.0008 (14) −0.0001 (13) 0.0004 (13) C5 0.0291 (18) 0.0122 (16) 0.0231 (18) 0.0020 (15) −0.0036 (14) 0.0027 (14) C6 0.042 (2) 0.0165 (18) 0.033 (2) 0.0034 (17) −0.0155 (17) −0.0002 (17) C7 0.0403 (19) 0.022 (2) 0.028 (2) 0.0061 (18) 0.0032 (16) 0.0026 (16) C8 0.0285 (16) 0.0156 (19) 0.0296 (19) 0.0021 (15) 0.0011 (14) 0.0029 (15) Geometric parameters (Å, º) O1—C1 1.236 (4) C2—H2 1.0000 O2—C3 1.416 (3) C3—H3A 0.9900 O2—H1O 0.84 (3) C3—H3B 0.9900 O3—C4 1.216 (3) C5—C8 1.523 (4) O4—C4 1.355 (3) C5—C6 1.523 (3) Geometric parameters (Å, º) O1—C1 1.236 (4) C2—H2 1.0000 O2—C3 1.416 (3) C3—H3A 0.9900 O2—H1O 0.84 (3) C3—H3B 0.9900 O3—C4 1.216 (3) C5—C8 1.523 (4) O4—C4 1.355 (3) C5—C6 1.523 (3) sup-6 supporting information O4—C5 1.482 (3) C5—C7 1.525 (4) N1—N2 1.421 (4) C6—H6A 0.9800 N1—H1N 0.91 (3) C6—H6B 0.9800 N1—H2N 0.911 (13) C6—H6C 0.9800 N2—C1 1.336 (5) C7—H7A 0.9800 N2—H3N 0.878 (18) C7—H7B 0.9800 N3—C4 1.352 (3) C7—H7C 0.9800 N3—C2 1.466 (4) C8—H8A 0.9800 N3—H4N 0.883 (14) C8—H8B 0.9800 C1—C2 1.531 (5) C8—H8C 0.9800 C2—C3 1.523 (4) C3—O2—H1O 102 (3) O3—C4—O4 124.9 (2) C4—O4—C5 119.0 (2) N3—C4—O4 110.6 (2) N2—N1—H1N 109 (3) O4—C5—C8 109.80 (19) N2—N1—H2N 107 (2) O4—C5—C6 102.46 (12) H1N—N1—H2N 114 (4) C8—C5—C6 110.49 (15) C1—N2—N1 122.7 (3) O4—C5—C7 109.8 (2) C1—N2—H3N 122 (3) C8—C5—C7 113.7 (2) N1—N2—H3N 114 (3) C6—C5—C7 109.99 (17) C4—N3—C2 119.3 (3) C5—C6—H6A 109.5 C4—N3—H4N 124 (3) C5—C6—H6B 109.5 C2—N3—H4N 110 (3) H6A—C6—H6B 109.5 O1—C1—N2 123.9 (3) C5—C6—H6C 109.5 O1—C1—C2 122.0 (3) H6A—C6—H6C 109.5 N2—C1—C2 114.0 (3) H6B—C6—H6C 109.5 N3—C2—C3 109.8 (2) C5—C7—H7A 109.5 N3—C2—C1 109.9 (3) C5—C7—H7B 109.5 C3—C2—C1 110.2 (2) H7A—C7—H7B 109.5 N3—C2—H2 109.0 C5—C7—H7C 109.5 C3—C2—H2 109.0 H7A—C7—H7C 109.5 C1—C2—H2 109.0 H7B—C7—H7C 109.5 O2—C3—C2 109.54 (19) C5—C8—H8A 109.5 O2—C3—H3A 109.8 C5—C8—H8B 109.5 C2—C3—H3A 109.8 H8A—C8—H8B 109.5 O2—C3—H3B 109.8 C5—C8—H8C 109.5 C2—C3—H3B 109.8 H8A—C8—H8C 109.5 H3A—C3—H3B 108.2 H8B—C8—H8C 109.5 O3—C4—N3 124.4 (2) N3—H4N 0.883 (14) C8—H8B 0.9800 C1—C2 1.531 (5) C8—H8C 0.9800 C2—C3 1.523 (4) C3—O2—H1O 102 (3) O3—C4—O4 124.9 (2) C4—O4—C5 119.0 (2) N3—C4—O4 110.6 (2) N2—N1—H1N 109 (3) O4—C5—C8 109.80 (19) N2—N1—H2N 107 (2) O4—C5—C6 102.46 (12) H1N—N1—H2N 114 (4) C8—C5—C6 110.49 (15) C1—N2—N1 122.7 (3) O4—C5—C7 109.8 (2) C1—N2—H3N 122 (3) C8—C5—C7 113.7 (2) N1—N2—H3N 114 (3) C6—C5—C7 109.99 (17) C4—N3—C2 119.3 (3) C5—C6—H6A 109.5 C4—N3—H4N 124 (3) C5—C6—H6B 109.5 C2—N3—H4N 110 (3) H6A—C6—H6B 109.5 O1—C1—N2 123.9 (3) C5—C6—H6C 109.5 O1—C1—C2 122.0 (3) H6A—C6—H6C 109.5 N2—C1—C2 114.0 (3) H6B—C6—H6C 109.5 N3—C2—C3 109.8 (2) C5—C7—H7A 109.5 N3—C2—C1 109.9 (3) C5—C7—H7B 109.5 C3—C2—C1 110.2 (2) H7A—C7—H7B 109.5 N3—C2—H2 109.0 C5—C7—H7C 109.5 C3—C2—H2 109.0 H7A—C7—H7C 109.5 C1—C2—H2 109.0 H7B—C7—H7C 109.5 O2—C3—C2 109.54 (19) C5—C8—H8A 109.5 O2—C3—H3A 109.8 C5—C8—H8B 109.5 C2—C3—H3A 109.8 H8A—C8—H8B 109.5 O2—C3—H3B 109.8 C5—C8—H8C 109.5 C2—C3—H3B 109.8 H8A—C8—H8C 109.5 H3A—C3—H3B 108.2 H8B—C8—H8C 109.5 O3—C4—N3 124.4 (2) Hydrogen-bond geometry (Å, º) D—H···A D—H H···A D···A D—H···A O2—H1o···N1i 0.84 (3) 1.94 (3) 2.776 (4) 174 (5) N1—H1n···O2i 0.91 (3) 2.24 (3) 3.121 (4) 162 (4) N1—H2n···O1ii 0.91 (1) 2.29 (2) 3.070 (4) 144 (3) Hydrogen-bond geometry (Å, º) D—H···A D—H H···A D···A D—H···A O2—H1o···N1i 0.84 (3) 1.94 (3) 2.776 (4) 174 (5) N1—H1n···O2i 0.91 (3) 2.24 (3) 3.121 (4) 162 (4) N1—H2n···O1ii 0.91 (1) 2.29 (2) 3.070 (4) 144 (3) sup-7 Acta Cryst. supporting information (2010). E66, o994 supporting information N2—H3n···O1iii 0.88 (2) 2.18 (2) 2.985 (4) 152 (3) N3—H4n···O3iv 0.88 (1) 2.02 (1) 2.892 (4) 172 (3) Symmetry codes: (i) −x, y+1/2, −z+1; (ii) −x+1, y−1/2, −z+1; (iii) x, y−1, z; (iv) x, y+1, z. supporting information N2—H3n···O1iii 0.88 (2) 2.18 (2) 2.985 (4) 152 (3) N3—H4n···O3iv 0.88 (1) 2.02 (1) 2.892 (4) 172 (3) Symmetry codes: (i) −x, y+1/2, −z+1; (ii) −x+1, y−1/2, −z+1; (iii) x, y−1, z; (iv) x, y+1, z. N2—H3n···O1iii N3—H4n···O3iv Symmetry codes: (i) −x, y+1/2, −z+1; (ii) −x+1, y−1/2, −z+1; (iii) x, y−1, z; (iv) x, y+1, z. sup-8 Acta Cryst. (2010). E66, o994
https://openalex.org/W4285381934	https://usv.ro/fisiere_utilizator/file/fefs/revista/vol_XIV_issue_1/20.%20QUALITY_OF_LIFE_IN_WOMEN_WITH_POSTMENOPAUSAL_OSTEOPOROSIS_DURING_THE_SARS-CoV-2_PANDEMIC_Holubiac_Iulian_%C8%98tefan.pdf	English	null	Quality of life in women with postmenopausal osteoporosis during the Sars-Cov-2 pandemic	Annals of the "Ştefan cel Mare" University. Physical Education and Sport Section. The Science and Art of Movement	2,021	cc-by-sa	2,017	Abstract Osteoporosis can affect the quality of life in women who suffer from this condition. The aim of the article was to observe the dynamics of quality of life from year to year among women with postmenopausal osteoporosis. Subjects included in the study completed the QUALEFFO- 41 questionnaire, a questionnaire specifically designed to measure quality of life. At the end of the study, worsening of the quality of life was observed in all areas covered by the QUALEFFO-41 questionnaire. Keywords: osteoporosis, quality of life, QUALEFFO-41, bone density Keywords: osteoporosis, quality of life, QUALEFFO-41, bone density Introduction Osteoporosis negatively affects patients' quality of life, by limiting their daily activities and independence. Chronic pain generated by osteoporosis can lead to anxiety, depression, frustration and social isolation. Back pain as a consequence of vertebral fractures has a significant impact on osteoporotic patients [11], [15], [8], [3], [17]. These pains to a greater or lesser extent hinder patients' ability to carry out their daily activities [20], [14]. Patients with back pain are sometimes unable to work [16], are limited in their social and recreational activities [9] and are emotionally stressed. The high number and severity of vertebral and hip fractures is associated with decreased quality of life [3], [17]. There are studies that confirm the need to exercise to improve the quality of life in people with osteoporosis [18], [2]; exercise plays an important role in improving cognitive function and mental health in the people concerned [19], [21], [4], [1], [7], [12], [13], [5], [6] increasing patients' confidence that they can independently perform daily tasks [22]. Methods QUALITY OF LIFE IN WOMEN WITH POSTMENOPAUSAL OSTEOPOROSIS DURING THE SARS-CoV-2 PANDEMIC Holubiac Iulian Ștefan Stefan cel Mare University of Suceava, Romania THE ANNALS OF THE "STEFAN CEL MARE" UNIVERSITY ISSN – 1844 – 9131, eISSN 2601 – 341X Volum XIV issue 1/ 2021 THE ANNALS OF THE "STEFAN CEL MARE" UNIVERSITY ISSN – 1844 – 9131, eISSN 2601 – 341X Volum XIV issue 1/ 2021 THE ANNALS OF THE "STEFAN CEL MARE" UNIVERSITY ISSN – 1844 – 9131, eISSN 2601 – 341X Volum XIV issue 1/ 2021 THE ANNALS OF THE "STEFAN CEL MARE" UNIVERSITY ISSN – 1844 – 9131, eISSN 2601 – 341X Volum XIV issue 1/ 2021 postmenopausal osteoporosis. Thirty women with osteopenia / osteoporosis (53.8 ± 3.9 years) were included in the study. A questionnaire for quality of life assessment (QUALEFFO-41) was applied to them before the outbreak of the pandemic and after the relaxation of emergencies. A score as low as possible on the QUALEFFO-41 test represents a better quality of life, while a high result represents a lower quality of life. Subjects did not participate in any exercise program during this period. The results obtained were interpreted using the SPSS program (v. 26). The Shapiro-Wilk test was performed for data distribution, and non-parametric Wilcoxon test was performed to compare the initial and final results. This questionnaire includes 41 questions, including six visual scales, covering the areas: pain, daily activities, activities carried out around the household, mobility, social activities, perception of general health and mental function. Methods The aim of the article is to highlight the effects of sedentary lifestyle on the quality of life in women with osteopenia / 146 Results and discussions For the pain domain, subjects experienced a worsening (Δ% = 32.07%) at the end of the study (M = 52.17, SD = 19.42), compared to the initial test (M = 39.50, SD = 18.54), the nonparametric Wilcoxon test showing that the difference is statistically insignificant, Z = -2.57, p = .010, r = 0.47. Also statistically significant was the difference observed for the ADL domain, where there was a worsening of 32.61 percent at the end of the study (12.71 ± 5.56) compared to the initial testing (9.58 ± 4.26), Z = -2.87, p = .004, r = 0.52. Table 1. Baseline and Post-Testing Results For QUALEFFO-41 Pre Post p ES Pain 39.50±18.54 52.17±19.42 .010 0.47 Activities of Daily Living 9.58±4.26 12.71±5.56 .004 0.52 Jobs Around the House 11.17±10.14 12.83±10.06 .013 0.45 Mobility 10.30±3.57 11.58±4.16 .017 0.52 Table 1. Baseline and Post-Testing Results For QUALEFFO-41 147 THE ANNALS OF THE "STEFAN CEL MARE" UNIVERSITY ISSN – 1844 – 9131, eISSN 2601 – 341X Volum XIV issue 1/ 2021 Leisure, Social Activities 58.42±6.06 62.84±4.91 .001 0.65 General Health Perception 45.83±16.63 58.89±8.74 .001 0.62 Mental Function 35.74±10.69 44.44±8.38 .001 0.64 Note. Results and represented as mean and standard deviation (±); ES = effect size. THE ANNALS OF THE "STEFAN CEL MARE" UNIVERSITY ISSN – 1844 – 9131, eISSN 2601 – 341X Volum XIV issue 1/ 2021 nonparametric Wilcoxon test showing that the difference is statistically significant, Z = -3.48, p < .001, r = 0.64. Conclusion In conclusion, the lack of exercise during the pandemic has greatly influenced the quality of life in patients with osteopenia / postmenopausal osteoporosis. THE ANNALS OF THE "STEFAN CEL MARE" UNIVERSITY ISSN – 1844 – 9131, eISSN 2601 – 341X Volum XIV issue 1/ 2021 Leisure, Social Activities 58.42±6.06 62.84±4.91 .001 0.65 General Health Perception 45.83±16.63 58.89±8.74 .001 0.62 Mental Function 35.74±10.69 44.44±8.38 .001 0.64 Note. Results and represented as mean and standard deviation (±); ES = effect size. Note. Results and represented as mean and standard deviation (±); ES = effect size. For Jobs Around the House domain, subjects recorded a 14.93 percent higher score after one year (12.83 ± 10.06) compared to the initial time (11.17 ± 10.14), Z = -2.49, p = .013, r = 0.45. p For the mobility field, the subjects registered a higher average at the final testing (M = 11.58, SD = 4.16) compared to the initial testing (M = 10.30, SD = 3.57), being an increase of 12.43%, the difference being statistically significant , Z = -2.83, p = .017, r = 0.52. And in the field of social activities, the subjects registered an increase of 7.58% at the end of the study (M = 62.84, SD = 4.91) compared to the initial testing (M = 58.42, SD = 6.06), the difference being statistically significant, Z = -3.56, p < .001, r = 0.65. Also, in the general health field, the subjects registered a statistically significant increase (Δ% = 28.49%), Z = -3.39, p = .001, r = 0.62. Figure 1. Pre and post-test results with a 95% confidence interval for the variables of the quality of life assessment questionnaire; The symbol () indicates intra-group difference (p < .05); The symbol (†) indicates intra-group difference (p < .001). Figure 1. Pre and post-test results with a 95% confidence interval for the variables of the quality of life assessment questionnaire; The symbol () indicates intra-group difference (p < .05); The symbol (†) indicates intra-group difference (p < .001). In the last domain (mental function), subjects experienced a worsening (Δ% = 24.35%) at the end of the study (M = 44.44, SD = 8.38) compared to the initial test (M = 35.74, SD = 10.69), the 148 THE ANNALS OF THE "STEFAN CEL MARE" UNIVERSITY ISSN – 1844 – 9131, eISSN 2601 – 341X Volum XIV issue 1/ 2021 nonparametric Wilcoxon test showing that the difference is statistically significant, Z = -3.48, p < .001, r = 0.64. nonparametric Wilcoxon test showing that the difference is statistically significant, Z = -3.48, p < .001, r = 0.64. References: [1] Ainsworth, B., Sternfeld, B., Slattery, M., Daguise, V., & Zahm, S. (1998). Physical activity and breast cancer: evaluation of physical activity assessment methods. Cancer, 83(3), 611-620. [2] Bonaiuti, D., Arioli, G., Diana, G., Franchignoni, F., Giustini, A., Monticone, M., . . . Maini, M. (2005). SIMFER rehabilitation treatment guidelines in postmenopausal and senile osteoporosis. Europa Medicophysica, 41(4), 315-337. [3] Boonen, S., Autier, P., Barette, M., Vanderschueren, D., Lips, P., & Haentjens, P. (2004). Functional outcome and quality of life following hip fracture in elderly women: a prospective controlled study. Osteoporosis International, 15(2), 87-94. y [4] Chlebowski, R. (2013). Nutrition and physical activity influence on breast cancer incidence and outcome. Breast, 22(2), S30-S37. [5] Curl, W. W. (2000). Aging and exercise: are they compatible in women? Clinical Orthopaedics and Related Research, 372, 151- 158. [6] DiPietro, L. (2001). Physical Activity in Aging: Changes in Patterns and Their Relationship to Health and Function. The Journals of Gerontology, 56(2), 13-22. [7] Eriksen, W., & Bruusgaard, D. (2004). Do physical leisure time activities prevent fatigue? A 15 month prospective study of nurses`aides. British Journal of Sports Medicine, 38(3), 331-336. [8] Fink, H. A., Ensrud, K. E., Nelson, D. B., Kerani, R. P., Schreiner, P. J., Zhao, Y., . . . Nevitt, M. C. (2003). Disability after clinical fracture in postmenopausal women with low bone density: the fracture intervention trial (FIT). Osteoporosis International, 14(1), 69-76. [9] Follick, M. J., Smith, T. W., & Ahern, D. K. (1985). The sickness impact profile: a global measure of disability in chronic low back pain. Pain, 21(1), 67-76. 149 THE ANNALS OF THE "STEFAN CEL MARE" UNIVERSITY ISSN – 1844 – 9131, eISSN 2601 – 341X Volum XIV issue 1/ 2021 THE ANNALS OF THE "STEFAN CEL MARE" UNIVERSITY ISSN – 1844 – 9131, eISSN 2601 – 341X Volum XIV issue 1/ 2021 [10] Gold, D. T., Stegmaier, K., Bales, C. W., Lyles, K. W., Westlund, R. E., & Drezner, M. K. (2009). Psychosocial Functioning and Osteoporosis in Late Life: Results of a Multidisciplinary Intervention. Journal of Womes's Health, 2(2), 149-155. [11] Hallberg, I., Rosenqvist, A. M., Kartous, L., Lofman, O., Wahlstrom, O., & Toss, G. (2004). Health-related quality of life after osteoporotic fractures. Osteoporosis International, 15(10), 834-841. [12] King, A., Oman, R., Brassington, G., Bliwise, D., & Haskell, W. (1997). Moderate-intensity exercise and self-rated quality of sleep in older adults. A randomized controlled trial. JAMA, 277(1), 32- 37. [13] McMurray, R., Ainsworth, B., Harrell, J., Griggs, T., & Williams, O. (1998). Is physical activity or aerobic power more influential on reducing cardiovascular disease risk factors? Medicine and Science in Sports and Exercise, 30(10), 1521-1529. [14] Nevitt, M. C., Ettinger, B., Black, D. M., Stone, K., Jamal, S. A., Ensrud, K., . . . Cummings, S. R. (1998). The Association of Radiographically Detected Vertebral Fractures with Back Pain and Function: A Prospective Study. Annals of Internal Medicine, 128(10), 793-800. [15] Oleksik, A. M., Ewing, S., Shen, W., Schoor, N. M., & Lips, P. (2005). Impact of incident vertebral fractures on health related quality of life (HRQOL) in postmenopausal women with prevalent vertebral fractures. Osteoporosis International, 16(8), 861-870. [16] Painter, J. R., Seres, J. L., & Newman, R. I. (1980). Assessing benefits of the pain center: Why some patients regress. Pain, 8(1), 101-113. [17] Papaioannou, A., Kennedy, C. C., Ioannidis, G., Sawka, A., Hopman, W. M., Pickard, L., . . . Adachi, J. D. (2009). The impact of incident fractures on health-related quality of life: 5 years of data from the Canadian Multicentre Osteoporosis Study. Osteoporosis International, 20(5), 703-714. [18] Papaioannou, A., Morin, S., Cheung, A. M., Atkinson, S., Brown, J. P., Feldman, S., . . . Leslie, W. D. (2010). 2010 clinical practice guidelines for the diagnosis and management of osteoporosis in Canada: summary. CMAJ, 182(17), 1864-1873. [19] Pines, A. (2009). Lifestyle and diet in postmenopausal women. Climacteric: The Journal of the International Menopause Society, 12(1), 62-65. 150 THE ANNALS OF THE "STEFAN CEL MARE" UNIVERSITY ISSN – 1844 – 9131, eISSN 2601 – 341X Volum XIV issue 1/ 2021 THE ANNALS OF THE "STEFAN CEL MARE" UNIVERSITY ISSN – 1844 – 9131, eISSN 2601 – 341X Volum XIV issue 1/ 2021 [20] Silverman, S. L., Minshall, M. E., Shen, W., Harper, K. D., & Xie, S. (2001). The relationship of health-related quality of life to prevalent and incident vertebral fractures in postmenopausal women with osteoporosis: results from the Multiple Outcomes of Raloxifene Evaluation Study. Arthritis & Rheumatology, 44(11), 2611-2619. [21] Takacs, J. (2014). Regular physical activity and mental health. The role of exercise in prevention and intervention of depressive disorders. Psychiatria Hungarica, 29(4), 386-397. [22] Villiers, T. d., Gass, M., Haines, C., Hall, J., Lobo, R., Pierroz, D., & Rees, M. (2013). Global Consensus Statement on menopausal hormone therapy. Maturitas, 74(4), 391-392. 151 151
https://openalex.org/W2044031753	https://europepmc.org/articles/pmc4409416?pdf=render	English	null	Polyandry in dragon lizards: inbred paternal genotypes sire fewer offspring	Ecology and evolution	2,015	cc-by	5,455	ª 2015 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. doi: 10.1002/ece3.1447 males or males with good genes (Tregenza and Wedell 2002; Bretman et al. 2009). But both experimental research and empirical research have shown that not all polyandrous females bias paternity against their relatives following copulation (e.g., lemon shark (Negaprion brevi- rostris), Feldheim et al. 2004; common shrews (Sorex araneus), Stockley 1997; and black ﬁeld crickets, (Teleogryllus commodus), Jennions et al. 2004). Avoiding breeding with a close relative may therefore be unneces- sary if there is no cost. However, when breeding with close relatives imposes ﬁtness costs (Keller 1998; Baer and Schmid-Hempel 1999; Frere et al. 2010; Walling et al. 2011; Nielsen et al. 2012), females may also be able to minimize inbreeding depression by biasing paternity Polyandry in dragon lizards: inbred paternal genotypes si fewer offspring Celine H. Frere1, Dani Chandrasoma2 & Martin J. Whiting2 1GeneCology Research Centre, University of the Sunshine Coast, Maroochydore DC, Sunshine Coast, Queensland 4558, Australia 2Department of Biological Sciences, Macquarie University, Sydney, New South Wales 2109, Australia Correspondence Correspondence Celine H. Frere, GeneCology Research Centre, University of the Sunshine Coast, Maroochydore DC, Qld 4558, Australia. Tel: +61 423 312 893; Fax: +61 7 5430 2881; Fax: +61 7 5430 2881; E-mail: cfrere@usc.edu.au Funding Information Permission to work in LNP was granted by the New South Wales Ofﬁce of the Environment and Heritage (Scientiﬁc licence number S13165), and procedures were approved by the Macquarie University Animal Ethics Committee (ARA2010/35). This work was funded by Macquarie. Received: 3 November 2014; Revised: 1 February 2015; Accepted: 8 February 2015 Received: 3 November 2014; Revised: 1 February 2015; Accepted: 8 February 2015 Received: 3 November 2014; Revised: 1 Received: 3 November 2014; Revised: 1 February 2015; Accepted: 8 February 2015 Ecology and Evolution 2015; 5(8): 1686– 1692 Ecology and Evolution 2015; 5(8): 1686– 1692 doi: 10.1002/ece3.1447 Keywords Cryptic female choice, genetic beneﬁts, genotype reconstruction, GERUD, inbreeding, polyandry, reptile, sexual selection, sperm competition. Multiple mating in female animals is something of a paradox because it can either be risky (e.g., higher probability of disease transmission, social costs) or provide substantial ﬁtness beneﬁts (e.g., genetic bet hedging whereby the likeli- hood of reproductive failure is lowered). The genetic relatedness of parental units, particularly in lizards, has rarely been studied in the wild. Here, we examined levels of multiple paternity in Australia’s largest agamid lizard, the eastern water dragon (Intellagama lesueurii), and determined whether male reproductive success is best explained by its heterozygosity coefﬁcient or the extent to which it is related to the mother. Female polyandry was the norm: 2/ 22 clutches (9.2%) were sired by three or more fathers, 17/22 (77.2%) were sired by two fathers, and only 3/22 (13.6%) clutches were sired by one father. Moreover, we reconstructed the paternal genotypes for 18 known mother–off- spring clutches and found no evidence that females were favoring less related males or that less related males had higher ﬁtness. However, males with greater heterozygosity sired more offspring. While the postcopulatory mechanisms underlying this pattern are not understood, female water dragons likely repre- sent another example of reproduction through cryptic means (sperm selection/ sperm competition) in a lizard, and through which they may ameliorate the effects of male-driven precopulatory sexual selection. Introduction Over the past four decades, experimental and empirical research has generated a large body of evidence for the genetic beneﬁts of polyandry (Baer and Schmid-Hempel 1999; Fisher et al. 2006; Eizaguirre et al. 2007; Klemme et al. 2008; Gowaty et al. 2010). From these studies, we have learnt that when females mate with multiple males, it may increase their ﬁtness (Tregenza and Wedell 1998; Fisher and Lara 1999; Byrne and Whiting 2008) and that of their offspring (Klemme et al. 2008; Firman 2011). An adaptive but largely untested explanation for polyandry is that females minimize inbreeding depression by biasing fertilization toward sperm from genetically compatible 1686 C. H. Frere et al. Polyandry in Dragon Lizards toward more heterozygous males. This hypothesis requires testing across a broad spectrum of taxa. display elaborate mating and social behaviors. Males dis- play alternative mating tactics (ARTs), switching between either aggressively defending a territory or adopting satel- lite behavior (Baird et al. 2012). They are also highly social and show nonrandom patterns of association within and between sexes that are independent of relatedness (Strick- land et al. 2014). The strongest bonds are found between females although males do form strong associations with females and both sexes at times avoid members of the same and opposite sex (Strickland et al. 2014). Female polyandry in reptiles is particularly common, yet our understanding of its potential beneﬁts remains limited (Uller 2008; Uller and Olsson 2008; Keogh et al. 2013). Typically, females that mate with multiple males stand to gain either direct or indirect beneﬁts, although there is currently no evidence for direct beneﬁts in lizards (Uller and Olsson 2008). Additionally, most reptiles lack parental care and, as such, beneﬁts of female polyandry are believed to have evolved as a mechanism to minimize inbreeding, minimize genetic incompatibility, and maximize the genetic quality and/or diversity of mates (Uller and Olsson 2008). While a few reptilian studies have shown that mul- tiple mating enhances female ﬁtness (Madsen et al. 1992; Olsson et al. 1994; Eizaguirre et al. 2007; Noble et al. 2013), only one study thus far has shown that male siring success correlated with the extent of genetic relatedness to the mother (Olsson et al. 1996). In the European sand liz- ard, females are promiscuous and mate with males as they encounter them. Introduction Because of the obvious risks of mating with a relative, especially given that relatedness may increase with spatial proximity, females are able to dis- criminate sperm and bias fertilization toward less closely related males (Olsson et al. 1994, 2004). Study system The eastern water dragon is a long-lived, large, semi-aqua- tic diurnal agamid lizard native to the east coast of Austra- lia (Thompson 1993). They often retreat to water when threatened and either sleep submerged up to their necks or on branches overhanging water (Courtice 1981; Thompson 1993). Males are larger (snout-vent length) than females, have relatively larger heads, and are conspicuously red ventrally, beginning in the neck region and including the limbs (Cuervo and Shine 2007). Eastern water dragons Study site We studied water dragons in Lane Cove National Park (LNP), which is situated in a bushland valley in northern metropolitan Sydney, Australia. The Lane Cove River courses through LNP and most water dragons occur within close proximity of the shoreline. The vegetation in the park consists mainly of casurina (Casurina glauca) woodlands along the riverbanks, isolated patches of Syd- ney blue gum (Eucalyptus saligna) forest, or areas con- taining a combination of blackbutt (E. pilularis), turpentine (Syncarpia glomulifera), and blue gum. The riverbank varies from densely vegetated to open, cleared areas consisting of wood chips and logs. We conducted ﬁeldwork along 2 km of river in LNP (beginning: 33°47029.54″S, 151° 9020.40″E, end: 33°47012.84″S, long: 151° 8053.20″E) from September 2010 to January 2012. Males establish territories along the riverbank, which is typically in close proximity to walking trails or picnic areas; consequently, they were accustomed to humans and allowed relatively close approach (ca. 2–10 m). Evolutionary theory predicts that females should favor males that are more distantly related and therefore either genetically dissimilar or genetically more compatible (e.g., (Slatyer et al. 2012). Unfortunately, very few studies have examined the link between parental relatedness and ﬁtness in polyandrous systems. This is particularly true for lizards: With the exception of the sand lizard (Lacerta agi- lis), we have a remarkably poor understanding of repro- ductive success as a simple function of genetic relatedness between parents. Here, we investigated the extent of mul- tiple paternity in the eastern water dragon (Intellagama lesueurii) and examined whether male siring success cor- related with (1) its heterozygosity coefﬁcient and/or (2) its extent of relatedness to the mother. Sample collection We collected blood samples from the caudal veins of 143 adult eastern water dragons and tail samples (ca. 3– 5 mm) from 169 offspring from 22 clutches. (Eastern water dragons have tails that are much longer than the body and are particularly thin in babies. Tail tips are frag- ile and break naturally as they grow.) Mothers were known for 18 clutches because we brought late-term gravid females into captivity and kept them in outdoor enclosures until they gave birth. The eggs were then incu- bated in the laboratory in damp river sand within plastic containers and checked daily. Blood sample was stored in 90% ethanol, and tail samples were stored in 75% etha- nol. Adults were caught by noosing or by hand. Microsatellite analysis We extracted DNA from tissue and blood samples using QIAGEN DNeasy extraction kit l (Qiagen, Shanghai, 1687 1687 ª 2015 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. C. H. Frere et al. Polyandry in Dragon Lizards China). Adults and offspring genotypes were obtained using nine microsatellite loci developed for eastern water dragons (Frere et al. 2011; : EWD6, 15, 16, 24, 34, 46, 51, 62 and 69). PCR ampliﬁcation procedures and conditions also followed Frere et al. (2011). All loci were found to be in Hardy–Weinberg equilibrium (HWE), and no link- age disequilibrium was detected. Loci were examined for patterns consistent with null alleles using Microchecker (The University of Hull, Hull, UK) (Frere et al. 2011), and no loci had null alleles. of parental relatedness and heterozygosity should not apply given that paternal genotypes used in this study were not identiﬁed using paternity analyses (Wang 2010) but were reconstructed using the maternal and offspring genotypes as described above. This allowed us to test whether the degree of parental genetic relatedness and paternal inbreeding had an impact on who sired the most offspring within a clutch. Male reproductive success (Rs) was measure by the number of sired offspring divided by clutch size. We investigated the relationship between Rs and the degree of parental genetic relatedness and pater- nal inbreeding ﬁtting a generalized linear model (GLM) using a binomial distribution. To account for heterogene- ity, we weighted the model by clutch size. Assessing multiple paternity using paternal genotype reconstruction To test for multiple paternity across our 22 clutches (18 with known mothers and 5 with unknown mothers), we used the program GERUD2.0 (Jones 2005). The exclusion probabilities for all nine microsatellites can be found in Table 1. GERUD2.0 determines the minimum number of father combinations to explain a given array of mother– offspring and ranks them by calculating their relative probabilities using patterns of Mendelian segregation and genotype frequencies measured from the population (in our case n = 143 adults). Using the most likely minimum-father combination, Gerud2.0 was used to reconstruct the paternal genotypes for the 18 known mother–offspring clutches. In addition, we investigated whether the probability of encounters between close paternal and maternal kin increased with geographic proximity. This is because we hypothesized that if females have high chances of encoun- tering close kin of the opposite sex, then this may inﬂu- ence the evolution of inbreeding avoidance mechanisms. We examined this possibility by investigating whether geographic proximity measured by the distance between captured locations correlated with pairwise relatedness measures calculated as above. ª 2015 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. 688 ª 2015 The Authors. Ecology and Evolution publishe Discussion Figure 1. Signiﬁcant relationship between males’ reproductive success (Rs) and their inbreeding coefﬁcient (F). Rs was measured by dividing the number of sired offspring by the size of the clutch. Signiﬁcance was assessed using GLM with binomial distribution. In this analysis, only clutches with known mothers were used (n = 18). From these, we used GERUD 2.0 to deduct the paternal genotype. When GERUD 2.0 assigned multiple sires to a clutch, we used the most likely minimum-father combination to assign paternal genotypes to clutch (see Table 2 for exclusion probabilities). Figure 1. Signiﬁcant relationship between males’ reproductive success (Rs) and their inbreeding coefﬁcient (F). Rs was measured by dividing the number of sired offspring by the size of the clutch. Signiﬁcance was assessed using GLM with binomial distribution. In this analysis, only clutches with known mothers were used (n = 18). From these, we used GERUD 2.0 to deduct the paternal genotype. When GERUD 2.0 assigned multiple sires to a clutch, we used the most likely minimum-father combination to assign paternal genotypes to clutch (see Table 2 for exclusion probabilities). Here, we present evidence that polyandrous female east- ern water dragons bias fertilization toward sperm from more heterozygous males rather than less genetically related males. This is consistent with ﬁndings from other systems such as splendid fairy-wrens (Malurus splendens) (Keith et al. 2005), sand lizards (Olsson et al. 1996), and eastern chipmunks (Tamias striatus) (Bergeron et al. 2011). Female mate choice for more heterozygous males Table 2. Presence of multiple paternity in the 22 clutches of eastern water dragons measured using GERUD 2.0. Statistical analysis When GERUD 2.0 assigned multiple sires to a clutch, we used the most likely minimum-father combination to assign paternal genotypes to clutch (see Table 2 for exclusion probabilities). Statistical analysis Paternal genotype reconstruction analysis conducted in GERUD2.0 (exclusion probability over all loci = 0.99, see Table 1) allowed us to ﬁnd strong evidence for multiple paternity in eastern water dragons with 13.6% (3/22) of clutches sired by a minimum of one father, 77.2% (17/ 22) of clutches sired by a minimum of two fathers, and 9.2% (2/22) of clutches sired by a minimum of three fathers (see Table 2 for details). We used the maternal and reconstructed paternal geno- types in COANCESTRY (V1.0.1.1) (Wang 2011) to mea- sure relatedness estimates between the maternal and paternal genotypes using Wang (Wang 2002) and the degree of paternal inbreeding coefﬁcient (F) using Ritland (Ritland 1996). Here, the biases introduced when the same markers are used to assign paternity and estimates Table 1. Measures of genetic diversity of the nine microsatellite loci derived from the 143 adults used in this study. All loci were found to be in Hardy–Weinberg equilibrium, and no linkage disequilibrium was detected. Locus Number of Alleles Observed Heterozygosity Expected Heterozygosity Exclusion Probability EWD 34 12 0.826 0.831 0.67 EWD 6 4 0.470 0.461 0.25 EWD 15 6 0.134 0.133 0.06 EWD 16 10 0.772 0.789 0.62 EWD 24 13 0.792 0.847 0.70 EWD 46 4 0.591 0.653 0.40 EWD 51 9 0.805 0.744 0.52 EWD 62 20 0.836 0.841 0.78 EWD 69 15 0.852 0.858 0.72 Over all loci 93 0.675 0.693 0.99 versity of the nine microsatellite loci derived from the 143 adults used in this study. All loci were found to be in no linkage disequilibrium was detected. Table 1. Measures of genetic diversity of the nine microsatellite loci derived from the 143 adults used in this study. All lo Hardy–Weinberg equilibrium, and no linkage disequilibrium was detected. ª 2015 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. 1688 C. H. Frere et al. Polyandry in Dragon Lizards 0 1 2 3 4 5 0.2 0.4 0.6 0.8 1.0 Inbreeding coefficient (F) Males reproductive success (Rs) Figure 1. Signiﬁcant relationship between males’ reproductive success (Rs) and their inbreeding coefﬁcient (F). Rs was measured by dividing the number of sired offspring by the size of the clutch. Signiﬁcance was assessed using GLM with binomial distribution. In this analysis, only clutches with known mothers were used (n = 18). From these, we used GERUD 2.0 to deduct the paternal genotype. The relationship between male reproductive success and heterozygosity, partner relatedness, and spatial overlap 0 1 2 3 4 5 0.2 0.4 0.6 0.8 1.0 Inbreeding coefficient (F) Males reproductive success (Rs) We found a male’s reproductive success (number of sired offspring/clutch size) signiﬁcantly correlated with their inbreeding coefﬁcient (GLM with binomial distribution: average effect = 0.3402, SE = 0.11, z-value = 3.068, P = 0.002). The more heterozygous males were, the more the offspring they sired (Fig. 1). However, we did not ﬁnd that males who sired the most offspring within a clutch were more distantly related to the females than males who sired fewer offspring. The extent of genetic similarity between male and female dragons was unrelated to spatial proximity at LNP based on capture locations (Fig. 2). Inbreeding coefficient (F) nd Evolution published by John Wiley & Sons Ltd. 1689 ª 2015 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. 1689 Discussion For example, a series of mating experiments in the promiscuous skink Eulamprus heatwolei found no evidence for indirect beneﬁts and the most parsimonious explanation for polyandry was high male encounter rates coupled with low mating costs for females (Keogh et al. 2013), an hypothesis highlighted in a recent review of multiple mating in reptiles (Uller and Olsson 2008). Eastern water dragons can occur at high densities, and mate encounter rates are high. For example, at Roma Street Parkland, there is a population in excess of 580 animals and females may overlap with up to 42 males (Gardiner et al. 2014). However, multiple matings as a consequence of frequent encounters may not be the only model that best explains water dragon mating dynamics because males control space and, as a result, females may be able to avoid particular males by closely associating with the dominant male. Furthermore, this model implies a low costs to reproduction which is likely not the case given the biased paternity we detected. Nev- ertheless, females may still be susceptible to coercion and ultimately, copulations that they may not favor. The suc- cess of males with higher levels of heterozygosity may be a result of two possible postcopulatory mechanisms of sexual selection. First, females may be capable of sperm selection and cryptically favor the sperm of more hetero- zygous males. Second, the sperm of more heterozygous males may fare better during sperm competition if sperm competition correlates with particular male genotypes that are also less heterozygous. This pattern is different to sand lizards, in which females favored less related males and this could be due to the differing probabilities of encountering kin in the two systems (high in sand lizards, low in water dragons). While the exact mechanisms driv- ing fertilization success remains elusive, our results are consistent with an indirect beneﬁts model of sexual selec- tion via multiple mating. I E d i i Figure 2. No evidence for a relationship between geographic proximity and relatedness between male and female eastern water dragons at Lane Cove National Park (LNP, females = 64, males = 79). has, however, been documented in birds such as the lek- king wire-tailed manakin (Pipra ﬁlicauda) (Ryder et al. 2009) and the cooperatively breeding white-browed spar- row weaver (Plocepasser mahali) (Harrison et al. 2013). Our study, however, provides the ﬁrst evidence for female bias fertilization toward more heterozygous males in liz- ards. Discussion Family Known mother Brood size Number of fathers Number of offspring sired by father 1 Number of offspring sired by father 2 Number of offspring sired by father 3 B1_1 Yes 8 2 6 2 B1_12 Yes 7 3 3 2 2 B1_14 Yes 11 2 9 2 B1_17 Yes 5 2 3 2 B1_18 Yes 8 2 6 2 B1_19 Yes 11 2 9 2 B1_2 Yes 9 2 7 2 B1_21 Yes 8 2 5 3 B1_24 Yes 9 1 9 B1_25 Yes 10 2 7 3 B1_26 Yes 13 2 8 5 B1_27 Yes 13 2 8 5 B1_4 Yes 7 2 6 1 B1_5 Yes 9 2 8 1 B1_8 Yes 8 2 6 2 B2_15 Yes 8 2 4 4 B2_17 Yes 4 1 4 B2_21 Yes 8 1 8 B1_10 No 8 3 3 2 3 B1_13 No 5 2 3 2 B1_15 No 5 2 3 2 B1_20 No 4 2 3 1 Table 2. Presence of multiple paternity in the 22 clutches of eastern water dragons measured using GERUD 2.0. ª 2015 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. 1689 C. H. Frere et al. Polyandry in Dragon Lizards –0.5 –0.4 –0.3 –0.2 –0.1 0 0.1 0.2 0.3 0.4 0.5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 Relatedness (R) Geographic proximity (m) Figure 2. No evidence for a relationship between geographic proximity and relatedness between male and female eastern water dragons at Lane Cove National Park (LNP, females = 64, males = 79). –0.5 –0.4 –0.3 –0.2 –0.1 0 0.1 0.2 0.3 0.4 0.5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 Relatedness (R) Geographic proximity (m) wire-tailed manakin, Ryder et al. 2009). While heterozy- gosity has been hypothesized as a plausible umbrella mechanism for the evolution of female polyandry (Ru- benstein 2007; Taylor et al. 2014), the beneﬁts of multiple mating in lizards have been difﬁcult to establish (Uller and Olsson 2008; Keogh et al. 2013). First, very few lizard species provide obvious parental care beyond simple par- ent–offspring associations (While et al. 2014), precluding direct beneﬁts beyond ejaculate products (Uller and Ols- son 2008). Indirect beneﬁts such as good genes have like- wise been difﬁcult to establish. ª 2015 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. Discussion There may be several reasons why we did not ﬁnd a correlation between male reproductive success and paren- tal relatedness. First, it may be that mating with close rel- atives does not incur major ﬁtness costs (e.g., naked mole rat Heterocephalus glaber, Reeve et al. 1990) and, as such, avoiding breeding with a close relative may be unneces- sary. Second, and in contrast to species with extended kinship (e.g., elephants, Archie et al. 2007), there may be no need for mechanisms to avoid inbreeding because the probability of encountering kin within close geographic proximity is low. While the probability of encountering close relatives within our study population is unknown, we expect it to be lower than species with extended family groups. Indeed, neither geographic proximity (our study) nor social preferences (see Strickland et al. 2014) correlate with relatedness. While further work is required to assess the extent (if any) of inbreeding depression in this spe- cies/population (Balloux et al. 2004), mechanisms to avoid inbreeding are only expected to evolve when the cost of tolerating inbreeding exceeds that of avoiding it (Waser et al. 1986). By favoring more heterozygous males, female eastern water dragons might simply seek to gain maximum genetic beneﬁts from polyandry. Heterozygosity has been, for instance, linked to greater disease resistance (Reid et al. 2007) and increased reproductive success (e.g., Ryder et al. 2009) and as such may be used as a proxy for the genetic quality of mates (e.g., the lekking In summary, Eastern water dragons experience intense precopulatory sexual selection in the form of male–male combat (Baird et al. 2012) and sexual coercion (pers. obs.). While sexual coercion by male water dragons may limit a female’s precopulatory mate choice, we provide evidence that postcopulatory mechanisms of sexual selec- tion are rife and may help level the sexual conﬂict playing 1690 C. H. Frere et al. Polyandry in Dragon Lizards ﬁeld. While we cannot be sure that the mechanism is sperm selection per se, as opposed to sperm competition, the outcome is the same and is likely to be adaptive although this requires future testing. Cuervo, J., and R. Shine. 2007. Hues of a dragon’s belly: morphological correlates of ventral coloration in water dragons. J. Zool. 273:298–304. Eizaguirre, C., D. Laloi, M. Massot, M. Richard, P. Federici, and J. Clobert. 2007. Condition dependence of reproductive strategy and the beneﬁts of polyandry in a viviparous lizard. Proc. R. References Frere, C. H., P. J. Prentis, T. Ezaz, and A. Georges. 2011. Isolation and characterisation of novel microsatellite and mitochondrial DNA markers for the Eastern Water Dragon (Physignathus lesueurii). Conserv. Genet. Resour. 4:1–4. Archie, E. A., J. A. Hollister-Smith, J. H. Poole, P. C. Lee, C. J. Moss, J. E. Maldonado, et al. 2007. Behavioural inbreeding avoidance in wild African elephants. Mol. Ecol. 16:4138– 4148. Gardiner, R., E. Doran, K. Strickland, and C. H. Frere. 2014. A face in the crowd: a non-invasive and cost effective photo-identiﬁcation methodology to understand the ﬁne scale movement of eastern water dragons. PLoS One 9: e9699. Baer, B., and P. Schmid-Hempel. 1999. Experimental variation in polyandry affects parasite loads and ﬁtness in a bumble- bee. Nature 397:151–154. Baird, T. A., T. D. Baird, and R. Shine. 2012. Aggressive transition between alternative male social tactics in a long- lived Australian dragon (Physignathus lesueurii) living at high density. PLoS One 7:e41819. Gowaty, P. A., Y.-K. Kimd, J. Rawlingsd, and W. W. Andersond. 2010. Polyandry increases offspring viability and mother productivity but does not decrease mother survival in Drosophila pseudoobscura. Proc. Natl Acad. Sci. USA 107:13771–13776. Balloux, F., W. Amos, and T. Coulson. 2004. Does heterozygosity estimate inbreeding in real populations? Mol. Ecol. 13:3021–3031. Harrison, X. A., J. E. York, D. L. Cram, and A. J. Young. 2013. Extra-group mating increases inbreeding risk in a cooperatively breeding bird. Mol. Ecol. 22:5700–5715. Bergeron, P., D. Reale, M. M. Humphries, and D. Garant. 2011. Evidence of multiple paternity and mate selection for inbreeding avoidance in wild eastern chipmunks. J. Evol. Biol. 24:1685–1694. Jennions, M. D., J. Hunt, R. Graham, and R. Brooks. 2004. No evidence for inbreeding avoidance through postcopulatory mechanisms in the black ﬁeld cricket, Teleogryllus commondus. Evolution 58:2472–2477. Bretman, A., D. Newcombe, and T. O. M. Tregenza. 2009. Promiscuous females avoid inbreeding by controlling sperm storage. Mol. Ecol. 18:3340–3345. Jones, A. 2005. GERUD 2.0: a computer program for the reconstruction of parental genotypes from half-sib progeny arrays with known or unknown parents. Mol. Ecol. Notes 5:708–711. Byrne, P. G., and M. J. Whiting. 2008. Simultaneous polyandry increases fertilization success in an African foam- nesting treefrog. Anim. Behav. 76:1157–1164. Courtice, G. 1981. The effect of temperature on bimodal gas exchange and the respiratory exchange ratio in the water dragon, Physignathus lesueurii. Comp. Biochem. Physiol. Part A 68:437–441. Keith, A. T., M. S. Webster, E. M. Tuttle, and S. Discussion Soc. B Biol. Sci. 274:425–430. Acknowledgments Permission to work in LNP was granted by the New South Wales Ofﬁce of the Environment and Heritage (Scientiﬁc licence number S13165), and procedures were approved by the Macquarie University Animal Ethics Committee (ARA2010/35). We thank Julie McInnes (Wildlife Licensing and Management Unit) and Andrew Duffy (Lane Cove National Park) for very helpful support during this project. For excellent assistance in the ﬁeld, we thank Daniel Noble, Danny Boerrigter, Ferdy Timmer- man, Martin Rossmanith, Katharina Hinz, and Sean Doo- dy. This work was funded by Macquarie and Queensland University. Feldheim, K. A., S. H. Gruber, and M. V. Ashley. 2004. Reconstruction of parental microsatellite genotypes reveals female polyandry and philopatry in the lemon shark, Negaprion brevirostris. Evolution 58:2332–2342. Negaprion brevirostris. Evolution 58:2332–2342. Firman, R. C. 2011. Polyandrous females beneﬁt by producing sons that achieve high reproductive success in a competitive environment. Proc. R. Soc. B Biol. Sci. 278:2823–2831. Fisher, D., and M. Lara. 1999. Effects of body size and home range on the access to mates paternity of male bridled nailtail wallabies. Anim. Behav. 58:121–130. Fisher, D. O., M. C. Double, S. P. Blomberg, M. D. Jennions, and A. Cockburn. 2006. Post-mating sexual selection increases lifetime ﬁtness of polyandrous females in the wild. Nature 444:89–92. Conﬂict of Interest Frere, C. H., M. Kr€utzen, A. M. Kopps, J. Mann, and W. B. Sherwin. 2010. Too close to home: inbreeding avoidance or tolerance in wild bottlenose dolphins (Tursiops sp.)? Proc. R. Soc. Lond. Biol. Sci. 277:2667–2673. None declared. References Pruett-Jones. 2005. Genetic similarity of social mates predicts the level of extrapair paternity in splendid fairy-wrens. Anim. Behav. 70:945–955. 1691 ª 2015 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. Polyandry in Dragon Lizards C. H. Frere et al. Keller, L. 1998. Inbreeding and its ﬁtness effects in an insular population of song sparrows (Melospiza melodia). Evolution 52:240–250. correlates of male mating success in a lek-breeding bird. Proc. R. Soc. B Biol. Sci. 276:2377–2384. Slatyer, R. A., B. S. Mautz, P. R. Y. Backwell, and M. D. Jennions. 2012. Estimating genetic beneﬁts of polyandry from experimental studies: a meta-analysis. Biol. Rev. 87:1–33. Keogh, J. S., K. D. L. Umbers, E. Wilson, J. Stapley, and M. J. Whiting. 2013. Inﬂuence of alternate reproductive tactics and pre- and postcopulatory sexual selection on paternity and offspring performance in a lizard. Behav. Ecol. Sociobiol. 67:629–638. Stockley, P.. 1997. No evidence of sperm selection by female common shrews. Proc. R. Soc. Lond. B Biol. Sci. 264:1497–1500. Strickland, K., R. Gardiner, A. Schultz, and C. H. Frere. 2014. The social life of eastern water dragons: sex differences, spatial overlap and genetic relatedness. Anim. Behav. 97:53–61. Klemme, I., H. Yl€onen, and J. A. Eccard. 2008. Long-term ﬁtness beneﬁts of polyandry in a small mammal, the bank vole Clethrionomys glareolus. Proc. R. Soc. B Biol. Sci. 275:1095–1100. Madsen, T., R. Shine, J. Loman, and T. Hakansson. 1992. Why do female adders copulate so frequently? Nature 355:440–441. Taylor, M. L., T. A. R. Price, and N. Wedell. 2014. Polyandry in nature: a global analysis. Trends Ecol. Evol. 29:376–383. Thompson, M.. 1993. Estimate of the population structure of the eastern water dragon, Physignathus lesueurii (Reptilia: Agamidae), along riverside habitat. Wildl. Res. 20:613–619. Nielsen, J. F., S. English, W. P. Goodall-Copestake, J. Wang, C. A. Walling, A. W. Bateman, et al. 2012. Inbreeding and inbreeding depression of early life traits in a cooperative mammal. Mol. Ecol. 21:2788–2804. Tregenza, T., and N. Wedell. 1998. Beneﬁts of multiple mates in the cricket Gryllus bimaculatus. Evolution 52:1726–1730. Noble, D. W. A., J. S. Keogh, and M. J. Whiting. 2013. Multiple mating in a lizard increases fecundity but provides no evidence for genetic beneﬁts. Behav. Ecol. 24:1128–1137. Tregenza, T., and N. Wedell. 2002. Polyandrous females avoid costs of inbreeding. Nature 415:71–73. Olsson, M., T. Madsen, R. Shine, A. Gullberg, and H. Tegelstrom. 1994. Rewards of promiscuity. Nature 372:230. Uller, T. 2008. ª 2015 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. References Developmental plasticity and the evolution of parental effects. Trends Ecol. Evol. 23:432–438. Olsson, M., R. Shine, T. Madsen, A. Gullberg, and H. Tegelstrom. 1996. Sperm selection by females. Nature 383:585. Uller, T., and M. Olsson. 2008. Multiple paternity in reptiles: patterns and processes. Mol. Ecol. 17:2566–2580. Olsson, M., T. Madsen, B. Ujvari, and E. Wapstra. 2004. Fecundity and MHC affects ejaculation tactics and paternity bias in sand lizards. Evolution 58:906–909. Walling, C. A., D. H. Nussey, A. Morris, T. H. Clutton- Brock, L. E. B. Kruuk, and J. M. Pemberton. 2011. Inbreeding depression in red deer calves. BMC Evol. Biol. 11:318–331. Reeve, H. K., D. F. Westnet, W. A. Noon, P. W. Sherman, and C. F. Aquadro. 1990. DNA ‘ﬁngerprinting’ reveals high levels of inbreeding in colonies of eusocial naked mole-rat. Proc. Natl Acad. Sci. USA 87:2496–2500. Wang, J. 2002. An estimator for pairwise relatedness using molecular markers. Genetics 160:1203–1215. Wang, J. 2010. Do marker-based paternity assignments favour heterozygous and unrelated males? Mol. Ecol. 19:1898–1913. Reid, J. M., P. Arcese, L. F. Keller, K. H. Elliott, L. Sampson, and D. Hasselquist. 2007. Inbreeding effects on immune response in free-living song sparrows (Melospiza melodia). Wang, J. 2011. COANCESTRY: a program for simulating, estimating and analysing relatedness and inbreeding coefﬁcients. Mol. Ecol. Resour. 11:141–145. Ritland, K. 1996. Estimators for pairwise relatedness and individual inbreeding coefﬁcients. Genet. Res. 67:175–185. Rubenstein, D. R. 2007. Female extrapair mate choice in a cooperative breeder: trading sex for help and increasing offspring heterozygosity. Proc. R. Soc. B Biol. Sci. 274: 1895–1903. Waser, P. M., S. N. Austad, and B. Keanne. 1986. When should animals tolerate inbreeding. Am. Nat. 128:529–537. Waser, P. M., S. N. Austad, and B. Keanne. 1986. When should animals tolerate inbreeding. Am. Nat. 128:529–537. While, G. M., B. Halliwell, and T. Uller. 2014. Reproductive Biology and Phylogeny of Lizards and Tuatara in J. L. Rheubert, D. S. Siegel, S. E. Trauth, eds. The evolutionary ecology of parental care in lizards. CRC Press, 590–619. While, G. M., B. Halliwell, and T. Uller. 2014. Reproductive Biology and Phylogeny of Lizards and Tuatara in J. L. Rheubert, D. S. Siegel, S. E. Trauth, eds. The evolutionary ecology of parental care in lizards. CRC Press, 590–619. Ryder, T. B., P. G. Parker, J. G. Blake, and B. A. Loiselle. 2009. It takes two to tango: reproductive skew and social 1692 ª 2015 The Authors. References Ecology and Evolution published by John Wiley & Sons Ltd.
https://openalex.org/W4288069746	https://liu.diva-portal.org/smash/get/diva2:1689630/FULLTEXT01	English	null	Protein-based luminescent aerogels with elastic properties	Green chemistry letters and reviews	2,022	cc-by	6,814	Green Chemistry Letters and Reviews Green Chemistry Letters and Reviews ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/tgcl20 Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=tgcl20 GREEN CHEMISTRY LETTERS AND REVIEWS 2022, VOL. 15, NO. 3, 508–518 https://doi.org/10.1080/17518253.2022.2102941 GREEN CHEMISTRY LETTERS AND REVIEWS 2022, VOL. 15, NO. 3, 508–518 https://doi.org/10.1080/17518253.2022.2102941 ABSTRACT ABSTRACT Gelatin is a famous gel-forming proteinaceous material with excellent hydrogelation properties. Herein, we report that protein nanoﬁbrils (PNFs) can be employed to form hybrids with gelatin that can be converted to aerogels with attractive mechanical properties. Moreover, we are able to inﬂuence the gelation behavior of PNFs by mechanochemical processing. A combination of mechanochemistry and aqueous self-assembly is employed to prepare PNFs functionalized with hydrophobic dyes. These materials are then mixed with gelatin and converted into aerogels by freeze drying. We ﬁnd that PNFs functionalized with PNFs lead to the formation of aerogels with more robust mechanical properties. Mechanochemical methodology as a green and scalable method can thus be employed for inﬂuencing the properties of protein-based aerogels. This represents a new and highly ﬂexible and novel strategy for tuning both properties and functionality of protein materials. This work opens a simple and feasible way to produce nontoxic and biodegradable aerogel materials with favorable mechanical strength. © 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. CONTACT Niclas Solin niclas.solin@liu.se Department of Physics, Chemistry, and Biology, Biomolecular and Organic Electronics, Linköping University, Linköping 581 83, Sweden Supplemental data for this article can be accessed online at https://doi.org/10.1080/17518253.2022.2102941 Protein-based luminescent aerogels with elastic properties Yusheng Yuan and Niclas Solin Department of Physics, Chemistry, and Biology, Biomolecular and Organic Electronics, Linköping University, Linköping, Sweden ARTICLE HISTORY Received 2 May 2022 Accepted 14 July 2022 KEYWORDS Self-assembly; protein ﬁbrils; gelatin; elastic aerogel; mechanochemistry Yusheng Yuan & Niclas Solin Yusheng Yuan & Niclas Solin To cite this article: Yusheng Yuan & Niclas Solin (2022) Protein-based luminescent aerogels with elastic properties, Green Chemistry Letters and Reviews, 15:3, 508-518, DOI: 10.1080/17518253.2022.2102941 To link to this article: https://doi.org/10.1080/17518253.2022.2102941 © 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group View supplementary material Published online: 27 Jul 2022. Submit your article to this journal Article views: 430 View related articles View Crossmark data 1. Introduction Mechanochemical methodology can accordingly be employed to tune and valorize various renewable protein materials—the hydrophobic mol- ecule/material may modify the physical properties of a protein while simultaneously valorizing the material by providing extra function. Herein we investigate if this methodology can be employed to inﬂuence the proper- ties of gelatin: PNF hydrogels and aerogels. aerogels include ultra-low-density, low optical index of refraction, low thermal conductivity, and low propa- gation speed of sound, and aerogels are investigated for a range of diﬀerent applications, including as materials for energy storage and conversion (15–17), sensors (18, 19), catalyst supports (20, 21), and environ- mental remediation materials (22, 23). Aerogels have been formed from a wide range of materials including Kevlar (24, 25) and bio-based materials (26–28). Many of the developed aerogels employ polymers ultimately derived from petroleum (13). In recent years, there has been an increased interest in developing aero- gels derived from biopolymers such as proteins or poly- saccharides. However, the resulting aerogels will often be brittle, and it is hence important to develop method- ology that can inﬂuence the mechanical properties of such materials. There are few reports presenting mechanically ﬂexible gelatin-based aerogels (29). It was demonstrated that chemical vapor deposition treatment with silanes resulted in ﬂexible aerogels (9, 30). More- over, Ti3C2Tx MXene/ gelatin nanocomposite aerogel formed for electromagnetic absorption (31). In addition, aerogels which gelatin cross-linked with polyvinyl alcohol to enhance the properties of compression and ﬂame retardancy (32). An interesting type of material for modiﬁcation of the properties of gelatin aerogels is protein nanoﬁbrils (PNFs) (33–35). PNFs can be formed by self-assembly in vitro from a wide range of proteins. PNFs are high aspect ratio particles with a typical diam- eter of 5–10 nm and lengths in the μm range. PNFs are related to amyloid ﬁbrils—a type of structure formed in vivo that can have both detrimental and beneﬁcial eﬀects on the organism (36, 37). PNFs have been inves- tigated regarding applications in diﬀerent areas and applications, including LEDs (38, 39), sensors (40–42), and drug delivery (43, 44). A recent example of the use of functional amyloid in materials science is the demon- stration that genetically modiﬁed Escherichia coli (E. coli.) bacteria produced curli (a functional amyloid) contain- ing bioﬁlms that could be processed into bioplastics (45). PNFs have also been converted into various types of aerogels (46). 1. Introduction been widely used in the pharmaceutical, food, cosmetic, and photographic industries because of its biodegrad- ability, biocompatibility, and non-immunogenicity (6–9). Gelatin can easily dissolve in water and form a physical thermally reversible hydrogel at low tempera- ture. However, the preparation of dry gelatin-based materials is challenging due to the brittleness of dry gelatin (10). This is unfortunate as hydrogels are gener- ally considered as attractive precursors for aerogels (11–13). A common way to convert a hydrogel into an aerogel involves freeze drying (14). The sample is ﬁrst frozen and water is then removed at reduced pressure. During the freezing step, ice formation will lead to defor- mations of the gel network, which can present a problem, but also an opportunity for tuning of aerogel structure and porosity. The unique properties of An important research topic is the replacement of materials derived from petroleum with materials obtain- able from renewable sources (1). Many proteins or protein-derived materials are formed as side products from industrial processes and are hence potential candi- dates for renewable raw materials (2–5). Proteins are extremely complex and have a rich supramolecular chemistry providing interesting opportunities when fab- ricating materials. Examples of such valuable character- istics employed in the present study are the ability of protein to self-assemble into ﬁbrils and tendencies of proteins or protein-derived materials to form gels. Gelatin is a low cost and widespread proteinaceous material—famous for its gel-forming capacity—which is derived from skin, tendons, and bone. Gelatin has GREEN CHEMISTRY LETTERS AND REVIEWS 509 is formed. Due to the hydrophobic eﬀect the dye will stay associated with the protein during PNF formation, the end result being an aqueous dispersion of PNFs functionalized with the hydrophobic dye (38, 39, 43, 50–55). We have reported that such functionalized PNFs have an increased tendency to aggregate, for example by forming ﬁlms at the air–water interface or forming abnormally large protein-spherulites (52, 56). The functionalization PNFs by hydrophobic dyes accord- ingly presents a convenient way to modify the colloidal properties of PNF dispersions. The methodology thus presents opportunities to modify the properties of protein materials in a cost eﬀective and green way— mill the protein with a hydrophobic molecule/material and then expose the protein to conditions that promote self-assembly of the protein into PNFs. The presence of the hydrophobic molecule/material may then inﬂuence the properties of the resulting macro- scopic materials. 1. Introduction y As a model protein, we employ hen egg white lyso- zyme (HEWL). As hydrophobic dye component, we investigate the luminescent dyes (2-butyl-6-(butyla- mino)-1H-benz[de]isoquinoline-1,3(2H)-dione, abbre- viated as F555); Fluorescent brighter 378 (F378); and 4-(dicyanomethylene)-2-methyl-6-(4-dimethylaminos- tyryl)-4H-pyran (DCM). The functionalized lysozyme protein nanoﬁbrils (LPNFs) are labeled as F555@LPNF, F378@LPNF and DCM@LPNF. Of these diﬀerent materials, the F555@LPNF material gives aerogels with the most attractive mechanical properties and our study therefore focus on the properties of the F555@LPNF system; however, for comparison, we also provide selected information for the gels derived from the F378@LPNF and DCM@LPNF systems. A sche- matic illustration of the materials preparation is shown in Figure 1 using F555 as an example. Brieﬂy, F555@LPNF and gelatin dispersions/solutions are mixed by stirring while heated. The resulting mixture is allowed to cool which results in the formation of a hydrogel. Next, the hydrogels are converted into aero- gels by a freeze-drying process. We ﬁnd that the pres- ence of hydrophobic dyes results in improved mechanical properties of the resulting aerogels. The fabricated F555@LPNF gel-based ﬂuorescent aerogel retains the typical features such as the amyloid ﬁbrils-based aerogels with lightweight (about As PNFs are built up by self-assembly their properties can be inﬂuenced by modifying the protein building block. There are many diﬀerent methods for modiﬁ- cation of proteins, for example being based on synthetic biology or click chemistry (47, 48). From a sustainability perspective methodology based on mechanochemistry, (49) should be considered. We have developed an alternative methodology for protein functionalization/ valorization based on mechanochemistry. By co-grind- ing of a protein, capable of self-assembly into PNFs, with hydrophobic dyes, a water-soluble hybrid material 510 Y. YUAN AND N. SOLIN Figure 1. Schematic illustration of aerogel-based formed from gelatin and LPNFs or F555@LPNFs. Figure 1. Schematic illustration of aerogel-based formed from gelatin and LPNFs or F555@LPNFs. 0.02 g·cm–3), high porosity (>93%), and speciﬁc surface area (>20 m2 g–1), and also exhibits the additional characteristic of shape recoverability. treated by the procedure described above in order to obtain lysozyme ﬁbrils (2% LPNF, w/v). 2.1. Materials Atomic force microscopy (AFM) measurements were obtained using a digital instruments dimension 3100 atomic force microscope. The initial reaction mixtures (LPNF, F555@LPNF, F378@LPNF, and DCM@LPNF) were diluted 100 times prior to applying to clean silica sub- strates (washed with TL-1 solution at 120°C for 20 min, Ammonia:H2O2:H2O = 1:1:5) and left to dry for 1 min. Excess ﬂuid was removed by applying a nitrogen gas ﬂow. The length distribution of PNFs was determined with the help of ImageJ software by counting 100 PNFs that were plotted as a histogram. (2-butyl-6-(butylamino)-1H-benzo[de]isoquinoline-1,3 (2H)-dione) (Fluorol 555, F555) was obtained from Exciton (Dayton, OH). 4-(dicyanomethylene)-2-methyl- 6-(4-dimethylaminostyryl)-4H-pyran (DCM) and Fluor- escent brighter 378 were obtained from TCl. HEWL and HCl (1M) were purchased from Sigma-Aldrich. Gelatin (GEL, Sinopharm Chemical Reagent Co., Ltd). All chemi- cals were used as received without further puriﬁcation, and deionized water (18.2 Ω) was used throughout. 2.3. AFM characterization of LPNFs and functionalized LPNFs 2. Materials and methods 2. Materials and methods GREEN CHEMISTRY LETTERS AND REVIEWS GREEN CHEMISTRY LETTERS AND REVIEWS 511 in diﬀerent ratios (while ensuring that the total amount of protein equaled 40 mg), then the mixture was stirred for 3 min at 60°C. The resulting mixture was maintained at 4°C for at least 2 h, forming emulsion-gels. F555@LPNF:GEL-x hydrogels were also prepared by mixing F555@LPNF dispersions and gelatin solutions in diﬀerent ratios. in diﬀerent ratios (while ensuring that the total amount of protein equaled 40 mg), then the mixture was stirred for 3 min at 60°C. The resulting mixture was maintained at 4°C for at least 2 h, forming emulsion-gels. F555@LPNF:GEL-x hydrogels were also prepared by mixing F555@LPNF dispersions and gelatin solutions in diﬀerent ratios. The micro-structures of the aerogels were imaged by scanning electron microscopy (SEM, Zeiss Sigma 300 VP, Germany) at an accelerating voltage of 2 kV, and the aerogels were cut with a scissor in the horizontal and vertical direction. After that the aerogels were sputter coated with Pt for 10 s at high vacuum (10−5 Pa), in order to avoid charge-up eﬀects during scanning elec- tron microscope (SEM) imaging. The resulting hydrogels (both LPNF:Gel and F555@LPNF:Gel) were placed on a steel plate containing with liquid nitrogen to directly freeze the hydrogels and were then dried under reduced pressure for 48 h to obtain the aerogels. 2.7. Young’s modulus of LPNF:GEL aerogels The characterization of Young’s modulus of LPNF matrix aerogels was performed by adding diﬀerent weight on the aerogels and observing the deformation. The stress s was calculated by the equation: 2.5. Rheology of LPNF:GEL hydrogels Rheological tests were performed using a TA HR-2 rhe- ometer equipped with 20 mm parallel stainless-steel plates. Rheological frequency sweeps from 0.1 to 100 Hz were performed with a shear strain of 1% in the parallel plate geometry at oscillatory rheology measurements. Rheological amplitude sweeps from 0.1% to 10,000% were assessed with a ﬁxed oscillatory frequency of 1 Hz. All samples were run in at least duplicates. s = F A where F is the applied weight on the aerogel and A is the contact area. The deformation of the aerogel at a given stress was measured and the strain calculated. The Young’s modulus was then obtained from the slope of a plot of stress against strain. where F is the applied weight on the aerogel and A is the contact area. The deformation of the aerogel at a given stress was measured and the strain calculated. The Young’s modulus was then obtained from the slope of a plot of stress against strain. 3. Results and discussion 3. Results and discussion (3) 2.4. Preparation of LPNF:GEL hydrogels and aerogels The dye (one of F555, F378, or DCM) (1 mg) and HEWL (100 mg) were ground with ceramic mortar and pestle for 10 min. The resulting mixture was dissolved in 5 mL 25 mM hydrochloric acid followed by ﬁltration through a 0.45 μm Polyethersulfone ﬁlter. The resulting dis- persion was heated at 80°C with magnetic stirring at 1000 rpm for 24 h to obtain F555@Lysozyme protein ﬁbrils (F555@LPNF). For comparison, the pure HEWL (100 mg) without F555 was ground for 10 min and Solutions of gelatin (GEL) in water (2% w/v) was pre- pared by dissolving 0.4 g gelatin in 20 ml deionized water, and the mixture was stirred for 1 h at 60°C until the gelatin was completely dissolved. LPNF:GEL hydro- gels named as LPNF:Gel-x (x represents the mass ratio of LPNF to gelatin, x = 2, 1.5, 1, 0.5, corresponding to ratios of 2:1, 1.5:1, 1:1, 0.5:1, respectively) were obtained by mixing the 2% LPNF solution and 2% gelatin solution 2.6. Porosity, speciﬁc surface area, and microstructure of LPNF:GEL aerogels A thermogravimetric analysis (TGA) instrument (Mettler Toledo) was used to evaluate the thermal properties of aerogel. The measurements were performed in the range of 30–900°C with a heating rate of 10°C/min in 900 μL alumina crucibles under nitrogen gas ﬂow (N2 ﬂow rate is 25 mL min−1). Before starting the measure- ments, the aerogel was kept at 100°C for 30 min to elim- inate adsorbed water. Furthermore, to estimate the residue content of the aerogel, it was maintained at 900°C at isothermal conditions for 30 min. The porosity of the obtained aerogels was calculated according to the following equation Porosity (%) = 1 −raerogel rs (1) (1) ρaerogel and ρs represent the apparent density and skel- etal density, respectively, of the aerogel, which can be calculated by the following equations raerogel = m V (2) rs = 1 rP (3) (2) 3.1. Morphology of LPNFs If the F555@LPNF:GEL-0.5, F378@LPNF:GEL-0.5 and On the other hand, for the samples functionalized with hydrophobic dyes, they tend to form gels also at a higher ﬁbril to gelatin ratio. F555@LPNF samples form gels at the F555@LPNF:GEL ratio of 1.5:1. However, during preliminary tests of the properties of the aerogels obtainable from the diﬀerent compositions (see below for further discussion of aerogel formation and struc- ture), it was found that a too high PNF ratio led to brittle samples. Therefore, the LPNF:GEL ratio of 0.5:1 (abbreviated as LPNF:GEL-0.5) was chosen for investi- gations involving aerogels (Figure 3(c)). In preliminary tests of the mechanical properties of the resulting aero- gels, a dramatic diﬀerence was found between aerogels made from LPNFs and aerogels made from LPNFs functionalized with hydrophobic dyes. All aerogels can withstand modest deformations but if the LPNF:GEL- 0.5 aerogel is compressed to about 50% of its original length cracks start to form, and if compressed to about 75% the sample shatters (Figure 3(e)). If the F555@LPNF:GEL-0.5, F378@LPNF:GEL-0.5 and sample shows a distribution shifted towards longer lengths, with the most common length being about 1.5 μm, meaning that F555 functionalized ﬁbrils tend to be longer than pure LPNFs. The length distributions of F378@LPNF and DCM@LPNF is more similar to that of the LPNF sample, with the most common length being about 1 μm (Figure S1, supporting information). 3.1. Morphology of LPNFs In order to characterize the length distribution of func- tionalized PNFs and normal PNFs (without hydrophobic dye), samples were prepared in an identical way (heating at 80°C with magnetic stirring at 1000 rpm for 24 h). Samples were then drop-casted onto substrates and AFM micrographs were recorded. The morphology of unmodiﬁed LPNFs as well as LPNFs functionalized with dyes (F555@LPNF, F378@LPNF, and DCM@LPNF) was investigated by AFM. As shown in Figure 2, the LPNF and F555@LPNF samples both have PNFs with lengths varying between 0.5 and 4 μm. The most common length of LPNF is about 1 μm, whereas the F555@LPNF where m and V are the weight and volume, respectively, of the ﬂuorescent aerogel. ρP represents the density of protein which was assumed to be 1.35 g cm–3 (57). The speciﬁc surface area of aerogels was measured at the relative pressure of P/Po = 0.995 by the Brunauer– Emmett–Teller (BET) method on a speciﬁc surface and pore analyzer (Micromeritics ASAP 2020). The nitrogen isothermal adsorption and desorption volumes were measured at −196°C with samples outgassed at 180°C for 5 h. The pore size distribution was calculated from the adsorption isotherm using the Barrett–Joyner– Halenda method (58). 512 Y. YUAN AND N. SOLIN Figure 2. AFM micrographs (a–b) and ﬁbril length distribution (c–d) of LPNF and F555@LPNF. Figure 2. AFM micrographs (a–b) and ﬁbril length distribution (c–d) of LPNF and F555@LPNF. On the other hand, for the samples functionalized with hydrophobic dyes, they tend to form gels also at a higher ﬁbril to gelatin ratio. F555@LPNF samples form gels at the F555@LPNF:GEL ratio of 1.5:1. However, during preliminary tests of the properties of the aerogels obtainable from the diﬀerent compositions (see below for further discussion of aerogel formation and struc- ture), it was found that a too high PNF ratio led to brittle samples. Therefore, the LPNF:GEL ratio of 0.5:1 (abbreviated as LPNF:GEL-0.5) was chosen for investi- gations involving aerogels (Figure 3(c)). In preliminary tests of the mechanical properties of the resulting aero- gels, a dramatic diﬀerence was found between aerogels made from LPNFs and aerogels made from LPNFs functionalized with hydrophobic dyes. All aerogels can withstand modest deformations but if the LPNF:GEL- 0.5 aerogel is compressed to about 50% of its original length cracks start to form, and if compressed to about 75% the sample shatters (Figure 3(e)). 3.3. Rheological characterization of LPNF:GEL hydrogels Rheological measurements were performed in order to evaluate the mechanical properties of the LPNF:gelatin gels and the F555@LPNF:gelatin gels. Frequency sweep tests are widely used to obtain information about the stability of the gel networks and to verify if their struc- tures correspond to the rheological deﬁnition of a gel. The storage (G′) and loss modulus (G′′) of a viscoelastic material, representing the deformation energy stored and dissipated during the shear process, respectively, were measured. Frequency sweep, measurements were carried out at diﬀerent oscillation frequencies with a constant oscillation amplitude (1%) and temperature (25°C). Within the angular frequency of 0.1–100 rad s−1, the value of the storage modulus (G′) is greater than the loss modulus (G′′) for both the F555@LPNF and LPNF hydrogel, indicating the formation of an elastic solid-like hydrogel (Figure 4). Interestingly, the stiﬀness of F555@LPNF hydrogel was about 10 times higher than that of pure LPNF hydrogel at the ratio of 2:1, and four times and two times higher for the 1.5:1 and 1:1 ratio separately. For the 0.5:1 ratio sample, the diﬀerence is small with a slightly higher stiﬀness for the F555@LPNF sample. PNFs can accordingly be employed to inﬂuence the mechanical properties of the hydrogels. The density of aerogels with a diﬀerent amount of LPNF is shown in Figure 7(a). The densities are 0.022 g cm−3 for the gelatin aerogel and 0.023 g cm−3 for LPNF:GEL-0.5 and 0.024, 0.025, and 0.026 g cm−3 for F378@LPNF:GEL-0.5, F555@LPNF:GEL-0.5, and DCM @LPNF:GEL-0.5 aerogels, respectively. The density of the aerogels incorporating various types and amounts of LPNF display only slight variations. The reason for the lower densities of gels incorporating LPNF is due to smaller volume shrinkage than for the GEL sample. The aerogels incorporating LPNF have a high porosity—up to 93% for the LPNF:GEL-0.5 and F555@LPNF:GEL0.5. In Figure 7(b) is shown photos of the diﬀerent aerogels under ambient light and under UV-light (365 nm), demonstrating the gels have ﬂuorescent properties. In addition, strain sweep experiments were performed to determine the linear viscoelastic regime of the hydro- gels (Figure 5). The breakage strain of the LPNF:GEL hydrogels increased from 235% to 553% by decreasing the ratio of LPNF to gelatin from 2:1 to 0.5:1. Interestingly, for the F555@LPNF:GEL hydrogels, the breakage strain increased from 29% to 221% when the PNF:GEL ratio was decreased from 2:1 to 0.5:1. 3.2. Initial tests of gelation and aerogel properties In the description below, the composite aerogels are coded as LPNF:GEL-x (where x is the mass ratio between LPNF (or dye@LPNF) and gelatin—the sample with a LPNF:GEL ratio of 0.5:1 is accordingly labeled as LPNF:GEL-0.5) and key parameters are summarized in Table 1. In order to convert the hydrogels into aerogels, we employed freeze drying. The samples were ﬁrst frozen by immersing the sample liquid nitrogen, and water was then removed under reduced pressure, result- ing in free standing aerogels. In Figure 6 is shown the SEM images of aerogels formed from gelatin, LPNF: GEL-0.5 as well as F555@LPNF:GEL-0.5 samples. The gelatin aerogel is built up from large ﬂake-like structures (Figure 6(a)). In the LPNF:GEL-0.5 sample there is still a ﬂake-like morphology but with some porosity. The mor- phology of the F555@LPNF:GEL-0.5 is dramatically diﬀerent with a relatively uniform and compact network with highly porous structures (Figure 6(c)). SEM images of the F378@LPNF:GEL-0.5 and DCM@LPNF:GEL-0.5 samples are shown in the support- ing information (Figure S2), and in both cases, a ﬂake- like morphology is observed but with much smaller dimensions than for the LPNF:GEL-0.5 sample. 3.2. Initial tests of gelation and aerogel properties To determine a suitable ratio between LPNF (and dye@LPNF) and gelatin, the two components were mixed at diﬀerent ratios and the sol-to-gel transition of LPNF:GEL was examined by the test tube inversion method (Figure 3(a,b)). Note that for the samples in Figure 3(a,b), the total amount of protein is always 40 mg, and the x:y ratio indicates the LPNF:GEL ratio. The mixtures employing unmodiﬁed LPNFs tend to form a gel when the LPNF:GEL ratio of is close to 1:1. Figure 3. (a–b) Photographs of samples of LPNF (or F555@LPNF) and gelatin mixed in diﬀerent ratios. (c) Photographs of aerogels formed from LPNF:GEL-0.5 and F555@LPNF:GEL-0.5. (d) Compression behavior of the F555@LPNF:GEL-0.5 aerogel. (e) Deformation behavior of LPNF:GEL-0.5 and dyes@LPNF:GEL-0.5 aerogels. GREEN CHEMISTRY LETTERS AND REVIEWS 513 GREEN CHEMISTRY LETTERS AND REVIEWS 513 513 Figure 3. (a–b) Photographs of samples of LPNF (or F555@LPNF) and gelatin mixed in diﬀerent ratios. (c) Photographs of aerogels formed from LPNF:GEL-0.5 and F555@LPNF:GEL-0.5. (d) Compression behavior of the F555@LPNF:GEL-0.5 aerogel. (e) Deformation behavior of LPNF:GEL-0.5 and dyes@LPNF:GEL-0.5 aerogels. Figure 3. (a–b) Photographs of samples of LPNF (or F555@LPNF) and gelatin mixed in diﬀerent ratios. (c) Photographs of aerogels formed from LPNF:GEL-0.5 and F555@LPNF:GEL-0.5. (d) Compression behavior of the F555@LPNF:GEL-0.5 aerogel. (e) Deformation behavior of LPNF:GEL-0.5 and dyes@LPNF:GEL-0.5 aerogels. Figure 3. (a–b) Photographs of samples of LPNF (or F555@LPNF) and gelatin mixed in diﬀerent ratios. (c) Photographs of aerogels formed from LPNF:GEL-0.5 and F555@LPNF:GEL-0.5. (d) Compression behavior of the F555@LPNF:GEL-0.5 aerogel. (e) Deformation behavior of LPNF:GEL-0.5 and dyes@LPNF:GEL-0.5 aerogels. Figure 4. Rheological measurements of dynamic frequency sweep of the LPNF:GEL (a) and F555@LPNF:GEL (b) co-assembled gels at a strain of 1% over a range of 0.1–100 Hz. Figure 4. Rheological measurements of dynamic frequency sweep of the LPNF:GEL (a) and F555@LPNF:GEL (b) co-assembled gels at a strain of 1% over a range of 0.1–100 Hz. Y. YUAN AND N. SOLIN 514 3.4. Porosity, speciﬁc surface area, and microstructure of LPNF:GEL aerogels DCM@LPNF:GEL-0.5 are compressed to 50% of their orig- inal length and the force is removed, the aerogels will revert to their original shape (Figure 3(d)). However, if compressed above about 75%, they will undergo a permanent deformation, but without breakage (Figure 3(e)). 3.3. Rheological characterization of LPNF:GEL hydrogels The breakage strain of the F555@LPNF:GEL hydrogels exhibited lower value compared to the LPNF:GEL samples indicating F555 doping increased the mechanical properties of the hydro- gels. This behavior may indicate that the F555@LPNF:GEL co-assembled structures are likely more rigid and hence unable to withstand higher strain values. The diﬀerence in modulus values indicated diﬀerent networks of micro/nanostructures inside the gels, potentially inﬂuen- cing their mechanical properties (59). The surface area and pore volume of the aerogels were investigated by N2 adsorption−desorption iso- therm analysis by using the BET method. As shown in Figure 7(c), the gelatin aerogel had a speciﬁc surface area of 6.7 m2 g−1, and for LPNF functionalized with F555 (F555@LPNF), it increased to 22.36 m2 g−1. Further- more, the speciﬁc surface area of F378@LPNF:GEL-0.5 and DCM@LPNF:GEL-0.5 aerogels were 14.59 and 18.37 m2 g−1, respectively. The average pore diameters of gelatin, F378@LPNF:GEL-0.5, DCM@LPNF:GEL-0.5, and F555@LPNF: GEL-0.5 aerogels were 377, 336, 312, and 349 nm, respectively. GREEN CHEMISTRY LETTERS AND REVIEWS 515 Figure 5. Rheological measurements of dynamic strain sweep of the LPNF gel (a) and F555@LPNF (b) co-assembled gels at a constant frequency of 1 Hz over a strain range of 0.1–10,000%. Figure 5. Rheological measurements of dynamic strain sweep of the LPNF gel (a) and F555@LPNF (b) co-assembled gels at a constant frequency of 1 Hz over a strain range of 0.1–10,000%. Table 1. Comparative results aerogels. Sample Density Pore diameter BET surface area Young’s modulus TOnset Tp (g cm−3) (nm) (m2 g−1) (kPa) (°C) (°C) GEL 0.022 377 6.7 Not given 265 310 LPNF:GEL-0.5 0.023 Not given Not given 3.27 262 306 F555@LPNF:GEL-0.5 0.025 336 22.63 2.94 257 295 F378@LPNF:GEL-0.5 0.024 312 14.59 3.53 282 324 DCM@LPNF:GEL-0.5 0.026 349 18.37 3.28 276 319 Table 1. Comparative results aerogels. in Table 1 (DTG curves were shown in supporting infor- mation, Figure S3). We further investigated the thermal stability by storing the diﬀerent samples at 200°C under air atmosphere for 24 h (See supporting information in Figure S4). This leads to an obvious discoloration but the aerogels containing LPNFs are able to retain their shape, showinga superior stability to temperature-induced defor- mation than many commercial-grade petroleum-based Thethermalstabilityoftheaerogelswasinvestigatedby TGA. As show in Figure 7(d), pristine gelatin and gelatin with protein matrix aerogels gave <10% weight loss at the beginning up to 100°C which can be attributed to loss of water. 3.3. Rheological characterization of LPNF:GEL hydrogels The LPNF:GEL-0.5 aerogel and F555@LPNF: Gel-0.5aerogelstartedtolosemassatabout270°C,indicat- ing a high thermal stability. The onset temperature (Tonset) and peak temperature (Tp) of diﬀerent materials are listed Figure 6. SEM images of aerogels. (a–c) Top-view of gelatin (a) LPNF:GEL-0.5 (b) F555@LPNF:GEL-0.5 (c) aerogels. (d–f) Cross-section of gelatin (d) LPNF:GEL-0.5 (e) F555@LPNF:GEL-0.5 (f) aerogels, scale bar is 10 μm. Figure 6. SEM images of aerogels. (a–c) Top-view of gelatin (a) LPNF:GEL-0.5 (b) F555@LPNF:GEL-0.5 (c) aerogels. (d–f) Cross-section of gelatin (d) LPNF:GEL-0.5 (e) F555@LPNF:GEL-0.5 (f) aerogels, scale bar is 10 μm. 516 Y. YUAN AND N. SOLIN Figure 7. (a) Density of the aerogels with the diﬀerent ratio of LPNF or Dyes@LPNF (F555, F378, DCM) and gelatin. (b) Photos of aero- gels under UV light (365 nm) and ambient light samples. (c) Nitrogen gas absorption–desorption curve of gelatin and LPNF matrix aerogels. (d) TGA curves of amyloid ﬁbril aerogel under nitrogen atmospheres. The residue obtained after the complete decompo- sition is remaining salts in the system. Figure 7. (a) Density of the aerogels with the diﬀerent ratio of LPNF or Dyes@LPNF (F555, F378, DCM) and gelatin. (b) Photos of aero- gels under UV light (365 nm) and ambient light samples. (c) Nitrogen gas absorption–desorption curve of gelatin and LPNF matrix aerogels. (d) TGA curves of amyloid ﬁbril aerogel under nitrogen atmospheres. The residue obtained after the complete decompo- sition is remaining salts in the system. thermoplastic foams that collapsed/melted after being subjected to a temperature of 150°C (60). References [16] Xiong, C.; Zou, Y.; Peng, Z.; Zhong, W. Synthesis of Morphology-Tunable Electroactive Biomass/Graphene Composites Using Metal Ions for Supercapacitors. Nanoscale 2019, 11, 7304–7316. [1] Tang, X.Z.; Kumar, P.; Alavi, S.; Sandeep, K.P. Recent Advances in Biopolymers and Biopolymer-based Nanocomposites for Food Packaging Materials. Crit. Rev. Food Sci. 2012, 52, 426–442. [17] Wang, D.C.; Yu, H.Y.; Qi, D.; Ramasamy, M.; Yao, J.; Tang, F.; Tam, K.M.C.; Ni, Q. Supramolecular Self-Assembly of 3D Conductive Cellulose Nanoﬁber Aerogels for Flexible Supercapacitors and Ultrasensitive Sensors. ACS Appl. Mater. Interfaces 2019, 11, 24435–24446. [2] Gautam, S.; Sharma, B.; Jain, P. Green Natural Protein Isolate Based Composites and Nanocomposites: A Review. Polym. Test 2021, 99, 106626. [3] Jafarzadeh, S.; Forough, M.; Amjadi, S.; Javan Kouzegaran, V.; Almasi, H.; Garavand, F.; Zargar, M. Plant Protein-Based Nanocomposite Films: A Review on the Used Nanomaterials, Characteristics, and Food Packaging Applications. Crit. Rev. Food Sci. 2022. doi:10.1080/ 10408398.2022.2070721 [18] Xiao, Z.; Zhou, W.; Zhang, N.; Zhang, Q.; Xia, X.; Gu, X.; Wang, Y.; Xie, S. All-Carbon Pressure Sensors with High Performance and Excellent Chemical Resistance. Small 2019, 15, 1804779. [19] Yang, L.; Liu, Y.; Filipe, C.D.; Ljubic, D.; Luo, Y.; Zhu, H.; Yan, J.; Zhu, S. Development of a Highly Sensitive, Broad- Range Hierarchically Structured Reduced Graphene Oxide/PolyHIPE Foam for Pressure Sensing. ACS Appl. Mater. Interfaces 2019, 11, 4318–4327. [4] Mihalca, V.; Kerezsi, A.D.; Weber, A.; Gruber-Traub, C.; Schmucker, J.; Vodnar, D.C.; Dulf, F.V.; Socaci, S.A.; Fărcaș, A.; Mureșan, C.I.; Suharoschi, R.; Pop, O.L. Protein-based Films and Coatings for Food Industry Applications. Polymers (Basel) 2021, 13, 769. [20] Qiu, J.; Fan, P.; Yue, C.; Liu, F.; Li, A. Multi-networked Nanoﬁbrous Aerogel Supported by Heterojunction Photocatalysts with Excellent Dispersion and Stability for Photocatalysis. J. Mater. Chem. A. 2019, 7, 7053–7064. [5] Giaveri, S.; Schmitt, A.M.; Roset Julia, L.; Scamarcio, V.; Murello, A.; Cheng, S.; Menin, L.; Ortiz, D.; Patiny, L.; Bolisetty, S. et al. Nature-inspired Circular-economy Recycling for Proteins: Proof of Concept. Adv. Mater. 2021, 33, 2104581. [21] Wu, Z.S.; Yang, S.; Sun, Y.; Parvez, K.; Feng, X.; Müllen, K. 3D Nitrogen-Doped Graphene Aerogel-Supported Fe3O4 Nanoparticles as Eﬃcient Electrocatalysts for the Oxygen Reduction Reaction. J. Am. Chem. Soc. 2012, 134, 9082–9085. [6] Douglas, S.J.; Davis, S.S.; Illum, L. Nanoparticles in Drug Delivery. Crit. Rev. Ther. Drug Carrier Syst. 1987, 3, 233– 261. [22] Feng, Z.Q.; Wu, F.; Jin, L.; Wang, T.; Dong, W.; Zheng, J. 4. Conclusion Herein, we have developed an all protein-based aerogel with favorable mechanical properties in a green and sus- tainable method by mechanochemistry. The properties of the aerogels can be adjusted by varying the relative ratio of PNFs and gelatin. The properties of PNFs can in turn be inﬂuenced by mechanochemical functionalization with hydrophobic dyes (dye@PNF). Aerogels prepared from gelatin and dye@PNF materials have better mechan- ical properties than aerogels prepared from gelatin and unmodiﬁed PNFs. These protein-based aerogels are pre- pared from edible components and may therefore ﬁnd applications related to e.g. food science. In addition, as the aerogels are functionalized with luminescent mol- ecules, they may ﬁnd applications as e.g. sensors or photo- nic materials. Herein, we have developed an all protein-based aerogel with favorable mechanical properties in a green and sus- tainable method by mechanochemistry. The properties of the aerogels can be adjusted by varying the relative ratio of PNFs and gelatin. The properties of PNFs can in turn be inﬂuenced by mechanochemical functionalization with hydrophobic dyes (dye@PNF). Aerogels prepared from gelatin and dye@PNF materials have better mechan- ical properties than aerogels prepared from gelatin and unmodiﬁed PNFs. These protein-based aerogels are pre- pared from edible components and may therefore ﬁnd applications related to e.g. food science. In addition, as the aerogels are functionalized with luminescent mol- ecules, they may ﬁnd applications as e.g. sensors or photo- nic materials. The pure gelatin aerogel is hard and we were not able to determine the Young’s modulus. The LPNF:GEL-0.5 aerogel has a Young’s modulus of 3.27 kPa. For aerogels obtained by mixing gelatin with LPNFs functionalized with the diﬀerent dyes (F555, F378 and DCM), the Young’s modulus were 2.94 kPa (F555@LPNF:GEL-0.5), 3.53 kPa (F378@LPNF:GEL-0.5), and 3.28 kPa (DCM@LPNF:GEL-0.5) The Young’s modulus, pore size and speciﬁc surface area as well as density for the diﬀerent aerogels are summarized in Table 1. LPNFs accordingly present an interesting material for tuning the properties of gelatin-derived aerogels. The mechanochemical processing provides an additional parameter to tune the properties of the aerogels. For the systems examined herein, the employment of diﬀerent dye@LPNF materials allows the formation of mechanically more robust aerogels that in contrast to aerogels incorporating LPNF (without dyes) do not undergo fracturing at large deformations. Acknowledgements We acknowledge Daniel Aili and the Laboratory of Molecular Materials at IFM, LiU for access to their rheometer. No potential conﬂict of interest was reported by the author(s). [14] Nyström, G.; Fong, W.K.; Mezzenga, R. Ice-Templated and Cross-Linked Amyloid Fibril Aerogel Scaﬀolds for Cell Growth. Biomacromolecules 2017, 18, 2858–2865. Funding [15] Wang, S.; Wang, R.; Zhao, Q.; Ren, L.; Wen, J.; Chang, J.; Fang, X.; Hu, N.; Xu, C. Freeze-Drying Induced Self- Assembly Approach for Scalable Constructing MoS2/ Graphene Hybrid Aerogels for Lithium-Ion Batteries. J. Colloid Interface Sci. 2019, 544, 37–45. Y. Y. acknowledges ﬁnancial support from the China Scholar- ship Council. Disclosure statement No potential conﬂict of interest was reported by the author(s). 4. Conclusion GREEN CHEMISTRY LETTERS AND REVIEWS 517 Structural Design: Functional Tailoring, Resource- Eﬃcient Applications and Sustainability Analysis. Prog. Mater. Sci. 2022, 125, 100915. Disclosure statement References Graphene Nanoﬁbrous Foam Designed as an Eﬃcient Oil Absorbent. Ind. Eng. Chem. Res. 2019, 58, 3000–3008. [7] Kim, H.W.; Kim, H.E.; Salih, V. Stimulation of Osteoblast Responses to Biomimetic Nanocomposites of Gelatin- Hydroxyapatite for Tissue Engineering Scaﬀolds. Biomaterials 2005, 26, 5221–5230. [23] Lin, Y.Z.; Zhong, L.B.; Dou, S.; Shao, Z.D.; Liu, Q.; Zheng, Y.M. Facile Synthesis of Electrospun Carbon Nanoﬁber/ Graphene Oxide Composite Aerogels for High Eﬃciency Oils Absorption. Environ. Int. 2019, 128, 37–45. [8] Liu, Y.; Chan-Park, M.B. Hydrogel Based on Interpenetrating Polymer Networks of Dextran and Gelatin for Vascular Tissue Engineering. Biomaterials 2009, 30, 196–207. [24] Liu, Z.; Lyu, J.; Fang, D.; Zhang, X. Nanoﬁbrous Kevlar Aerogel Threads for Thermal Insulation in Harsh Environments. ACS Nano. 2019, 13, 5703–5711. [9] Wang, J.; Zhao, D.; Shang, K.; Wang, Y.T.; Ye, D.D.; Kang, A.H.; Liao, W.; Wang, Y.Z. Ultrasoft Gelatin Aerogels for Oil Contaminant Removal. J. Mater. Chem. A. 2016, 4, 9381–9389. [25] Cheng, B.; Wu, P. Scalable Fabrication of Kevlar/Ti3C2Tx MXene Intelligent Wearable Fabrics with Multiple Sensory Capabilities. ACS Nano. 2021, 15, 8676–8685. [10] Kozlov, P.V.; Burdygina, G.I. The Structure and Properties of Solid Gelatin and the Principles of Their Modiﬁcation. Polymer 1983, 24, 651–666. [26] Si, Y.; Wang, X.; Dou, L.; Yu, J.; Ding, B. Ultralight and Fire- Resistant Ceramic Nanoﬁbrous Aerogels with Temperature-Invariant Superelasticity. Sci. Adv. 2018, 4, 8925. [11] Zhao, S.; Malfait, W.J.; Guerrero-Alburquerque, N.; Koebel, M.M.; Nyström, G. Biopolymer Aerogels and Foams: Chemistry, Properties, and Applications. Angew. Chem. Int. Ed. 2018, 57, 7580–7608. [27] Song, J.; Chen, C.; Yang, Z.; Kuang, Y.; Li, T.; Li, Y.; Huang, H.; Kierzewski, I.; Liu, B.; He, S. et al. Highly Compressible, Anisotropic Aerogel with Aligned Cellulose Nanoﬁbers. ACS Nano. 2018, 12, 140–147. [12] Qian, Z.; Wang, Z.; Zhao, N.; Xu, J. Aerogels Derived from Polymer Nanoﬁbers and Their Applications. Macromol. Rapid Commun. 2018, 39, 1700724. [28] Li, Y.Q.; Samad, Y.A.; Polychronopoulou, K.; Liao, K. Lightweight and Highly Conductive Aerogel-Like [13] Wei, G.; Zhang, J.; Usuelli, M.; Zhang, X.; Liu, B.; Mezzenga, R. Biomass vs Inorganic and Plastic-Based Aerogels: Y. YUAN AND N. SOLIN 518 Amyloid Nanostructure; Amyloid Hydrogels as Drug Delivery Vehicles. Chem. Commun. 2013, 49, 5082– 5084. Carbon from Sugarcane with Superior Mechanical and EMI Shielding Properties. ACS Sustainable Chem. Eng. 2015, 3, 1419–1427. [45] [29] Rigueto, C.V.T.; Nazari, M.T.; Massuda, L.Á.; Ostwald, B.E.P.; Piccin, J.S.; Dettmer, A. References Production and Environmental Applications of Gelatin-Based Composite Adsorbents for Contaminants Removal: A Review. Environ. Chem. Lett. 2021, 19, 2465–2486. [45] Duraj-Thatte, A.M.; Manjula-Basavanna, A.; Courchesne, N.M.D.; Cannici, G.I.; Sánchez-Ferrer, A.; Frank, B.P.; van’t Hag, L.; Cotts, S.K.; Fairbrother, D.H.; Mezzenga, R.; Joshi, N.S. Water-Processable, Biodegradable and Coatable Aquaplastic from Engineered Bioﬁlms. Nat. Chem. Biol. 2021, 17, 732–738. [30] Li, S.L.; Wang, J.; Zhao, H.B.; Cheng, J.B.; Zhang, A.N.; Wang, T.; Cao, M.; Fu, T.; Wang, Y.Z. Ultralight Biomass Aerogels with Multifunctionality and Superelasticity Under Extreme Conditions. ACS Appl. Mater. Interfaces 2021, 13, 59231–59242. [46] Nyström, G.; Fernández-Ronco, M.P.; Bolisetty, S.; Mazzotti, M.; Mezzenga, R. Amyloid Templated Gold Aerogels. Adv. Mater. 2016, 28, 472–478. [47] Altamura, L.; Horvath, C.; Rengaraj, S.; Rongier, A.; Elouarzaki, K.; Gondran, C.; Maçon, A.L.; Vendrely, C.; Bouchiat, V.; Fontecave, M. et al. A Synthetic Redox Bioﬁlm Made from Metalloprotein–Prion Domain Chimera Nanowires. Nat. Chem. 2017, 9, 157–163. [31] Yang,M.;Yuan,Y.;Li,Y.;Sun,X.;Wang,S.;Liang,L.;Ning,Y.;Li, J.; Yin, W.; Li, Y. Anisotropic Electromagnetic Absorption of Aligned Ti3C2Tx MXene/Gelatin Nanocomposite Aerogels. ACS Appl. Mater. Interfaces 2020, 12, 33128–33138. [48] Guttenplan, A.P.; Young, L.J.; Matak-Vinkovic, D.; Kaminski, C.F.; Knowles, T.P.; Itzhaki, L.S. Nanoscale Click-Reactive Scaﬀolds from Peptide Self-Assembly. J. Nanobiotechnol. 2017, 15, 1–8. [32] Wang, Y.T.; Zhao, H.B.; Degracia, K.; Han, L.X.; Sun, H.; Sun, M.; Wang, Y.Z.; Schiraldi, D.A. Green Approach to Improving the Strength and Flame Retardancy of Poly (Vinyl Alcohol)/Clay Aerogels: Incorporating Biobased Gelatin. ACS Appl. Mater. Interfaces 2017, 9, 42258–42265. [49] Friščić, T.; Mottillo, C.; Titi, H.M. Mechanochemistry for Synthesis. Angew. Chem. Int. Ed. 2020, 132, 1030–1041. [33] Lendel, C.; Solin, N. Protein Nanoﬁbrils and Their Use as Building Blocks of Sustainable Materials. RSC Adv. 2021, 11, 39188–39215. [50] Rizzo, A.; Inganäs, O.; Solin, N. Preparation of Phosphorescent Amyloid-Like Protein Fibrils. Chem-Eur. J. 2010, 16, 4190–4195. [34] Knowles, T.P.; Mezzenga, R. Amyloid Fibrils as Building Blocks for Natural and Artiﬁcial Functional Materials. Adv. Mater. 2016, 28, 6546–6561. [51] Bäcklund, F.G.; Wigenius, J.; Westerlund, F.; Inganäs, O.; Solin, N. Amyloid Fibrils as Dispersing Agents for Oligothiophenes: Control of Photophysical Properties Through Nanoscale Templating and Flow Induced Fibril Alignment. J. Mater. Chem. C. 2014, 2, 7811–7822. [35] Ke, P.C.; Zhou, R.; Serpell, L.C.; Riek, R.; Knowles, T.P.; Lashuel, H.A.; Gazit, E.; Hamley, I.W.; Davis, T.P.; Fändrich, M. et al. Half a Century of Amyloids: Past, Present and Future. Chem. Soc. Rev. 2020, 49, 5473–5509. [52] Bäcklund, F.G.; Pallbo, J.; Solin, N. References Controlling Amyloid Fibril Formation by Partial Stirring. Biopolymers 2016, 105, 249–259. [36] Knowles, T.P.; Vendruscolo, M.; Dobson, C.M. The Amyloid State and Its Association with Protein Misfolding Diseases. Nat. Rev. Mol. Cell Bio. 2014, 15, 384–396. [53] Bäcklund, F.G.; Solin, N. Development and Application of Methodology for Rapid Screening of Potential Amyloid Probes. ACS Comb. Sci. 2014, 16, 721–729. [37] Chiti, F.; Dobson, C.M. Protein Misfolding, Functional Amyloid, and Human Disease. Annu. Rev. Biochem. 2006, 75, 333–366. [54] Wang, L.; Solin, N. Preparation of Functionalized Protein Materials Assisted by Mechanochemistry. J. Mater. Sci. 2018, 53, 13719–13732. [38] Rizzo, A.; Solin, N.; Lindgren, L.J.; Andersson, M.R.; Inganas, O. White Light with Phosphorescent Protein Fibrils in OLEDs. Nano Lett. 2010, 10, 2225–2230. [55] Bäcklund, F.G.; Solin, N. Tuning the Aqueous Self- Assembly Process of Insulin by a Hydrophobic Additive. RSC Adv. 2015, 5, 92254–92262. [39] Yuan, Y.; Solin, N. Mechanochemical Preparation and Self- Assembly of Protein: Dye Hybrids for White Luminescence. ACS Appl. Poly. Mater. 2021, 3, 4825–4836. [56] Wang, L.; Bäcklund, F.G.; Yuan, Y.; Nagamani, S.; Hanczyc, P.; Sznitko, L.; Solin, N. Air–Water Interface Assembly of Protein Nanoﬁbrils Promoted by Hydrophobic Additives. ACS Sustainable Chem. Eng. 2021, 9, 9289–9299. [40] Hu, J.; Wang, L.; Zhang, X.; Yu, W.; Gao, H.W.; Solin, N.; Hu, Z.; Uvdal, K. Selective Colorimetric Detection of Copper (II) by a Protein-Based Nanoprobe. Spectrochim. Acta A. 2021, 252, 119462. [57] Fischer, H.; Polikarpov, I.; Craievich, A.F. Average Protein Density Is a Molecular-Weight-Dependent Function. Protein Sci. 2004, 13, 2825–2828. [41] Han, Y.; Cao, Y.; Bolisetty, S.; Tian, T.; Handschin, S.; Lu, C.; Mezzenga, R. Amyloid Fibril-Templated High- Performance Conductive Aerogels with Sensing Properties. Small 2020, 16, 2004932. [58] Kruk, M.; Jaroniec, M.; Sayari, A. Application of Large Pore MCM-41 Molecular Sieves to Improve Pore Size Analysis Using Nitrogen Adsorption Measurements. Langmuir 1997, 13, 6267–6273. p [42] Yuan Y.; Solin N. Protein-Based Flexible Conductive Aerogels for Piezoresistive Pressure Sensors. ACS Appl. Bio Mater. 2022, 5, 3360–3370. doi:10.1021/acsabm.2c00348 [59] Colquhoun, C.; Draper, E.R.; Schweins, R.; Marcello, M.; Vadukul, D.; Serpell, L.C.; Adams, D.J. Controlling the Network Type in Self-Assembled Dipeptide Hydrogels. Soft. Matter. 2017, 13, 1914–1919. [43] Yuan, Y.; Wang, L.; Porcheddu, A.; Colacino, E.; Solin, N. Mechanochemical Preparation of Protein: Hydantoin Hybrids and Their Release Properties. Chem. Sus. Chem. 2022, 15, e202102097. [60] Ye, X.; Capezza, A.J.; Xiao, X.; Lendel, C.; Hedenqvist, M.S.; Kessler, V.G.; Olsson, R.T. References Protein Nanoﬁbrils and Their Hydrogel Formation with Metal Ions. ACS Nano. 2021, 15, 5341–5354. [44] Mains, J.; Lamprou, D.A.; McIntosh, L.; Oswald, I.D.; Urquhart, A.J. Beta-Adrenoceptor Antagonists Aﬀect
https://openalex.org/W2162540985	https://hal.inrae.fr/hal-02646428/document	English	null	Repertoire of Escherichia coli agonists sensed by innate immunity receptors of the bovine udder and mammary epithelial cells	Veterinary research	2,012	cc-by	7,609	Adeline Porcherie, Patricia Cunha, Angélina Trotereau, Perrine Roussel, Florence Gilbert, Pascal P. Rainard, Pierre Germon Adeline Porcherie, Patricia Cunha, Angélina Trotereau, Perrine Roussel, Florence Gilbert, Pascal P. Rainard, Pierre Germon To cite this version: Adeline Porcherie, Patricia Cunha, Angélina Trotereau, Perrine Roussel, Florence Gilbert, et al.. Repertoire of Escherichia coli agonists sensed by innate immunity receptors of the bovine udder and mammary epithelial cells. Veterinary Research, 2012, 43, pp.14. 10.1186/1297-9716-43-14. hal- 02646428 Distributed under a Creative Commons Attribution 4.0 International License © 2012 Porcherie et al; 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. Repertoire of Escherichia coli agonists sensed by innate immunity receptors of the bovine udder and mammary epithelial cells Adeline Porcherie1,2, Patricia Cunha1,2, Angelina Trotereau1,2, Perrine Roussel1,2, Florence B Gilbert1,2, Pascal Rainard1,2 and Pierre Germon1,2* Abstract Escherichia coli is a frequent cause of clinical mastitis in dairy cows. It has been shown that a prompt response of the mammary gland after E. coli entry into the lumen of the gland is required to control the infection, which means that the early detection of bacteria is of prime importance. Yet, apart from lipopolysaccharide (LPS), little is known of the bacterial components which are detected by the mammary innate immune system. We investigated the repertoire of potential bacterial agonists sensed by the udder and bovine mammary epithelial cells (bMEC) during E. coli mastitis by using purified or synthetic molecular surrogates of bacterial agonists of identified pattern-recognition receptors (PRRs). The production of CXCL8 and the influx of leucocytes in milk were the readouts of reactivity of stimulated cultured bMEC and challenged udders, respectively. Quantitative PCR revealed that bMEC in culture expressed the nucleotide oligomerization domain receptors NOD1 and NOD2, along with the Toll-like receptors TLR1, TLR2, TLR4, and TLR6, but hardly TLR5. In line with expression data, bMEC proved to react to the cognate agonists C12-iE-DAP (NOD1), Pam3CSK4 (TLR1/2), Pam2CSK4 (TLR2/ 6), pure LPS (TLR4), but not to flagellin (TLR5). As the udder reactivity to NOD1 and TLR5 agonists has never been reported, we tested whether the mammary gland reacted to intramammary infusion of C12-iE-DAP or flagellin. The udder reacted to C12-iE-DAP, but not to flagellin, in line with the reactivity of bMEC. These results extend our knowledge of the reactivity of the bovine mammary gland to bacterial agonists of the innate immune system, and suggest that E. coli can be recognized by several PRRs including NOD1, but unexpectedly not by TLR5. The way the mammary gland senses E. coli is likely to shape the innate immune response and finally the outcome of E. coli mastitis. CXCL8 and C5a and the proinflammatory cytokines IL-1ß, IL-6 and TNFa [6,7]. * Correspondence: Pierre.germon@tours.inra.fr 1INRA, UMR 1282 Infectiologie et Santé Publique, F-37380 Nouzilly, France Full list of author information is available at the end of the article HAL Id: hal-02646428 https://hal.inrae.fr/hal-02646428v1 Submitted on 29 May 2020 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Porcherie et al. Veterinary Research 2012, 43:14 http://www.veterinaryresearch.org/content/43/1/14 Porcherie et al. Veterinary Research 2012, 43:14 VETERINARY RESEARCH VETERINARY RESEARCH RESEARCH Open Access Repertoire of Escherichia coli agonists sensed by innate immunity receptors of the bovine udder and mammary epithelial cells Experimentally induced mastitis Healthy mid-lactating Holstein cows of the experimental herd of the Institute at Nouzilly were selected on the basis of an absence of detectable bacterial growth from two weekly consecutive aseptically collected milk sam- ples in their four mammary quarters and less than 100 000 cells/mL in milk. The cows were in their sec- ond or third lactation and between 2 and 6 months in lactation; they were milked twice a day, at 0800 and 1600 h. The use and care of the cows in this study were approved by the Regional Committee of Ethics for Ani- mal Experimentation (CREEA) (approval CL2007-47). At time zero, five cows received in the lumen of quar- ters through the teat canal either 0.5 mL RPMI 1640 medium (Gibco), C12-iE-DAP (10 μg), Tri-DAP (50 μg). The fourth quarter was left untreated. A second group of five cows received, at time zero, either 0.5 mL RPMI 1640 medium (Gibco) or purified flagellin (5, 1 or 0.1 μg). The infused quarters and the control quarter were aseptically sampled just before the morning milk- ing, and infusion was carried out within 30 min post milking. The quarters were then sampled at 4, 8, 12, 24, 32, 48 and, in the case of NOD1 agonists, 72 h post infusion (hpi). Cells in milk were counted with an auto- mated cell counter (Fossomatic model90; Foss Food Technology, Hillerod, Denmark) as described previously [26]. The occurrence of new infections in the quarters under experiment was verified by plating milk samples onto blood agar plates. Results from quarters infected during the experiment were discarded leading to the removal of results from one quarter in the control group of the each experiment, one quarter infused with C12-iE-DAP and one infused with flagellin (1 μg). Of these agonists that trigger inflammation of the udder, only LPS and MDP can be produced by E. coli strains and contribute to the inflammation observed during bovine mastitis. Yet, E. coli can also produce other agonists that can contribute to this inflammation by triggering TLR2 or other PRR such as TLR5 or NOD1. Actually, lipoproteins from the E. coli envelope can be detected by the TLR2 receptor in combination with either TLR1 (for tri-acylated lipoproteins) or TLR6 (for di-acylated lipoproteins) [14]. In addition, TLR5 was shown to recognize flagellin, the major constituent of flagella [19]. Reagents C12-iE-DAP (C12-D-g-Glu-mDAP), Tri-DAP (L-Ala-g- D-Glu-mDAP), synthetic diacylated (Pam2CSK4) and triacylated (Pam3CSK4) lipopeptide, ultra pure LPS from E. coli K-12, bacterial flagellin from S. enterica ser- ovar Typhimurium were obtained lyophilized from Invi- voGen (Invivogen, France). Homologues of human TLR receptors 1-10 have been identified in bovine and were shown to be expressed at different levels in the skin [15,16]. In the udder, the expression of TLR2, TLR4 and NOD2 has been demon- strated by several laboratories [17,18]. In agreement with the expression of these receptors, infusion of puri- fied bacterial compounds recognized by these PRR lead to an inflammation of the udder mimicking the initial response to experimental infections with live bacteria. For example, infusion of LTA, muramyl-dipeptide (MDP) and LPS have already been shown to induce a neutrophil recruitment and to provoke symptoms simi- lar, yet not identical, to those caused by live bacteria [1,18]. Most importantly, it seems that these bacterial agonists can act synergistically and increase the response of the udder [18]. Agonists were made soluble in sterile pyrogen-free water and then diluted in cell culture medium. Experimentally induced mastitis In human, NOD1 recognizes peptidoglycan fragments composed of gamma-D-gluta- myl-meso-diaminopimelic acid (iE-DAP) or N-acetylmura- myl-L-alanyl-g-D-glutamyl- meso-diaminopimelic acid (mur-TriDAP) [20]. Recognition of both flagellin and peptidoglycan frag- ments is an important issue given the role these MAMPs can play during an infection. Indeed, flagellin is a major player in the recognition of mucosal pathogens [19]. NOD1 has also been demonstrated as an important med- iator in controlling infections by, for example, Listeria monocytogenes, Helicobacter pylori or entero-invasive E. coli [21-23]. Furthermore, it was recently shown that NOD1 could play a role in the maturation of the immune system and that its stimulation could modify the response of the host in a case of neutrophilic inflammation [24,25]. In order to better characterize how E. coli is recog- nized by bMEC, we investigated the repertoire of E. coli MAMPs that could be recognized by bMEC. MAMPs tested are known agonists of different innate immune receptors such as TLR2/TLR1 or TLR2/TLR6 heterodi- mers, TLR4, TLR5, NOD1 and NOD2. Recognition of both flagellin and peptidoglycan frag- ments is an important issue given the role these MAMPs can play during an infection. Indeed, flagellin is a major player in the recognition of mucosal pathogens [19]. NOD1 has also been demonstrated as an important med- iator in controlling infections by, for example, Listeria monocytogenes, Helicobacter pylori or entero-invasive E. coli [21-23]. Furthermore, it was recently shown that NOD1 could play a role in the maturation of the immune system and that its stimulation could modify the response of the host in a case of neutrophilic inflammation [24,25]. Introduction Mastitis is an important pathology in the dairy industry, both in terms of economic impact and animal health. Escherichia coli is among the major mastitis pathogens responsible for clinical mastitis in cows [1-4]. The infec- tion is initiated by the entry of the bacteria through the teat canal and, after a short incubation period, is charac- terized by an important inflammatory response and an important influx of neutrophils into the udder [1,5]. A contribution of Mammary Epithelial Cells (MEC) to the production of these different mediators has been suggested by different authors [8-11]. Indeed, the incu- bation of primary cell cultures of bovine Mammary Epithelial Cells (bMEC) obtained from healthy animals with bacteria, either E. coli or S. aureus, induces a strong response [11-13]. Initiation of the inflammation is accompanied by the production in milk of several molecules in the early stages of infection such as the neutrophil chemo-attractants Recognition of bacteria by host cells, for example macrophages, dendritic cells or epithelial cells, relies upon so called Pattern Recognition Receptors (PRR) [14]. Such receptors belong to three different families namely the Toll-like (TLR), NOD-like (NLR) and RIG- 1-like (RLR) receptors. Each of these receptors * Correspondence: Pierre.germon@tours.inra.fr 1INRA, UMR 1282 Infectiologie et Santé Publique, F-37380 Nouzilly, France Full list of author information is available at the end of the article Page 2 of 8 Porcherie et al. Veterinary Research 2012, 43:14 http://www.veterinaryresearch.org/content/43/1/14 Page 2 of 8 Porcherie et al. Veterinary Research 2012, 43:14 http://www.veterinaryresearch.org/content/43/1/14 Materials and methods Reagents recognizes a set of bacterial motifs or Microbe Asso- ciated Molecular Patterns (MAMPs). For example, TLR4, TLR2 and TLR5 are respectively involved in sensing lipopolysaccharide (LPS), lipoteichoic acid (LTA) (and lipoproteins) and flagellin [14]. Quantification of CXCL8 by ELISA Statistical analyses of the concentrations of CXCL8 or SCC in milk were performed with the nonparametric Friedman test. Comparisons of CXCL8 concentrations in bMEC supernatants were done using the Kruskal and Wallis test followed by multiple comparisons with a Bonferoni correction. A P value < 0.05 was considered significant. CXCL8 concentrations were measured in skim milk by ELISA as described [11,28]. The lowest limit of detec- tion and level of quantification in milk were 0.01 and 0.03 ng/mL for CXCL8, respectively. Culture and stimulation of bMEC bMEC were isolated from five lactating cows as previously described and cryopreserved in liquid nitrogen [10]. Cells were used at their third passage and cultivated as described [27]. Briefly, cells were seeded into 24-well tissue culture plates at a density of 105 cells/well and cultured until con- fluence in a growth medium made up of D-MEM-F12 advanced medium with 2 mM L-glutamine, 20 mM HEPES, 50 μg/mL IGF-1, 10 μg/mL FGF, 10 μg/mL EGF and 1 μg/mL hydrocortisone as additives. The growth medium was then replaced with stimulation medium made up of Advanced D-MEM-F12 medium with 2 mM L-gluta- mine, 20 mM HEPES, and 4 ng/mL hydrocortisone as additives. Stimulations with bacterial agonists were carried Reverse transcription and qPCR analysis Reverse transcription and qPCR analysis Total RNA was extracted from bMEC by using the NucleoSpin RNA II extraction kit (Macherey-Nagel, Düren, Germany), and the residual genomic DNA was removed by using DNase digestion with RNase-free DNase (Macherey-Nagel). The total RNA quantity and quality were assessed by using a NanoDrop spectrophot- ometer (NanoDrop Technologies, Wilmington, DE, Porcherie et al. Veterinary Research 2012, 43:14 http://www.veterinaryresearch.org/content/43/1/14 Page 3 of 8 Page 3 of 8 Porcherie et al. Veterinary Research 2012, 43:14 http://www.veterinaryresearch.org/content/43/1/14 out 16-24 h later. The medium was removed, and agonists (uLPS, C12-iE-DAP, Pam2CSK4, Pam3CSK4, flagellin) were added at the desired concentration in 1 mL of stimu- lation medium. Control wells were treated with stimulation medium only. 5 h after exposure to the MAMP, culture medium was aspirated and stored at -20°C. USA). Total RNA (1 μg) was then reverse transcribed to cDNA using random hexamers and SuperScript RT III (Invitrogen) according to the manufacturer’s instructions. Diluted cDNA samples were stored at 4°C until use. Pri- mers used in this study are listed in Table 1. Relative quantities of gene transcripts were measured as described previously [27]. USA). Total RNA (1 μg) was then reverse transcribed to cDNA using random hexamers and SuperScript RT III (Invitrogen) according to the manufacturer’s instructions. Diluted cDNA samples were stored at 4°C until use. Pri- mers used in this study are listed in Table 1. Relative quantities of gene transcripts were measured as described previously [27]. Multiple PRRs are expressed by bMEC In a first attempt to characterize the repertoire of bacter- ial agonists recognized by the bovine udder, we investi- gated the expression of different Pattern Recognition Receptors (PRR) by RT-qPCR. PRR investigated are those homologous to mammalian receptors involved in recog- nition of LPS (TLR4), peptidoglycan (NOD1 and NOD2), lipoproteins (TLR2, TLR1 and TLR6) and flagellin (TLR5). Expression of all these PRRs was observed in the bMEC preparations isolated from the five different cows (Figure 1). Yet, expression of TLR5 was found to be Table 1 List of primers used in this study Gene Primer ID Primer sequence Size of amplicon Annealing temperature (°C) 18S 18sF CGGGGAGGTAGTGACGAAA 196 bp 62 18sR CCGCTCCCAAGATCCAACTA ACTB ACTB f ACGGGCAGGTCATCACCATC 166 bp 64 ACTB r AGCACCGTGTTGGCGTAGAG PPIA PPIA Fq TCCGGGATTTATGTGCCAGGG 206 bp 66 PPIA Rq GCTTGCCATCCAACCACTCAG TLR1 TLR1 Fq ACCCTACTCTGAACCTCAAG 142 bp 62 TLR1 Rq GACTGCACACTGGATTTCTG TLR2 TLR2 Fq ACTGGGTGGAGAACCTCATGGTCC 307 bp 62 TLR2 Rq ATCTTCCGCAGCTTACAGAAGC TLR4 TLR4 F2q GCATGGAGCTGAATCTCTAC 238 bp 62 TLR4 R2q CAGGCTAAACTCTGGATAGG TLR5 TLR5 Fq TTCCTGCAACCTCACCCAAG 192 bp 62 TLR5 Rq CTGAGATTGGGCAGGTTTCG TLR6 TLR6 Fq CTCCGGGAGATAGTCACTTC 297 bp 62 TLR6 Rq GGCCCTGGATTCTATTATGG NOD1 NOD1 F2q TGGTCACTCACATCCGAAAC 218 bp 62 NOD1 F2q AGGCCTGAGATCCACATAAG NOD2 NOD2-f CCCAGGGGCTCAGAACTAACA 238 bp 62 NOD2-r CCTTCATCCTGGACGTGGTTC Table 1 List of primers used in this study Porcherie et al. Veterinary Research 2012, 43:14 http://www.veterinaryresearch.org/content/43/1/14 Page 4 of 8 Figure 1 Expression of PRR by bMEC. Quantitative RT-PCR was performed on RNA obtained from bMEC isolated from 5 different cows. ΔCt values (Ct target–Ct for ß-actin gene) for each bMEC are represented on the graph. PCR efficiency was above 90% for all genes and Ct were obtained for an identical fluorescence threshold for all genes. peptidoglycan fragments and lipoproteins, we investi- gated whether this was correlated to response to these compounds. Our results demonstrate that the NOD1 agonist C12-iE-DAP induces the production of CXCL8 in a dose dependent manner starting at 100 ng/mL C12-iE-DAP (Figure 2). bMEC also responded to Pam3CSK4 and Pam2CSK4, two lipoprotein analogs respectively agonists of the heterodimer receptors TLR1/2 and TLR2/6 (Figure 2). Interestingly, a strong response was observed with as little as 10 ng/mL of Pam2CSK4 and this response was not increased when using 100 ng/mL or 1000 ng/mL concentrations. Multiple PRRs are expressed by bMEC Con- sistent with the low transcription of TLR5 by bMEC, these cells failed to respond to flagellin in a significant manner: indeed, even at a high concentration (1000 ng/ mL), a very low production of CXCL8 was observed (Figure 2). much lower than that for other TLRs: indeed, although the PCR efficiencies were similar for all genes analyzed (above 90%), the ΔCt value for TLR5 was on average between 3 and 9 cycles above that of other genes. On the contrary, TLR4 and NOD1 were the most highly expressed receptors. Altogether, these results indicate that, in addition to LPS, bMEC are able to sense and respond to TLR2, NOD1 and NOD 2 agonists but not to TLR5 agonists. The response of the udder to bacterial agonists mimics that of bMEC bMEC respond to stimulation by LPS, peptidoglycan fragments but not flagellin bMEC respond to stimulation by LPS, peptidoglycan fragments but not flagellin Twenty-four hours prior to stimulation, the culture medium was replaced by stimulation medium. Cells were incubated in the presence of different purified agonists at the indicated concentrations. CXCL8 concentration in the supernatant was measured by ELISA 5 h after initiation of the stimulation. Data are median values and interquartile ranges. statistical significance (P < 0.05) of values from stimulated versus unstimulated bMEC. Figure 2 CXCL8 secretion by bMEC after stimulation with different bacterial agonists. bMEC from five different cows were seeded in 24-well plates at a concentration of 105 cells/well. Twenty-four hours prior to stimulation, the culture medium was replaced by stimulation medium. Cells were incubated in the presence of different purified agonists at the indicated concentrations. CXCL8 concentration in the supernatant was measured by ELISA 5 h after initiation of the stimulation. Data are median values and interquartile ranges. statistical significance (P < 0.05) of values from stimulated versus unstimulated bMEC. bMEC respond to stimulation by LPS, peptidoglycan fragments but not flagellin Among the above tested agonists, Pam2CSK4 and Pam3CSK4 are supposed to act through stimulation of TLR2, a receptor that is known to be both expressed in vivo at the surface of mammary epithelial cells and functional as demonstrated by intramammary infusion of LTA [11,17]. We therefore focused our in ubero stu- dies on receptors whose activation had never been stu- died previously in the cow. To investigate whether these transcription results trans- lated into functional differences we investigated the response of bMEC to the different agonists recognized by these PRRs. Results obtained confirmed previous stu- dies showing that bMEC respond to stimulation by ago- nists such as LPS, MDP or LTA by the production of CXCL8 (Figure 2 and data not shown) [18]. As all bMEC used in this study also express receptors for We therefore tested the capacity of TLR5 and NOD1 agonists to induce inflammation when infused in the udder. C12-iE-DAP was infused in the mammary gland of 5 cows and we monitored the somatic cell count (Figure 3a) as well as the CXCL8 production in milk (Figure 3b). Our results clearly establish that this agonist induced cellular recruitment and CXCL8 production. Increased SCC com- pared to the control quarter was observed as early as 12 hpi. On the contrary, the use of another NOD1 agonist, Tri-DAP, failed to induce both a significant cellular recruit- ment and CXCL8 production in milk. Figure 2 CXCL8 secretion by bMEC after stimulation with different bacterial agonists. bMEC from five different cows were seeded in 24-well plates at a concentration of 105 cells/well. Twenty-four hours prior to stimulation, the culture medium was replaced by stimulation medium. Cells were incubated in the presence of different purified agonists at the indicated concentrations. CXCL8 concentration in the supernatant was measured by ELISA 5 h after initiation of the stimulation. Data are median values and interquartile ranges. statistical significance (P < 0.05) of values from stimulated versus unstimulated bMEC. Flagellin was infused in the udder of 5 cows at three different doses, up to 5 μg: in all cases, no significant cellular recruitment was observed, consistent with the lack of TLR5 expression and response of bMEC to fla- gellin (Figure 4). Figure 2 CXCL8 secretion by bMEC after stimulation with different bacterial agonists. bMEC from five different cows were seeded in 24-well plates at a concentration of 105 cells/well. Discussion SCC and CXCL8 were quantified in milk 4, 8, 12, 24, 32, 48 h post-infusion. Data are median values and interquartile ranges. statistical significance (P < 0.05) of values before and after treatment. Figure 4 Somatic cell counts (SCC) in quarters infused with flagellin 0.1 μg (☐), flagellin 1 μg (△); flagellin 5 μg (×) or control quarter (◊). Purified agonists were infused into the udder of five different cows. SCC and CXCL8 were quantified in milk 4, 8, 12, 24, 32, 48 h post-infusion. Data are median values and interquartile ranges. statistical significance (P < 0.05) of values before and after treatment. Figure 4 Somatic cell counts (SCC) in quarters infused with flagellin 0.1 μg (☐), flagellin 1 μg (△); flagellin 5 μg (×) or control quarter (◊). Purified agonists were infused into the udder of five different cows. SCC and CXCL8 were quantified in milk 4, 8, 12, 24, 32, 48 h post-infusion. Data are median values and interquartile ranges. statistical significance (P < 0.05) of values before and after treatment. from a role in triggering the innate immune response, activation of NOD1 could also contribute to a more effi- cient reduction of the pathogen load by neutrophils as was shown in other infections [29]. Yet, we only observed inflammation of the udder with C12-iE-DAP and not with Tri-DAP. The major difference between these two agonists is that C12-iE-DAP is rendered membrane permeable by a lipophylic moiety which allows the molecule to better enter the host cell and activate NOD1 [30]. Indeed, the NOD1 receptor is cyto- plasmic and its activation requires that the agonist is transported into the host cell [31]. Figure 3 A–Somatic cell counts (SCC) in quarters infused with C12-iE-DAP 10 μg (☐) Tri-DAP 50 μg (△) or control quarter (◊) Figure 3 A–Somatic cell counts (SCC) in quarters infused with C12-iE-DAP 10 μg (☐), Tri-DAP 50 μg (△) or control quarter (◊). B–CXCL8 concentration in milk from quarters infused with C12-iE- DAP 10 μg (☐), Tri-DAP 50 μg (△) or control quarter (◊). Purified agonists were infused into the udder of five different cows. SCC and CXCL8 were quantified in milk 4, 8, 12, 24, 32, 48 h post-infusion. Data are median values and interquartile ranges. statistical significance (P < 0.05) of values before and after treatment. Discussion μg ( ), μg ( ) q ( ) B–CXCL8 concentration in milk from quarters infused with C12-iE- DAP 10 μg (☐), Tri-DAP 50 μg (△) or control quarter (◊). Purified agonists were infused into the udder of five different cows. SCC and CXCL8 were quantified in milk 4, 8, 12, 24, 32, 48 h post-infusion. Data are median values and interquartile ranges. statistical significance (P < 0.05) of values before and after treatment. The failure to induce inflammation of the udder with Tri-DAP is therefore likely to result from the lack of access of this agonist to the cytoplasm of host cells. Nevertheless, during the course of mastitis, it is possible that peptidoglycan fragments recognized by NOD1 can reach the cytoplasm of bovine cells following invasion of epithelial cells by E. coli. Indeed, invasion of mammary epithelial cells has been described for some strains of E. coli involved in recurrent mastitis cases [32]. Further- more, expression of membrane transporters under parti- cular circumstances like inflammation could transport peptidoglycan fragments, as was shown for MDP, a NOD2 agonist [18,33]. several studies that showed that these cells were able to respond to different bacterial stimuli. In this study we investigated in more details the bacterial compounds that could be recognized by bMEC and whether these compounds were able to induce inflammation when infused into the udder. By a combination of transcrip- tion data, in vitro stimulation of bMEC and in ubero experiments we confirmed previous studies and showed for the first time that NOD1 could play a role in bovine mastitis while, on the contrary, TLR5 is not likely to be an important contributor to the inflammation of the udder. Most interestingly, we demonstrated that the TLR5 gene was only weakly transcribed and, consistently, that bacterial flagellin was not recognized by bMEC and did not induce inflammation of the udder. The failure of purified flagellin to induce inflammation of the udder was not expected since it is considered an important target of mucosal immunity [34]. Although the flagellin used in this study was purified from S. enterica serovar More precisely, our results show for the first time that peptidoglycan fragments known to activate the NOD1 receptor in other mammals could be recognized by bMEC and are able to induce inflammation in ubero. This result is all the more important given the multiple activities that the NOD1 receptor is likely to play. Discussion This study was undertaken in order to understand how E. coli can be sensed by the mammary gland and to bet- ter characterize the potential contribution of bMEC to the inflammation of the udder. The role of bMEC in initiating the inflammation has been suggested by Porcherie et al. Veterinary Research 2012, 43:14 http://www.veterinaryresearch.org/content/43/1/14 Page 5 of 8 Figure 3 A–Somatic cell counts (SCC) in quarters infused with C12-iE-DAP 10 μg (☐), Tri-DAP 50 μg (△) or control quarter (◊). B–CXCL8 concentration in milk from quarters infused with C12-iE- DAP 10 μg (☐), Tri-DAP 50 μg (△) or control quarter (◊). Purified agonists were infused into the udder of five different cows. SCC and CXCL8 were quantified in milk 4, 8, 12, 24, 32, 48 h post-infusion. Data are median values and interquartile ranges. statistical significance (P < 0.05) of values before and after treatment. from a role in triggering the innate immune response activation of NOD1 could also contribute to a more effi cient reduction of the pathogen load by neutrophils a was shown in other infections [29]. Yet, we only observed inflammation of the udder with C12-iE-DAP and not with Tri-DAP. The major difference between these two agonists is that C12-iE-DAP is rendered membrane permeable by a lipophylic moiety which allows the molecule to better enter the host cell and activate NOD1 [30]. Indeed, the NOD1 receptor is cyto plasmic and its activation requires that the agonist i transported into the host cell [31]. The failure to induce inflammation of the udder with T i DAP i h f lik l l f h l k Figure 3 A–Somatic cell counts (SCC) in quarters infused with C12-iE-DAP 10 μg (☐), Tri-DAP 50 μg (△) or control quarter (◊). B–CXCL8 concentration in milk from quarters infused with C12-iE- DAP 10 μg (☐), Tri-DAP 50 μg (△) or control quarter (◊). Purified agonists were infused into the udder of five different cows. SCC and CXCL8 were quantified in milk 4, 8, 12, 24, 32, 48 h post-infusion. Data are median values and interquartile ranges. statistical significance (P < 0.05) of values before and after treatment. Figure 4 Somatic cell counts (SCC) in quarters infused with flagellin 0.1 μg (☐), flagellin 1 μg (△); flagellin 5 μg (×) or control quarter (◊). Purified agonists were infused into the udder of five different cows. Discussion Apart Page 6 of 8 Porcherie et al. Veterinary Research 2012, 43:14 http://www.veterinaryresearch.org/content/43/1/14 Regarding the specificity of bovine PRRs, one should bear in mind that the specificity of these receptors might not be strictly identical to that of human/mouse PRRs, as reviewed by Werling et al. [40]. These authors actually state that bovine TLR1/TLR2 heterodimers could sense both Pam2CSK4 and Pam3CSK4. Concerning TLR5, stu- dies with Salmonella Typhimurium flagellin indicated that it could activate bovine TLR5 expressing macrophages [41]. The specificities of bovine NOD1 and NOD2 recep- tors have not been characterized so far. Typhimurium, it is known to activate most mammalian TLR5 receptors. Furthermore, the region of flagellin recognized by TLR5 is located in one of the two con- served regions of flagellin and is present both in Salmo- nella Typhimurium and E. coli flagellin [19,35]. It is therefore likely that, as for Salmonella Typhimurium fla- gellin, E. coli flagellin will not be detected by bMEC and does not contribute to the inflammation of the udder during E. coli mastitis. Still, it is possible that TLR5 contributes to the response of the host when the infection worsens. In other pathologies, stimulation of TLR5 in epithelial cells is pre- cluded by the basolateral localization of TLR5 and only occurs as a consequence of tissue damage whereby flagel- lin gets in contact with TLR5 receptors [36]. This situa- tion is often the result of invasive pathogens and, in some instances, could occur in severe E. coli mastitis cases. One could also speculate that expression of TLR5 could be induced upon activation of other PRRs. Aside from detection by TLR5, flagellin, when translocated to the cytoplasm of the host cell, is also known to activate the inflammasome through stimulation of NLRC4 (also called IPAF) [37]. However, the contribution of this path- way to the response of the host to flagellin is still under debate [34]. The udder unresponsiveness to flagellin also suggests that milk macrophages were either too few or not reactive enough to set up inflammation. Investigations described in this manuscript were obtained with purified agonists used alone. Our recent results with MDP and LTA indicate that agonists can act synergistically. Because a bacterium expresses several MAMPs at a time such a synergy is likely to best mimic what bMEC are exposed to during E. coli mastitis. Discussion Synergy between MAMPs has already been observed in other mod- els: in mice, for example, inoculation of LPS after an MDP treatment increases the immune response [42]. Synergy has also been observed with human monocytes and den- dritic cells [43]. Our preliminary data indicate that such a synergy may exist in ubero between LPS and MDP (data not shown). The relevance of these potential synergies is important in that it is likely to improve the recognition of an udder pathogen and lead to an early response of the host, which impacts the outcome of infection. A corollary would be to study how different E. coli strains stimulate each of these different PRRs. Because our results were obtained with purified generic compounds, they do not necessarily reflect the full diversity of E. coli MAMPs that can be recognized. Indeed, the E. coli species is very diverse: recent genomic studies have shown that of the 4500-5000 genes that one E. coli strain might contain only approx. 1800 genes are found in all E. coli strains [44]. Mastitis E. coli isolates has only been incompletely characterized. The presence of different genes character- ized as virulence factors in other E. coli pathotypes has been investigated by several authors but none has been shown to be highly prevalent in mastitis E. coli strains. Recently, a preliminary study showed that the prototypical E. coli mastitis strain P4 contains several unique regions [45]. Although no potential virulence genes have been identified as specific of mastitis E. coli strains, the way E. coli stimulates the host cells might play a role in the inflammation of the udder. Altogether, the above results support the idea that flagel- lin is not a critical motif for the early detection of E. coli in the healthy udder, neither through the activation of TLR5 nor that of NLRC4. Interestingly, proteomic analysis of the mastitis E. coli strain P4 have shown that expression of fla- gellin is decreased when bacteria are grown in milk com- pared to conventional laboratory growth medium [38]. Our results also show that bMEC are also able to sense the presence of lipoproteins through the TLR1/2 and TLR2/6 heterodimers. Both agonists used in this study induced a significant response of bMEC, Pam2CSK4 being more potent than Pam3CSK4 at inducing CXCL8 secretion by bMEC. Authors’ contributions PRa and FBG conceived the study and participated in its design and coordination; PRa, FBG, AT and PC carried out collection and analysis of milk samples. AP, PG and PC performed and analyzed stimulation of bMEC and expression of PRR data. PG wrote the manuscript. AP, FBG, PRo and PRa helped writing the final version of the manuscript. All authors read and approved the final manuscript. 19. Ramos HC, Rumbo M, Sirard JC: Bacterial flagellins: mediators of pathogenicity and host immune responses in mucosa. Trends Microbiol 2004, 12:509-517. 20. Chaput C, Boneca IG: Peptidoglycan detection by mammals and flies. Microbes Infect 2007, 9:637-647. 20. Chaput C, Boneca IG: Peptidoglycan detection by mammals and flies. Microbes Infect 2007, 9:637-647. Author details 1 1INRA, UMR 1282 Infectiologie et Santé Publique, F-37380 Nouzilly, France. 2Université François Rabelais, UMR 1282, Tours, France. 18. Bougarn S, Cunha P, Harmache A, Fromageau A, Gilbert FB, Rainard P: Muramyl dipeptide synergizes with Staphylococcus aureu lipoteichoic acid to recruit neutrophils in the mammary gland and to stimulate mammary epithelial cells. Clin Vaccine Immunol 2010, 17:1797-1809. Acknowledgements l g P. Roussel is recipient of a grant from the « Microbiology and Food Chain » and « Animal Health » divisions of INRA. This project was funded in part by Pfizer AH. 16. Menzies M, Ingham A: Identification and expression of Toll-like receptors 1-10 in selected bovine and ovine tissues. Vet Immunol Immunopathol 2006, 109:23-30. 16. Menzies M, Ingham A: Identification and expression of Toll-like receptors 1-10 in selected bovine and ovine tissues. Vet Immunol Immunopathol 2006, 109:23-30. 17. Petzl W, Zerbe H, Gunther J, Yang W, Seyfert HM, Nurnberg G, Schuberth HJ: Escherichia col, but not Staphylococcus aureu triggers an early increased expression of factors contributing to the innate immune defense in the udder of the cow. Vet Res 2008, 39:18. Discussion Jungi TW, Farhat K, Burgener IA, Werling D: Toll-like receptors in domestic animals. Cell Tissue Res 2011, 343:107-120. 15. Jungi TW, Farhat K, Burgener IA, Werling D: Toll-like receptors in domestic animals. Cell Tissue Res 2011, 343:107-120. References 1. Burvenich C, Van Merris V, Mehrzad J, Diez-Fraile A, Duchateau L: Severity of E. col mastitis is mainly determined by cow factors. Vet Res 2003, 34:521-564. 23. Viala J, Chaput C, Boneca IG, Cardona A, Girardin SE, Moran AP, Athman R, Memet S, Huerre MR, Coyle AJ, DiStefano PS, Sansonetti PJ, Labigne A, Bertin J, Philpott DJ, Ferrero RL: Nod1 responds to peptidoglycan delivered by the Helicobacter pylori ca pathogenicity island. Nat Immunol 2004, 5:1166-1174. 2. Botrel MA, Haenni M, Morignat E, Sulpice P, Madec JY, Calavas D: Distribution and antimicrobial resistance of clinical and subclinical mastitis pathogens in dairy cows in Rhone-Alpes, France. Foodborne Pathog Dis 2010, 7:479-487. 2. Botrel MA, Haenni M, Morignat E, Sulpice P, Madec JY, Calavas D: Distribution and antimicrobial resistance of clinical and subclinical mastitis pathogens in dairy cows in Rhone-Alpes, France. Foodborne Pathog Dis 2010, 7:479-487. 24. Bouskra D, Brezillon C, Berard M, Werts C, Varona R, Boneca IG, Eberl G: Lymphoid tissue genesis induced by commensals through NOD1 regulates intestinal homeostasis. Nature 2008, 456:507-510. 3. Bradley AJ, Leach KA, Breen JE, Green LE, Green MJ: Survey of the incidence and aetiology of mastitis on dairy farms in England and Wales. Vet Rec 2007, 160:253-257. 25. Clarke TB, Davis KM, Lysenko ES, Zhou AY, Yu Y, Weiser JN: Recognition of peptidoglycan from the microbiota by Nod1 enhances systemic innate immunity. Nat Med 2010, 16:228-231. 4. Kalmus P, Aasmae B, Karssin A, Orro T, Kask K: Udder pathogens and their resistance to antimicrobial agents in dairy cows in Estonia. Acta Vet Scand 2011, 53:4. 4. Kalmus P, Aasmae B, Karssin A, Orro T, Kask K: Udder pathogens and their resistance to antimicrobial agents in dairy cows in Estonia. Acta Vet Scand 2011, 53:4. 26. Bannerman DD, Paape MJ, Lee JW, Zhao X, Hope JC, Rainard P: Escherichia col and Staphylococcus aureu elicit differential innate immune responses following intramammary infection. Clin Diagn Lab Immunol 2004, 11:463-472. 5. Vangroenweghe F, Rainard P, Paape M, Duchateau L, Burvenich C: Increase of Escherichia col inoculum doses induces faster innate immune response in primiparous cows. J Dairy Sci 2004, 87:4132-4144. 5. Vangroenweghe F, Rainard P, Paape M, Duchateau L, Burvenich C: Increase of Escherichia col inoculum doses induces faster innate immune response in primiparous cows. J Dairy Sci 2004, 87:4132-4144. 6. Competing interests Th h d l h 21. Kim JG, Lee SJ, Kagnoff MF: Nod1 is an essential signal transducer in intestinal epithelial cells infected with bacteria that avoid recognition by toll-like receptors. Infect Immun 2004, 72:1487-1495. The authors declare that they have no competing interests. The authors declare that they have no competing interests. The authors declare that they have no competing interests. Received: 5 October 2011 Accepted: 13 February 2012 Published: 13 February 2012 Received: 5 October 2011 Accepted: 13 February 2012 Published: 13 February 2012 Received: 5 October 2011 Accepted: 13 February 2012 Published: 13 February 2012 22. Mosa A, Trumstedt C, Eriksson E, Soehnlein O, Heuts F, Janik K, Klos A, Dittrich-Breiholz O, Kracht M, Hidmark A, Wigzell H, Rottenberg ME: Nonhematopoietic cells control the outcome of infection with Listeria monocytogene in a nucleotide oligomerization domain 1-dependent manner. Infect Immun 2009, 77:2908-2918. Discussion Such results are not surprising since expression of the TLR2 receptor has already been shown in bMEC and was increased in infected udders [8,17]. In addition, infusion of the TLR2 agonist LTA in the udder of cows was shown to induce inflammation [18]. A new finding of this study is that bMEC reacted strongly to much lower concentrations of Pam2CSK4 than to Pam3CSK4. This higher reactivity is in line with the higher expression of TLR6 compared to TLR1 by bMEC. Nevertheless, although di-acyl lipoproteins can be found in E. coli, most E. coli lipoproteins are tri-acylated pro- teins [39]. It is therefore likely that the response of bMEC to Pam3CSK4 resembles most to that induced by E. coli lipoproteins. In conclusion, bMEC are equipped with innate immu- nity receptors that allow them to sense several E. coli MAMPs. Overall, expression of PRRs as expressed by qPCR mirrored the reactivity of bMEC as assessed by the production of CXCL8, and the results obtained in bMEC in vitro were in good agreement with the in ubero studies. Such findings are in line with the concept that bMEC are key players in initiating neutrophil inflammation during Page 7 of 8 Page 7 of 8 Porcherie et al. Veterinary Research 2012, 43:14 http://www.veterinaryresearch.org/content/43/1/14 Porcherie et al. Veterinary Research 2012, 43:14 http://www.veterinaryresearch.org/content/43/1/14 11. Rainard P, Fromageau A, Cunha P, Gilbert FB: Staphylococcus aureu lipoteichoic acid triggers inflammation in the lactating bovine mammary gland. Vet Res 2008, 39:52. E. coli mastitis. Recognition of several MAMPs at a time could contribute to the onset of an early response of the cow after infection by E. coli. These results pave the way to a better understanding of the early steps of the inflam- mation triggered by E. coli entrance into the udder. The precise contribution of the different PRR to this response is now necessary and deserves further studies. 12. Rinaldi M, Li RW, Capuco AV: Mastitis associated transcriptomic disruptions in cattle. Vet Immunol Immunopathol 2010, 138:267-279. 13. Gunther J, Koczan D, Yang W, Nurnberg G, Repsilber D, Schuberth HJ, Park Z, Maqbool N, Molenaar A, Seyfert HM: Assessment of the immune capacity of mammary epithelial cells: comparison with mammary tissue after challenge with Escherichia col. Vet Res 2009, 40:31. 14. Kumar H, Kawai T, Akira S: Pathogen recognition by the innate immune system. Int Rev Immunol 2011, 30:16-34. 15. Porcherie et al. Veterinary Research 2012, 43:14 http://www.veterinaryresearch.org/content/43/1/14 32. Dogan B, Klaessig S, Rishniw M, Almeida RA, Oliver SP, Simpson K, Schukken YH: Adherent and invasive Escherichia col are associated with persistent bovine mastitis. Vet Microbiol 2006, 116:270-282. 32. Dogan B, Klaessig S, Rishniw M, Almeida RA, Oliver SP, Simpson 33. Vavricka SR, Musch MW, Chang JE, Nakagawa Y, Phanvijhitsiri K, Waypa TS, Merlin D, Schneewind O, Chang EB: hPepT1 transports muramyl dipeptide, activating NF-kappaB and stimulating IL-8 secretion in human colonic Caco2/bbe cells. Gastroenterology 2004, 127:1401-1409. 34. Vijay-Kumar M, Gewirtz AT: Flagellin: key target of mucosal innate immunity. Mucosal Immunol 2009, 2:197-205. 35. Jacchieri SG, Torquato R, Brentani RR: Structural study of binding of flagellin by Toll-like receptor 5. J Bacteriol 2003, 185:4243-4247. 35. Jacchieri SG, Torquato R, Brentani RR: Structural study of binding of flagellin by Toll-like receptor 5. J Bacteriol 2003, 185:4243-4247. 36. Rhee SH, Im E, Riegler M, Kokkotou E, O’Brien M, Pothoulakis C: Pathophysiological role of Toll-like receptor 5 engagement by bacterial flagellin in colonic inflammation. Proc Natl Acad Sci USA 2005, 102:13610-13615. 37. Miao EA, Andersen-Nissen E, Warren SE, Aderem A: TLR5 and Ipaf: dual sensors of bacterial flagellin in the innate immune system. Semin Immunopathol 2007, 29:275-288. p 38. Lippolis JD, Bayles DO, Reinhardt TA: Proteomic changes in Escherichia col when grown in fresh milk versus laboratory media. J Proteome Res 2009, 8:149-158. 39. Kovacs-Simon A, Titball RW, Michell SL: Lipoproteins of bacterial pathogens. Infect Immun 2011, 79:548-561. 39. Kovacs-Simon A, Titball RW, Michell SL: Lipoproteins of bacterial pathogens. Infect Immun 2011, 79:548-561. 40. Werling D, Jann OC, Offord V, Glass EJ, Coffey TJ: Variation matters: TLR structure and species-specific pathogen recognition. Trends Immunol 2009, 30:124-130. 41. Metcalfe HJ, Best A, Kanellos T, La Ragione RM, Werling D: Flagellin expression enhances Salmonell accumulation in TLR5-positive macrophages. Dev Comp Immunol 2010, 34:797-804. 42. Shikama Y, Kuroishi T, Nagai Y, Iwakura Y, Shimauchi H, Takada H, Sugawara S, Endo Y: Muramyldipeptide augments the actions of lipopolysaccharide in mice by stimulating macrophages to produce pro- IL-1ß and by down-regulation of suppressor of cytokine signaling 1 (SOCS1). Innate Immun 2011, 17:3-15. 43. Fritz JH, Girardin SE, Fitting C, Werts C, Mengin-Lecreulx D, Caroff M, Cavaillon JM, Philpott DJ, Adib-Conquy M: Synergistic stimulation of human monocytes and dendritic cells by Toll-like receptor 4 and NOD1- and NOD2-activating agonists. Eur J Immunol 2005, 35:2459-2470. 44. References Shuster DE, Kehrli ME Jr, Rainard P, Paape M: Complement fragment C5a and inflammatory cytokines in neutrophil recruitment during intramammary infection with Escherichia col. Infect Immun 1997, 65:3286-3292. 6. Shuster DE, Kehrli ME Jr, Rainard P, Paape M: Complement fragment C5a and inflammatory cytokines in neutrophil recruitment during intramammary infection with Escherichia col. Infect Immun 1997, 65:3286-3292. 27. Bougarn S, Cunha P, Gilbert FB, Meurens F, Rainard P: Technical note: validation of candidate reference genes for normalization of quantitative PCR in bovine mammary epithelial cells responding to inflammatory stimuli. J Dairy Sci 2011, 94:2425-2430. 7. Riollet C, Rainard P, Poutrel B: Differential induction of complement fragment C5a and inflammatory cytokines during intramammary infections with Escherichia col and Staphylococcus aureu. Clin Diagn Lab Immunol 2000, 7:161-167. 28. Rainard P: Consequences of interference of milk with chemoattractants for enzyme-linked immunosorbent assay quantifications. Clin Vaccine Immunol 2010, 17:848-852. 29. Lysenko ES, Clarke TB, Shchepetov M, Ratner AJ, Roper DI, Dowson CG, Weiser JN: Nod1 signaling overcomes resistance of S. pneumoniae to opsonophagocytic killing. PLoS Pathog 2007, 3:e118. 8. Goldammer T, Zerbe H, Molenaar A, Schuberth HJ, Brunner RM, Kata SR, Seyfert HM: Mastitis increases mammary mRNA abundance of beta- defensin 5, toll-like-receptor 2 (TLR2), and TLR4 but not TLR9 in cattle. Clin Diagn Lab Immunol 2004, 11:174-185. 30. Masumoto J, Yang K, Varambally S, Hasegawa M, Tomlins SA, Qiu S, Fujimoto Y, Kawasaki A, Foster SJ, Horie Y, Mak TW, Núñez G, Chinnaiyan AM, Fukase K, Inohara N: Nod1 acts as an intracellular receptor to stimulate chemokine production and neutrophil recruitment in vivo. J Exp Med 2006, 203:203-213. 9. Strandberg Y, Gray C, Vuocolo T, Donaldson L, Broadway M, Tellam R: Lipopolysaccharide and lipoteichoic acid induce different innate immune responses in bovine mammary epithelial cells. Cytokine 2005, 31:72-86. 31. Girardin SE, Boneca IG, Carneiro LA, Antignac A, Jehanno M, Viala J, Tedin K, Taha MK, Labigne A, Zahringer U, Coyle AJ, DiStefano PS, Bertin J, Sansonetti PJ, Philpott DJ: Nod1 detects a unique muropeptide from gram-negative bacterial peptidoglycan. Science 2003, 300:1584-1587. 10. Lahouassa H, Moussay E, Rainard P, Riollet C: Differential cytokine and chemokine responses of bovine mammary epithelial cells to Staphylococcus aureu and Escherichia col. Cytokine 2007, 38:12-21. Page 8 of 8 Page 8 of 8 Porcherie et al. Veterinary Research 2012, 43:14 http://www.veterinaryresearch.org/content/43/1/14 doi:10.1186/1297-9716-43-14 Cite this article as: Porcherie et al.: Repertoire of Escherichia coli agonists sensed by innate immunity receptors of the bovine udder and mammary epithelial cells. Veterinary Research 2012 43:14. Porcherie et al. Veterinary Research 2012, 43:14 http://www.veterinaryresearch.org/content/43/1/14 Touchon M, Hoede C, Tenaillon O, Barbe V, Baeriswyl S, Bidet P, Bingen E, Bonacorsi S, Bouchier C, Bouvet O, Calteau A, Chiapello H, Clermont O, Cruveiller S, Danchin A, Diard M, Dossat C, Karoui ME, Frapy E, Garry L, Ghigo JM, Gilles AM, Johnson J, Le Bouguénec C, Lescat M, Mangenot S, Martinez-Jéhanne V, Matic I, Nassif X, Oztas S, et al: Organised genome dynamics in the Escherichia col species results in highly diverse adaptive paths. PLoS Genet 2009, 5:e1000344. 45. Dufour D, Germon P, Brusseaux E, Le Roux Y, Dary A: First evidence of the presence of genomic islands in Escherichia col P4, a mammary pathogen frequently used to induce experimental mastitis. J Dairy Sci 2011, 94:2779-2793. doi:10.1186/1297-9716-43-14 Cite this article as: Porcherie et al.: Repertoire of Escherichia coli agonists sensed by innate immunity receptors of the bovine udder and mammary epithelial cells. Veterinary Research 2012 43:14. doi:10.1186/1297-9716-43-14 Cite this article as: Porcherie et al.: Repertoire of Escherichia coli agonists sensed by innate immunity receptors of the bovine udder and mammary epithelial cells. Veterinary Research 2012 43:14. doi:10.1186/1297-9716-43-14 Cite this article as: Porcherie et al.: Repertoire of Escherichia coli agonists sensed by innate immunity receptors of the bovine udder and mammary epithelial cells. Veterinary Research 2012 43:14. 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https://openalex.org/W3214222039	https://journals.ajsrp.com/index.php/jis/article/download/3830/3625	Arabic	null	loyalty in the Holy Quran: الولاء في القرآن الكريم	Mağallaẗ al-ʻulūm al-islāmiyyaẗ	2,021	cc-by	19,160	وزارة ا ألوقاف والشؤون واملقدسات اإلسالمية\|\| األردن املستخلص :تناول ت هذه الدراسة موضوع الوالء في القرآن الكريم ،ملا له من أهمية بالغة في عقيدة املسلم ،وما يترتب عليها من أثر ، وملَّا كان هذا الوالء يتناول أصنافًا عديدة ،تفاوت كثير من الناس في مشروعيتها ، أو في التوسع فيها دون ضوابط ، هدفت هذه الدراسة إلى بيان معنى الوالء وأنواعه في القرآن الكريم ،ومعرفة أهم مظاهره ، .والضوابط التي رسمها القرآن الكريم لكل نوع من هذه األنواع واتبع الباحث لتحقيق هذه األهداف :(املنهج االستقرائي) لجمع واستقراء لفظة الوالء في القرآن الك ريم ، واملنهج (التحليلي) لبيان معنى هذه اللفظة من خالل سياقها في القرآن ، .واملنهج (االستنباطي) الستنباط دالالت هذه اللفظة من خالل السياق القرآني وتوصلت هذه الدراسة إلى بعض النتائج كان من أهمها : قد يكون الوالء لغير هللا بمفهومه الدنيوي ، وال يكون بمعناه.املطلق إال هلل تعالى الكلمات املفتاحية :الوالء ،القرآن ،مظاهر ،ضوابط. Journal of Islamic Sciences Volume (4), Issue (2): 30 Jun 2021 P: 17 - 34 ISSN: 2664-4347 مجلة العلوم اإلسالمية ( املجلد4) ، ( العدد2) : 30 يونيو2021م ص : 17 - 34 loyalty in the Holy Quran Salama Awad Al-Hosan Ministry of Endowments and Islamic Affairs and Holy Places \|\| Jordan Journal of Islamic Sciences Volume (4), Issue (2): 30 Jun 2021 P: 17 - 34 ISSN: 2664-4347 مجلة العلوم اإلسالمية ( املجلد4) ، ( العدد2) : 30 يونيو2021م ص : 17 - 34 loyalty in the Holy Quran Salama Awad Al-Hosan Ministry of Endowments and Islamic Affairs and Holy Places \|\| Jordan Journal of Islamic Sciences Volume (4), Issue (2): 30 Jun 2021 P: 17 - 34 ISSN: 2664-4347 مجلة العلوم اإلسالمية ( املجلد4) ، ( العدد2) : 30 يونيو2021م ص : 17 - 34 مجلة العلوم اإلسالمية ( املجلد4) ، ( العدد2) : 30 يونيو2021م ص : 17 - 34 Journal of Islamic Sciences Volume (4), Issue (2): 30 Jun 2021 P: 17 - 34 ISSN: 2664-4347 سالمه عوض الحصان سالمه عوض الحصان مقدمة. الحمد هلل الذي نزّل القرآن على رسوله-  - تبياناً لكل ش يء ،قال تعالى : ﴿ ِّونَزَّلْنَا عَلَيْكَ الْكِ تَابَ تِبْيَاناً لِّكُل َش َ يْءٍ وَهُ دً ى وَرَ حْ مَ ةً وَبُشْ رَى لِلْمُسْ لِمِين﴾ :[النحل89 ] ،والصالة والسالم على الهادي البشير سيدنا محمد-  - وعلى آله وصحبه أجمعين وبعد : فإن القرآن الكريم كتاب هللا تعالى الذي ال تنقض ي عجائبه وأسراره ،وال تنتهي إلى حدٍ كنوزه وعلومه ، وهو املنهاج الذي يزيل ما غم على قلوب البشر ،وما أشكل على عقولهم ، وملا أشكل على الناس حقيقة الوالء ، كان ال بد Salama Awad Al-Hosan Abstract: This study deals with the issue of loyalty in the Holy Quran. Because of its great importance in the Muslim faith and the impact it entails, and since this loyalty deals with many types, many people differed in its legitimacy, or expanded without restrictions, this study aimed to clarify the meaning and types of loyalty in the Holy Qur’an, and to know its most important aspects and the controls set by the Holy Quran for each of these types. To achieve these goals, the researcher followed: (the inductive approach) to collect and extrapolate the word loyalty in the Noble Qur’an, the (analytical) method to clarify the meaning of this word through its context in the Qur’an, and the (deductive) approach to derive the connotations of this word through the Qur’an context. And this study reached some results, the most important of which are: that loyalty may be to someone other than God in its worldly sense, and it may only be in its absolute meaning to God Almighty. Keywords: Loyalty, The Quran, Appearances, Regulations. الوالء في القرآن الكريم سالمه عوض الحصان وزارة ا ألوقاف والشؤون واملقدسات اإلسالمية\|\| األردن الوالء في القرآن الكريم سالمه عوض الحصان وزارة ا ألوقاف والشؤون واملقدسات اإلسالمية\|\| األردن أهمية الدراسة تكمن أهمية هذه الدراسة في : 1- أن هذا املوضوع قد تأوله كثير من الناس ،فمنهم من بالغ في تأويله ومنهم من تساهل ، فجرد الوالء من .ضوابطه وقيوده تكمن أهمية هذه الدراسة في : 1- أن هذا املوضوع قد تأوله كثير من الناس ،فمنهم من بالغ في تأويله ومنهم من تساهل ،فجرد الوال .ضوابطه وقيوده 1- أن هذا املوضوع قد تأوله كثير من الناس ،فمنهم من بالغ في تأويله ومنهم من تساهل ، فجرد الوالء من .ضوابطه وقيوده 2- .اللبس الحاصل عند كثير من الناس بين الوالء والتعايش السلمي وخاصة مع غير املسلم لذا كان ال بد من : 2- .اللبس الحاصل عند كثير من الناس بين الوالء والتعايش السلمي وخاصة مع غير املسلم لذا كان ال بد من : أ- جمع اآليات التي حملت هذا املعنى ،للوصول إلى الهدف املنشود من الدراسة ، وهو بيان وجوه ومظاهر .الوالء في القرآن الكريم أ- جمع اآليات التي حملت هذا املعنى ،للوصول إلى الهدف املنشود من الدراسة ، وهو بيان وجوه ومظاهر .الوالء في القرآن الكريم أ- جمع اآليات التي حملت هذا املعنى ،للوصول إلى الهدف املنشود من الدراسة ، وهو بيان وجوه ومظاهر .الوالء في القرآن الكريم ب- مناقشة وجوه ومظاهر الوالء في القرآن الكريم مناقشة علمية ، للوصول إلى معنى واضح كل الوضوح للوالء .وضوابطه ب- مناقشة وجوه ومظاهر الوالء في القرآن الكريم مناقشة علمية ، للوصول إلى معنى واضح كل الوضوح للوالء .وضوابطه أهداف الدراسة أهداف الدراسة تهدف هذه الدراسة إلى تحقيق األهداف اآلتية : 1- .معرفة املعنى املراد من لفظة الوالء في القرآن الكريم 2- .معرفة أنواع الوالء في القرآن الكريم 3- .بيان مظاهر الوالء 4- بيان ومعرفة ضوابط الوالء في القرآن الكريم. 4- بيان ومعرفة ضوابط الوالء في القرآن الكريم. 4- بيان ومعرفة ضوابط الوالء في القرآن الكريم. //www.ajsrp.com : https : Available at //www.ajsrp.com : https : Available at //www.ajsrp.com : https : Available at ( 17 ) المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم اإلسالمية ــ المجلد ال رابع ــ العدد ال ثاني ــ يونيو 21 20 م لنا من الرجوع إلى الكالم الفصل في هذا األمر من القرآن الكريم ، ( فقد وردت مادة يول ) في القرآن في مواطن كثيرة ، .لذا جاء هذا البحث لدراستها دراسة موضوعية لنا من الرجوع إلى الكالم الفصل في هذا األمر من القرآن الكريم ، ( فقد وردت مادة يول ) في القرآن في مواطن كثيرة ، .لذا جاء هذا البحث لدراستها دراسة موضوعية مشكلة الدراسة وأسئلتها ملّا كان موضوع الوالء من املوضوعات التي أشك ل على الناس حدودها وضوابطها ، كان من األهمية أن نبحث في املواضع التي جاءت متضمنة مادة هذه اللفظة لتجيب عن األسئلة اآلتية : 1- .ما املقصود بالوالء في القرآن الكريم 2- ما أنواع الوالء التي أشار إليها القرآن الكريم؟ 3- ما مظاهر الوالء في القرآن الكريم؟ 4- ما ضوابط الوالء التي أقرها القرآن الكريم؟ أهداف الدراسة تهدف هذه الدراسة إلى تحقيق األهداف اآلتية : 1- .معرفة املعنى املراد من لفظة الوالء في القرآن الكريم 2- .معرفة أنواع الوالء في القرآن الكريم 3- .بيان مظاهر الوالء المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم اإلسالمية ــ المجلد ال رابع ــ العدد ال ثاني ــ يونيو 21 20 م سميح الكراسنه وآخرون 2- كتاب " مفهوم الوالء والبراء في القرآن والسنة " علي بن نايف الشحود 3- كتاب " الوالء والبراء " محمد بن سعيد القحطاني 4- مقاالت في مفهوم الوالء ، .والبراء وبعد االطالع على هذه املؤلفات تبين أن جميع هذه املؤلفا ت لم تتناول املوضوع بدراسة علمية شاملة من خالل جميع مواضعها في القرآن الكريم ، فبحث االنتماء والوالء الوطني في القرآن والسنة كان تركيزه منصباً على .االنتماء والوالء الوطني مركزاً على الجانب التربوي في ذلك وأما كتاب الوالء والبراء لعلي بن نايف الشحود ،فكان ت ركيزه على تحديد الوالء هلل تعالى والبراءة من الكافرين واملنافقين ،دون أن يتعرض لآليات ودالالتها في بيان الوالء وأنواعه ،وكذلك فعل القحطاني ، وكذلك املقاالت .التي لم ترتقي إلى املستوى الذي تحاكي فيه أسلوب القرآن ومنهجه في عرض الوالء في القرآن وبعد االطالع على هذه املؤلفات تبين أن جميع هذه املؤلفا ت لم تتناول املوضوع بدراسة علمية شاملة من خالل جميع مواضعها في القرآن الكريم ، فبحث االنتماء والوالء الوطني في القرآن والسنة كان تركيزه منصباً على .االنتماء والوالء الوطني مركزاً على الجانب التربوي في ذلك اا وأما كتاب الوالء والبراء لعلي بن نايف الشحود ،فكان ت ركيزه على تحديد الوالء هلل تعالى والبراءة من الكافرين واملنافقين ،دون أن يتعرض لآليات ودالالتها في بيان الوالء وأنواعه ،وكذلك فعل القحطاني ، وكذلك املقاالت .التي لم ترتقي إلى املستوى الذي تحاكي فيه أسلوب القرآن ومنهجه في عرض الوالء في القرآن منهجية الدراسة: ويقوم منهج البحث على ما يلي : 1- )املنهج االستقرائي من خالل تتبع جميع اآليات التي ورد بها لفظة مادة (ولى ، ومن ثم االكتفاء باآليات التي .تخدم املوضوعات ذات الصلة بالدراسة 2- املنهج التحليلي من خالل النظر في تفاسير املتقدمين واملتأخرين والوصول إلى ما تبرزه هذه النصوص في خدمة .موضوع الدراسة مااا 1- )املنهج االستقرائي من خالل تتبع جميع اآليات التي ورد بها لفظة مادة (ولى ، ومن ثم االكتفاء باآليات التي .تخدم املوضوعات ذات الصلة بالدراسة 2- املنهج التحليلي من خالل النظر في تفاسير املتقدمين واملتأخرين والوصول إلى ما تبرزه هذه النصوص في خدمة .موضوع الدراسة م م 2- املنهج التحليلي من خالل النظر في تفاسير املتقدمين واملتأخرين والوصول إلى ما تبرزه هذه النصوص في خدمة .موضوع الدراسة 3- املنهج االستنباطي الستنباط الضوابط التي تحدد أنواع الوالء ومظاهره وحدوده ،وما هو مباح وما هو مح المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم اإلسالمية ــ المجلد ال رابع ــ العدد ال ثاني ــ يونيو 21 20 م المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم اإلسالمية ــ المجلد ال رابع ــ العدد ال ثاني ــ يونيو 21 20 م 1- بحث االنتماء والوالء الوطني في الكتاب والسنة النبوية د. سميح الكراسنه وآخرون 2- كتاب " مفهوم الوالء والبراء في القرآن والسنة " علي بن نايف الشحود 3- كتاب " الوالء والبراء " محمد بن سعيد القحطاني 1- بحث االنتماء والوالء الوطني في الكتاب والسنة النبوية د. حدود البحث : حدود البحث : تعالج هذه الدراسة موضوع الوالء في القرآن الك ريم ، (من خالل تتبع مادة يول ) في القرآن الكريم ، ومن ثم .االستعانة بكتب اللغة والتفسير والعقيدة والحديث الشريف ر بعد البحث واالطالع في الدراسات السابقة التي تناولت املوضوع ، لم يجد الباحث حسب اطالعه إال الدراسات اآلتية : الوالء في القرآن الكريم ( 18 ) الحصان ( 18 ) الوالء في القرآن الكريم تمهيد : بداية وقبل الشروع في املوضوع يجب أن نقدم للموضوع بتعريف ملفهوم الوالء في اللغة واالصطالح ، حتى تكون الدراسة مبنية على منهج واضح ال تختلط فيه املفاهيم وال تتعارض مع مفاهيم أخرى ت خرج الدراسة عن .مسارها الصحيح أوالا :مفهوم الوالء لغة : والوالء ال يكون باإلكراه؛ ألن األصل فيه مشاعر وجدانية تتحرك بدافع املحبة ، فمن ال يحب ال يخلص وال يناصر؛ لذلك جعل هللا تعالى املؤم نين بعضهم أولياء بعض ،قال تعالى﴿ : ٍوَاملْ ُؤْمِنُونَ وَاملْ ُؤْمِنَاتُ بَعْضُهُمْ أَوْلِيَاء بَعْض َيَأْمُ رُونَ بِاملْ َعْرُوفِ وَيَنْهَوْنَ عَنِ املْ ُنكَ رِ وَيُقِيمُ ونَ الصَّالَةَ وَيُؤْتُونَ الزَّكَاةَ وَيُطِ يعُونَ اللّهَ و ُرَ سُ ولَهُ أُوْلَـئِكَ سَ يَرْحَ مُه َّمُ اللّهُ إِن ٌاللّهَ عَزِيزٌ حَ كِ يم﴾ [التوبة : 71 ]؛ وسبب هذه الوالية ألن ديدنهم املحبة والتناصح والتراحم واألمر باملعروف والنهي عن .املنكر الوالء لغةا :يأتي تعريف الوالء بمعانٍ متقاربة منها : ُ"[ولى] الوَ لي : القربُ والدنو ،يقال :تباعد بعد ولى ، و" وكل َمما يَليك ، "أي مما يقاربك( 1 ( ) 2) ،وكذلك تأتي بمعنى :والء "محبَّة وصداقة ، معاهدة"والء( 3) ، ومنها الولي الذي يأتي بمعنى :"الصَّدِ يق والنَّصِ ير ،قال ابْنُ األَعرابي( 4) : الوِلي التَّابِعُ املْ ُحِ ب. ُ.. واملُواالة :ضِ دّ املُعاداة ، والوَ لي : "ِّضِ د الْعَدُ و( 5). والولي من أسماء هللا تعالى ويأتي بمعنى : ُ"الناصِ ر ، َوَقِيل : ِ ّاملُ َتَوَ ل ي ألُمور الْعَالَمِ وَالْ خَ الَ ئِقِ القائمُ بِهَا ، ْوَمِن َّأَسمائه عَزَّ وَجَ ل :الْوَالِي ،وَهُ وَ مالِكُ األَشياء جَ مِيعِهَا املُتَصَرِّفُ فِيهَا ،قَالَ ابْنُ األَثير( 6) : وكأَن الوِالية تُشعر بالتَّدْ بير والقُ درة والفِعل ، َوَمَ ا لَمْ يَجْ تَمِعْ ذَلِك"فِيهَا لَمْ يَنْطَلِقْ عَلَيْهِ اسْ مُ الْوَالِي( 7) ،والوالية :" بالكسر :السلطان. والوَالية : النصرة ،يقال :هم على والية ، " أي مجتمعون في النصرة( 8). ا م ي ي ويتضح من التعريفات السابقة أن الوالء هو النصرة والصداقة واملحبة والقرب والدنو ، ويصح بمفهومه السابق على الخ الق واملخلوق ،غير أن الوالء ال يكون مطلقاً إال هلل تعالى؛ ألنه مالك األشياء كلها وهو متولي تدبيرها ، ومن معرفة اسم هللا تعالى (الولي) يتبين لنا صفات الولي الحق وهي : .التدبير والقدرة والفعل كما قال ابن األثير ثانياا : الوالء اصطالحاا :"هو مشاعر الفرد وأحاسيسه "اإليجابية باملحبة والنصرة تجاه موضوع معين( 9) ، وبما أن الوالء مشاعر وأحاسيس فالبد من أن تكون هنالك عالقة قوية انتمائية بين الفرد والطرف االخر ، تحرك مشاعر الفرد وتدفعه إلى إخالص الوالء بقناعة تامة للطرف اآلخر ،ويظهر ذلك في قوله تعالى : ﴿ ْقُلْ إِن كُ نتُم َتُحِ ب ون ٌاللّهَ فَاتَّبِعُونِي يُحْ بِبْكُ مُ اللّهُ وَيَغْفِرْ لَكُ مْ ذُنُوبَكُ مْ وَاللّهُ غَفُ ورٌ رَّحِ يم﴾ [آل عمران : 31 ] . أوالا :مفهوم الوالء لغة : اا الوالء لغةا :يأتي تعريف الوالء بمعانٍ متقاربة منها : ُ"[ولى] الوَ لي : القربُ والدنو ،يقال :تباعد بعد ولى ، و" وكل َمما يَليك ، "أي مما يقاربك( 1 ( ) 2) ،وكذلك تأتي بمعنى :والء "محبَّة وصداقة ، معاهدة"والء( 3) ، ومنها الولي الذي يأتي بمعنى :"الصَّدِ يق والنَّصِ ير ،قال ابْنُ األَعرابي( 4) : الوِلي التَّابِعُ املْ ُحِ ب. ُ.. واملُواالة :ضِ دّ املُعاداة ، والوَ لي : "ِّضِ د الْعَدُ و( 5). والولي من أسماء هللا تعالى ويأتي بمعنى : ُ"الناصِ ر ، َوَقِيل : ِ ّاملُ َتَوَ ل ي ألُمور الْعَالَمِ وَالْ خَ الَ ئِقِ القائمُ بِهَا ، ْوَمِن َّأَسمائه عَزَّ وَجَ ل :الْوَالِي ،وَهُ وَ مالِكُ األَشياء جَ مِيعِهَا املُتَصَرِّفُ فِيهَا ،قَالَ ابْنُ األَثير( 6) : وكأَن الوِالية تُشعر بالتَّدْ بير والقُ درة والفِعل ، َوَمَ ا لَمْ يَجْ تَمِعْ ذَلِك"فِيهَا لَمْ يَنْطَلِقْ عَلَيْهِ اسْ مُ الْوَالِي( 7) ،والوالية :" بالكسر :السلطان. والوَالية : النصرة ،يقال :هم على والية ، " أي مجتمعون في النصرة( 8). ويتضح من التعريفات السابقة أن الوالء هو النصرة والصداقة واملحبة والقرب والدنو ، ويصح بمفهومه السابق على الخ الق واملخلوق ،غير أن الوالء ال يكون مطلقاً إال هلل تعالى؛ ألنه مالك األشياء كلها وهو متولي تدبيرها ، ومن معرفة اسم هللا تعالى (الولي) يتبين لنا صفات الولي الحق وهي : .التدبير والقدرة والفعل كما قال ابن األثير ثانياا : الوالء اصطالحاا :"هو مشاعر الفرد وأحاسيسه "اإليجابية باملحبة والنصرة تجاه موضوع معين( 9) ، وبما أن الوالء مشاعر وأحاسيس فالبد من أن تكون هنالك عالقة قوية انتمائية بين الفرد والطرف االخر ، تحرك مشاعر الفرد وتدفعه إلى إخالص الوالء بقناعة تامة للطرف اآلخر ،ويظهر ذلك في قوله تعالى : ﴿ ْقُلْ إِن كُ نتُم َتُحِ ب ون ٌاللّهَ فَاتَّبِعُونِي يُحْ بِبْكُ مُ اللّهُ وَيَغْفِرْ لَكُ مْ ذُنُوبَكُ مْ وَاللّهُ غَفُ ورٌ رَّحِ يم﴾ [آل عمران : 31 ] . (1) الفارابي ،الصحاح تاج اللغة وصحاح العربية ،ج :6 ،ص : 2528 . خطَّة البحث : اشتمل هذا البحث على مقدمة و تمهيد ومبحثين وخاتمة ،على النحو اآلتي :  :املقدمة .وتضمنت ما سبق  تمهيد : .ًويشمل تعريف الوالء لغة واصطالحا  املبحث األول : الوالء الديني  املطلب األول :الوالء هلل تعالى.  املطلب الثاني :مظاهر الوالء هلل تعالى وثمراته  املطلب الثالث : الوالء للنبي-  -  املبحث الثاني : الوالء الدنيوي  املطلب األول : .الوالء بين املؤمنين  املطلب الثاني : .الوالء لألسرة والقبيلة  املطلب الثالث : .الوالء لولي األمر  املطلب الرابع : .الوالء املحرم  .الخاتمة: أهم النتائج، التوصيات واملقترحات وهللا اسأل أن يكون هذا العمل خالصاً لوجهه الكريم وأن يتقبله وأن ينفع به ، .وصلى هللا على سيدنا محمد وعلى آله وصحبه أجمعين مب ربع امرم و  .الخاتمة: أهم النتائج، التوصيات واملقترحات وهللا اسأل أن يكون هذا العمل خالصاً لوجهه الكريم وأن يتقبله وأن ينفع به ، .وصلى هللا على سيدنا محمد وعلى آله وصحبه أجمعين ( 19 ) الوالء في القرآن الكريم الحصان الحصان المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم اإلسالمية ــ المجلد ال رابع ــ العدد ال ثاني ــ يونيو 21 20 م تمهيد : (1) الفارابي ،الصحاح تاج اللغة وصحاح العربية ،ج :6 ،ص : 2528 . (2) عمر ،بمساعدة فريق عمل ،معجم اللغة العربية املعاصرة ،ج :3 ،ص : 2498 . (3) هو أبو عبد الله محمد بن زياد املعروف بابن اإلعرابي ،من أكابر أئمة اللغة ، (ت : 231 هـ). ينظر- األنباري ، نزهة األلباء في طبقات األدباء ،ص : 119 - 122 . (4) أحمد ،معجم اللغة العربية املعاصرة ،ج : 15 ،ص : 411 . (5) محمد بن محمد بن عبد الكريم بن عبد الواحد الشيباني املعروف بابن األثير(ت : 630 )هـ ،ينظر _ ابن األثير ، أسد الغابة في معرفة الصحابة ،ص :8. (6) ينظر- ابن منظور ،لسان العرب ،ج : 15 ،ص : 407 . (7) الفارابي ،الصحاح تاج اللغة وصحاح العربية ،ج :3 ،ص : 2530 . (8) السرحان ،الوالء واالنتماء لدى الشباب األردني وأثره في بناء الشخصية ،ص : 41 . (9) الشنقيطي ،أضواء البيان في إيضاح القرآن بالقرآن ،ج :2 ،ص : 42 . (2) عمر ،بمساعدة فريق عمل ،معجم اللغة العربية املعاصرة ،ج :3 ،ص : 2498 . (3) هو أبو عبد الله محمد بن زياد املعروف بابن اإلعرابي ،من أكابر أئمة اللغة ، (ت : 231 هـ). ينظر- األنباري ، نزهة األلباء في طبقات األدباء ،ص : 119 - 122 . املبحث األول :ال والء هلل تعالى وللنبي-  -. أملها والوالء في القرآن الكريم ظهر في أبرز مالمحه عندما بيّن هللا تعالى أن الوالء الحقيقي هو عهد ما بينه وبين عباده ، ًأخذه منهم أزال ،قال تعالى﴿ : وَإِذْ أَخَ ذَ رَب كَ مِن بَنِ ي آدَمَ مِن ظُهُورِهِ مْ ذُرِّيَّتَهُمْ وَأَشْ هَدَ هُ مْ عَلَ ى ُأَنفُ سِ هِمْ أَلَسْ ت َبِرَبِّكُ مْ قَالُواْ بَلَ ى شَ هِدْ نَا أَن تَقُ ولُواْ يَوْمَ الْقِيَامَ ةِ إِنَّا كُ نَّا عَنْ هَ ذَ ا غَافِلِين﴾ [األعراف : 172 ] ، ومعنى إشهادهم على أنفسهم "إنما هو بما نصب لهم من األدلة القاطعة بأنه ربهم املستحق منهم ألن يعبدوه وحده ، وعليه)فمعنى (قالوا بلى ،أي : " قالوا ذلك بلسان حالهم لظهور األدلة عليه ، ًفهذا العهد ملزم للناس جميعا ، إال أن منهم من كان إقراره بهذا العهد ًطوعاً ومنهم من كان كرها– النفاق- .؛ لذلك نجد أن كثيرًا من الناس تنكروا لهذا العهد وسوف أبين الوالء هلل تعالى ولرسوله الك ريم وفق املطالب اآلتية : املطلب األول : .الوالء هلل تعالى والوالء هلل تعالى يكون باإليمان في ألوهيته وربوبيته وأسمائه وصفاته ، وقد أولى القرآن الكريم قضية الوالء هلل جل العناية؛ ألنها أساس العقيدة الصحيحة ،فال إسالم وال إيمان من غير والء مطلق هلل تعالى ،فإذا كانت الوالية استحقاقاً؛ فاهلل تعالى هو املستحق فله الحاكمية والسلطان املطلق الذي ال يقهر ،قال تعالى﴿ : ًقُلْ أَغَيْرَ اللّهِ أَبْغِ ي رَبّا ِوَهُ وَ رَب كُلِّ ش َ يْءٍ وَالَ تَكْ سِ بُ كُل نَفْ سٍ إِالَّ عَلَيْهَا وَالَ تَزِرُ وَازِرَ ةٌ وِزْ رَ أُخْ رَى ثُمَّ إ ِلَى رَبِّكُ م مَّ رْجِ عُكُ مْ فَيُنَبِّئُكُ م بِمَا كُ نتُمْ فِيه َتَخْ تَلِفُ ون﴾ [األنعام : 164 .] أ والنقل والعقل يحكم بأن الوالء املطلق هلل وحده ، فكيف تنصرف العقول عن الوالء ملن فطر السموات واألرض ،وتكفل بأسباب ديمومة الكائنات في هذه األرض ،إلى من ال يملك من أس ًباب الدنيا شيئا ،قال تعالى﴿ : ْقُل ْأَغَيْرَ اللّهِ أَتَّخِ ذُ وَلِيّاً فَاطِ رِ السَّ مَاوَاتِ وَاألَرْضِ وَهُ وَ يُطْعِمُ وَالَ يُطْعَمُ قُلْ إِنِّيَ أُمِرْتُ أَن َأَكُونَ أَوَّلَ مَنْ أَسْ لَمَ وَالَ تَكُونَنَّ مِن َاملْ ُشْ رِكَ ين﴾ [األنعام : 14 ]"أي منطق يسمح بأن يتخذ غير هللا وليا؟ إن كان يتواله لينصره ويعينه ، فاهلل هو فاطر السماوات واألرض ،فله السلطان في السماوات واألرض ،وإن كان يتواله ليرزقه ويطعمه ، فاهلل هو الرازق املطعم ملن في السماوات ومن في األرض ،ففيم الوالء لغير صاحب السلطان الرزاق؟"( 10 ). (10) قطب ،في ظالل القرآن ،ج :2 ،ص : 1054 . المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم اإلسالمية ــ المجلد ال رابع ــ العدد ال ثاني ــ يونيو 21 20 م المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم اإلسالمية ــ المجلد ال رابع ــ العدد ال ثاني ــ يونيو 21 20 م ًثالثا :ورود لفظ الوالء في القرآن الكريم : ٍورد لفظ مادة (ولى) في القرآن الكريم صراحة في أكثر من تسعة وسبعين موضعًا، بصيغٍ مختلفة ،جاءت تفيد املحبة والنصرة في أكثر من ستةٍ وأربعين موضعًا ، وبمعنى الحفيظ واملانع في أكثر من ثالثة عشر موضعًا ،وبمعنى األرباب واألصنام وما يعبد من دون هللا في تسعة مواضع تقريبًا ، وبمعنى األتباع واألعوان واألقران في أربعة مواضع ،وبمعنى االبن وال وريث والقريب في تسعة مواضع ، وجاءت هذه الصيغ للتأكيد على هذا املعنى من خالل عدة محاور ،من أهمها :إثبات الوالية املطلقة هلل تعالى ،كقوله تعالى﴾ : أَمِ اتَّخَ ذُ وا َ مِنْ دُونِهِ أَوْلِيَاءَ فَاللَّهُ هُ وَ الْوَ لِي وَهُ وَ يُحْ يِ املْ َوْتَى وَهُ وَ عَلَ ى كُلِّ ش ٌيْءٍ قَدِ ير﴿[الشورى :9 .] وجاءت معاني هذه اللفظة تلميحً ا في القرآن الكريم في أكثر من ثمانية عشر موضعًا ، حيث جاءت بمعنى املحبة والنصرة في أربعة عشر موضعًا ،وبمعنى األرباب في موضعين ،وبمعنى الحفيظ في موضعٍ واحد ، وبمعنى العهد .في موضعٍ واحد أوالا :مفهوم الوالء لغة : ﴾ والوالء ال يكون باإلكراه؛ ألن األصل فيه مشاعر وجدانية تتحرك بدافع املحبة ، فمن ال يحب ال يخلص وال يناصر؛ لذلك جعل هللا تعالى املؤم نين بعضهم أولياء بعض ،قال تعالى﴿ : ٍوَاملْ ُؤْمِنُونَ وَاملْ ُؤْمِنَاتُ بَعْضُهُمْ أَوْلِيَاء بَعْض َيَأْمُ رُونَ بِاملْ َعْرُوفِ وَيَنْهَوْنَ عَنِ املْ ُنكَ رِ وَيُقِيمُ ونَ الصَّالَةَ وَيُؤْتُونَ الزَّكَاةَ وَيُطِ يعُونَ اللّهَ و ُرَ سُ ولَهُ أُوْلَـئِكَ سَ يَرْحَ مُه َّمُ اللّهُ إِن ٌاللّهَ عَزِيزٌ حَ كِ يم﴾ [التوبة : 71 ]؛ وسبب هذه الوالية ألن ديدنهم املحبة والتناصح والتراحم واألمر باملعروف والنهي عن .املنكر (1) الفارابي ،الصحاح تاج اللغة وصحاح العربية ،ج :6 ،ص : 2528 . (2) عمر ،بمساعدة فريق عمل ،معجم اللغة العربية املعاصرة ،ج :3 ،ص : 2498 . (3) هو أبو عبد الله محمد بن زياد املعروف بابن اإلعرابي ،من أكابر أئمة اللغة ، (ت : 231 هـ). ينظر- األنباري ، نزهة األلباء في طبقات األدباء ،ص : 119 - 122 . أ (4) أحمد ،معجم اللغة العربية املعاصرة ،ج : 15 ،ص : 411 . أ (4) أحمد ،معجم اللغة العربية املعاصرة ،ج : 15 ،ص : 411 . م (5) محمد بن محمد بن عبد الكريم بن عبد الواحد الشيباني املعروف بابن األثير(ت : 630 )هـ ،ينظر _ ابن األثير ، أسد الغابة في معرفة الصحابة ص:8 (9) الشنقيطي ،أضواء البيان في إيضاح القرآن بالقرآن ،ج :2 ،ص : 42 . الوالء في القرآن الكريم الوالء في القرآن الكريم ( 20 ) الحصان الحصان (10) قطب ،في ظالل القرآن ،ج :2 ،ص : 1054 . (11) البخاري ، صحيح البخاري ،كتاب :التوحيد ،باب : ِمَ ا جَ اءَ فِي دُعَاء النَّبِ يِّ صَلَّ ى هللاُ عَلَيْهِ وَسَ لَّمَ أُمَّ تَهُ إِلَى تَوْحِ يدِ اللَّهِ تَبَارَكَ وَتَعَالَى ،ج : 9 ،ص : 114 ،رقم : 7373 . (12) البقرة : 257 / األعراف :3 - 196 / هود : 113 / يوسف : 101 . (13) البيضاوي ،أنوار التنزيل وأسرار التأويل ،ج :2 ،ص : 132 . (14) البقاعي ، نظم الدرر في تناسب اآليات والسور ،ج :6 ،ص : 193 . (15) السعدي ،تيسير الكريم الرحمن ،ص631 . املبحث األول :ال والء هلل تعالى وللنبي-  -. ق هم﴿ز يؤو﴾اق إ إ ثم كانت الحجة على من نقض امليثاق الذي قطعه مع هللا وهو في عالم الذر ، ببيان حال من اتخذ من دون هللا أولياء ،فقال تعالى﴿ :مَثَلُ الَّذِ ينَ اتَّخَ ذُ وا مِن دُونِ اللَّهِ أَوْلِيَاء كَ مَثَلِ ا ُلْعَنكَ بُوتِ اتَّخَ ذَ تْ بَيْتاً وَإِنَّ أَوْهَ نَ الْبُيُوتِ لَبَيْت َالْعَنكَ بُوتِ لَوْ كَانُوا يَعْلَمُ ون﴾ [العنكبوت : 41 ] ، وهنا يمثل هللا تعالى حال من اتخذ أولياء من دون هللا يرجون نفعهم ونصرهم ،" هذا مثل ضربه هللا ملن عبد معه غيره ، َّيقصد به التعزز والت قَ وِّي والنفع ،وأن األمر بخالف مقصوده ، فإن مثله كمثل العنكبوت ،اتخذت بيتا يقيها من الحر والبرد واآلفات﴿ ، ِوَإِنَّ أَوْهَ نَ الْبُيُوت﴾ أضعفها وأوهاها﴿ ُلَبَيْت ِالْعَنْكَ بُوت﴾ فالعنكبوت من الحيوانات الضعيفة ،وبيتها من أضعف البيوت ،فما ازدادت باتخاذه إال ضعف ا ، كذلك هؤالء الذين يتخذون من دونه أولياء ،فقراء عاجزون من جميع الوجوه ، وحين اتخذوا األولياء من دونه يتعززون بهم ويستنصرونهم ،ازدادوا ضعفا إلى ضعفهم ، "ووهنا إلى وهنهم( 15 ). املبحث األول :ال والء هلل تعالى وللنبي-  -. ِأَشَ د حُ بّاً لِّلّه﴾ [البقرة : 165 ] ؛ لذلك عبّر هللا تعالى عن التزام املؤمنين باملحبة الصادقة والعزيمة القوية بأنهم أشد حباً هلل وذلك باتباع أوامره ونواهيه ، واملحبة تقتض ي الطاعة ، .فعلى قدر املحبة تكون الطاعة أما النصرة فتتمثل بعد االمتثال والطاعة بتحقيق التوحيد الخالص هلل تعالى ،فعن معاذ بن جبل-  - قال : قال النبي-  - :"يا معاذ أتدري ما حق هللا على العباد؟ ،قال :هللا ورسوله أعلم ،قال :أن يعبدوه وال يشركوا به شيئا ، أتدري ما حق م عل ه؟ قال:هللا ور وله أعلم قال:أن ال يعذ م"( 11 )وتكون بنش تعال ه الت أنزل إل النا ِأَشَ د حُ بّاً لِّلّه﴾ [البقرة : 165 ] ؛ لذلك عبّر هللا تعالى عن التزام املؤمنين باملحبة الصادقة والعزيمة القوية بأنهم أشد حباً هلل وذلك باتباع أوامره ونواهيه ، واملحبة تقتض ي الطاعة ، .فعلى قدر املحبة تكون الطاعة أما النصرة فتتمثل بعد االمتثال والطاعة بتحقيق التوحيد الخالص هلل تعالى ،فعن معاذ بن جبل-  - قال : قال النبي-  - :"يا معاذ أتدري ما حق هللا على العباد؟ ،قال :هللا ورسوله أعلم ،قال :أن يعبدوه وال يشركوا به شيئا ، أتدري ما حقهم عليه؟ ،قال :هللا ورسوله أعلم ،قال :أن ال يعذبهم"( 11 ) ، وتكون بنشر تعاليمه التي أنزل إلى الناس كافة ،والدفاع عن هذا الدين بكل الوسائل املشروعة ،وحماية املقدسات ،ونشر القيم اإلسالمية السامية ، وتحقيق العدل واألمان ،قال تعالى﴿ : َيَا أَي هَا الَّذِ ين ْآمَ نُوا إِن تَنصُرُوا اللَّهَ يَنصُرْكُ مْ وَيُثَبِّتْ أَقْدَ امَ كُ م﴾ [محمد :7]. أ وقد أثبت هللا تعالى لنفسه الوالية املطلقة في كتابه العزيز ، ونفاها عمن سواه في مواطن كثيرة من القرآن( 12 ) ، ثم بين ملن تكون له الوالية بعده– جل جالله- من عباده ،قال تعالى﴿ : َّإِن َمَا وَلِي كُ مُ اللّهُ وَرَ سُ ولُهُ وَالَّذِ ين َآمَ نُواْ الَّذِ ينَ يُقِيمُ ونَ الصَّالَةَ وَيُؤْتُونَ الزَّكَاةَ وَهُ مْ رَاكِ عُون﴾ [املائدة : 55 ] ،وفي إفراد لفظة (وليكم) إشارة "للتنبيه على أن الوالية هلل سبحانه وتعالى على األصالة ، ولرسوله-  - وللمؤمنين على ا"لتبع( 13 ) ،فبين أن الوالية أصالة هلل تعالى ، ثم للنبي-  - على التبع ،ثم الذين امنوا ،وتعرض لصفاتهم التي استحقوا بموجبها الوالية ،فقال تعالى﴿ : والذين آمنوا﴾ أي :"أوجدوا اإليمان وأقروا به ،ثم وصفهم بما يصدق دعواهم اإليمان فقال﴿ :والذين يقيمون الصالة﴾ أي تمكيناً لصلتهم بالخالق﴿ويؤتون الزكاة﴾ "إحساناً إلى الخالئق( 14 ) . املبحث األول :ال والء هلل تعالى وللنبي-  -. وال يكمن الوهن ببناء البيت من النسيج فقط ،بل يتعدى ذلك إلى ما هو أبعد من ذلك ، فوهن البيت هنا تكمن في الضعف في فساد العالقة بين أنثى العنكبوت وذكرها ، ففي خضوع ذكر العنكبوت لألنثى وامتثاله لها جعل منه لقمة سائغة لألنثى ،وهذا ما أظهره العلم الحديث ،حيث قال أستاذ في علم الحشرات ، ِفي كُلِّيةِ العلومِ في جامعة ِالقاهرةِ في بعض كتبه العلميّة :"إنّ في قوله تعالى﴿ : ًكَ مَ ثَلِ العنكبوت اتخذت بَيْتا﴾ إعجازاً علميّاً؛ حيث إنّ التي تبني البيتَ هي األنثى ، ِفجاءت تاءُ التأنيثِ في قوله﴿ : ًكَ مَ ثَلِ العنكبوت اتخذت بَيْتا﴾ َاألنثى هي التي تغزِلُ البيت ، وهي التي ِتُرغِّبُ الذَّ كَ رَ في الدخولِ إلى البيت ،حيث ت ٍقومُ أمامه بحركاتٍ مُ غرية ، ِوتُسمِعُه بعضَ األلحانِ الطنّانة ، فيأوي إلى بيتِها ،وبعدَ التلقيحِ تأكلُه إنْ لم يفرّ ويهرب ،وتفترسُ ه ،وتأكلُ أوالدَها من بعدُ إنْ لم يفرّوا ، ًويأكلُ بعضُ أوالدِ ها بعضا ، ثم كانت الحجة على من نقض امليثاق الذي قطعه مع هللا وهو في عالم الذر ، ببيان حال من اتخذ من دون هللا أولياء ،فقال تعالى﴿ :مَثَلُ الَّذِ ينَ اتَّخَ ذُ وا مِن دُونِ اللَّهِ أَوْلِيَاء كَ مَثَلِ ا ُلْعَنكَ بُوتِ اتَّخَ ذَ تْ بَيْتاً وَإِنَّ أَوْهَ نَ الْبُيُوتِ لَبَيْت َالْعَنكَ بُوتِ لَوْ كَانُوا يَعْلَمُ ون﴾ [العنكبوت : 41 ] ، وهنا يمثل هللا تعالى حال من اتخذ أولياء من دون هللا يرجون نفعهم ونصرهم ،" هذا مثل ضربه هللا ملن عبد معه غيره ، َّيقصد به التعزز والت قَ وِّي والنفع ،وأن األمر بخالف مقصوده ، فإن مثله كمثل العنكبوت ،اتخذت بيتا يقيها من الحر والبرد واآلفات﴿ ، ِوَإِنَّ أَوْهَ نَ الْبُيُوت﴾ أضعفها وأوهاها﴿ ُلَبَيْت ِالْعَنْكَ بُوت﴾ فالعنكبوت من الحيوانات الضعيفة ،وبيتها من أضعف البيوت ،فما ازدادت باتخاذه إال ضعف ا ، كذلك هؤالء الذين يتخذون من دونه أولياء ،فقراء عاجزون من جميع الوجوه ، وحين اتخذوا األولياء من دونه يتعززون بهم ( 15 ) صرو هم وي إ ى ض هم و ض ز إ ى و هم وو وال يكمن الوهن ببناء البيت من النسيج فقط ،بل يتعدى ذلك إلى ما هو أبعد من ذلك ، فوهن البيت هنا تكمن في الضعف في فساد العالقة بين أنثى العنكبوت وذكرها ، ففي خضوع ذكر العنكبوت لألنثى وامتثاله لها جعل منه لقمة سائغة لألنثى ،وهذا ما أظهره العلم الحديث ،حيث قال أستاذ في علم الحشرات ، ِفي كُلِّيةِ العلومِ في جامعة ِالقاهرةِ في بعض كتبه العلميّة :"إنّ في قوله تعالى﴿ : ًكَ مَ ثَلِ العنكبوت اتخذت بَيْتا﴾ إعجازاً علميّاً؛ حيث إنّ التي تبني البيتَ هي األنثى ، ِفجاءت تاءُ التأنيثِ في قوله﴿ : ًكَ مَ ثَلِ العنكبوت اتخذت بَيْتا﴾ َاألنثى هي التي تغزِلُ البيت ، وهي التي ِتُرغِّبُ الذَّ كَ رَ في الدخولِ إلى البيت ،حيث ت ٍقومُ أمامه بحركاتٍ مُ غرية ، ِوتُسمِعُه بعضَ األلحانِ الطنّانة ، فيأوي إلى بيتِها ،وبعدَ التلقيحِ تأكلُه إنْ لم يفرّ ويهرب ،وتفترسُ ه ،وتأكلُ أوالدَها من بعدُ إنْ لم يفرّوا ، ًويأكلُ بعضُ أوالدِ ها بعضا ، (11) البخاري ، صحيح البخاري ،كتاب :التوحيد ،باب : ِمَ ا جَ اءَ فِي دُعَاء النَّبِ يِّ صَلَّ ى هللاُ عَلَيْهِ وَسَ لَّمَ أُمَّ تَهُ إِلَى تَوْحِ يدِ اللَّهِ تَبَارَكَ وَتَعَالَى ،ج : 9 ،ص : 114 ،رقم : 7373 . املبحث األول :ال والء هلل تعالى وللنبي-  -. وإذا كان الوالء يعني املحبة والنصرة؛ فإن محبة هللا تعالى هي الغاية التي يجد من أجلها املجدون ، ويتهجد من أجلها املتهجدون ،واملحبة هي اإلتباع واالستسالم واالنقياد الصادق ملا أمر أو نهى ، وكل ما خالف ذلك هو من قبيل صرف الوالء لغير هللا لقوله تعالى﴿ :وَمِنَ النَّاسِ مَ ن ْيَتَّخِ ذُ مِن دُونِ اللّهِ أَندَ اداً يُحِ ب ونَهُمْ كَ حُ بِّ اللّهِ وَالَّذِ ينَ آمَنُوا والنقل والعقل يحكم بأن الوالء املطلق هلل وحده ، فكيف تنصرف العقول عن الوالء ملن فطر السموات واألرض ،وتكفل بأسباب ديمومة الكائنات في هذه األرض ،إلى من ال يملك من أس ًباب الدنيا شيئا ،قال تعالى﴿ : ْقُل ْأَغَيْرَ اللّهِ أَتَّخِ ذُ وَلِيّاً فَاطِ رِ السَّ مَاوَاتِ وَاألَرْضِ وَهُ وَ يُطْعِمُ وَالَ يُطْعَمُ قُلْ إِنِّيَ أُمِرْتُ أَن َأَكُونَ أَوَّلَ مَنْ أَسْ لَمَ وَالَ تَكُونَنَّ مِن َاملْ ُشْ رِكَ ين﴾ [األنعام : 14 ]"أي منطق يسمح بأن يتخذ غير هللا وليا؟ إن كان يتواله لينصره ويعينه ، فاهلل هو فاطر السماوات واألرض ،فله السلطان في السماوات واألرض ،وإن كان يتواله ليرزقه ويطعمه ، فاهلل هو الرازق املطعم ملن في السماوات ومن في األرض ،ففيم الوالء لغير صاحب السلطان الرزاق؟"( 10 ). وإذا كان الوالء يعني املحبة والنصرة؛ فإن محبة هللا تعالى هي الغاية التي يجد من أجلها املجدون ، ويتهجد من أجلها املتهجدون ،واملحبة هي اإلتباع واالستسالم واالنقياد الصادق ملا أمر أو نهى ، وكل ما خالف ذلك هو من قبيل صرف الوالء لغير هللا لقوله تعالى﴿ :وَمِنَ النَّاسِ مَ ن ْيَتَّخِ ذُ مِن دُونِ اللّهِ أَندَ اداً يُحِ ب ونَهُمْ كَ حُ بِّ اللّهِ وَالَّذِ ينَ آمَنُوا (10) قطب ،في ظالل القرآن ،ج :2 ،ص : 1054 . (15) السعدي ،تيسير الكريم الرحمن ،ص631 . املبحث األول :ال والء هلل تعالى وللنبي-  -. ( 21 ) الوالء في القرآن الكريم الوالء في القرآن الكريم الوالء في القرآن الكريم الحصان المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم اإلسالمية ــ المجلد ال رابع ــ العدد ال ثاني ــ يونيو 21 20 م المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم اإلسالمية ــ المجلد ال رابع ــ العدد ال ثاني ــ يونيو 21 20 م ِأَشَ د حُ بّاً لِّلّه﴾ [البقرة : 165 ] ؛ لذلك عبّر هللا تعالى عن التزام املؤمنين باملحبة الصادقة والعزيمة القوية بأنهم أشد حباً هلل وذلك باتباع أوامره ونواهيه ، واملحبة تقتض ي الطاعة ، .فعلى قدر املحبة تكون الطاعة أما النصرة فتتمثل بعد االمتثال والطاعة بتحقيق التوحيد الخالص هلل تعالى ،فعن معاذ بن جبل-  - قال : قال النبي-  - :"يا معاذ أتدري ما حق هللا على العباد؟ ،قال :هللا ورسوله أعلم ،قال :أن يعبدوه وال يشركوا به شيئا ، أتدري ما حقهم عليه؟ ،قال :هللا ورسوله أعلم ،قال :أن ال يعذبهم"( 11 ) ، وتكون بنشر تعاليمه التي أنزل إلى الناس كافة ،والدفاع عن هذا الدين بكل الوسائل املشروعة ،وحماية املقدسات ،ونشر القيم اإلسالمية السامية ، وتحقيق العدل واألمان ،قال تعالى﴿ : َيَا أَي هَا الَّذِ ين ْآمَ نُوا إِن تَنصُرُوا اللَّهَ يَنصُرْكُ مْ وَيُثَبِّتْ أَقْدَ امَ كُ م﴾ [محمد :7]. وقد أثبت هللا تعالى لنفسه الوالية املطلقة في كتابه العزيز ، ونفاها عمن سواه في مواطن كثيرة من القرآن( 12 ) ، ثم بين ملن تكون له الوالية بعده– جل جالله- من عباده ،قال تعالى﴿ : َّإِن َمَا وَلِي كُ مُ اللّهُ وَرَ سُ ولُهُ وَالَّذِ ين َآمَ نُواْ الَّذِ ينَ يُقِيمُ ونَ الصَّالَةَ وَيُؤْتُونَ الزَّكَاةَ وَهُ مْ رَاكِ عُون﴾ [املائدة : 55 ] ،وفي إفراد لفظة (وليكم) إشارة "للتنبيه على أن الوالية هلل سبحانه وتعالى على األصالة ، ولرسوله-  - وللمؤمنين على ا"لتبع( 13 ) ،فبين أن الوالية أصالة هلل تعالى ، ثم للنبي-  - على التبع ،ثم الذين امنوا ،وتعرض لصفاتهم التي استحقوا بموجبها الوالية ،فقال تعالى﴿ : والذين آمنوا﴾ أي :"أوجدوا اإليمان وأقروا به ،ثم وصفهم بما يصدق دعواهم اإليمان فقال﴿ :والذين يقيمون الصالة﴾ أي تمكيناً لصلتهم بالخالق﴿ويؤتون الزكاة﴾ "إحساناً إلى الخالئق( 14 ) . (16) النابلس ي ،موسوعة اإلعجاز العلمي في القرآن والسنة ،ج2 ،ص234 . (17) الزمخشري ،الكشاف عن حقائق غوامض التنزيل ،ج :4 ،ص : 514 . (18) الطبري ،جامع البيان في تأويل القرآن ،ج : 13 ،ص : 323 . املبحث األول :ال والء هلل تعالى وللنبي-  -. فال والية محبة أو نصرة ملن حادّ هللا ورسوله ،مهما بلغت درجة القرابة أو تزايدت أواصر املحبة ، فاملحبة في هللا والبغض في هللا ،قال تعالى﴿ : ِالَ تَجِ دُ قَوْماً يُؤْمِنُونَ بِاللَّه وَالْيَوْمِ اآلْ خِ رِ يُوَاد ونَ مَ نْ حَ ادَّ اللَّهَ وَرَ سُ ولَهُ وَلَوْ كَانُوا ِيمَ انَ وَأَيَّدَ هُ م آبَاءهُ مْ أَوْ أَبْنَاءهُ مْ أَوْ إِخْ وَانَهُمْ أَوْ عَشِ يرَتَهُمْ أُوْلَئِكَ كَ تَبَ فِي قُلُوبِهِمُ اإلْ ُبِرُوحٍ مِّنْه﴾ [املجادلة : 22 ] ، فاملؤمن الحق ال يو الي من أعلن العداء هلل تعالى مهما بلغت درجة القرابة ، فكل صلة ووشيجة ال تقوم على أساس محبة هللا .تعالى فهي منبوذة مذمومة ومثال ذلك تبرؤ األنبياء من والية غير هللا تعالى ،وها هو النبي-  - يتبرأ من والية الكفار واملشركين ، قال تعالى﴿ : َّإِنَّ وَلِيِّـيَ اللّهُ ال َذِ ي نَزَّلَ الْكِ تَابَ وَهُ وَ يَتَوَ لَّى الصَّالِ حِ ين﴾ [األعراف : 196 ] ، أي أن "نصيري ومعيني وظهيري عليكم (هللا الذي نزل الكتاب) عليّ بالحق ، "وهو الذي يتولى من صلح عمله بطاعته من خلقه( 18 ) ، مبيناً لهم في ذات الوقت عجز من يتولونهم من األوثان وغيرها ،قال تعال ى﴿ : ْوَالَّذِ ينَ تَدْ عُونَ مِن دُونِهِ الَ يَسْ تَطِ يعُونَ نَصْرَكُ مْ وَال أَنفُ سَ هُم َيَنْصُرُون﴾ [األعراف : 197 ] ،فإذا كان لكم من دون هللا أولياء ، فهل يستطيعون نفعكم أو دفع البالء عنكم؟ كال "ال يستطيعون نصركم ،وال هم مع عجزهم عن نصرتكم يقدرون على نصرة أنفس هم ، فأي هذين أولى بالعبادة وأحق 1- التبرؤ من كل ش يء يخالف الوالء هلل تعالى ، والبراءة من الشرك واإلقرار بوحدانية هللا تعالى من أوليات الوالء هلل ،فال محبة ملن ال يقر بوحدانية هللا تعالى ،وال نصرة ملن عادى هللا ورسوله ،قال تعالى﴿ : ٌقَدْ كَانَتْ لَكُ مْ أُسْ وَة حَ سَ نَةٌ فِي إِبْرَاهِ يمَ وَالَّذِ ينَ مَ عَهُ إِذْ قَالُوا لِقَ وْمِهِمْ إِنَّا بُرَاء مِنكُ مْ وَمِمَّ ا تَعْبُدُ ونَ مِن دُو نِ اللَّهِ كَ فَ رْنَا بِكُ مْ وَبَدَ ا بَيْنَنَا ُوَبَيْنَكُ مُ الْعَدَ اوَةُ وَالْبَغْضَاء أَبَداً حَ تَّى تُؤْمِنُوا بِاللَّهِ وَحْ دَ ه... ﴾ [املمتحنة : 4] ، وقد صرحوا "بأن سبب عداوتهم وبغضائهم ليس إال كفرهم باهلل ،وما دام هذا السبب قائمًا كانت العداوة قائمة ، حتى إن أزالوه وآمنوا باهلل وحده انقلبت العداوة مواالة ، "والبغضاء محبة( 17 ). املبحث األول :ال والء هلل تعالى وللنبي-  -. (11) البخاري ، صحيح البخاري ،كتاب :التوحيد ،باب : ِمَ ا جَ اءَ فِي دُعَاء النَّبِ يِّ صَلَّ ى هللاُ عَلَيْهِ وَسَ لَّمَ أُمَّ تَهُ إِلَى تَوْحِ يدِ اللَّهِ تَبَارَكَ الوالء في القرآن الكريم الوالء في القرآن الكريم الوالء في القرآن الكريم ( 22 ) الحصان الحصان الحصان المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم اإلسالمية ــ المجلد ال رابع ــ العدد ال ثاني ــ يونيو 21 20 م فضعفُ بيتِ العنكبوت إضافة إلى ضعفِ بنيتِه ،هو ضعيف في عال ِقاتِه الداخلية ، ٍوقد يُجمعُ الضَّعفانِ في ضعف " ٍواحد( 16 ). َ فضعفُ بيتِ العنكبوت إضافة إلى ضعفِ بنيتِه ،هو ضعيف في عال ِقاتِه الداخلية ، ٍوقد يُجمعُ الضَّعفانِ في ضعف " ٍواحد( 16 ). املبحث األول :ال والء هلل تعالى وللنبي-  -. َ وهذا هو حال من يتخذ من دون هللا وليًا فيركن إليه ، فقد شبَّه هللا تعالى حال هؤالء بحال وهاء بيت العنكبوت من عدم وفاء األنثى لزوجها وأسرتها ،وهذا ما عبَّر عنه هللا تعالى بقوله : ﴿ َوَمِنَ النَّاسِ مَ نْ ي ِتَّخِ ذُ مِنْ دُون اللَّهِ أَنْدَ ادًا يُحِ ب ونَهُمْ كَ حُ بِّ اللَّهِ وَالَّذِ ينَ آمَ نُوا أَشَ د حُ بًّا لِلَّهِ وَلَوْ يَرَى الَّذِ ينَ ظَلَمُ وا إِذْ يَرَوْ نَ الْعَذَ ابَ أَنَّ الْقُ وَّةَ لِلَّهِ جَ مِيعًا ( ِوَأَنَّ اللَّهَ شَ دِ يدُ الْعَذَ اب165 َّ) إِذْ تَبَر( ُأَ الَّذِ ينَ ات بِعُوا مِنَ الَّذِ ينَ اتَّبَعُوا وَرَ أَوُا الْعَذَ ابَ وَتَقَ طَّعَتْ بِهِمُ األْ َسْ بَاب166 َ) وَقَال ْالَّذِ ينَ اتَّبَعُوا لَوْ أَنَّ لَنَا كَ رَّةً فَنَتَبَرَّأَ مِنْهُمْ كَ مَا تَبَرَّ ءُوا مِنَّا كَ ذَ لِكَ يُرِيهِمُ اللَّهُ أَع َمَالَهُمْ ح َسَ رَاتٍ عَلَيْهِمْ وَمَ ا هُ مْ بِخَ ارِجِ ينَ مِن ِالنَّار﴾[البقرة : 165 - 166 ] ثم يبين هللا تعالى أن الولي الحق هو القادر الذي يحيي املوتى وهو على كل ش يء قدير ، ال يعجزه ش يء في السموات واألرض ،قال تعالى﴿ : َأَمِ اتَّخَ ذُ وا مِن دُونِهِ أَوْلِيَاء فَاللَّهُ هُ و ٌالْوَ لِي وَهُ وَ يُحْ يِ ي املَوْتَى وَهُ وَ عَلَ ى كُلِّ ش َ يْءٍ قَدِ ير﴾ [الشورى :9 .] املطلب الثاني :مظاهر الوالء هلل تعالى وثمراته ومن مظاهر الوالء هلل تعالى ما يأتي : 1- التبرؤ من كل ش يء يخالف الوالء هلل تعالى ، والبراءة من الشرك واإلقرار بوحدانية هللا تعالى من أوليات الوالء هلل ،فال محبة ملن ال يقر بوحدانية هللا تعالى ،وال نصرة ملن عادى هللا ورسوله ،قال تعالى﴿ : ٌقَدْ كَانَتْ لَكُ مْ أُسْ وَة حَ سَ نَةٌ فِي إِبْرَاهِ يمَ وَالَّذِ ينَ مَ عَهُ إِذْ قَالُوا لِقَ وْمِهِمْ إِنَّا بُرَاء مِنكُ مْ وَمِمَّ ا تَعْبُدُ ونَ مِن دُو نِ اللَّهِ كَ فَ رْنَا بِكُ مْ وَبَدَ ا بَيْنَنَا ُوَبَيْنَكُ مُ الْعَدَ اوَةُ وَالْبَغْضَاء أَبَداً حَ تَّى تُؤْمِنُوا بِاللَّهِ وَحْ دَ ه... ﴾ [املمتحنة : 4] ، وقد صرحوا "بأن سبب عداوتهم وبغضائهم ليس إال كفرهم باهلل ،وما دام هذا السبب قائمًا كانت العداوة قائمة ، حتى إن أزالوه وآمنوا باهلل وحده انقلبت العداوة مواالة ، "والبغضاء محبة( 17 ). املبحث األول :ال والء هلل تعالى وللنبي-  -. فال والية محبة أو نصرة ملن حادّ هللا ورسوله ،مهما بلغت درجة القرابة أو تزايدت أواصر املحبة ، فاملحبة في هللا والبغض في هللا ،قال تعالى﴿ : ِالَ تَجِ دُ قَوْماً يُؤْمِنُونَ بِاللَّه وَالْيَوْمِ اآلْ خِ رِ يُوَاد ونَ مَ نْ حَ ادَّ اللَّهَ وَرَ سُ ولَهُ وَلَوْ كَانُوا ِيمَ انَ وَأَيَّدَ هُ م آبَاءهُ مْ أَوْ أَبْنَاءهُ مْ أَوْ إِخْ وَانَهُمْ أَوْ عَشِ يرَتَهُمْ أُوْلَئِكَ كَ تَبَ فِي قُلُوبِهِمُ اإلْ ُبِرُوحٍ مِّنْه﴾ [املجادلة : 22 ] ، فاملؤمن الحق ال يو الي من أعلن العداء هلل تعالى مهما بلغت درجة القرابة ، فكل صلة ووشيجة ال تقوم على أساس محبة هللا .تعالى فهي منبوذة مذمومة امأاهلل املطلب الثاني :مظاهر الوالء هلل تعالى وثمراته ومن مظاهر الوالء هلل تعالى ما يأتي : 1- التبرؤ من كل ش يء يخالف الوالء هلل تعالى ، والبراءة من الشرك واإلقرار بوحدانية هللا تعالى من أوليات الوالء هلل ،فال محبة ملن ال يقر بوحدانية هللا تعالى ،وال نصرة ملن عادى هللا ورسوله ،قال تعالى﴿ : ٌقَدْ كَانَتْ لَكُ مْ أُسْ وَة حَ سَ نَةٌ فِي إِبْرَاهِ يمَ وَالَّذِ ينَ مَ عَهُ إِذْ قَالُوا لِقَ وْمِهِمْ إِنَّا بُرَاء مِنكُ مْ وَمِمَّ ا تَعْبُدُ ونَ مِن دُو نِ اللَّهِ كَ فَ رْنَا بِكُ مْ وَبَدَ ا بَيْنَنَا ُوَبَيْنَكُ مُ الْعَدَ اوَةُ وَالْبَغْضَاء أَبَداً حَ تَّى تُؤْمِنُوا بِاللَّهِ وَحْ دَ ه... ﴾ [املمتحنة : 4] ، وقد صرحوا "بأن سبب عداوتهم وبغضائهم ليس إال كفرهم باهلل ،وما دام هذا السبب قائمًا كانت العداوة قائمة ، حتى إن أزالوه وآمنوا باهلل وحده انقلبت العداوة مواالة ، "والبغضاء محبة( 17 ). املبحث األول :ال والء هلل تعالى وللنبي-  -. ا فال والية محبة أو نصرة ملن حادّ هللا ورسوله ،مهما بلغت درجة القرابة أو تزايدت أواصر املحبة ، فاملحبة في هللا والبغض في هللا ،قال تعالى﴿ : ِالَ تَجِ دُ قَوْماً يُؤْمِنُونَ بِاللَّه وَالْيَوْمِ اآلْ خِ رِ يُوَاد ونَ مَ نْ حَ ادَّ اللَّهَ وَرَ سُ ولَهُ وَلَوْ كَانُوا ِيمَ انَ وَأَيَّدَ هُ م آبَاءهُ مْ أَوْ أَبْنَاءهُ مْ أَوْ إِخْ وَانَهُمْ أَوْ عَشِ يرَتَهُمْ أُوْلَئِكَ كَ تَبَ فِي قُلُوبِهِمُ اإلْ ُبِرُوحٍ مِّنْه﴾ [املجادلة : 22 ] ، فاملؤمن الحق ال يو الي من أعلن العداء هلل تعالى مهما بلغت درجة القرابة ، فكل صلة ووشيجة ال تقوم على أساس محبة هللا .تعالى فهي منبوذة مذمومة ومثال ذلك تبرؤ األنبياء من والية غير هللا تعالى ،وها هو النبي-  - يتبرأ من والية الكفار واملشركين ، قال تعالى﴿ : َّإِنَّ وَلِيِّـيَ اللّهُ ال َذِ ي نَزَّلَ الْكِ تَابَ وَهُ وَ يَتَوَ لَّى الصَّالِ حِ ين﴾ [األعراف : 196 ] ، أي أن "نصيري ومعيني وظهيري عليكم (هللا الذي نزل الكتاب) عليّ بالحق ، "وهو الذي يتولى من صلح عمله بطاعته من خلقه( 18 ) ، مبيناً لهم في ذات الوقت عجز من يتولونهم من األوثان وغيرها ،قال تعال ى﴿ : ْوَالَّذِ ينَ تَدْ عُونَ مِن دُونِهِ الَ يَسْ تَطِ يعُونَ نَصْرَكُ مْ وَال أَنفُ سَ هُم َيَنْصُرُون﴾ [األعراف : 197 ] ،فإذا كان لكم من دون هللا أولياء ، فهل يستطيعون نفعكم أو دفع البالء عنكم؟ كال "ال يستطيعون نصركم ،وال هم مع عجزهم عن نصرتكم يقدرون على نصرة أنفس هم ، فأي هذين أولى بالعبادة وأحق (16) النابلس ي ،موسوعة اإلعجاز العلمي في القرآن والسنة ،ج2 ،ص234 . (17) الزمخشري ،الكشاف عن حقائق غوامض التنزيل ،ج :4 ،ص : 514 . (18) الطبري ،جامع البيان في تأويل القرآن ،ج : 13 ،ص : 323 . الوالء في القرآن الكريم ( 23 ) الحصان المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم اإلسالمية ــ المجلد ال رابع ــ العدد ال ثاني ــ يونيو 21 20 م باأللوهة؟ أمن ينصر وليه ويمنع نفسه ممن أراده ، أم من ال يستطيع نصر وليه ويعجز عن منع نفسه ممن أراده وبَغاه بمكروه(" 19 ). املبحث األول :ال والء هلل تعالى وللنبي-  -. 2- الطاعة :والوالء يكون بالقول والعمل ، فمن أعلن الوالء هلل وجب عليه أن يعبر عن ذلك بالتزام طاعة هللا تعالى فيما أمر أو نهى وتحكيم كتابه ،وهذه الطاعة إلزامية لقوله تعالى﴿ : ْيَا أَي هَا الَّذِ ينَ آمَ نُواْ أَطِ يعُواْ اللّهَ وَأَطِ يعُوا ُالرَّسُ ولَ وَأُوْ لِي األَمْرِ مِنكُ مْ فَإِن تَنَازَعْتُمْ فِي ش َ يْءٍ فَرُد وهُ إِلَى اللّهِ وَالرَّسُ ولِ إِن كُ نتُمْ تُؤْمِن َون َبِاللّهِ وَالْيَوْمِ اآلخِ رِ ذَلِك ًخَ يْرٌ وَأَحْ سَ نُ تَأْوِيال﴾ [النساء : 59 ] ،وطاعة هللا تعالى تتمثل في إقامة شعائره وفرائضه التي فرضها ، فتحقيق العبادات له تعالى هي من مضامين الطاعة ،فإقامة الصالة وإيتاء الزكاة وصوم رمضان وحج البيت ، واالبتعاد عما حرّم م ن الفواحش ،وتنفيذ أوامره وإن خالفت هوى النفس ،فقد ضرب لنا إبراهيم-  - أروع صور الطاعة في االمتثال ألمر هللا تعالى بذبح ابنه ،قال تعالى﴿ : فَلَمَّ ا بَلَغَ مَ عَهُ السَّ عْيَ قَالَ يَا بُنَيَّ إِنِّي أَرَى فِي املْ َنَامِ أَنِّي َأَذْبَحُ كَ فَانظُرْ مَ اذَا تَرَى ق َالَ يَا أَبَتِ افْعَلْ مَا تُؤْمَرُ سَ تَجِ دُ نِي إِن شَ اء اللَّهُ مِنَ الصَّابِرِين﴾ [الصافات : 102 ] ، فكان الجواب من ابنه :أن يا أبتي أمر هللا أولى بالنفاذ "إنه يتلقى األمر ال في طاعة واستسالم فحسب ، ولكن في رض ى كذلك وفي يقين (يا أبت) في مودة وقربى ،(افعل م)ا تؤمر... (19) الطبري ،املصدر السابق ،ج : 13 ،ص : 323 . (20) قطب ،في ظالل القرآن ،ج :5 ،ص : 2995 . (21) ابن عطية ،املحرر الوجيز في تفسير الكتاب العزيز ،ج :1 ،ص : 422 . ا (21) ابن عطية ،املحرر الوجيز في تفسير الكتاب العزيز ،ج :1 ،ص : 422 . (19) الطبري ،املصدر السابق ،ج : 13 ،ص : 323 . (20) قطب ،في ظالل القرآن ،ج :5 ،ص : 2995 . (21) ابن عطية ،املحرر الوجيز في تفسير الكتاب العزيز ،ج :1 ،ص : 422 . (19) الطبري ،املصدر السابق ،ج : 13 ،ص : 323 . املبحث األول :ال والء هلل تعالى وللنبي-  -. فهو يحس ما أحسه من قبل قلب أبيه ، يحس أن الرؤيا إشارة ،وأن اإلشارة أمر ثم هو األدب مع هللا ، ومعرفة حدود قدرته وطاقته في االحتمال واالستعانة بربه على ضعفه ونسبة الفضل إليه في إعانته على التضحية ،ومساعدته على الطاعة﴾ : ستجدني إن شاء هللا من الصابرين"﴿ ( 20 ) ، .ًهذا هو الوالء الحق هلل تعالى في أروع صوره امتثاال واحتسابا ﴿ا يلها 3- الدفاع عن الدين :ويكون بإبراز الصورة الحقيقية املشرقة لهذا الدين الحنيف ، إحقاقاً للحق وتنفيذً ا ألمر هللا تعالى في إيصال هذه الدعوة إلى جميع الناس ،بالطريقة التي أراد هللا تعالى ،من الحكمة واملوعظة الحسنة واللين واملجادلة بالتي هي أحسن ،قال تعالى﴿ : َادْعُ إِلِى سَ بِيلِ رَبِّكَ بِالْ حِ كْ مَةِ وَاملْ َوْعِظَةِ الْ حَ سَ نَةِ وَجَ ادِ لْهُم بِالَّتِ ي هِ ي َأَحْ سَ نُ إِنَّ رَبَّكَ هُ وَ أَعْلَمُ بِمَن ضَلَّ عَن سَ بِيلِهِ وَهُ وَ أَعْلَمُ بِاملْ ُهْت َدِ ين﴾ [النحل : 125 .] ويتضمن الدفاع أن ننتصر لدين هللا تعالى من الطغاة والبغاة ، سواء كانوا ممن ينتمون لهذا الدين أو من غيرهم؛ ألن من واجب األمة الدفاع عن هذا الدين ومقدساته ، واالنتصار للمسلمين الذين اضطهدوا في مشارق األرض ومغاربها؛ حتى ال يفتنوا في دينهم ،ق ال تعالى﴿ : َوَقَاتِلُوهُ مْ حَ تَّى الَ تَكُونَ فِتْنَةٌ وَيَكُونَ الدِّ ينُ كُل هُ لِلّه فَإِنِ انتَهَوْاْ فَإِنَّ اللّه ٌبِمَ ا يَعْمَ لُونَ بَصِ ير﴾ [األنفال : 39 .] .ثمرات الوالء هلل تعالى 1- محبة هللا تعالى : وهي الغاية التي يسعى إليها من عرف هللا تعالى حق املعرفة ، فتحصل محبة هللا تعالى لعبده عندما يخلص الوالء له ، ًوينقاد ألمره رهباً ورغبا ، وتظهر عالمات محبة هللا تعالى للعبد "أن يرى العبد مهديًا مسددًا ذا قبول في األرض ، "فلطف هللا بالعبد ورحمته إياه هي ثمرة محبته( 21 ) ،قال تعالى﴿ : ُيَا أَي هَا الَّذِ ينَ آمَن ْوا َمَ ن يَرْتَدَّ مِنكُ مْ عَن دِ ينِهِ فَسَ وْفَ يَأْتِي اللّهُ بِقَ وْمٍ يُحِ ب هُمْ وَيُحِ ب ونَهُ أَذِ لَّةٍ عَلَ ى املْ ُؤْمِنِين َأَعِزَّةٍ عَلَ ى الْكَافِرِينَ يُجَ اهِ دُ ون َفِي سَ بِيلِ اللّهِ وَالَ يَخَ افُونَ لَوْمَةَ آلئِمٍ ذَلِكَ فَضْلُ اللّهِ يُؤْتِيهِ م ٌن يَشَ اءُ وَاللّهُ وَاسِ عٌ عَلِيم﴾ [املائدة : 54 ] ، وهذه اآلية تحذير من هللا تعالى للمؤمنين من الوقوع في شرك االفتتان عن الدين ،بأن هؤالء ال وزن لهم عند هللا تعالى ، لذا (21) ابن عطية ،املحرر الوجيز في تفسير الكتاب العزيز ،ج :1 ،ص : 422 . (22) رضا ، )تفسير القرآن الحكيم (تفسير املنار ،ج :5 ،ص : 376 . (23) ينظر- السعدي ،تيسير الكريم الرحمن في تفسير كالم املنان ،ج :1 ،ص : 236 . (24) البخاري ، صحيح البخاري ،كتاب اإليمان ، ِبَابُ حَ الَوَةِ اإلِيمَ ان ،ج :1 ،ص : 12 ،رقم : 16 . (25) السعدي ،تيسير الكريم الرحمن في تفسير كالم املنان ،ج :1 ،ص : 659 . المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم اإلسالمية ــ المجلد ال رابع ــ العدد ال ثاني ــ يونيو 21 20 م 3- حصول السعادة : ومن ثمار الو الء املطلق هلل تعالى أن يشعر املوالي هلل تعالى بالرضا والطمأنينة والسعادة؛ ألنه أعلن الوالية الحقة الخالصة هلل تعالى فعن أنس بن مالك-  -عن النبي-  - قال : "ثالث من كن فيه وجد حالوة اإليمان :أن يكون هللا ورسوله أحب إليه مما سواهما ، وأن يحب املرء ال يحبه إال لله ، وأن يكره أن يعود في الكفر "كما يكره أن يقذف في النار( 24 ). المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم اإلسالمية ــ المجلد ال رابع ــ العدد ال ثاني ــ يونيو 21 20 م المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم اإلسالمية ــ المجلد ال رابع ــ العدد ال ثاني ــ يونيو 21 20 م يستبدلهم بمن هم أعز عليه منهم ،يدافعون عن دينه ألنهم يحبون هللا تعالى ويحبهم فكتب لهم ا لعزة والكرامة ، وقد تحقق هذا في زمن خالفة أبي بكر الصديق-  - عندما حاولت فيه فئة ضالة أن تفرق بين أركان اإلسالم وقواعده ،وتبث جذور الفتنة بين املسلمين بعد وفاة النبي-  - ، وهذه اآلية وإن كانت قد تحققت في أبي بكر والصحابة الذين قاتلوا معه املرتدين ، إال أنها تنطبق في كل زمان أوجد هللا تعالى فئة صابرة على الحق ، مدافعة عنه ،غايتهم في ذلك محبة هللا تعالى ورسوله-  -. المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم اإلسالمية ــ المجلد ال رابع ــ العدد ال ثاني ــ يونيو 21 20 م 2- العزة والغلبة : ًإن من استجار باهلل ولجأ إليه ال يضره بعد ذلك شيئا ،ألن هللا هو املعز وهو املذل ، وهو القادر على كل ش يء ،فال تقهره قوة وال يمنعه مانع ،ق ال تعالى﴿ : َالَّذِ ينَ يَتَّخِ ذُ ونَ الْكَافِرِينَ أَوْلِيَاء مِن دُونِ املْ ُؤْمِنِين ًأَيَبْتَغُونَ عِندَ هُ مُ الْعِزَّةَ فَإِنَّ العِزَّةَ لِلّهِ جَ مِيعا﴾ [النساء : 139 ] ، وليس أعجب ممن يوالي أعداء هللا ويبتغي عندهم العزة ،وقد سلبت منهم ، وهنا "استفهام تقريع و توبيخ ، إن كانوا يبتغون عندهم العزة وهي املنعة والغلبة ورفعة القدر فإن العزة هلل جميعًا ،فهو يؤتيها من يشاء ، فكان عليهم أن يطلبوها منه بصدق اإليمان والسير على سنته تعالى واتباع هداية وحيه الذي يرشدهم إلى طرقها ، "ويبين أسبابها( 22 ) ، فو هللا لن ينالهم إال الخزي والذل الذي .استمرأته نفوسهم الضعيفة َ فالذين يبتغون العزة من هللا تعالى سينالون العزة ،وسيكون لهم الغلبة ،قال تعالى﴿ : َوَمَ ن يَتَوَلَّ اللّه َوَرَ سُ ولَهُ وَالَّذِ ينَ آمَ نُواْ فَإِنَّ حِ زْبَ اللّهِ هُ مُ الْغَالِبُون﴾ [املائدة : 56 ] ، وهنا اضاف هللا تعالى هذا الحزب "إضافة عبودية ووالية ، "وحزبه هم الغالبون الذين لهم العاقبة في الدنيا واآلخرة( 23 ) ، وفي تذييل اآلية بما جاء بها من املؤكدات بشرى .من هللا تعالى ألوليائه بأنهم منصورون ال محالة وإن استبطأت نفوسهم ذلك فالذين يبتغون العزة من هللا تعالى سينالون العزة ،وسيكون لهم الغلبة ،قال تعالى﴿ : َوَمَ ن يَتَوَلَّ اللّه َوَرَ سُ ولَهُ وَالَّذِ ينَ آمَ نُواْ فَإِنَّ حِ زْبَ اللّهِ هُ مُ الْغَالِبُون﴾ [املائدة : 56 ] ، وهنا اضاف هللا تعالى هذا الحزب "إضافة عبودية ووالية ، "وحزبه هم الغالبون الذين لهم العاقبة في الدنيا واآلخرة( 23 ) ، وفي تذييل اآلية بما جاء بها من املؤكدات بشرى .من هللا تعالى ألوليائه بأنهم منصورون ال محالة وإن استبطأت نفوسهم ذلك 3- حصول السعادة : ومن ثمار الو الء املطلق هلل تعالى أن يشعر املوالي هلل تعالى بالرضا والطمأنينة والسعادة؛ ألنه أعلن الوالية الحقة الخالصة هلل تعالى فعن أنس بن مالك-  -عن النبي-  - قال : "ثالث من كن فيه وجد حالوة اإليمان :أن يكون هللا ورسوله أحب إليه مما سواهما ، وأن يحب املرء ال يحبه إال لله ، وأن يكره أن يعود في الكفر "كما يكره أن يقذف في النار( 24 ). املبحث األول :ال والء هلل تعالى وللنبي-  -. (21) ابن عطية ،املحرر الوجيز في تفسير الكتاب العزيز ،ج :1 ،ص : 422 . الوالء في القرآن الكريم الوالء في القرآن الكريم ( 24 ) الحصان (26) مسلم ،صحيح مسلم ،تحقيق :محمد فؤاد عبد الباقي ،( دار إحياء التراث العربي– )بيروت ،كتاب اإليمان ، ِبَابُ وُجُ وبِ مَ حَ بَّةِ رَسُ ول ِهللاِ صلَّ ى هللا عَلَيْهِ وَسَ لَّمَ أَكْ ثَرَ مِنَ األْ َهْ لِ وَالْوَلَد ، َوَالْوَالِدِ والنَّاسِ أجْ مَ عِين ، َ ِْيمَ انِ عَلَ ى مَنْ لمْ يُحِ بَّهُ هَ ذِ هِ امل وَإِطْالَ قِ عَدَ مِ اإلْ َحَ بَّة ،ج : 1 ، ص67 ،رقم : 44 . ( 27 ) البخاري ، صحيح البخاري ، كتاب األيمان والنذور ، ٌبَاب : َكَ يْفَ كَانَتْ يَمِينُ النَّبِ يِّ صَلَّ ى هللاُ عَلَيْهِ وَسَ لَّم ،ج :8 ،ص129 ،رقم : 6632 . ( 28 ) ابن عاشور ،التحرير والتنوير ،ج : 5 ،ص : 97 . ( 29 ) الخازن ، لباب التأويل في معاني التنزيل ،ج :4 ،ص : 176 . ( 30 ) أبن حنبل ،مسند اإلمام أحمد بن حنبل ،مسند النساء ،مُ سْ نَدُ الصّ ِ دِّ يقَ ةِ عَائِشَ ةَ بِنْتِ الصّ ِ دِّ يقِ رَض ِ يَ هللاُ عَنْهَا ،ج : 41 ،ص : 148 ، رقم : 24601 . املطلب الثالث :الوالء للنبي-  - . ًومن مظاهر محبته أيضا ،توقيره وتبجيله ،قال تعالى﴿ : ِيَا أَي هَا الَّذِ ينَ آمَ نُوا الَ تَرْفَعُوا أَصْوَاتَكُ مْ فَوْقَ صَوْت َالنَّبِ يِّ وَالَ تَجْ هَرُوا لَهُ بِالْقَ وْلِ كَ جَ هْرِ بَعْضِ كُ مْ لِبَعْضٍ أَن تَحْ بَطَ أَعْمَ الُكُ مْ وَأَنتُمْ ال َتَشْ عُرُون﴾ [الحجرات :2] ، وذلك حتى في رفع الصوت في حضرة النبي-  - والتأدب في مخاطبته ، لذلك جاء األمر اإللهي "أن يبجلوه ويفخموه ويعظموه وال "يرفعوا أصواتهم عنده وال ينادوه كما ينادي بعضهم بعضا فيقول يا محمد بل يقولون يا رسول هللا يا نبي( 29 ). ومن مظاهر محبة النبي-  - أيضاً االقتداء به في كل حركاته وسكناته ، وخاصة في الجانب التشريعي لقوله تعالى﴿ : َِ َن كَانَ يَرْجُ و اللَّهَ وَالْيَوْمَ اآلْ خِ رَ وَذَكَ رَ اللَّه لَقَ دْ كَانَ لَكُ مْ فِي رَسُ ولِ اللَّهِ أُسْ وَةٌ حَ سَ نَةٌ ملّ ًكَ ثِيرا﴾ [األحزاب : 21 ] ، وهذا التأس ي واالقت داء به ألنه كان يمثل الجانب العملي للقرآن الكريم كما جاء في حديث أم املؤمنين عائشة عندما سُ ئلت عن خلق النبي-  - قالت :"كان خلقه القرآن.".. املطلب الثالث :الوالء للنبي-  - . ( 30 ) ؛ وألن االقتداء به هو الطريق املوصل إلى الجنة "ألنه حكم وهذا عمر بن الخطاب-  - يبين للنبي-  - مدى محبته له ،إال أنه تفاجأ من رد النبي-  - الذي بيّن أن الوالء الحقيقي واملحبة الصادقة له ال تكون إال إذا بلغت مبلغاً يتجاوز حدود محبة النفس ،فعن عبد هللا بن هشام ، قال :"كنا مع النبي-  - وهو آخذ بيد عمر بن الخطاب ،فقال له عمر :يا رسول هللا ، ألنت أحب إلي من كل ش يء إال من نفس ي ،فقال النبي-  - :ال ،والذي نفس ي بيده ،حتى أكون أحب إليك من نفسك ،فقال له ع مر :فإنه اآلن ،وهللا ، ألنت أحب إلي من نفس ي ،فقال النبي-  - : "اآلن يا عمر( 27 ) ، فما كان من عمر إال أن انخلع من محبة نفسه وآثر محبة النبي-  - .؛ ألنه أولى باملؤمنين من أنفسهم ومن مظاهر محبة النبي-  - إتباعه فيما أمر أو نهى؛ ألن اتباع النبي هو اتباع هلل تعالى ،قال تعالى﴿ : قُلْ إِن ٌكُ نتُمْ تُحِ ب ونَ اللّهَ فَاتَّبِعُونِي يُحْ بِبْكُ مُ اللّهُ وَيَغْفِرْ لَكُ مْ ذُنُوبَكُ مْ وَاللّهُ غَفُ ورٌ رَّحِ يم﴾ [آل عمران : 31 ] ، وطاعته فيما يبلغ عن ربه تعالى؛ ألنه ال ينطق عن الهوى؛ بل هو وحيٌ من هللا تعالى ،وطاعته واجبة لقوله تعالى﴿ : َقُلْ أَطِ يعُواْ اللّه َوَالرَّسُ ولَ فإِن تَوَلَّوْاْ فَإِنَّ اللّهَ الَ يُحِ ب الْكَافِرِين﴾ [آل عمران : 32 ] ،وقوله تعالى﴿ : وَأَطِ يعُوا اللَّهَ وَأَطِ يعُوا الرَّسُ ولَ فَإِن ُتَوَلَّيْتُمْ فَإِنَّمَ ا عَلَ ى رَسُ ولِنَا الْبَالَ غُ املْ ُبِين﴾ [التغابن : 12 ]، وجاء التأكيد بطاعة الرسول بعد طاعة هللا تعالى "لينبه على وجوب طاعته فيما يأمر به ، ولو كان أمره غير مقترن بقرائن تبليغ الوحي لئال يتوهم السامع أن طاعة الرسول املأمور بها ترجع إلى طاعة هللا فيما يبلغه عن هللا دون ما يأمر به في غير التشريع ،فإن امتثال أمره ك"له خير( 28 ). ( 30 ) أبن حنبل ،مسند اإلمام أحمد بن حنبل ،مسند النساء ،مُ سْ نَدُ الصّ ِ دِّ يقَ ةِ عَائِشَ ةَ بِنْتِ الصّ ِ دِّ يقِ رَض ِ يَ هللاُ عَنْهَا ،ج : 41 ،ص : 148 ، رقم : 24601 . املطلب الثالث :الوالء للنبي-  - . ًومن مظاهر محبته أيضا ،توقيره وتبجيله ،قال تعالى﴿ : ِيَا أَي هَا الَّذِ ينَ آمَ نُوا الَ تَرْفَعُوا أَصْوَاتَكُ مْ فَوْقَ صَوْت َالنَّبِ يِّ وَالَ تَجْ هَرُوا لَهُ بِالْقَ وْلِ كَ جَ هْرِ بَعْضِ كُ مْ لِبَعْضٍ أَن تَحْ بَطَ أَعْمَ الُكُ مْ وَأَنتُمْ ال َتَشْ عُرُون﴾ [الحجرات :2] ، وذلك حتى في رفع الصوت في حضرة النبي-  - والتأدب في مخاطبته ، لذلك جاء األمر اإللهي "أن يبجلوه ويفخموه ويعظموه وال "يرفعوا أصواتهم عنده وال ينادوه كما ينادي بعضهم بعضا فيقول يا محمد بل يقولون يا رسول هللا يا نبي( 29 ). ومن مظاهر محبة النبي-  - أيضاً االقتداء به في كل حركاته وسكناته ، وخاصة في الجانب التشريعي لقوله تعالى﴿ : َِ َن كَانَ يَرْجُ و اللَّهَ وَالْيَوْمَ اآلْ خِ رَ وَذَكَ رَ اللَّه لَقَ دْ كَانَ لَكُ مْ فِي رَسُ ولِ اللَّهِ أُسْ وَةٌ حَ سَ نَةٌ ملّ ًكَ ثِيرا﴾ [األحزاب : 21 ] ، وهذا التأس ي واالقت داء به ألنه كان يمثل الجانب العملي للقرآن الكريم كما جاء في حديث أم املؤمنين عائشة عندما سُ ئلت عن خلق النبي-  - قالت :"كان خلقه القرآن.".. ( 30 ) ؛ وألن االقتداء به هو الطريق املوصل إلى الجنة "ألنه حكم ومن مظاهر محبة النبي-  - إتباعه فيما أمر أو نهى؛ ألن اتباع النبي هو اتباع هلل تعالى ،قال تعالى﴿ : قُلْ إِن ٌكُ نتُمْ تُحِ ب ونَ اللّهَ فَاتَّبِعُونِي يُحْ بِبْكُ مُ اللّهُ وَيَغْفِرْ لَكُ مْ ذُنُوبَكُ مْ وَاللّهُ غَفُ ورٌ رَّحِ يم﴾ [آل عمران : 31 ] ، وطاعته فيما يبلغ عن ربه تعالى؛ ألنه ال ينطق عن الهوى؛ بل هو وحيٌ من هللا تعالى ،وطاعته واجبة لقوله تعالى﴿ : َقُلْ أَطِ يعُواْ اللّه َوَالرَّسُ ولَ فإِن تَوَلَّوْاْ فَإِنَّ اللّهَ الَ يُحِ ب الْكَافِرِين﴾ [آل عمران : 32 ] ،وقوله تعالى﴿ : وَأَطِ يعُوا اللَّهَ وَأَطِ يعُوا الرَّسُ ولَ فَإِن ُتَوَلَّيْتُمْ فَإِنَّمَ ا عَلَ ى رَسُ ولِنَا الْبَالَ غُ املْ ُبِين﴾ [التغابن : 12 ]، وجاء التأكيد بطاعة الرسول بعد طاعة هللا تعالى "لينبه على وجوب طاعته فيما يأمر به ، ولو كان أمره غير مقترن بقرائن تبليغ الوحي لئال يتوهم السامع أن طاعة الرسول املأمور بها ترجع إلى طاعة هللا فيما يبلغه عن هللا دون ما يأمر به في غير التشريع ،فإن امتثال أمره ك"له خير( 28 ). املطلب الثالث :الوالء للنبي-  - . ما والوالء للنبي-  - من لوازم شهادة التوحيد ،التي بها ينتقل اإلنسان من حالة الكفر إلى نور اإليمان ، ووالية النبي-  -حق ،فهو أولى بنا من أنفسنا لقوله تعالى﴿ : النَّبِ ي ... ْأَوْ لَى بِاملْ ُؤْمِنِينَ مِنْ أَنفُ سِ هِم﴾ [األحزاب : 6 ] وهذه األحقية في الوالية قدمت على والية النفس "ألنه-  - بذل لهم من النصح ،والشفقة ،والرأفة ،ما كان به أرحم الخلق ، وأرأفهم ،فرسول هللا أعظم الخلق مِنَّةً عليهم من كل أحد ،فإنه لم يصل إليهم مثقال ذرة من الخير ، وال اندفع عنهم مثقال ذرة من الشر ، "إال على يديه وبسببه( 25 ). الوالء في القرآن الكريم ( 25 ) الحصان المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم اإلسالمية ــ المجلد ال رابع ــ العدد ال ثاني ــ يونيو 21 20 م ومن مظاهر الوالء للنبي-  - 1- محبته :ومحبة النبي-  - واجبة على كل مسلم ، وهي ليست كمحبتنا ألنفسنا أو أوالدنا أو آباءنا أو محبتنا لرغبات النفس؛ بل هي محبة من نوع آخر ال يساويها وال يدانيها محبة لقوله-  - :" ِالَ يُؤْمِنُ أَحَ دُ كُ مْ حَ تَّى أَكُونَ أَحَ بَّ إِلَيْه " َمِنْ وَلَدِ هِ وَوَ الِدِ هِ وَالنَّاسِ أَجْ مَ عِين( 26 ). النفس؛ بل هي محبة من نوع آخر ال يساويها وال يدانيها محبة لقوله-  - :" ِالَ يُؤْمِنُ أَحَ دُ كُ مْ حَ تَّى أَكُونَ أَحَ بَّ إِلَيْه " َمِنْ وَلَدِ هِ وَوَ الِدِ هِ وَالنَّاسِ أَجْ مَ عِين( 26 ). املطلب الثالث :الوالء للنبي-  - . وهذا عمر بن الخطاب-  - يبين للنبي-  - مدى محبته له ،إال أنه تفاجأ من رد النبي-  - الذي بيّن أن الوالء الحقيقي واملحبة الصادقة له ال تكون إال إذا بلغت مبلغاً يتجاوز حدود محبة النفس ،فعن عبد هللا بن هشام ، قال :"كنا مع النبي-  - وهو آخذ بيد عمر بن الخطاب ،فقال له عمر :يا رسول هللا ، ألنت أحب إلي من كل ش يء إال من نفس ي ،فقال النبي-  - :ال ،والذي نفس ي بيده ،حتى أكون أحب إليك من نفسك ،فقال له ع مر :فإنه اآلن ،وهللا ، ألنت أحب إلي من نفس ي ،فقال النبي-  - : "اآلن يا عمر( 27 ) ، فما كان من عمر إال أن انخلع من محبة نفسه وآثر محبة النبي-  - .؛ ألنه أولى باملؤمنين من أنفسهم ومن مظاهر محبة النبي-  - إتباعه فيما أمر أو نهى؛ ألن اتباع النبي هو اتباع هلل تعالى ،قال تعالى﴿ : قُلْ إِن ٌكُ نتُمْ تُحِ ب ونَ اللّهَ فَاتَّبِعُونِي يُحْ بِبْكُ مُ اللّهُ وَيَغْفِرْ لَكُ مْ ذُنُوبَكُ مْ وَاللّهُ غَفُ ورٌ رَّحِ يم﴾ [آل عمران : 31 ] ، وطاعته فيما يبلغ عن ربه تعالى؛ ألنه ال ينطق عن الهوى؛ بل هو وحيٌ من هللا تعالى ،وطاعته واجبة لقوله تعالى﴿ : َقُلْ أَطِ يعُواْ اللّه َوَالرَّسُ ولَ فإِن تَوَلَّوْاْ فَإِنَّ اللّهَ الَ يُحِ ب الْكَافِرِين﴾ [آل عمران : 32 ] ،وقوله تعالى﴿ : وَأَطِ يعُوا اللَّهَ وَأَطِ يعُوا الرَّسُ ولَ فَإِن ُتَوَلَّيْتُمْ فَإِنَّمَ ا عَلَ ى رَسُ ولِنَا الْبَالَ غُ املْ ُبِين﴾ [التغابن : 12 ]، وجاء التأكيد بطاعة الرسول بعد طاعة هللا تعالى "لينبه على وجوب طاعته فيما يأمر به ، ولو كان أمره غير مقترن بقرائن تبليغ الوحي لئال يتوهم السامع أن طاعة الرسول املأمور بها ترجع إلى طاعة هللا فيما يبلغه عن هللا دون ما يأمر به في غير التشريع ،فإن امتثال أمره ك"له خير( 28 ). ( 31 ) الشاطبي ،االعتصام ،ج :2 ،ص : 854 . ( 32 ) ابن ماجة ،سنن ابن ماجه ،ج :1 ،ص : 16 ،رقم : 43. صححه األلباني في سلسلة األحاديث الصحيحة وش يء من فقهها وفوائدها ،ج : 2 ،ص : 610 ،رقم : 937 . ( 33 ) البخاري ، صحيح البخاري ،كتاب حديث األنبياء ، َبَابُ مَ ا ذُكِ رَ عَنْ بَنِ ي إِسْ رَائِيل ،ج :4 ،ص : 170 ،رقم : 3461 . ( 34 ) أبو داود ،سنن أبي داود ،ج :3 ،ص : 322 ،رقم : 3660). صححه األلباني في صحيح الجامع الصغير وزياداته ،ج :2 ،ص : 1145 ،رقم : 2309 . ( 35 ) البغوي ،معالم التنزيل ،ج :7 ،ص : 341 . ( 36 ) البخاري ، صحيح البخاري ،كتاب اإليمان ،باب : َِخِ يهِ مَ ا يُحِ ب لِنَفْ سِ ه مِنَ اإلِيمَ انِ أَنْ يُحِ بَّ ألِ ،ج : 1 ،ص : 12 ،رقم : 13 .مسلم ، صحيح مسلم ،كتاب اإليمان ،باب :الدليل على أن من خصال اإليمان أن يحب ألخيه املسلم ما يحب لنفسه من الخير ،ج : 1ص : 67 ،رقم : 45 . المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم اإلسالمية ــ المجلد ال رابع ــ العدد ال ثاني ــ يونيو 21 20 م المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم اإلسالمية ــ المجلد ال رابع ــ العدد ال ثاني ــ يونيو 21 20 م الوحي على نفسه ،حتى صار في علمه وعمله على وفقه ، فكان الوحي ًحاكمً ا وافقً ا قائال ، وكان هو عليه الصالة والسالم "مذعنًا ملبيًا نداءه واقفً ا عند حكمه( 31 ). َ الوحي على نفسه ،حتى صار في علمه وعمله على وفقه ، فكان الوحي ًحاكمً ا وافقً ا قائال ، وكان هو عليه الصالة والسالم "مذعنًا ملبيًا نداءه واقفً ا عند حكمه( 31 ). َ 2- نصرته :ونصرة النبي-  - واجبة على كل مسلم لقوله تعالى﴿ : ُإِالَّ تَنصُرُوهُ فَقَ دْ نَصَرَهُ اللّه... ﴾ [التوبة : 40 ] ، وتكون نصرة النبي-  - بالدفاع عن سنته والتمسك بها ،فعن العرباض بن سارية ،قال : "وعظنا رسول هللا-  - موعظة ذرفت منها العيون ،ووجلت منها القلوب ،فقلنا :يا رسول هللا ،إن هذه ملوعظة مودع ، فماذا تعهد إلينا؟ قال :قد تركتكم على البيضاء ليلها كنهارها ،ال يزيغ عنها بعدي إال هالك ،من يعش منكم فسيرى اختالفا كثيرا ، فعل يكم بما عرفتم من سنتي ،وسنة الخلفاء الراشدين املهديين ، "عضوا عليها بالنواجذ( 32 ) ، نشر دعوته إلى الناس كافة لقوله-  - : ""بلغوا عني ولو آية( 33 ) ، وقد أثنى النبي-  - على من قام بتبليغ رسالته على أكمل وجه فقال-  - "نَضَّرَ اللَّهُ امْ رَأً سَ مِعَ مِنَّا حَ دِ يثًا ، ُفَحَ فِظَهُ حَ تَّى يُبَلِّغَه ، ُفَرُبَّ حَ امِلِ فِقْ هٍ إِلَى مَ نْ هُ وَ أَفْقَ هُ مِنْه ، ِوَرُ بَّ حَ امِل " ٍفِقْ هٍ لَيْسَ بِفَ قِيه( 34 ). املبحث الثاني : الوالء الدنيوي وهذه الوجوه من الوالء منها ما هو موافق لضوابط الشرع ،ومتى خرج عن هذه الضوابط أصبح هذا الوالء ًمذموما ،وقد يصل حكمه إلى التحريم ،ومنه ما هو مذموم وحكمه التحريم ،وال بد له من توبة نصوح ، وهذه الوجوه ندرسها وفق املطالب اآلتية : املطلب األول : .الوالء بين املؤمنين أمما والوالء بين املؤمنين له أصوله في الشريعة اإلسالمية ،قال تعالى﴿ : َوَاملْ ُؤْمِنُون ٍوَاملْ ُؤْمِنَاتُ بَعْضُهُمْ أَوْلِيَاء بَعْض َيَأْمُ رُونَ بِاملْ َعْرُوفِ وَيَنْهَوْنَ عَنِ املْ ُنكَ رِ وَيُقِيمُ ونَ الصَّالَةَ وَيُؤْتُونَ الزَّكَاةَ وَيُطِ يعُونَ اللّهَ و َّرَ سُ ولَهُ أُوْلَـئِكَ سَ يَرْحَ مُهُمُ اللّهُ إِن ٌاللّهَ عَزِيزٌ حَ كِ يم﴾ [الحجرات : 10 ] ، وهذه األخوة هي أخوة في الدين والوالية كما قال البغوي- رحمه هللا تعالى-( 35 ) ، وهذه املواالة لها ركنان ،هما :املحبة والنصرة ، والنصوص الشرعية أكثرت من ذكر هذه الوالية ملا لها من أثر في وحدة األمة اإلسالمية ،ففي جانب املحبة وبعد أن بيّن أن املؤمنين إخوة ،جع ل هذه املحبة جزء من كمال اإليمان لقول النبي-  - : ْ"الَ يُؤْمِنُ أَحَ دُ كُ م ، " َِخِ يهِ مَ ا يُحِ ب لِنَفْ سِ ه حَ تَّى يُحِ بَّ ألِ( 36 ) ،وهذه املحبة تقتض ي عدم ظلمه واحتقاره وغيبته ، وستره وأن يكون في حاجة أخيه لقوله-  - : َ"املُسْ لِمُ أَخُ و املُسْ لِمِ الَ يَظْلِمُ هُ و ُالَ يُسْ لِمُ ه ، َوَمَنْ كَانَ فِي حَ اجَ ةِ أَخِ يهِ كَان ِاللَّهُ فِي حَ اجَ تِه ، ًوَمَنْ فَرَّجَ عَنْ مُسْ لِمٍ كُ رْبَة ، ِفَرَّجَ اللَّهُ عَنْهُ كُ رْبَةً مِنْ كُ رُبَاتِ يَوْمِ القِيَامَ ة ، َوَمَنْ سَ تَرَ مُ سْ لِمًا سَ تَرَهُ اللَّهُ يَوْم ( 31 ) الشاطبي ،االعتصام ،ج :2 ،ص : 854 . ( 32 ) ابن ماجة ،سنن ابن ماجه ،ج :1 ،ص : 16 ،رقم : 43. صححه األلباني في سلسلة األحاديث الصحيحة وش يء من فقهها وفوائدها ،ج : 2 ،ص : 610 ،رقم : 937 . ( 33 ) البخاري ، صحيح البخاري ،كتاب حديث األنبياء ، َبَابُ مَ ا ذُكِ رَ عَنْ بَنِ ي إِسْ رَائِيل ،ج :4 ،ص : 170 ،رقم : 3461 . ( 34 ) أبو داود ،سنن أبي داود ،ج :3 ،ص : 322 ،رقم : 3660). صححه األلباني في صحيح الجامع الصغير وزياداته ،ج :2 ،ص : 1145 ،رقم : 2309 . ( 35 ) البغوي ،معالم التنزيل ،ج :7 ،ص : 341 . ( 32 ) ابن ماجة ،سنن ابن ماجه ،ج :1 ،ص : 16 ،رقم : 43. صححه األلباني في سلسلة األحاديث الصحيحة وش يء من فقهها وفوائدها ،ج : 2 ،ص : 610 ،رقم : 937 . ( 31 ) الشاطبي ،االعتصام ،ج :2 ،ص : 854 . املطلب الثالث :الوالء للنبي-  - . ًأ﴿َ َ ُ َأ َ َا آ ُ َّ أَ (26) مسلم ،صحيح مسلم ،تحقيق :محمد فؤاد عبد الباقي ،( دار إحياء التراث العربي– )بيروت ،كتاب اإليمان ، ِبَابُ وُجُ وبِ مَ حَ بَّةِ رَسُ ول ِهللاِ صلَّ ى هللا عَلَيْهِ وَسَ لَّمَ أَكْ ثَرَ مِنَ األْ َهْ لِ وَالْوَلَد ، َوَالْوَالِدِ والنَّاسِ أجْ مَ عِين ، َ ِْيمَ انِ عَلَ ى مَنْ لمْ يُحِ بَّهُ هَ ذِ هِ امل وَإِطْالَ قِ عَدَ مِ اإلْ َحَ بَّة ،ج : 1 ، ص67 ،رقم : 44 . َََ ( 30 ) أبن حنبل ،مسند اإلمام أحمد بن حنبل ،مسند النساء ،مُ سْ نَدُ الصّ ِ دِّ يقَ ةِ عَائِشَ ةَ بِنْتِ الصّ ِ دِّ يقِ رَض ِ يَ هللاُ عَنْهَا ،ج : 41 ،ص : 148 ، رقم : 24601 . الوالء في القرآن الكريم ( 26 ) الحصان المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم اإلسالمية ــ المجلد ال رابع ــ العدد ال ثاني ــ يونيو 21 20 م المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم اإلسالمية ــ المجلد ال رابع ــ العدد ال ثاني ــ يونيو 21 20 م " ِالقِيَامَ ة( 37 ) ،وإبدا ء املشورة والنصح له ملا فيه الخير والرشاد لقوله-  - ""وإذا استنصحك فانصح له( 38 ) ، وكذلك من مظاهر املحبة الدعاء ألخيه املؤمن بظاهر الغيب لقوله تعالى﴿ : وَالَّذِ ينَ جَ اؤُوا مِن بَعْدِ هِ مْ يَقُ ولُونَ رَبَّنَا اغْفِرْ لَنَا وَإلِ ِخْ وَانِنَا الَّذِ ينَ سَ بَقُ ونَا ٌِيمَانِ وَالَ تَجْ عَلْ فِي قُلُوبِنَا غِالًّ لِّلَّذِ ينَ آمَنُوا رَبَّنَا إِنَّكَ رَؤُ وفٌ رَّحِ يم بِاإلْ﴾ [الحشر : 10 ]. وأما النصرة للمؤمن فتكون أوالً في دينه إذا احتاج إلى مناصرة ،قال تعالى : ﴿ ِوَإِنِ اسْ تَنصَرُوكُ مْ فِي الدِّ ين َّفَعَلَيْكُ مُ النَّصْرُ إِال ٌعَلَ ى قَوْمٍ بَيْنَكُ مْ وَبَيْنَهُم مِّيثَاقٌ وَاللّهُ بِمَ ا تَعْمَلُونَ بَصِ ير﴾ [األنفال : 72 ] وكذلك تكون النصرة بإرشاده إلى طريق الحق وردعه عن طريق الباطل لقوله-  - : ""انْصُرْ أَخَ اكَ ظَاملِ ًا أَوْ مَ ظْلُومً ا ،قَالُوا : ِيَا رَسُ ولَ اللَّه ، ُهَ ذَ ا نَنْصُر ُه مَ ظْلُومً ا ، َفَكَ يْفَ نَنْصُرُهُ ظَاملِ ًا؟ قَال : ِتَأْخُ ذُ فَوْقَ يَدَ يْه( 39 ) ،وتقتض ي أيضًا الذب عن عرضه عند الوقوع في عرضه : ْ"مَن "ِذَبَّ عَنْ عِرْضِ أَخِ يه بِاملْ َغِيبِ كَانَ حَ قًّ ا عَلَ ى هللاِ عَزَّ وَجَ لَّ أَنْ يَعْتِقَ هُ مِنَ النَّار( 40 ). المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم اإلسالمية ــ المجلد ال رابع ــ العدد ال ثاني ــ يونيو 21 20 م املطلب الثاني : الوالء لألسرة والقبيلة واألسرة هي الركن األساس لبناء املجتمع ،وهي اللبنة التي يقوم عليها تكوين شخصية الفرد ، ومن ثم بناء الحضارات ،وملا لها من أثر كبير في تقرير الغاية التي خلق من أجلها اإلنسان ، فقد أعطت الشريعة اإلسالمية أهمية بارزة لألسرة ، فجعلت النصوص التي تنظم شؤون األسرة نصوصاً تفصيلية ، تعالج جميع املشكالت التي قد تؤثر على .جو األسرة من محبة وألفة وتناصر وتناصح والقرآن يبين القيمة الحقيقية لألسرة من خالل األساس الذي تقوم عليه من السكينة والطمأنينة واملودة والرحمة ،قال تعالى﴿ : َوَمِنْ آيَاتِهِ أ نْ خَ لَقَ لَكُ م مِّنْ أَنفُ سِ كُ مْ أَزْوَ اجاً لِّتَسْ كُ نُوا إِلَيْهَا وَجَ عَلَ بَيْنَكُ م مَّ وَدَّةً وَرَ حْ مَ ةً إِنَّ فِي َذَلِكَ آلَ يَاتٍ لِّقَ وْمٍ يَتَفَ كَّ رُون﴾ [الروم : 21 ] ،وكذلك يبين إن أولى الناس بالوالء بعد هللا تعالى ورسوله هما الوالدان ، وأكثر من ذك ر فضلهما( 41 ) ،ومنها قوله تعالى﴿ : َوَقَض َ ى رَب كَ أَالَّ تَعْبُدُ واْ إِالَّ إِيَّاهُ وَبِالْوَالِدَ يْنِ إِحْ سَ اناً إِمَّا يَبْلُغَنَّ عِندَ كَ الْكِ بَر ًأَحَ دُ هُ مَا أَوْ كِ الَهُ مَا فَالَ تَقُ ل لَّهُمَا أُفٍّ وَالَ تَنْهَرْهُ مَ ا وَقُل لَّهُمَا قَوْالً كَ رِيما﴾ [اإلسراء : 23 ] ، والسبب في أن هللا تعالى قرن عبادته ببر الوالدين يعود إلى أن "السبب الحقيقي لوجود اإلنسان هو تخليق هللا تعالى وإيجاده ، والسبب الظاهري هو األبوان ،فأمر بتعظيم السبب الحقيقي ، "ثم أتبعه باألمر بتعظيم السبب الظاهري( 42 ) ،وجاءت السنة النبوية مؤك دة لنصوص القرآن التي تحث على املواالة بين أفراد األسرة فقال-  - : َِهْ لِه "خَ يْرُكُ مْ خَ يْرُكُ مْ ألِ ، "َهْ لِ ي وَأَنَا مِنْ خَ يْرِكُ مْ ألِ( 43 ). ( 37 ) البخاري ، صحيح البخاري ،املصدر السابق ،كتاب املظالم والغصب ،باب : ُالَ يَظْلِمُ املُسْ لِمُ املُسْ لِمَ وَالَ يُسْ لِمُه ،ج : 3 ،ص : 128 ،رقم : 2442 . املطلب األول : .الوالء بين املؤمنين ( 36 ) البخاري ، صحيح البخاري ،كتاب اإليمان ،باب : َِخِ يهِ مَ ا يُحِ ب لِنَفْ سِ ه مِنَ اإلِيمَ انِ أَنْ يُحِ بَّ ألِ ،ج : 1 ،ص : 12 ،رقم : 13 .مسلم ، صحيح مسلم ،كتاب اإليمان ،باب :الدليل على أن من خصال اإليمان أن يحب ألخيه املسلم ما يحب لنفسه من الخير ،ج : 1ص : 67 ،رقم : 45 . ( 33 ) البخاري ، صحيح البخاري ،كتاب حديث األنبياء ، َبَابُ مَ ا ذُكِ رَ عَنْ بَنِ ي إِسْ رَائِيل ،ج :4 ،ص : 170 ،رقم : 3461 . ( 33 ) البخاري ، صحيح البخاري ،كتاب حديث األنبياء ، َبَابُ مَ ا ذُكِ رَ عَنْ بَنِ ي إِسْ رَائِيل ،ج :4 ،ص : 170 ،رقم : 3461 . ( 34 ) أبو داود ،سنن أبي داود ،ج :3 ،ص : 322 ،رقم : 3660). صححه األلباني في صحيح الجامع الصغير وزياداته ،ج :2 ،ص : 1145 ،رقم : 2309 . وي م ج ص ( 36 ) البخاري ، صحيح البخاري ،كتاب اإليمان ،باب : َِخِ يهِ مَ ا يُحِ ب لِنَفْ سِ ه مِنَ اإلِيمَ انِ أَنْ يُحِ بَّ ألِ ،ج : 1 ،ص : 12 ،رقم : 13 .مسلم ، صحيح مسلم ،كتاب اإليمان ،باب :الدليل على أن من خصال اإليمان أن يحب ألخيه املسلم ما يحب لنفسه من الخير ،ج : 1ص : 67 ،رقم : 45 . الوالء في القرآن الكريم ( 27 ) الحصان ( 27 ) الوالء في القرآن الكريم الحصان الحصان المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم اإلسالمية ــ المجلد ال رابع ــ العدد ال ثاني ــ يونيو 21 20 م ق آ فا ( 44 ) أبو حيان ،البحر املحيط في التفسير ،ج :8 ،ص : 414 . ( 45 ) البخاري ، صحيح البخاري ،مصدر سابق ،كتاب األدب ، ِبَابُ صِ لَةِ الوَالِدِ املُشْ رِك ،ج :8 ،ص :4 ،رقم : 5978 . ( (46الواحدي ،أسباب النزول ،ص : 424. رواه الحاكم في صحيحه ،قال محققه : وأصل القصة ثابت في الصحيحين وغيرهما دون ذكر .اآلية ( 47 ) البخاري ، صحيح البخاري ،مصدر سابق ،كتاب األدب ،بَابُ تُبَل الرَّحِ مُ بِبَالَلِهَا ،ج :8 ،ص :6 ،رقم : 5990 . ( 44 ) أبو حيان ،البحر املحيط في التفسير ،ج :8 ،ص : 414 . ( 45 ) البخاري ، صحيح البخاري ،مصدر سابق ،كتاب األدب ، ِبَابُ صِ لَةِ الوَالِدِ املُشْ رِك ،ج :8 ،ص :4 ،رقم : 5978 . ( (46الواحدي ،أسباب النزول ،ص : 424. رواه الحاكم في صحيحه ،قال محققه : وأصل القصة ثابت في الصحيحين وغيرهما دون ذكر .اآلية َاأ ُ المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم اإلسالمية ــ المجلد ال رابع ــ العدد ال ثاني ــ يونيو 21 20 م أما والية اآلباء لألبناء فال تختلف في جوهرها عن والية األبناء لآلباء ، وقد أوجز هللا تعالى لنا حكم والية األبناء الخارجين عن عقيدة التوحيد في قوله تعالى : ﴿ َّوَنَادَى نُوحٌ ر بَّهُ فَقَ الَ رَبِّ إِنَّ ابُنِ ي مِنْ أَهْ لِ ي وَإِنَّ وَعْدَ كَ الْ حَ ق َوَأَنتَ أَحْ كَ مُ الْ حَ اكِ مِين ﴿ 45 ﴾ ِّقَالَ يَا نُوحُ إِنَّهُ لَيْسَ مِنْ أَهْ لِكَ إِنَّهُ عَمَ لٌ غَيْرُ صَالِ حٍ فَالَ تَسْ أَلْنِ مَ ا لَيْسَ لَكَ بِهِ عِلْمٌ إِن ي َأَعِظُكَ أَن تَكُونَ مِنَ الْ جَ اهِ لِين﴾ [هود : 45 – 46 ]. ِ ِ ِ ِ﴾ وال ينفك القرآن من التحذير من والية غير هللا تعالى ، مذكراً بأن الوالء الخالص هو هلل تعالى وحده ، لا ينازعه في ذلك أحد ،قال تعالى﴿ : ِيَا أَي هَا الَّذِ ينَ آمَ نُواْ الَ تَتَّخِ ذُ واْ آبَاءكُ مْ وَإِخْ وَانَكُ مْ أَوْلِيَاء إَنِ اسْ تَحَ ب واْ الْكُ فْ رَ عَلَ ى اإل ِيمَان َوَمَ ن يَتَوَلَّهُم مِّنكُ مْ فَأُوْلَـئِكَ هُ مُ الظَّاملِ ُون﴾ [ا لتوبة : 23 ]؛ لذلك تبرأ النبي-  - من والية النسب والقرابة ، التي بنيت على أصول حمية الجاهلية األولى ،ليعلن الوالء هلل تعالى ولعباده الذين ارتض ى ،وجاء ذلك في حديث عمرو بن العاص-  - قال : "سمعت النبي-  - جهارًا غير سر يقول : " " إن آل أبى- قال عمرو في كتاب محمد ب ن جعفر بياض- ليسوا بأوليائي ،إنما وليي هللا وصالح املؤمنين ، "ولكن لهم رحم أبلها بباللها( 47 ). المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم اإلسالمية ــ المجلد ال رابع ــ العدد ال ثاني ــ يونيو 21 20 م ًولم يترك اإلسالم هذا الوالء مطلقا ،بل جعل له ضوابط تبين كيفية هذه العالقة ، فجعل الوالء بين االبن ًووالديه يقوم أساسا على سالمة العقيدة؛ ولكن إذا تعذر ذلك أبقى على والء املحبة دون النصرة امتثاالً ألمر هللا تعالى﴿ : ًوَإِن جَ اهَ دَ اكَ عَلى أَن تُشْ رِكَ بِي مَ ا لَيْسَ لَكَ بِهِ عِلْمٌ فَالَ تُطِ عْهُمَ ا وَصَاحِ بْهُمَ ا فِي الد نْيَا مَ عْرُوفا ْوَاتَّبِعْ سَ بِيلَ مَن ِأَنَابَ إ َلَيَّ ثُمَّ إِلَيَّ مَ رْجِ عُكُ مْ فَأُنَبِّئُكُ م بِمَ ا كُ نتُمْ تَعْمَ لُون﴾ [لقمان : 15 ] ، واملصاحبة في الدنيا تعني البر فيما أمر هللا تعالى ( 37 ) البخاري ، صحيح البخاري ،املصدر السابق ،كتاب املظالم والغصب ،باب : ُالَ يَظْلِمُ املُسْ لِمُ املُسْ لِمَ وَالَ يُسْ لِمُه ،ج : 3 ،ص : 128 ،رقم : 2442 . ( 38 ) مسلم ،صحيح مسلم ،املصدر السابق ،كتاب السالم ،باب من حق املسلم للمسلم رد السالم ،ج :4 ،ص : 1704 رقم : 2162 . ( 39 ) البخاري ، صحيح البخاري ،املصدر سابق ،كتاب املظالم والغصب ،بَاب :أَعِنْ أَخَ اكَ ظَاملِ ًا أَوْ مَ ظْلُومً ا ،ج :3 ،ص : 128 ،رقم : 2443 . ( 40 ) الطبراني ،املعجم الكبير ،مسند النساء ،باب :األلف : ِأَسْ مَ اءُ بِنْتُ يَزِيدَ بْنِ السَّ كَ نِ األْ َنْصَارِيَّة ،ج : 24 ،ص : 176 ،رقم : 443 . صححه األلباني في صحيح الجامع الصغير وزياداته ،ج :2 ،ص : 1071 ،رقم : 6240 . ( 43 ) ابن حبان (املتوفى : 354 )هـ ،صحيح ابن حبان بترتيب ابن بلبان ،ج : 9 ،ص : 401 ،رقم : 4186 . صححه األلباني في سلسلة األحاديث الصحيحة وش يء من فقهها وفوائدها ،ج :1 ،ص : 575 ،رقم : 285 . الوالء في القرآن الكريم الوالء في القرآن الكريم الحصان الحصان ( 28 ) المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم اإلسالمية ــ المجلد ال رابع ــ العدد ال ثاني ــ يونيو 21 20 م لهما "وهو إطعامهما وكسوتهما وعدم جفائهما وانتهارهما ،وعيادتهما إذا مرضا ، "ومواراتهما إذا ماتا( 44 ) ، وقد ورد في الحديث الصحيح أن أسماء بنت أبي بكر قالت :"أتتني أمي راغبة ،في عهد النبي-  - ،فسألت النبي - :آصلها؟ قال : "نعم( 45 ). المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم اإلسالمية ــ المجلد ال رابع ــ العدد ال ثاني ــ يونيو 21 20 م والوالء للقبيلة والعشيرة ضرورة ال غنى عنها ، تأبى املروءة أن تتنصل منها وها هم قوم شعيب عزّ عليهم قومهم من التعرض له ،قال تعالى﴿ : قَالُواْ يَا شُ عَيْبُ مَ ا َنَفْ قَ هُ كَ ثِيراً مِّمَّ ا تَقُ ولُ وَإِنَّا لَنَرَاكَ فِينَا ضَعِيفاً وَلَوْالَ رَهْ طُك ٍلَرَجَ مْ نَاكَ وَمَا أَنتَ عَلَيْنَا بِعَزِيز﴾ [هود : 91 ] ، وكذلك انحياز بنو هاشم مسلمهم وكافرهم مع النبي وأبي طالب في شعب مكة بعد محاصرة اهل مكة لهم ،فأحق الناس بمحبة ون صرة الرجل هم قومه؛ لذلك كان األمر اإللهي للنبي-  - أن يبدأ دعوته بقومه ،قال تعالى﴿ : َوَأَنذِ رْ عَشِ يرَتَكَ األْ َقْرَبِين﴾ [الشعراء : 214 ] ،فقد روى أبو هريرة-  - أنه ملا نزلت هذه اآلية ، قال رسول هللا-  - :"يا معشر قريش ،اشتروا أنفسكم من هللا ،ال أغني عنكم من هللا شيئا ، يا بني عبد وال ينفك القرآن من التحذير من والية غير هللا تعالى ، مذكراً بأن الوالء الخالص هو هلل تعالى وحده ، لا ينازعه في ذلك أحد ،قال تعالى﴿ : ِيَا أَي هَا الَّذِ ينَ آمَ نُواْ الَ تَتَّخِ ذُ واْ آبَاءكُ مْ وَإِخْ وَانَكُ مْ أَوْلِيَاء إَنِ اسْ تَحَ ب واْ الْكُ فْ رَ عَلَ ى اإل ِيمَان َوَمَ ن يَتَوَلَّهُم مِّنكُ مْ فَأُوْلَـئِكَ هُ مُ الظَّاملِ ُون﴾ [ا لتوبة : 23 ]؛ لذلك تبرأ النبي-  - من والية النسب والقرابة ، التي بنيت على أصول حمية الجاهلية األولى ،ليعلن الوالء هلل تعالى ولعباده الذين ارتض ى ،وجاء ذلك في حديث عمرو بن العاص-  - قال : "سمعت النبي-  - جهارًا غير سر يقول : " " إن آل أبى- قال عمرو في كتاب محمد ب ن جعفر بياض- ليسوا بأوليائي ،إنما وليي هللا وصالح املؤمنين ، "ولكن لهم رحم أبلها بباللها( 47 ). المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم اإلسالمية ــ المجلد ال رابع ــ العدد ال ثاني ــ يونيو 21 20 م أمّ ا إذا ترتب على اختالف العقيدة العداوة هلل ولرسوله وللمسلمين ،فعندئذٍ تحرم املوالة لقوله تعالى : ﴿ إِنَّمَا ْيَنْهَاكُ مُ اللَّهُ عَنِ الَّذِ ينَ قَاتَلُوكُ مْ فِي الدِّ ينِ وَأَخْ رَجُ وكُ مْ مِنْ دِ يَارِكُ مْ وَظَاهَ رُوا عَلَ ى إِخْ رَاجِ كُ م ْأَنْ تَوَلَّوْهُ مْ وَمَنْ يَتَوَلَّهُم َفَأُولَئِكَ هُ مُ الظَّاملِ ُون﴾[املمتحنة :9] وال شك أن اإلسالم ما جاء ليقطع وشائج األرحام والقرابة ، بل دعا إلى املحافظة عليها وإن اختلفت العقيدة مالم يتبع ذلك عداوة هللا تعالى ولرسوله وللمسلمين ،فقال تعالى : ﴿ ْالَ يَنْهَاكُ مُ اللَّهُ عَنِ الَّذِ ينَ لَمْ يُقَ اتِلُوكُ مْ فِي الدِّ ينِ وَلَم َيُخْ رِجُ وكُ مْ مِنْ دِ يَارِكُ مْ أَنْ تَبَر وهُ مْ وَتُقْ سِ طُوا إِلَيْهِمْ إِنَّ اللَّهَ يُحِ ب املْ ُقْ سِ طِ ين﴾ [املمتحنة : 8] ، فعن عامر بن عبد هللا بن الزبير ،عن أبي ه قال :" قدمت قتيلة بنت عبد العزى على ابنتها أسماء بنت أبي بكر بهدايا :ضباب وسمن وأقط ، فلم تقبل هداياها ولم تدخلها منزلها ، فسألت لها عائشة النبي- صلى هللا عليه وسلم- عن ذلك ،فقال﴿ : ال ينهاكم هللا عن الذين لم يقاتلوكم في الدين﴾ اآلية. فأدخلتها منزلها وقبل"ت منها هداياها( 46 ) . أمّ ا إذا ترتب على اختالف العقيدة العداوة هلل ولرسوله وللمسلمين ،فعندئذٍ تحرم املوالة لقوله تعالى : ﴿ إِنَّمَا ْيَنْهَاكُ مُ اللَّهُ عَنِ الَّذِ ينَ قَاتَلُوكُ مْ فِي الدِّ ينِ وَأَخْ رَجُ وكُ مْ مِنْ دِ يَارِكُ مْ وَظَاهَ رُوا عَلَ ى إِخْ رَاجِ كُ م ْأَنْ تَوَلَّوْهُ مْ وَمَنْ يَتَوَلَّهُم َفَأُولَئِكَ هُ مُ الظَّاملِ ُون﴾[املمتحنة :9] أما والية اآلباء لألبناء فال تختلف في جوهرها عن والية األبناء لآلباء ، وقد أوجز هللا تعالى لنا حكم والية األبناء الخارجين عن عقيدة التوحيد في قوله تعالى : ﴿ َّوَنَادَى نُوحٌ ر بَّهُ فَقَ الَ رَبِّ إِنَّ ابُنِ ي مِنْ أَهْ لِ ي وَإِنَّ وَعْدَ كَ الْ حَ ق َوَأَنتَ أَحْ كَ مُ الْ حَ اكِ مِين ﴿ 45 ﴾ ِّقَالَ يَا نُوحُ إِنَّهُ لَيْسَ مِنْ أَهْ لِكَ إِنَّهُ عَمَ لٌ غَيْرُ صَالِ حٍ فَالَ تَسْ أَلْنِ مَ ا لَيْسَ لَكَ بِهِ عِلْمٌ إِن ي َأَعِظُكَ أَن تَكُونَ مِنَ الْ جَ اهِ لِين﴾ [هود : 45 – 46 ]. المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم اإلسالمية ــ المجلد ال رابع ــ العدد ال ثاني ــ يونيو 21 20 م لهما "وهو إطعامهما وكسوتهما وعدم جفائهما وانتهارهما ،وعيادتهما إذا مرضا ، "ومواراتهما إذا ماتا( 44 ) ، وقد ورد في الحديث الصحيح أن أسماء بنت أبي بكر قالت :"أتتني أمي راغبة ،في عهد النبي-  - ،فسألت النبي - :آصلها؟ قال : "نعم( 45 ). وال شك أن اإلسالم ما جاء ليقطع وشائج األرحام والقرابة ، بل دعا إلى املحافظة عليها وإن اختلفت العقيدة مالم يتبع ذلك عداوة هللا تعالى ولرسوله وللمسلمين ،فقال تعالى : ﴿ ْالَ يَنْهَاكُ مُ اللَّهُ عَنِ الَّذِ ينَ لَمْ يُقَ اتِلُوكُ مْ فِي الدِّ ينِ وَلَم َيُخْ رِجُ وكُ مْ مِنْ دِ يَارِكُ مْ أَنْ تَبَر وهُ مْ وَتُقْ سِ طُوا إِلَيْهِمْ إِنَّ اللَّهَ يُحِ ب املْ ُقْ سِ طِ ين﴾ [املمتحنة : 8] ، فعن عامر بن عبد هللا بن الزبير ،عن أبي ه قال :" قدمت قتيلة بنت عبد العزى على ابنتها أسماء بنت أبي بكر بهدايا :ضباب وسمن وأقط ، فلم تقبل هداياها ولم تدخلها منزلها ، فسألت لها عائشة النبي- صلى هللا عليه وسلم- عن ذلك ،فقال﴿ : ال ينهاكم هللا عن الذين لم يقاتلوكم في الدين﴾ اآلية. فأدخلتها منزلها وقبل"ت منها هداياها( 46 ) . َ وال شك أن اإلسالم ما جاء ليقطع وشائج األرحام والقرابة ، بل دعا إلى املحافظة عليها وإن اختلفت العقيدة مالم يتبع ذلك عداوة هللا تعالى ولرسوله وللمسلمين ،فقال تعالى : ﴿ ْالَ يَنْهَاكُ مُ اللَّهُ عَنِ الَّذِ ينَ لَمْ يُقَ اتِلُوكُ مْ فِي الدِّ ينِ وَلَم َيُخْ رِجُ وكُ مْ مِنْ دِ يَارِكُ مْ أَنْ تَبَر وهُ مْ وَتُقْ سِ طُوا إِلَيْهِمْ إِنَّ اللَّهَ يُحِ ب املْ ُقْ سِ طِ ين﴾ [املمتحنة : 8] ، فعن عامر بن عبد هللا بن الزبير ،عن أبي ه قال :" قدمت قتيلة بنت عبد العزى على ابنتها أسماء بنت أبي بكر بهدايا :ضباب وسمن وأقط ، فلم تقبل هداياها ولم تدخلها منزلها ، فسألت لها عائشة النبي- صلى هللا عليه وسلم- عن ذلك ،فقال﴿ : ال ينهاكم هللا عن الذين لم يقاتلوكم في الدين﴾ اآلية. فأدخلتها منزلها وقبل"ت منها هداياها( 46 ) . املطلب الثالث : الوالء لولي األمر أم ي يم وجعل اإلسالم للعالقة بين الحاكم واملحكوم ضوابط منها :ال طاعة مطلقة إال هلل تعالى ، فطاعة والة األمر مرتبطة بطاعتهم هلل تعالى ،لقوله-  - :"ال طاعة ملخلوق في معصية هللا عز وجل"( 52 ) ،فمن أطاع هللا تعا لى وجبت طاعته ومن عصاه فال سمع وال طاعة له ،ومن األدلة على وجوب طاعة والة األمر في غير معصية ،قوله-  - : "من أطاعني فقد أطاع هللا ،ومن عصاني فقد عص ى هللا ،ومن أطاع أميري فقد أطاعني ، "ومن عص ى أميري فقد عصاني( 53 ). 53 ) ومن هذه الضوابط أيضًا عدم الخروج على والة األم ر إال إذا ظهر منهم الكفر الصريح ، ويخرج من ذلك تقصيرهم في بعض األمور التي ال حول لهم بها وال قوة ،لقوله-  - : ُ"وَأَنْ الَ نُنَازِعَ األَمْ رَ أَهْ لَه ،إِالَّ أَنْ تَرَوْ ا كُ فْ رًا بَوَاحً ا ، " ٌعِنْدَ كُ مْ مِنَ اللَّهِ فِيهِ بُرْهَ ان( 54 ). ولهم حق النصيحة بالحسنى ،فالحاكم يحتاج إلى من يعينه في الحق ،ال من يزين له الباطل ، فإذا سكت الناس عن النصيحة تركوا الحكام للبطانة الفاسدة، ًيعيثون في األمة فسادا،والنصيحة لهم واجبة لحديث تميم ومن هذه الضوابط أيضًا عدم الخروج على والة األم ر إال إذا ظهر منهم الكفر الصريح ، ويخرج من ذلك تقصيرهم في بعض األمور التي ال حول لهم بها وال قوة ،لقوله-  - : ُ"وَأَنْ الَ نُنَازِعَ األَمْ رَ أَهْ لَه ،إِالَّ أَنْ تَرَوْ ا كُ فْ رًا بَوَاحً ا ، " ٌعِنْدَ كُ مْ مِنَ اللَّهِ فِيهِ بُرْهَ ان( 54 ). أاا تقصيرهم في بعض األمور التي ال حول لهم بها وال قوة ،لقوله-  - : ُ"وَأَنْ الَ نُنَازِعَ األَمْ رَ أَهْ لَه ،إِالَّ أَنْ تَرَوْ ا كُ فْ رًا بَوَاحً ا ، " ٌعِنْدَ كُ مْ مِنَ اللَّهِ فِيهِ بُرْهَ ان( 54 ). ولهم حق النصيحة بالحسنى ،فالحاكم يحتاج إلى من يعينه في الحق ،ال من يزين له الباطل ، فإذا سكت الناس عن النصيحة تركوا الحكام للبطانة الفاسدة ، ًيعيثون في األمة فسادا ، والنصيحة لهم واجبة لحديث تميم الداري أن النبي-  - قال :"الدين النصيحة ،قلنا :ملن؟ قال :هلل ولكتابه ولرسوله وألئمة امل"سلمين وعامتهم( 55 ) . المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم اإلسالمية ــ المجلد ال رابع ــ العدد ال ثاني ــ يونيو 21 20 م المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم اإلسالمية ــ المجلد ال رابع ــ العدد ال ثاني ــ يونيو 21 20 م املطلب ،ال أغني عنكم من هللا شيئا ،يا عباس بن عبد املطلب ،ال أغني عنك من هللا شيئا ،يا صفية عمة رسول هللا ، ال أغني عنك من هللا شيئا ،يا فاطمة بنت رسول هللا ، "سليني بما شئت ال أغني عنك من هللا شيئا( 48 ). كذلك نصرتهم في كل عمل يدعو إلى الفضيلة وينهى عن الرذيلة كما نصر النبي-  - قبيلته قريش في حلف الفضول ،الذي يدعوا إلى نصرة املظلوم وردع الظالم فقال-  - : "لقد شهدت في دار عبد هللا بن جدعان حلفا ما "أحب أن لي به حمر النعم ولو أدعى به في اإلسالم ألجبت( 49 ) ،هكذا تكون نصرة القبيلة أن ترتقي بها ف ي معالي األخالق والفضيلة ،واالبتعاد عن عصبية الجاهلية املقيتة قال-  - :"قد أذهب هللا عنكم عُبيّة الجاهلية وفخرَها باآلباء ، ّمؤمن تقي ، ّوفاجر شقي ، "والناس بنو آدم وآدم من تراب( 50 ). ( 48 ) مسلم ،صحيح مسلم ،كتاب اإليمان ،باب في قوله تعالى :{وأنذر عشيرتك األقربين} [الشعراء : 214 ] ،ج :1 ،ص : 192 ،رقم : 206 . ( 49 ) ابن هشام ،السيرة النبوية البن هشام ،ج :1 ،ص : 134 . ( 50 ) الترمذي ،سنن الترمذي ،أبواب املناقب ،باب ،ج :5 ،ص : 735 ،رقم : 3956 . ( 51 ) ابن عاشور ،التحرير والتنوير ،ج : 5 ،ص : 96 . ( 52 ) ابن حنبل ،مسند اإلمام أحمد بن حنبل ،مصدر سابق ،مسند علي بن أبي طالب ،ج :2 ،ص : 67 ،رقم : 1094 . ( 53 ) البخاري ، صحيح البخاري ،مصدر سابق ،كتاب األحكام ،باب :بَابُ قَوْلِ اللَّهِ تَعَالَى و }ْ{أَطِ يعُوا اللَّهَ وَأَطِ يعُوا الرَّسُ ولَ وَأُولِي األَمْ رِ مِنْكُ م [النساء : 59 ] ،ج :9 ،ص : 61 ،رقم : 7137 . ( 54 ) البخاري ،املصدر السابق ،كتاب الفتن ،باب : ِّبَابُ قَوْلِ النَّبِ ي ﷺ :« َسَ تَرَ وْ ن بَعْدِ ي أُمُ ورً ا تُنْكِ رُونَهَا» ،ج :9 ،ص : 47 ،رقم : 7056 . ( 55 ) مسلم ،صحيح مسلم ،مصدر سابق ،كتاب اإليمان ،باب بيان أن الدين النصيحة ،ج1 ،ص74 ،رقم : 55 . المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم اإلسالمية ــ المجلد ال رابع ــ العدد ال ثاني ــ يونيو 21 20 م والوالء للقبيلة والعشيرة ضرورة ال غنى عنها ، تأبى املروءة أن تتنصل منها وها هم قوم شعيب عزّ عليهم قومهم من التعرض له ،قال تعالى﴿ : قَالُواْ يَا شُ عَيْبُ مَ ا َنَفْ قَ هُ كَ ثِيراً مِّمَّ ا تَقُ ولُ وَإِنَّا لَنَرَاكَ فِينَا ضَعِيفاً وَلَوْالَ رَهْ طُك ٍلَرَجَ مْ نَاكَ وَمَا أَنتَ عَلَيْنَا بِعَزِيز﴾ [هود : 91 ] ، وكذلك انحياز بنو هاشم مسلمهم وكافرهم مع النبي وأبي طالب في شعب مكة بعد محاصرة اهل مكة لهم ،فأحق الناس بمحبة ون صرة الرجل هم قومه؛ لذلك كان األمر اإللهي للنبي-  - أن يبدأ دعوته بقومه ،قال تعالى﴿ : َوَأَنذِ رْ عَشِ يرَتَكَ األْ َقْرَبِين﴾ [الشعراء : 214 ] ،فقد روى أبو هريرة-  - أنه ملا نزلت هذه اآلية ، قال رسول هللا-  - :"يا معشر قريش ،اشتروا أنفسكم من هللا ،ال أغني عنكم من هللا شيئا ، يا بني عبد ( 29 ) الوالء في القرآن الكريم الوالء في القرآن الكريم الحصان املطلب الثالث : الوالء لولي األمر مأ يا ومن صور الوالء التي أشار إليها القرآن ،الوال ء لولي األمر لقوله تعالى﴿ : َيَا أَي هَا الَّذِ ينَ آمَ نُواْ أَطِ يعُواْ اللّه ُوَأَطِ يعُواْ الرَّسُ ولَ وَأُوْ لِي األَمْ رِ مِنكُ مْ فَإِن تَنَازَعْتُمْ فِي ش َ يْءٍ فَرُد وهُ إِلَى اللّهِ وَالرَّسُ ولِ إِن ك ِنتُمْ تُؤْمِنُونَ بِاللّهِ وَالْيَوْمِ اآلخِ ر َذَلِك ًخَ يْرٌ وَأَحْ سَ نُ تَأْوِيال﴾ [النساء : 59 ] ،وطاعة ولي األمر واجبة مالم يحكم بغير ما أنزل هللا تعالى ، فإذا ما أقام حكم الله ، وأقام العدل فله حق الطاعة "فطاعة الرسول تشتمل على احترام العدل املشرع لهم وعلى تنفيذه ، وطاعة والة األمور تنفيذ للعدل ، وأشار بهذا"التعقيب إلى أن الطاعة املأمور بها هي الطاعة في املعروف( 51 ). مأا ومن صور الوالء التي أشار إليها القرآن ،الوال ء لولي األمر لقوله تعالى﴿ : َيَا أَي هَا الَّذِ ينَ آمَ نُواْ أَطِ يعُواْ اللّه ُوَأَطِ يعُواْ الرَّسُ ولَ وَأُوْ لِي األَمْ رِ مِنكُ مْ فَإِن تَنَازَعْتُمْ فِي ش َ يْءٍ فَرُد وهُ إِلَى اللّهِ وَالرَّسُ ولِ إِن ك ِنتُمْ تُؤْمِنُونَ بِاللّهِ وَالْيَوْمِ اآلخِ ر َذَلِك ًخَ يْرٌ وَأَحْ سَ نُ تَأْوِيال﴾ [النساء : 59 ] ،وطاعة ولي األمر واجبة مالم يحكم بغير ما أنزل هللا تعالى ، فإذا ما أقام حكم الله ، وأقام العدل فله حق الطاعة "فطاعة الرسول تشتمل على احترام العدل املشرع لهم وعلى تنفيذه ، وطاعة والة األمور تنفيذ للعدل ، وأشار بهذا"التعقيب إلى أن الطاعة املأمور بها هي الطاعة في املعروف( 51 ). وجعل اإلسالم للعالقة بين الحاكم واملحكوم ضوابط منها :ال طاعة مطلقة إال هلل تعالى ، فطاعة والة األمر مرتبطة بطاعتهم هلل تعالى ،لقوله-  - :"ال طاعة ملخلوق في معصية هللا عز وجل"( 52 ) ،فمن أطاع هللا تعا لى وجبت طاعته ومن عصاه فال سمع وال طاعة له ،ومن األدلة على وجوب طاعة والة األمر في غير معصية ،قوله-  - : "من أطاعني فقد أطاع هللا ،ومن عصاني فقد عص ى هللا ،ومن أطاع أميري فقد أطاعني ، "ومن عص ى أميري فقد عصاني( 53 ). ا ( 50 ) الترمذي ،سنن الترمذي ،أبواب املناقب ،باب ،ج :5 ،ص : 735 ،رقم : 3956 . ( 48 ) مسلم ،صحيح مسلم ،كتاب اإليمان ،باب في قوله تعالى :{وأنذر عشيرتك األقربين} [الشعراء : 214 ] ،ج :1 ،ص : 192 ،رقم : 206 . ( 49 ) ابن هشام ،السيرة النبوية البن هشام ،ج :1 ،ص : 134 . ( 56 ) قطب ،في ظالل القرآن ،مرجع سابق ،ج :2 ،ص : 909 . ( (57 الطبري ،جامع البيان في تأويل القرآن ،ج : 20 ،ص : 439 . املطلب الثالث : الوالء لولي األمر ولهم حق النصيحة بالحسنى ،فالحاكم يحتاج إلى من يعينه في الحق ،ال من يزين له الباطل ، فإذا سكت الناس عن النصيحة تركوا الحكام للبطانة الفاسدة ، ًيعيثون في األمة فسادا ، والنصيحة لهم واجبة لحديث تميم الداري أن النبي-  - قال :"الدين النصيحة ،قلنا :ملن؟ قال :هلل ولكتابه ولرسوله وألئمة امل"سلمين وعامتهم( 55 ) . ( 48 ) مسلم ،صحيح مسلم ،كتاب اإليمان ،باب في قوله تعالى :{وأنذر عشيرتك األقربين} [الشعراء : 214 ] ،ج :1 ،ص : 192 ،رقم : 206 . ( 49 ) ابن هشام ،السيرة النبوية البن هشام ،ج :1 ،ص : 134 . ا ( 50 ) الترمذي ،سنن الترمذي ،أبواب املناقب ،باب ،ج :5 ،ص : 735 ،رقم : 3956 . إ ( 53 ) البخاري ، صحيح البخاري ،مصدر سابق ،كتاب األحكام ،باب :بَابُ قَوْلِ اللَّهِ تَعَالَى و }ْ{أَطِ يعُوا اللَّهَ وَأَطِ يعُوا الرَّسُ ولَ وَأُولِي األَمْ رِ مِنْكُ م [النساء : 59 ] ،ج :9 ،ص : 61 ،رقم : 7137 . َ ( 54 ) البخاري ،املصدر السابق ،كتاب الفتن ،باب : ِّبَابُ قَوْلِ النَّبِ ي ﷺ :« َسَ تَرَ وْ ن بَعْدِ ي أُمُ ورً ا تُنْكِ رُونَهَا» ،ج :9 ،ص : 47 ،رقم : 7056 . ( 55 ) مسلم ،صحيح مسلم ،مصدر سابق ،كتاب اإليمان ،باب بيان أن الدين النصيحة ،ج1 ،ص74 ،رقم : 55 . ( 30 ) الوالء في القرآن الكريم الحصان الحصان المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم اإلسالمية ــ المجلد ال رابع ــ العدد ال ثاني ــ يونيو 21 20 م املطلب الرابع :الوالء املحرم مما وهذا الوالء محرم قطعاً ألنه يخرج صاحبه من امللة ، ملخالفته النهي الصريح بعدم مواالة أعداء هللا تعالى ومن هؤالء : ََ وهذا الوالء محرم قطعاً ألنه يخرج صاحبه من امللة ، ملخالفته النهي الصريح بعدم مواالة أعداء هللا تعالى ومن هؤالء : ََ ومن هؤالء : أوالا :الكافرين وعلى رأسهم اليهود والنصارى لقوله تعالى في حق الكافرين﴿ : َيَا أَي هَا الَّذِ ين ْآمَ نُواْ الَ تَتَّخِ ذُ وا ًالْكَافِرِينَ أَوْلِيَاء مِن دُونِ املْ ُؤْمِنِينَ أَتُرِيدُ ونَ أَن تَجْ عَلُواْ لِلّهِ عَلَيْكُ مْ سُ لْطَاناً م بِينا﴾ [النساء : 144 ] ، وهنا النهي صريح عن مواالة الكافرين ،لكن ما املقصود باملواالة هنا؟ هل هي مواالتهم في دينهم؟ أم ف ي محبتهم ومناصرتهم؟ يقول قطب : "إنها تعني التناصر والتحالف معهم ،وال تتعلق بمعنى اتباعهم في دينهم ، فبعيد جدً ا أن يكون بين املسلمين من يميل إلى اتباع اليهود والنصارى في الدين ، "إنما هو والء التحالف والتناصر( 56 ). وقد بين هللا تعالى أن ال مواالة للكافرين ومنهم اليهود والنصارى ،إنما مواالتهم بينهم ، وقد حذّ ر من عدم االنتهاء عن ذلك بأن عواقبه وخيمة ،قال تعالى﴿ : ِوَالَّذينَ كَ فَ رُواْ بَعْضُهُمْ أَوْلِيَاء بَعْضٍ إِالَّ تَفْ عَلُوهُ تَكُ ن فِتْنَةٌ فِي األَرْض ٌوَفَسَ ادٌ كَ بِير﴾[األنفال : 73 ] ، وقد رأينا كيف عمّ الفساد وحلّ الخراب في البالد والعباد بعد أن تحالف املسلمون مع أعداء هللا تعالى ،فأصبحت األمة ممسوخة الهوية ،ليس لها مهابة وال مكانة بين األمم ، وأي فسادٍ اكثر من ذلك! وقد أبيحت الدماء وانتهكت األعراض ، حتى أصبحنا منهم في عدم االحساس والشعور ،أصبحنا نحن األدوات التي يستخدمونها في هدم الدين ،قال تعالى﴿ : ٍيَا أَي هَا الَّذِ ينَ آمَ نُواْ الَ تَتَّخِ ذُ واْ الْيَهُودَ وَالنَّصَارَى أَوْلِيَاء بَعْضُهُمْ أَوْلِيَاء بَعْض َوَمَ ن يَتَوَلَّهُم مِّنكُ مْ فَإِنَّهُ مِنْهُمْ إِنَّ اللّهَ الَ يَهْدِ ي الْقَ وْمَ الظَّاملِِين﴾ [املائد ة : 51 ]. ويخرج من هذا التحريم ما كان على سبيل التعايش السلمي مع املسالم من اليهود والنصارى وغيرهم لقوله تعالى﴿ : ْالَ يَنْهَاكُ مُ اللَّهُ عَنِ الَّذِ ينَ لَمْ يُقَ اتِلُوكُ مْ فِي الدِّ ينِ وَلَمْ يُخْ رِجُ وكُ م مِّن دِ يَارِكُ مْ أَن تَبَر وهُ م ْوَتُقْ سِ طُوا إِلَي َهِمْ إِنَّ اللَّه َيُحِ ب املْ ُقْ سِ طِ ين﴾ [املمتحنة :8]. ثانياا : الوالء للشيطان ا جاء التحذير من مواالة الشيطان بصورة أعظم من األمر بعدم مواالته ، فقد عبّر هللا تعالى بطريقة أخرى وهي بيان أن الشيطان عدو ، ًثم األمر باتخاذه عدوا ،قال تعالى﴿ : َإِنَّ الشَّ يْطَان لَكُ مْ عَدُ وٌّ فَاتَّخِ ذُ وهُ عَدُ وّاً إِنَّمَ ا يَدْ عُو ِحِ زْبَهُ لِيَكُونُوا مِنْ أَصْ حَ ابِ السَّ عِير﴾ [فاطر : 6] ، أي "فأنزلوه من أنفسكم منزلة العدو منكم واحذروه بطاعة هللا واستغشاشكم إياه حذركم من عدوكم الذي تخافون غائلته على أنفسكم ، فال تطيعوه وال تتبعو ا خطواته ، فإنه إنما يدعو حزبه إلى طاعته والقبول منه ، )ِوالكفر باهلل (لِيَكُونُوا مِنْ أَصْ حَ ابِ السَّ عِير"( 57 ). ثم بعد هذه العداوة كان من يتخذ الشياطين أولياء من الخاسرين قال تعالى﴿ : وَمَ ن يَتَّخِ ذِ الشَّ يْطَانَ وَلِيّاً مِّن ْدُونِ اللّهِ فَقَ دْ خَ سِ رَ خُ س ًرَاناً م بِينا﴾ [النساء : 119 ] ،ووصف هللا تعالى أنصار الشيطان وأوليائه بعدم اإليمان ، فقال تعالى﴾ : َإِنَّا جَ عَلْنَا الشَّ يَاطِ ينَ أَوْلِيَاء لِلَّذِ ينَ الَ يُؤْمِنُون﴾ [األعراف : 27 ] ، أي ال يمكن أن تكون هنالك والية بين مؤمن وشيطان ، وملا كانوا غير مؤمنين فقد حقت عليهم الضاللة ،قال تعالى﴿ : ُفَرِيقاً هَ دَ ى وَفَرِيقاً حَ قَّ عَلَيْهِمُ الضَّالَلَةُ إِنَّهُم َاتَّخَ ذُ وا الشَّ يَاطِ ينَ أَوْلِيَاء مِن دُونِ اللّهِ وَيَحْ سَ بُونَ أَنَّهُم م هْتَدُ ون﴾ [األعراف : 30 ] ، واآليات التي تحذر من مواالة الكافرين واليهود والن صارى والشياطين كثيرة ،لذا اكتفيت بما يؤدي الغرض منها. ا جاء التحذير من مواالة الشيطان بصورة أعظم من األمر بعدم مواالته ، فقد عبّر هللا تعالى بطريقة أخرى وهي بيان أن الشيطان عدو ، ًثم األمر باتخاذه عدوا ،قال تعالى﴿ : َإِنَّ الشَّ يْطَان لَكُ مْ عَدُ وٌّ فَاتَّخِ ذُ وهُ عَدُ وّاً إِنَّمَ ا يَدْ عُو ِحِ زْبَهُ لِيَكُونُوا مِنْ أَصْ حَ ابِ السَّ عِير﴾ [فاطر : 6] ، أي "فأنزلوه من أنفسكم منزلة العدو منكم واحذروه بطاعة هللا واستغشاشكم إياه حذركم من عدوكم الذي تخافون غائلته على أنفسكم ، فال تطيعوه وال تتبعو ا خطواته ، فإنه إنما يدعو حزبه إلى طاعته والقبول منه ، )ِوالكفر باهلل (لِيَكُونُوا مِنْ أَصْ حَ ابِ السَّ عِير"( 57 ). ال خاتمة : الحمد هلل والصالة والسالم على رسول هللا ،وبعد : فقد اكتملت دراستي عن موضوع الوالء في القرآن الكريم ، وقد حاولت قدر استطاعتي أن اجتهد في الوصول إلى املعاني املرادة من ذكر هذا املوضوع في القرآن ، ويبقى هذا الجهد من اجتهادات البشر وقد يعتريه ما يعتريه من القصور ،فما فيه من خطأ او قصور فمن نفس ي والشيطان ، .واستغفره تعالى على ذلك الحمد هلل والصالة والسالم على رسول هللا ،وبعد : فقد اكتملت دراستي عن موضوع الوالء في القرآن الكريم ، وقد حاولت قدر استطاعتي أن اجتهد في الوصول إلى املعاني املرادة من ذكر هذا املوضوع في القرآن ، ويبقى هذا الجهد من اجتهادات البشر وقد يعتريه ما يعتريه من القصور ،فما فيه من خطأ او قصور فمن نفس ي والشيطان ، .واستغفره تعالى على ذلك وبعد هذه الدراسة أرى أن أذكر بعض النتائج التي توصلت إليها الدراسة : 1- جاءت مادة (ول ي ) في القرآن في عدة معانٍ منها املحبة والنص رة ،والوريث والقريب ، .وكذلك األرباب واألصنام 2- بينت الدراسة أن الوالء بمعناه االصطالحي يختلف عن التعايش السلمي الذي أقره اإلسالم للتعامل مع غير املسلم املسالم 3- .أن الوالء بمعناه املطلق ال يكون إال هلل تعالى 4- أن مواالة النبي ال تكون إال باتباع ما جاء به والنتهاء عما نهى عنه 5- .بينت الدراسة أن مواالة املسلمين ال تكون إال فيما بينهم 6- تحريم مواالة أعداء اإلسالم وبعد هذه الدراسة أرى أن أذكر بعض النتائج التي توصلت إليها الدراسة : 1- جاءت مادة (ول ي ) في القرآن في عدة معانٍ منها املحبة والنص رة ،والوريث والقريب ، .وكذلك األرباب واألصنام 2- بينت الدراسة أن الوالء بمعناه االصطالحي يختلف عن التعايش السلمي الذي أقره اإلسالم للتعامل مع غير املسلم املسالم 3- .أن الوالء بمعناه املطلق ال يكون إال هلل تعالى 4- أن مواالة النبي ال تكون إال باتباع ما جاء به والنتهاء عما نهى عنه 5- .بينت الدراسة أن مواالة املسلمين ال تكون إال فيما بينهم 6- تحريم مواالة أعداء اإلسالم غير املسلم املسالم 3- .أن الوالء بمعناه املطلق ال يكون إال هلل تعالى 4- أن مواالة النبي ال تكون إال باتباع ما جاء به والنتهاء عما نهى عنه 5- .بينت الدراسة أن مواالة املسلمين ال تكون إال فيما بينهم 6- تحريم مواالة أعداء اإلسالم 6- تحريم مواالة أعداء اإلسالم ثانياا : الوالء للشيطان ََ جاء التحذير من مواالة الشيطان بصورة أعظم من األمر بعدم مواالته ، فقد عبّر هللا تعالى بطريقة أخرى وهي بيان أن الشيطان عدو ، ًثم األمر باتخاذه عدوا ،قال تعالى﴿ : َإِنَّ الشَّ يْطَان لَكُ مْ عَدُ وٌّ فَاتَّخِ ذُ وهُ عَدُ وّاً إِنَّمَ ا يَدْ عُو ِحِ زْبَهُ لِيَكُونُوا مِنْ أَصْ حَ ابِ السَّ عِير﴾ [فاطر : 6] ، أي "فأنزلوه من أنفسكم منزلة العدو منكم واحذروه بطاعة هللا واستغشاشكم إياه حذركم من عدوكم الذي تخافون غائلته على أنفسكم ، فال تطيعوه وال تتبعو ا خطواته ، فإنه إنما يدعو حزبه إلى طاعته والقبول منه ، )ِوالكفر باهلل (لِيَكُونُوا مِنْ أَصْ حَ ابِ السَّ عِير"( 57 ). ثم بعد هذه العداوة كان من يتخذ الشياطين أولياء من الخاسرين قال تعالى﴿ : وَمَ ن يَتَّخِ ذِ الشَّ يْطَانَ وَلِيّاً مِّن ْدُونِ اللّهِ فَقَ دْ خَ سِ رَ خُ س ًرَاناً م بِينا﴾ [النساء : 119 ] ،ووصف هللا تعالى أنصار الشيطان وأوليائه بعدم اإليمان ، َََفقال و بول و زب إ ى ي)ِِير هل(ِي وو ِن ص بِ رب و ثم بعد هذه العداوة كان من يتخذ الشياطين أولياء من الخاسرين قال تعالى﴿ : وَمَ ن يَتَّخِ ذِ الشَّ يْطَانَ وَلِيّاً مِّن ْدُونِ اللّهِ فَقَ دْ خَ سِ رَ خُ س ًرَاناً م بِينا﴾ [النساء : 119 ] ،ووصف هللا تعالى أنصار الشيطان وأوليائه بعدم اإليمان ، فقال تعالى﴾ : َإِنَّا جَ عَلْنَا الشَّ يَاطِ ينَ أَوْلِيَاء لِلَّذِ ينَ الَ يُؤْمِنُون﴾ [األعراف : 27 ] ، أي ال يمكن أن تكون هنالك والية بين مؤمن وشيطان ، وملا كانوا غير مؤمنين فقد حقت عليهم الضاللة ،قال تعالى﴿ : ُفَرِيقاً هَ دَ ى وَفَرِيقاً حَ قَّ عَلَيْهِمُ الضَّالَلَةُ إِنَّهُم َاتَّخَ ذُ وا الشَّ يَاطِ ينَ أَوْلِيَاء مِن دُونِ اللّهِ وَيَحْ سَ بُونَ أَنَّهُم م هْتَدُ ون﴾ [األعراف : 30 ] ، واآليات التي تحذر من مواالة الكافرين واليهود والن صارى والشياطين كثيرة ،لذا اكتفيت بما يؤدي الغرض منها. ( 31 ) الوالء في القرآن الكريم الوالء في القرآن الكريم المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم اإلسالمية ــ المجلد ال رابع ــ العدد ال ثاني ــ يونيو 21 20 م التوصيات .واملقترحات استنادا لنتائج البحث توص ي ال باحثة وتقترح بما يأتي : 1- أن تخصص دراسات للتفريق بين.مفهوم الوالء وبين مفهوم التعايش والسلم 2- .أن تخصص دراسات في ضوابط الوالء ومظاهره وهللا اسأل أن يتقبل هذا العمل لوجهه الكريم ، .والحمد هلل رب العاملين - ابن هشام ، عبد امللك بن أيوب الحميري (املتوفى : 213 )هـ ،السيرة النبوية البن هشام ،تحقيق :مص وآخرون( ،مكتبة ومطبعة مصطفى البابي الحلبي وأوالده بمصر ، 1375 هـ- 1955 م) ط2. - ابن هشام ، عبد امللك بن أيوب الحميري (املتوفى : 213 )هـ ،السيرة النبوية البن هشام ،تحقيق : مصطفى السقا وآخرون( ،مكتبة ومطبعة مصطفى البابي الحلبي وأوالده بمصر ، 1375 هـ- 1955 م) ط2. - ابن هشام ، عبد امللك بن أيوب الحميري (املتوفى : 213 )هـ ،السيرة النبوية البن هشام ،تحقيق : مصطفى السقا وآخرون( ،مكتبة ومطبعة مصطفى البابي الحلبي وأوالده بمصر ، 1375 هـ- 1955 م) ط2. - أبو حيان ،محمد بن يوسف (املتوفى : 745 )هـ ،البحر املحيط في التفسير ،تحقيق :صدقي محمد جميل( ، دار الفكر– بيروت ، 1420 )هـ ، .د.ط - أبو حيان ،محمد بن يوسف (املتوفى : 745 )هـ ،البحر املحيط في التفسير ،تحقيق :صدقي محمد جميل( ، دار الفكر– بيروت ، 1420 )هـ ، .د.ط - أبو داود ،سليمان بن األشعث (املتوفى : 275 )هـ ،سنن أبي داود ،تحقيق :محمد محيي الدين عبد الحميد( ، املكتبة العصرية ، صيدا– )بيروت. - أبو داود ،سليمان بن األشعث (املتوفى : 275 )هـ ،سنن أبي داود ،تحقيق :محمد محيي الدين عبد الحميد( ، املكتبة العصرية ، صيدا– )بيروت. - األلباني ،محمد ناصر الدين (املتوفى : 1420 )هـ ،سلسلة األحاديث الصحيحة وش يء من فقهها وفوائدها( ، مكتبة املعارف للنشر والتوزيع ،الرياض ، 1415هـ- 1995 .م) ط1 - األلباني ،محمد ناصر الدين (املتوفى : 1420 )هـ ،سلسلة األحاديث الصحيحة وش يء من فقهها وفوائدها( ، مكتبة املعارف للنشر والتوزيع ،الرياض ، 1415هـ- 1995 .م) ط1 - األلباني ،محمد ناصر الدين (املتوفى : 1420 )هـ ، صحيح الجامع الصغير وزياداته ( .)املكتب اإلسالمي - األنباري ،عبد الرحمن بن محمد (املتوفى : 577 )هـ ،نزهة األلباء في طبقات األدباء ،تحقيق :إبراهيم السامرائي ، (مكتبة املنار ، الزرقاء- األردن ، 1405هـ- 1985 )م ،ط3 ،ص : 119 - 122 . - األلباني ،محمد ناصر الدين (املتوفى : 1420 )هـ ، صحيح الجامع الصغير وزياداته ( .)املكتب اإلسالمي - األنباري ،عبد الرحمن بن محمد (املتوفى : 577 )هـ ،نزهة األلباء في طبقات األدباء ،تحقيق :إبراهيم (مكتبة املنار ، الزرقاء- األردن ، 1405هـ- 1985 )م ،ط3 ،ص : 119 - 122 . - األنباري ،عبد الرحمن بن محمد (املتوفى : 577 )هـ ،نزهة األلباء في طبقات األدباء ،تحقيق :إبراهيم السامرائي ، (مكتبة املنار ، الزرقاء- األردن ، 1405هـ- 1985 )م ،ط3 ،ص : 119 - 122 . - ابن هشام ، عبد امللك بن أيوب الحميري (املتوفى : 213 )هـ ،السيرة النبوية البن هشام ،تحقيق :مص وآخرون( ،مكتبة ومطبعة مصطفى البابي الحلبي وأوالده بمصر ، 1375 هـ- 1955 م) ط2. - البخاري ،محمد بن اسماعيل (ت : 256 )هـ ، صحيح البخاري ،تحقيق :محمد زهير بن ناصر الناصر( ، دار طوق النجاة ، 1422 )ه( ، مصورة عن)السلطانية بإضافة ترقيم محمد فؤاد عبد الباقي ،ط1. - البخاري ،محمد بن اسماعيل (ت : 256 )هـ ، صحيح البخاري ،تحقيق :محمد زهير بن ناصر الناصر( ، دار طوق النجاة ، 1422 )ه( ، مصورة عن)السلطانية بإضافة ترقيم محمد فؤاد عبد الباقي ،ط1. - البغوي ،الحسين بن مسعود (املتوفى : 510 )هـ ،معالم التنزيل في تفسير القرآن ،تحقيق :عبد الرزاق املهدي( ، دار إحياء التراث العربي–بيروت ، 1420 )ه ،ط1. - البغوي ،الحسين بن مسعود (املتوفى : 510 )هـ ،معالم التنزيل في تفسير القرآن ،تحقيق :عبد الرزاق املهدي( ، دار إحياء التراث العربي–بيروت ، 1420 )ه ،ط1. - البقاعي ،إبراهيم بن عمر (املتوفى : 885 )هـ ،نظم الدرر في تناسب ا آليات والسور( ،دار الكتاب اإلسالمي ، .)القاهرة - البيضاوي ،عبد هللا بن عمر (املتوفى : 685 )هـ ،ـ ،أنوار التنزيل وأسرار التأويل ،تحقيق : محمد عبد الرحمن املرعشلي( ،دار إحياء التراث العربي– بيروت1418 )ه ،ط1. - البقاعي ،إبراهيم بن عمر (املتوفى : 885 )هـ ،نظم الدرر في تناسب ا آليات والسور( ،دار الكتاب اإلسالمي ، .)القاهرة - البيضاوي ،عبد هللا بن عمر (املتوفى : 685 )هـ ،ـ ،أنوار التنزيل وأسرار التأويل ،تحقيق : محمد عبد الرحمن املرعشلي( ،دار إحياء التراث العربي– بيروت1418 )ه ،ط1. م - الترمذي ،محمد بن عيس ى (املتوفى : 279 )هـ ،سنن الترمذي ،تحقيق :أحمد محمد شاكر وآخرون( ، شركة مكتبة ومطبعة مصطفى البابي الحلبي– مصر ، 1395هـ- 1975م) ط2. - الترمذي ،محمد بن عيس ى (املتوفى : 279 )هـ ،سنن الترمذي ،تحقيق :أحمد محمد شاكر وآخرون( ، شركة مكتبة ومطبعة مصطفى البابي الحلبي– مصر ، 1395هـ- 1975م) ط2. - حسين ،سيد قطب إبراهيم (املتوفى : 1385 )هـ ،في ظالل القرآن( ،دار الشروق- بيروت- القاهرة ، 1412ه ، ) ، ط17 . - حسين ،سيد قطب إبراهيم (املتوفى : 1385 )هـ ،في ظالل القرآن( ،دار الشروق- بيروت- القاهرة ، 1412ه ، ) ، ط17 . - الخازن ،علي بن محمد (املتوفى : 741 )هـ ، لباب التأويل في معاني التنزيل ،تصحيح :محمد علي شاهين( ، دار الكتب العلمية- بيروت ، 1415 )هـ ،ط1. - الخازن ،علي بن محمد (املتوفى : 741 )هـ ، لباب التأويل في معاني التنزيل ،تصحيح :محمد علي شاهين( ، دار الكتب العلمية- بيروت ، 1415 )هـ ،ط1. املصادر واملراجع : املصادر واملراجع : - ابن األثير ،علي بن أبي الكرم محمد (املتوفى : 630 )هـ ،أسد الغابة في معرفة الصحابة تحقيق :عل ي محمد معوض- عادل أحمد عبد املوجود ،الناشر( :دار الكتب العلمية1415 هـ- 1994 )م ،ط1 . م - ابن حبان ،محمد بن حبان (املتوفى : 354 )هـ ،صحيح ابن حبان بترتيب ابن بلبان ،تحقيق شعيب األرناؤوط ، (مؤسسة الرسالة-بيروت ، 1414 – 1993 ) ،ط2. - ابن حبان ،محمد بن حبان (املتوفى : 354 )هـ ،صحيح ابن حبان بترتيب ابن بلبان ،تحقيق شعيب األرناؤوط ، (مؤسسة الرسالة-بيروت ، 1414 – 1993 ) ،ط2. - ابن حنبل أحمد بن محمد (املتوفى : 241 )هـ ،مسند اإلمام أحمد بن حنبل ،تحقيق :شعيب األرنؤوط وآخرون ، (مؤسسة الرسالة ، 1421 هـ- 2001 )م ،ط1. - ابن حنبل أحمد بن محمد (املتوفى : 241 )هـ ،مسند اإلمام أحمد بن حنبل ،تحقيق :شعيب األرنؤوط وآخرون ، (مؤسسة الرسالة ، 1421 هـ- 2001 )م ،ط1. - ابن عاشور ،محمد الطاهر بن محمد (املتوفى : 1393 )هـ ،التحرير والتنوير( ،الدار التونسية للنشر– تونس ، 1984 .)هـ - ابن عطية ،عبد الحق بن غالب (املتوفى : 542 )هـ ، املحرر الوجيز في تفسير الكتاب العزيز ،تحقيق : عبد السالم عبد الشافي( ،دار الكتب العلمية– بيروت ، 1422 )ه ،ط1. - ابن عطية ،عبد الحق بن غالب (املتوفى : 542 )هـ ، املحرر الوجيز في تفسير الكتاب العزيز ،تحقيق : عبد السالم عبد الشافي( ،دار الكتب العلمية– بيروت ، 1422 )ه ،ط1. - ابن ماجة ،محمد بن يزيد (املتوفى : 273 )هـ ،سنن ابن ماجه ،تحقيق : شعيب األرنؤوط- وآخرون( ، دار الرسالة العاملية ، 1430هـ- 2009 )م ،ط1. - ابن ماجة ،محمد بن يزيد (املتوفى : 273 )هـ ،سنن ابن ماجه ،تحقيق : شعيب األرنؤوط- وآخرون( ، دار الرسالة العاملية ، 1430هـ- 2009 )م ،ط1. - ابن منظور ،محمد بن مكرم بن ع لى (املتوفى : 711 )هـ ،لسان العرب( ،دار صادر– بيروت ، 1414 )ه ،ط3 - ابن منظور ،محمد بن مكرم بن ع لى (املتوفى : 711 )هـ ،لسان العرب( ،دار صادر– بيروت ، 1414 )ه، الوالء في القرآن الكريم ( 32 ) الحصان الوالء في القرآن الكريم ( 32 ) الحصان المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم اإلسالمية ــ المجلد ال رابع ــ العدد ال ثاني ــ يونيو 21 20 م المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم اإلسالمية ــ المجلد ال رابع ــ العدد ال ثاني ــ يونيو 21 20 م المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم اإلسالمية ــ المجلد ال رابع ــ العدد ال ثاني ــ يونيو 21 20 م المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم اإلسالمية ــ المجلد ال رابع ــ العدد ال ثاني ــ يونيو 21 20 م - ال شاطبي ،إبراهيم بن موس ى (املتوفى : 790 )هـ ،االعتصام ،تحقيق :سليم بن عيد الهاللي( ،دار ابن عفان ، السعودية ، 1412 هـ- 1992 )م ،ط1. - ال شاطبي ،إبراهيم بن موس ى (املتوفى : 790 )هـ ،االعتصام ،تحقيق :سليم بن عيد الهاللي( ،دار ابن عفان ، السعودية ، 1412 هـ- 1992 )م ،ط1. - الشنقيطي ،محمد األمين بن محمد املختار (املتوفى : 1393 )هـ ،أضواء البيان في إيضاح القرآن بالقرآن( ، دار الفكر للطباعة و النشر و التوزيع بيروت– لبنان ، 1415هـ- 1995 .)مـ - الشنقيطي ،محمد األمين بن محمد املختار (املتوفى : 1393 )هـ ،أضواء البيان في إيضاح القرآن بالقرآن( ، دار الفكر للطباعة و النشر و التوزيع بيروت– لبنان ، 1415هـ- 1995 .)مـ - الطبراني ،سليمان بن أحمد (املتوفى : 360 )هـ ،املعجم الكبير ،تحقيق :حمدي بن عبد املجيد السلفي( ، دار إحياء التراث العربي ، 1983م) ط2. - الطبراني ،سليمان بن أحمد (املتوفى : 360 )هـ ،املعجم الكبير ،تحقيق :حمدي بن عبد املجيد السلفي( ، دار إحياء التراث العربي ، 1983م) ط2. - الطبري ،محمد بن جرير (املتوفى : 310 )هـ ،جامع البيان في تأويل القرآن ،تحقيق :أحمد محمد شاكر( ،م ؤسسة الرسالة ، 1420 هـ- 2000 .)م - الطبري ،محمد بن جرير (املتوفى : 310 )هـ ،جامع البيان في تأويل القرآن ،تحقيق :أحمد محمد شاكر( ،م ؤسسة الرسالة ، 1420 هـ- 2000 .)م - عمر ،أحمد مختار عبد الحميد (املتوفى : 1424هـ) بمساعدة فريق عمل ،معجم اللغة العربية املعاصرة( ، عالم الكتب ، 1429 هـ- 2008 ) م ،ط1. - عمر ،أحمد مختار عبد الحميد (املتوفى : 1424هـ) بمساعدة فريق عمل ،معجم اللغة العربية املعاصرة( ، عالم الكتب ، 1429 هـ- 2008 ) م ،ط1. - الفارابي ،إسماعيل بن حماد (املتوفى : 393 )هـ ،الصحاح تاج اللغة وصحاح العربية ،تحقيق : أحمد عبد الغفور عطار( ،دار العلم للماليين– بيروت ، 1407هـ - 1987م) ط4. - الفارابي ،إسماعيل بن حماد (املتوفى : 393 )هـ ،الصحاح تاج اللغة وصحاح العربية ،تحقيق : أحمد عبد الغفور عطار( ،دار العلم للماليين– بيروت ، 1407هـ - 1987م) ط4. - ابن هشام ، عبد امللك بن أيوب الحميري (املتوفى : 213 )هـ ،السيرة النبوية البن هشام ،تحقيق :مص وآخرون( ،مكتبة ومطبعة مصطفى البابي الحلبي وأوالده بمصر ، 1375 هـ- 1955 م) ط2. - الرازي ،محمد بن عمر (املتوفى : 606 )هـ ،مفاتيح الغيب( ،دار إحياء التراث العربي ،بيروت- 1420هـ) ط الرازي ،محمد بن عمر (املتوفى : 606 )هـ ،مفاتيح الغيب( ،دار إحياء التراث العربي ،بيروت- 1420هـ) ط3. - رضا ،محمد رشيد (املتوفى : 1354 )هـ ،تفسير القرآن الحكيم ( )تفسير املنار( ،الهيئة املصرية ال عامة للكتاب ، 1990 .)م - رضا ،محمد رشيد (املتوفى : 1354 )هـ ،تفسير القرآن الحكيم ( )تفسير املنار( ،الهيئة املصرية ال عامة للكتاب ، 1990 .)م - الزمخشري ، محمود بن عمرو (املتوفى : 538 )هـ ،الكشاف عن حقائق غوامض التنزيل( ،دار الكتاب العربي– بيروت ، 1407 )ه ،ط3. - الزمخشري ، محمود بن عمرو (املتوفى : 538 )هـ ،الكشاف عن حقائق غوامض التنزيل( ،دار الكتاب العربي– بيروت ، 1407 )ه ،ط3. - السرحان ،محمد قضام ،الوالء واالنتماء لدى الشباب األردني واثره في بناء الشخصية( ، مطبعة التوفيق– عمان2003 .)م - السرحان ،محمد قضام ،الوالء واالنتماء لدى الشباب األردني واثره في بناء الشخصية( ، مطبعة التوفيق– عمان2003 .)م - السعدي ، عبد الرحمن بن ناصر (املتوفى : 1376 )هـ ،تيسير الكريم الرحمن في تفسير كالم املنان ،تحقيق : عبد الرحمن بن معال اللويحق( ،مؤسسة الرسالة ، 1420 هـ- 2000 )م ،ط1. - سيد قطب إبراهيم (املتوفى : 1385 )هـ ،في ظالل القرآن( ،دار الشروق- بيروت- القاهرة ، 1412 )ه ،ط17 ،ج : 2 ، ص : 909 . - سيد قطب إبراهيم (املتوفى : 1385 )هـ ،في ظالل القرآن( ،دار الشروق- بيروت- القاهرة ، 1412 )ه ،ط17 ،ج : 2 ، ص : 909 . الوالء في القرآن الكريم ( 33 ) الحصان الوالء في القرآن الكريم ( 33 ) الحصان المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم اإلسالمية ــ المجلد ال رابع ــ العدد ال ثاني ــ يونيو 21 20 م ا - النابلس ي ،محمد راتب ،موسوعة اإلعجاز العلمي في القرآن والسنة ، دار املكتبي- سورية- دمشق- الحلبوني- جادة ابن سينا ،الطبعة : الثانية1426 هـ- 2005 م - النابلس ي ،محمد راتب ،موسوعة اإلعجاز العلمي في القرآن والسنة ، دار املكتبي- سورية- دمشق- الحلبوني- جادة ابن سينا ،الطبعة : الثانية1426 هـ- 2005 م - النيسابوري ،مسلم بن الحجاج (املتوفى : 261 )هـ ،صحيح مس لم ،تحقيق :محمد فؤاد عبد الباقي( ، دار إحياء التراث العربي– .)بيروت - النيسابوري ،مسلم بن الحجاج (املتوفى : 261 )هـ ،صحيح مس لم ،تحقيق :محمد فؤاد عبد الباقي( ، دار إحياء التراث العربي– .)بيروت - الواحدي ،علي بن أحمد (املتوفى : 468 )هـ ،أسباب نزول القرآن ،تحقيق :عصام بن عبد املحسن الحميدان ، الناشر : دار اإلصالح- الدمام ،الطبعة :الثانية ، 1412 هـ- 1992 م - الواحدي ،علي بن أحمد (املتوفى : 468 )هـ ،أسباب نزول القرآن ،تحقيق :عصام بن عبد املحسن الحميدان ، الناشر : دار اإلصالح- الدمام ،الطبعة :الثانية ، 1412 هـ- 1992 م ( 34 ) الوالء في القرآن الكريم الحصان الوالء في القرآن الكريم
https://openalex.org/W2596535500	https://zenodo.org/records/1099020/files/10000396.pdf	English	null	Modeling and Simulation of Standalone Photovoltaic Charging Stations for Electric Vehicles	Zenodo (CERN European Organization for Nuclear Research)	2,015	cc-by	7,206	I. INTRODUCTION Fig. 1 EV charging/discharging: H2V2H structure LECTRIC vehicles (EVs) are green transports, and they are good means to solve transport and environmental problems. The environmental benefits of EVs increase with the produced electricity from green sources such as (wind, solar, small scale hydroelectricity). Green sources are also a good solution and alternative enabling to avoid the problem of reliability and safely which can be encountered when grid power is used to charge EVs [1]. Charging stations provide power supply for EVs; so, the deployment of complete infrastructures with sophistic equipments is indispensable for promoting EVs. However, EV charging process takes much time than charging process using fuel car [2], and batteries should be efficiently used since the propulsion of EVs depend on their energy storage capacity [3], [23]. E E This paper focuses on the modeling and sizing of EV charging stations using solar energy. More precisely, we address a PV panel mathematical model whose parameters are extracted from PV tests. We address also the voltage modeling of lead-acid battery we use in our system H2V. In fact, the battery state information allows optimal control for battery charging/discharging process, reduces the risk of overcharge and deep charging. In addition, it is possible to extend the battery life and reach its optimal usage. To do so, we focus on the modeling of the lead-acid battery voltage by developing a mathematical model enabling to reach these objectives. The proposed model describes the profile of battery voltage considering time and electrical parameters. These parameters can be found using manufacturer data or experimental tests. Afterward, a mathematical model is deduced using a curve- fitting approach. Many parameters such as the state of charge (SOC), time of charging and discharging, current of charging and discharging should be taken into account during charging and discharging processes. EVs can be charged by either plugging into electrical outlets or by means of on-board electricity generation. There are two main places where the batteries of EVs can be charged: either on a car park, corporate or public, or at home. With this last charging solution, a system H2V is designed to charge BEVs using green sources such as photovoltaic systems as illustrated in Fig. 1. In a H2V system, EV can be charged with direct current from photovoltaic panels (PV) or with current from a Lead-Acid battery installed at home (see Fig. 1). World Academy of Science, Engineering and Technology International Journal of Computer and Information Engineering Vol:9, No:1, 2015 World Academy of Science, Engineering and Technology International Journal of Computer and Information Engineering Vol:9, No:1, 2015 I. INTRODUCTION Conversely, the system can provide the electric energy from the EV to the household when this is needed (V2H). The study of these charging/discharging processes H2V and V2H The remainder of the paper is structured as follows. Section II introduces the energy storage sources. Section III presents the adopted modeling approach. PV and Lead-Acid battery modeling as well as DC/DC converter topologies are presented in this section. Section IV concerns the system sizing. The obtained results and a comparison study of simulation results with datasheet and experimental results are given in Section V. Last section concludes the paper and gives some future directions of this work. R. Mkahl is with the laboratory OPERA/FCLAB FR-CNRS3539, University of Technology Belfort-Montbéliard, Belfort, France (corresponding author, phone 0033695015520; e-mail: rania.mkahl@utbm.fr). A. Nait-Sidi-Moh is with Laboratory of Innovative Technologies, University of Picardie Jules Verne, Saint-Quentin, France (e-mail: ahmed.nait-sidi-moh@u-picardie.fr). International Science Index, Computer and Information Engineering Vol:9, No:1, 2015 waset.org/Public Keywords—Electric vehicles, photovoltaic energy, lead-acid batteries, charging process, modeling, simulation, experimental tests. R. Mkahl is with the laboratory OPERA/FCLAB FR-CNRS3539, University of Technology Belfort-Montbéliard, Belfort, France (corresponding author, phone 0033695015520; e-mail: rania.mkahl@utbm.fr). A. Nait-Sidi-Moh is with Laboratory of Innovative Technologies, University of Picardie Jules Verne, Saint-Quentin, France (e-mail: ahmed.nait-sidi-moh@u-picardie.fr). M. Wack is with the laboratory OPERA/FCLAB FR-CNRS3539, University of Technology Belfort-Montbéliard, Belfort, France (e-mail: maxime.wack@utbm.fr). International Science Index, Computer and Information Engineering Vol:9, No:1, 2015 waset.org/Publication/10000396 Fig. 1 EV charging/discharging: H2V2H structure M. Wack is with the laboratory OPERA/FCLAB FR-CNRS3539, University of Technology Belfort-Montbéliard, Belfort, France (e-mail: maxime.wack@utbm.fr). Modeling and Simulation of Standalone Photovoltaic Charging Stations for Electric Vehicles R. Mkahl, A. Nait-Sidi-Moh, M. Wack systems requires adequate models in order to ensure an optimal energy management and effective usage of the system components. This modeling is based on mathematical equations that govern the system in order to predict and evaluate its behavior. systems requires adequate models in order to ensure an optimal energy management and effective usage of the system components. This modeling is based on mathematical equations that govern the system in order to predict and evaluate its behavior. Abstract—Batteries of electric vehicles (BEV) are becoming more attractive with the advancement of new battery technologies and promotion of electric vehicles. BEV batteries are recharged on board vehicles using either the grid (G2V for Grid to Vehicle) or renewable energies in a stand-alone application (H2V for Home to Vehicle). This paper deals with the modeling, sizing and control of a photovoltaic stand-alone application that can charge the BEV at home. The modeling approach and developed mathematical models describing the system components are detailed. Simulation and experimental results are presented and commented. International Science Index, Computer and Information Engineering Vol:9, No:1, 2015 waset.org/Publication/10000396 Fig. 3 Equivalent circuit of a solar cell Fig. 2 Ragone plot of energy storage Fig. 3 Equivalent circuit of a solar cell This diagram shows that supercapacitors (Scaps) have a very high power density but a very low energy density. The batteries have a low power density and high energy density. The batteries are "reversible" generators; they can store electrical energy in chemical form and return it at any time when it is needed. The nature of electric vehicles is governed primarily by the characteristics of the used batteries. The weight and volume of batteries are usually a decisive factor. A BEV must satisfy certain conditions: a good specific power for high accelerations, good specific energy synonymous with good battery life, stable voltage generating regular performances, a long lifetime and low maintenance and recyclable [6]. The equation of PV module witch expresses the characteristic current–voltage (I-V) of PV module is given as follow [13]:       × + −     − × + − × = ) ) ( ) ( ( 1 )) / ) ( ) ( ( exp( ) ( ) ( 0 SHm p sm m s p Sm m L p m R n R t I t V nkT n n R t I t V q I t I n t I (1) (1) n: diode ideality factor (1 for an ideal diode); ns: cells in series np: cells in parallel; Im: current of PV module; V: voltage of PV module; RSm: series resistance of PV module; RSHm: shunt resistance of PV module; IL : photovoltaic current; I0: reverse saturation current; : terminal potential (0.0259V at 25C°). Batteries currently used in electric vehicles are: Lead-Acid (LA) battery, Lithium-Ion (Li-Ion) battery, Nickel Metal Hybrdrure [7], Nickel Cadmium battery [6], and Zink-Air. Li- Ion battery is the main hope for electric vehicles [8]. It holds a higher voltage per element, good volume and mass performance, and a very high specific energy compared to other types of batteries. It is worth noting that the specific systems of stationary electricity storage are very different from the specific systems of embedded systems for which energies and unit powers are weak. In stationary systems, the amount of energy and the total power available then become largely predominant. III. MODELING OF THE SYSTEM COMPONENTS B. Lead-Acid Battery Modeling In this section, we focus on the modeling of the main components of charging station. To do so, we present PV and Lead-Acid battery models and DC/DC converter topologies. II. ENERGY STORAGE SOURCES Energy storage system includes different storage technologies, electrochemical technology (battery), electrical scholar.waset.org/1307-6892/10000396 International Scholarly and Scientific Research & Innovation 9(1) 2015 72 World Academy of Science, Engineering and Technology International Journal of Computer and Information Engineering Vol:9, No:1, 2015 International Science Index, Computer and Information Engineering Vol:9, No:1, 2015 waset.org/Publication/10000396 Lead-Acid batteries have high availability and they are the least expensive storage batteries for any application, while still providing reasonable performance and life characteristics. They are widely used like the stationary electricity storage in PV systems as given in [9]. Generally, unless otherwise specified by the manufacturer, PV module consists of thirty six cells in series (ns =36 cells) and np=1 cell. The PWX500-12V module is chosen for this study. The parameters I0, RSm, RSHm of this module are not given in the data sheet, so we have determined it by using the functions 'fittype' and 'fit' of Matlab. The parametric identification is considered by using current-voltage (I-V) characteristic curve of the module extracted from experimental data. The PV panel parameters are reported in the Table I. TABLE I PV PARAMETERS PV parameters Digital values RSm 0.2297 Ω RSHm 60.78 Ω I0 3.886e-009 A B. Lead-Acid Battery Modeling A. PV Modeling technology (supercapacitor), and mechanical technology (a flywheel) as detailed in [4]. To compare the energy storage sources many parameters can be considered. Among these parameters we underline energy density, power density, cycle life performance, cost, environmental impact [5]. Fig. 2 illustrates the Ragone diagram of different energy storages. Several studies are developed about the mathematical modeling of the PV cell. A model of the photovoltaic cell with double diode is developed in [10]. This model consists of a current source representing the light flux, in parallel with two diodes. The diode represents the cell behavior in darkness. Tow resistances, shunt and series resistance are added to present internal losses. The current-voltage (I-V) equation can be simplified by considering the shunt resistance as infinity [11]. Another works have been proposed to simplify the PV cell by using single-model PV cell with one diode [12]. Like this last research work, we use a single-model PV cell in order to formalize the PV solar cell. The equivalent electrical circuit is presented in Fig. 3. Fig. 2 Ragone plot of energy storage 1. Voltage Modeling In the literature the battery behavior has been described by many models. In fact, several research works proposed some scholar.waset.org/1307-6892/10000396 International Scholarly and Scientific Research & Innovation 9(1) 2015 73 World Academy of Science, Engineering and Technology International Journal of Computer and Information Engineering Vol:9, No:1, 2015 World Academy of Science, Engineering and Technology International Journal of Computer and Information Engineering Vol:9, No:1, 2015 technique as detailed in [21]. technique as detailed in [21]. battery models which describing battery behavior for specifics purpose, from the battery design and performance estimation to the circuit simulation. According to these researches, the battery models can be classified into three categories: Based on the curves showing the evolution of the terminal voltage discharging, we deduce an expression of the voltage discharging battery by an extrapolation of the points which have been raised. The electrochemical model: this model is used to optimize the physical design aspects of batteries, to characterize the fundamental mechanisms of power generation and lay electric parameters of the battery with chemical parameters [14]. Shepherd model which can be considered as the best battery model for using for the hybrid vehicle analysis is one example of electrochemical model [15]. For charging curve, the datasheet does not give sufficient curves. So, we charge the battery with constant current in order to obtain the curve enabling to describe the relationship between the terminal battery voltages during charging versus charging time. The battery is charged with constant current I1 until the battery voltage reaches overcharge voltage. Then it discharges at constant current until the battery voltage dropped to the deep-discharge protection point. We repeat these processes with currents I2, I3,..., In. Finally, the obtained curves, from performed tests, describe the variation of the battery terminal voltage during charging/discharging process over the time. Based on the test data we deduce an expression of the voltage charging battery by an extrapolation of the point which we have raised. The mathematical model is the second category. This model is mostly too abstract to embody any practical meaning but it is useful to system designers [16]. Ciemat model is one example of a mathematical model. This model consists of an ideal voltage in series with resistance, the electromotive force and resistance which are expressed according to the battery SOC as detailed in [17]. 1. Voltage Modeling International Science Index, Computer and Information Engineering Vol:9, No:1, 2015 waset.org/Publication/10000396 The third category is electric model. This last lies between electrochemical and mathematical model. Electric model consists of voltage source in series with a resistance which is not function of battery SOC [18]. From the datasheet, we obtain the curves and with applying the method of extrapolation of the curve, we obtain a polynomial equation of 3rd degree. The goal of this section is to determine a relationship between the battery voltage and the battery time charging/discharging. This expression takes into account the electrical parameters. During cycles of charging and discharging, the characteristics of the battery depend on its SOC, charging/discharging current and charging/discharging time. As given in [19], the SOC of the battery is defined by: D Ct Bt At t V + + + = 2 3 ) ( (3) (3) (3) where A, B, C, and D are the parameters of the battery whose expressions are detailed in the following subsection. It is worth noting that the parameters A, B, and C depend on the battery current. The parameter D depends on the battery current and the open circuit voltage. ∫ − = dt t i c soc soc n ) ( 1 0 (2) (2) 2. Identification of Parameters A, B, C, D Using basic fitting technique, the parameters A, B and C are given as polynomial functions of 7th degree depending on the battery current I. These parameters can be determined by: where: SOC0: initial state of charging percentage; Cn: battery capacity in Ampere-hours; i(t): battery current. - Case of charging: A= ae-07; B = b; C = c. - Case of charging: A= ae-07; B = b; C = c. - Case of discharging: A = (4, 63e-13)a; - Case of discharging: A = (4, 63e-13)a; Fig. 4 Voltage E versus SOC 0 20 40 60 80 100 11.2 11.4 11.6 11.8 12 12.2 12.4 12.6 12.8 13 13.2 soc% E[V] given limits approximation given limits Fig. 4 Voltage E versus SOC 0 20 40 60 80 100 11.2 11.4 11.6 11.8 12 12.2 12.4 12.6 12.8 13 13.2 soc% E[V] given limits approximation given limits B = (2,7778e-009)b; C = 0, 016667c where a, b and c are given by: where a, b and c are given by: a b c M V (4) (4) where the vector V is given by: where the vector V is given by: Fig. 4 Voltage E versus SOC V I, I, I, I, I, I, I, 1 In the H2V concept the Lead-Acid battery YUASA SW280 was used, the nominal voltage is 12V and the capacity Cn is 7,6 Ah, for n = 20. The effect of temperature on discharging curves is not given by the datasheets, so the modeling is done at a temperature of 20°C. The matrix M is given by Least Square Fitting as detailed in [21]. The parameter D represents the starting voltage (voltage at the beginning of charging/discharging). Experimentally, the expression of this parameter is given by: As exposed in [20], datasheets give the relationship between E (open circuit voltage) and SOC (State of Charge). For the battery YUASA SW280 this relationship is linear as illustrated in Fig. 4. A mathematical equation of SOC according to E is established using a linear approximation dv E D ± = (5) dv E D ± = (5) International Scholarly and Scientific Research & Innovation 9(1) 2015 scholar.waset.org/1307-6892/10000396 74 World Academy of Science, Engineering and Technology International Journal of Computer and Information Engineering Vol:9, No:1, 2015 Fig. 6 Combined recharging cycle Yes DC DC VLA VLi-Ion Current control Voltage control VLi-Ion ref 220V iLi-Ion ref 50A SOCLi-Ion >80% No α where E is the open circuit voltage, dv is the initial voltage drop at the switching on process in the battery. We use the sign "+" (respectively "-") in the case of the battery charging (resp. discharging). The initial voltage drop at the switching on process in the battery is given by: RI dv = (6) (6) R corresponds to the battery resistance which is a function of the current I. Using the technique of basic fitting we can find the relationship between R and I. We measure the values of the open circuit voltage E and the values of the starting voltage V0 of the battery, then we find dv = E-V0. For several values of dv, using (6), we calculate the resistance values for each corresponding values of dv, and then we find the relationship between R and I using interpolation method. This calculation is detailed in [21]. Fig. 6 Combined recharging cycle A. Ev Sizing The power demand is due to speed variations, tire friction dissipation, aerodynamics dissipation, and mass elevation. C. DC/DC Charger Topologies Solar energy is converted to electric energy by the PV panels. Electric energy is used for recharging Lead-Acid batteries via DC/DC Boost converter (see Fig. 5(a)). Boost converter is controlled by maximum power point tracking algorithm (MPPT) for obtaining always the PV maximum power at PV panel output. International Science Index, Computer and Information Engineering Vol:9, No:1, 2015 waset.org/Publica where V: vehicle speed; M=1000 (kg): vehicle mass; α = 0 (radians): road angle with a horizontal line; g = 9.81M·s−2: acceleration of gravity; Cr = 0.01: friction coefficients; Cx = 0.30: aerodynamic coefficients; ρ = 1.225 (km. m3): air density; S = 2.5(m2): front surface area. In addition, a Buck- Boost (current-bidirectional converter) is used for charging embedded Li-Ion battery into the EV from home energy (H2V). This converter can be used also for charging Lead-Acid batteries from vehicle battery (V2H) when it is needed. Fig. 5 (b) corresponds to a DC-DC current- bidirectional converter. Fig. 7 NEDC driving cycle 0 200 400 600 800 1000 1200 0 50 100 150 Time (s) Speed (Km/h) 0 200 400 600 800 1000 1200 -5000 0 5000 10000 15000 20000 Time (s) Power demande(W) (a) boost (b) Buck-Boost Fig. 5 DC-DC charger topologies International Science Index, Computer and Information Eng Fig. 7 NEDC driving cycle So the vehicle energy demand equals to 17kWh. This amount of energy needs to be embedded and stored in the Li- Ion batteries pack. For testing vehicles, driving cycles have been normalized. European light-duty vehicles have to face the New European Driving Cycle (NEDC). The NEDC consists of 4 repeated urban cycles (ECE-15) and an Extra- Urban driving cycle. Fig. 7 shows the NEDC driving cycle with the speed and the power demand of the small car. Fig. 5 DC-DC charger topologies Fig. 6 shows the fast recharge control technique consisting on a current control from low SOCLi-Ion to 80% and then switching to a voltage control from 80% to 100% of SOCLi-Ion. where the vector V is given by: This power demand can be expressed as in [22] by: dex, Computer and Information Engineering Vol:9, No:1, 2015 waset.org/Publication/10000396 International Science Index, Computer and Information Engineering Vol:9, No:1, 2015 waset.org/Publication/10000396       + + + = 2 2 1 ) sin( ) cos( V SC dt dV M Mg Mg C V P x r ρ α α (7) (7) V. SIMULATION AND EXPERIMENTAL RESULTS The simulation platform of PV system consists of PV panels, Lead-Acid batteries, Boost converter and MPPT P&Q as illustrated in Fig. 8. Hereafter, we present simulation results of PV model and we report a benchmarking with datasheets and experimental results. C. Sizing of Lead-Acid Battery Current [A] As underlined previously we use of YUASA SW280 Lead- Acid battery as the embedded battery at home. Its characteristics are reported in Table V, and sizing results are presented in Table VI. TABLE V CHARACTERISTICS OF LEAD-ACID BATTERIES YUASA SW280 YUASA SW280 Characteristics Digital values Voltage 12 V Volume 151x65x99 mm Weight Nominal capacity Energy / Volume Energy/Weight Internal resistance of charged battery Maximum discharge current in minute 2.7 kg 7.6 Ah 93 Wh/l 30 Wh/Kg 15 Ω 50A TABLE VI SIZING OF LEAD-ACID BATTERIES YUASA SW280 Sizing Digital values N° in series 4 N° in parallel Pack voltage Pack capacity Pack mass 78 48 V 588 Ah 842.4 kg V S E R ernational Science Index, Computer and Information Engineering Vo Fig. 9 Comparison of simulation results with datasheets for several levels of radiation and at cell temperature of 25°C International Science Index, Computer and Informatio Fig. 9 shows that the simulation results match with those of the datasheet. After comparing the simulation results with the datasheet, we compared the simulation results with experimental results of current-voltage (I-V) characteristics and power-voltage (P-V) characteristics. The used materials for experimental tests are PWX500-12V module, variable load, current sensor, voltage sensor, contact thermometer, and light meter as illustrated in Fig. 10. The aim of these tests is to determine the I-V, P-V curves of the PV panel and find the PV panel electric parameters from the I-V curve. Fig. 10 Test bench of PV panel International Science Index, Computer and Information Engineering Vol:9, No:1, 2015 waset.org/Publication/10000396 e Index, Computer and Information Engineering Vol:9, No:1, 2015 waset.org/Publication/10000396 TABLE IV SIZING OF SOLAR PANEL PWX500-12V PWX500-12V Sizing Digital values Number of PV panels Surface of PV Power max Weight 148 70 m2 7.4 kWc 1361 kg . Sizing of Lead-Acid Battery Fig. 8 PV system controlled by MPPT algorithm Fig. 8 PV system controlled by MPPT algorithm Fig. 9 Comparison of simulation results with datasheets for several levels of radiation and at cell temperature of 25°C 0 5 10 15 20 25 0 0.5 1 1.5 2 2.5 3 3.5 Voltage [V] Current [A] 500W/m² 400W/m² 600W/m² 800W/m² 1000W/m² 0 5 10 15 20 25 0 0.5 1 1.5 2 2.5 3 3.5 Voltage [V] Current [A] 500W/m² 400W/m² 600W/m² 800W/m² 1000W/m² IV. SYSTEM SIZING Data of PV sizing are given in Table II. Characteristics and sizing PV panels are reported in Tables III and IV. PV panels are supposed to be installed on the roof and have a good orientation for capturing a maximum solar radiation. In this section, we focus on the sizing of charging station for small car following a flat road. scholar.waset.org/1307-6892/10000396 International Scholarly and Scientific Research & Innovation 9(1) 2015 75 World Academy of Science, Engineering and Technology International Journal of Computer and Information Engineering Vol:9, No:1, 2015 TABLE II DATA VALUES FOR PV SIZING PV PARAMETERS Data Values Device EV Power of receiver Usage time Factor Corrector Radiation 4704 W 6 h 1.3 5 k Wh/m².j TABLE III CHARACTERISTICS OF SOLAR PANEL PWX500-12V PWX500-12V Characteristics Digital values Power max Optimal current Optimal voltage Short circuit cure Open circuit volt 50 W 2.9 A 17.2 V 3.2 A 21.6 V TABLE IV SIZING OF SOLAR PANEL PWX500-12V PWX500-12V Sizing Digital values Number of PV panels Surface of PV Power max Weight 148 70 m2 7.4 kWc 1361 kg Sizing of Lead-Acid Battery d li d i l f ASA S 280 datasheets for several levels of the radiation. Fig. 9 shows the superposition of the characteristic curves of PV panels I = f(V) with a cell temperature = 25°C and solar radiation at 5 levels: 1000W/m2, 800W/m2, 600W/m2, 500W/m2, 400W/m2. TABLE II DATA VALUES FOR PV SIZING PV PARAMETERS Fig. 8 PV system controlled by MPPT algorithm Fig. 9 Comparison of simulation results with datasheets for several levels of radiation and at cell temperature of 25°C 0 5 10 15 20 25 0 0.5 1 1.5 2 2.5 3 3.5 Voltage [V] Current [A] 500W/m² 400W/m² 600W/m² 800W/m² 1000W/m² Fig. 8 PV system controlled by MPPT algorithm Fig. 8 PV system controlled by MPPT algorithm A. PV Simulation Results 12 Comparing P-V curves of PV model and experimental results 0 5 10 15 20 0 2 4 6 8 10 12 14 16 18 Voltage(V) Power(W) E=280W/m², T=22C° E= 415.625 W/m², T= 18.9C° E=400 W/m², T=20C° E=342W/m², T=26.671C° 0 5 10 15 20 0 2 4 6 8 10 12 14 16 18 Voltage(V) Power(W) E=280W/m², T=22C° E= 415.625 W/m², T= 18.9C° E=400 W/m², T=20C° E=342W/m², T=26.671C° Fig. 12 Comparing P-V curves of PV model and experimental results 0 5 10 15 20 0 2 4 6 8 10 12 14 16 18 Voltage(V) Power(W) E=280W/m², T=22C° E= 415.625 W/m², T= 18.9C° E=400 W/m², T=20C° E=342W/m², T=26.671C° Fig. 12 Comparing P-V curves of PV model and experimental results Fig. 13 PV module current, voltage and power applying MPPT algorithm 0 5 10 15 20 0 2 4 6 8 10 12 14 16 18 Voltage(V) Power(W) E=280W/m², T=22C° E= 415.625 W/m², T= 18.9C° E=400 W/m², T=20C° E=342W/m², T=26.671C° 0 100 200 0 1 2 3 4 5 Time (s) Module current (A) 0 100 200 0 5 10 15 Time (s) Module voltage (v) 0 50 100 150 200 0 20 40 60 Time (s) Power (W) ience Index, Computer and Information Engineering Vol:9, No:1, 2015 waset.org/Publication/10000396 Based on the obtained results, absolute error and relative error are reported in Table VII. The parameter C represents the battery nominal capacity with C = 7.6 Ah. nce Index, Computer and Information Engineering Vol:9, No:1, 2015 waset.org/Publication/10000396 International Science Index, Computer and Information Engineering Vol:9, No:1, 2015 waset.org/Publication/10000396 Power(W) TABLE VII ERRORS BETWEEN DATASHEETS AND SIMULATION RESULTS Discharging current Absolute error Relative error TABLE VII ERRORS BETWEEN DATASHEETS AND SIMULATION RESULTS Discharging current Absolute error Relative error 0.05C 0.1C 0.2C 0.4C 0.6C 1C 2C 0.05 0.01 0.003 8.95.e-4 0.006 0.05 0.276 0.44% 0.1% 0.03% 0.008% 0.05% 0.43% 2.83% International Science Index, Computer and Information Engineering Vol:9, No:1, 2015 waset.org/Publica Fig. 12 Comparing P-V curves of PV model and experimental results Fig. 13 PV module current, voltage and power applying MPPT algorithm 0 100 200 0 1 2 3 4 5 Time (s) Module current (A) 0 100 200 0 5 10 15 Time (s) Module voltage (v) 0 50 100 150 200 0 20 40 60 Time (s) Power (W) The average error represents good model accuracy for each rate of discharging current. A. PV Simulation Results I-V characteristics of a PWX500-12V module are simulated according to (1). The simulation results are compared with Fig. 10 Test bench of PV panel scholar.waset.org/1307-6892/10000396 International Scholarly and Scientific Research & Innovation 9(1) 2015 76 Fig. 14 Comparing the simulation of discharge voltage with the datasheet (dashed line refers to datasheet) 10 0 10 1 10 2 10 3 8 9 10 11 12 13 Discharge time [min] Battery voltage [V] data sheet simu 2C 1C 0.6C 0.4C 0.2C 0.05C 0.1C gineering and Technology and Information Engineering 2015 Comparison I-V curves of PV model and experimental results Fig. 14 Comparing the simulation of discharge voltage with the datasheet (dashed line refers to datasheet) 0 5 10 15 20 0 0.5 1 1.5 Voltage (V) E= 400 W/m², T= 20C° E= 415.6 W/m², T= 18.9C° E= 342 W/m², T= 26.671C° E= 280 W/m², T= 22C° 18 10 0 10 1 10 2 10 3 8 9 10 11 12 13 Discharge time [min] Battery voltage [V] data sheet simu 2C 1C 0.6C 0.4C 0.2C 0.05C 0.1C World Academy of Science, Engineering and Technology International Journal of Computer and Information Engineering Vol:9, No:1, 2015 World Academy of Science, Engineering and Technology International Journal of Computer and Information Engineering Vol:9, No:1, 2015 10 0 10 1 10 2 10 3 8 9 10 11 12 13 Discharge time [min] Battery voltage [V] data sheet simu 2C 1C 0.6C 0.4C 0.2C 0.05C 0.1C Fig. 11 Comparison I-V curves of PV model and experimental results 0 5 10 15 20 0 0.5 1 1.5 Voltage (V) Current(A) E= 400 W/m², T= 20C° E= 415.6 W/m², T= 18.9C° E= 342 W/m², T= 26.671C° E= 280 W/m², T= 22C° Fig. 11 Comparison I-V curves of PV model and experimental results 0 5 10 15 20 0 0.5 1 1.5 Voltage (V) Current(A) E= 400 W/m², T= 20C° E= 415.6 W/m², T= 18.9C° E= 342 W/m², T= 26.671C° E= 280 W/m², T= 22C° 0 5 10 15 20 0 0.5 1 1.5 Voltage (V) Current(A) E= 400 W/m², T= 20C° E= 415.6 W/m², T= 18.9C° E= 342 W/m², T= 26.671C° E= 280 W/m², T= 22C° Current(A) Fig. 11 Comparison I-V curves of PV model and experimental results Fig. 14 Comparing the simulation of discharge voltage with the datasheet (dashed line refers to datasheet) Fig. A. PV Simulation Results The simulation curves coincide with datasheets, which allows concluding that the analytical expression represents well the evolution of the terminal voltage battery YUASA SW280. The comparison of obtained simulation results with experimental measurements for validation of the battery model accuracy is reported. Experimentations are carried out under ambient temperature. To perform the experimental tests, some materials are required such as regulated power supply for constant charge current, electronic load to discharge with constant current, acquisition card with voltage probe, PC with Labview and a current sensor. Figs. 15 and 16 show the test bench of battery test. It is worth to note that the performed tests for battery model and PV model are carried out within the laboratory FEMOT-ST/FCLAB in Belfort, a small city in the north east of France. We underline also that the PV tests are done in the first week of November 2011. International Science Index, Computer and I Fig. 13 PV module current, voltage and power applying MPPT algorithm Figs. 11 and 12 show the superposition of the experimental and simulation results for the I-V and P-V characteristic curves respectively. The comparison study of the two results shows a good accuracy of this model. Fig. 15 Test bench of charge battery The MPPT algorithm is applied to obtain the PV maximum power. Fig. 13 shows PV module current, voltage and power applying MPPT algorithm. B. Simulation Results of Battery Lead-Acid 17 Comparison of simulation and experimental results of discharging terminal voltage 10 0 10 1 10 2 10 3 10 4 10 10.5 11 11.5 12 12.5 13 Time [s] Voltage [v] exp simu I=3.22A I=2.9A 10 0 10 1 10 2 10 3 10 4 10 10.5 11 11.5 12 12.5 13 Time [s] Voltage [v] exp simu I=3.22A I=2.9A 0 2000 4000 6000 8000 10000 12000 -200 -150 -100 -50 0 Time (s) Lead-Acid batteries current(A) Fig. 19 Simulation of charging voltage and current pack of batteries from PV current applying MPPT algorithm Fig. 17 Comparison of simulation and experimental results of discharging terminal voltage (a) Combined charge cycle vs time (b) SoC vs time (c) Alpha vs time Fig. 20 Li-Ion batteries charge from Lead-Acid batteries using the combined charge cycle (a) Combined charge cycle vs time In Fig. 17, we show that the experimental curves match well with those given by the analytical expression. This leads to the conclusion that the analytical expression represents well the evolution of the battery voltage Yuasa SW280. International Science Index, Computer and Information Engine Fig. 18 Supervision system of charging Lead-Acid batteries from PV panels Yes No Lead-Acid pack Voltage < 57.7 Stop charging process Continue charging process (a) Combined charge cycle vs time (a) Combined charge cycle vs time International Science Index, Computer and (b) SoC vs time Fig. 18 Supervision system of charging Lead-Acid batteries from PV panels (b) SoC vs time (b) SoC vs time (c) Alpha vs time Voltage and current of Lead-Acid battery with MPPT algorithm are presented in Fig. 19. W remark that the battery voltage increases until reaching the voltage of full charge and then the current switched off. The pack of batteries is fully charged for nearly 3 hours. Fig. 20 presents the control performance in terms of current and voltage control. We can observe that the Li-Ion current is tracking its reference allowing a fast Li-Ion batteries charge (the Li-Ion battery is charged until 80% in less than 30min), than the last 20% are charged very slowly according to the Li- Ion battery characteristics (about 5 hours to obtain a full charge (100%)). Fig. 20 (b) corresponds to the Li-Ion SoC (starting from an arbitrary initial condition, 40% in this case). (c) Alpha vs time Fig. B. Simulation Results of Battery Lead-Acid The work presented in this section is focused on the battery model results. Once the parameters A, B, C, and D are determined, the voltage is plotted using (3) for several current rates on the same graph compared with the curves of the datasheets, Fig. 14. Fig. 15 Test bench of charge battery scholar.waset.org/1307-6892/10000396 International Scholarly and Scientific Research & Innovation 9(1) 2015 77 World Academy of Science, Engineering and Technology International Journal of Computer and Information Engineering Vol:9, No:1, 2015 Fig. 16 Test bench of discharge battery The combined recharge cycle control is illustrated in Fig. 20 (a) where we can see the first value control corresponding to the current until 80% of SoC and the voltage control from 80% to 100%. Once the SoC is reaching 100%, the Li-Ion recharge is stopped and the control equals to 0 (the value of iLi- Ion(A) in Fig. 20 (a), and the value of Alpha in Fig. 20 (c). Alpha is the temperature coefficient of the short-circuit current). Fig. 19 Simulation of charging voltage and current pack of batteries from PV current applying MPPT algorithm (a) Combined charge cycle vs time (b) SoC vs time (c) Alpha vs time Fig. 20 Li-Ion batteries charge from Lead-Acid batteries using the combined charge cycle 0 2000 4000 6000 8000 10000 12000 25 30 35 40 45 50 55 60 Time (s) Lead-Acid batteries voltage (v) 0 2000 4000 6000 8000 10000 12000 -200 -150 -100 -50 0 Time (s) Lead-Acid batteries current(A) Fig. 19 Simulation of charging voltage and current pack of batteries from PV current applying MPPT algorithm 0 2000 4000 6000 8000 10000 12000 25 30 35 40 45 50 55 60 Time (s) Lead-Acid batteries voltage (v) 0 2000 4000 6000 8000 10000 12000 -200 -150 -100 -50 0 Time (s) Lead-Acid batteries current(A) 0 2000 4000 6000 8000 10000 12000 25 30 35 40 45 50 55 60 Time (s) Lead-Acid batteries voltage (v) Fig. 16 Test bench of discharge battery The proposed model enables to predict the voltage battery for discharging rate other than those given in datasheet. Fig. 17 shows results of battery discharging for I=3.22A and 2.9A. Fig. VI. CONCLUSION VI. CONCLUSION Solutions for Sustainable Environment (CESSE), vol. 18, pp. 538–544, 2012. Solutions for Sustainable Environment (CESSE), vol. 18, pp. 538–544, 2012. To properly coordinate the operations of all elements of charging/discharging system H2V and V2H, a modeling and sizing of the whole system is required to describe and simulate the behavior of each element. In this paper, we performed a mathematical modeling of charging station for EV using solar energy; model parameters of PV panel and Lead-acid battery are identified using fitting methodology. PV and Lead-Acid models are validated experimentally. MPPT control method for obtaining PV maximum power is applied. System sizing is proposed and finally home batteries pack is charged by PV current until reaching full charging voltage. The obtained results show that the batteries Li-Ion can be charged promptly from the current of Lead-Acid batteries using buck/boost converter. In our future work, we will focus on the energy management in both directions H2V and V2H with a particular attention to bring to the battery as mean of energy storage. [15] W. Zhoua, H. Yanga, and Z. Fang, “Battery behaviour prediction and battery working states analysis of a hybrid solar–wind power generation system,” Siencedirecte, Re-newable, vol. 33, pp. 1413–1423, 2008. [16] R.A Jackey, “A Simple effective lead-acid battery modelling process for electrical system component selection,” SAE Technical Paper, 2007. [17] O. Geraud, G. Robin, B. Multon, and H. Ben Ahmed, “Energy modeling of a lead-acid battery within hybrid wind/photovoltaic systems,” European Power Electronic Conference 2003, Toulouse, 2003. [18] M. Dürr, A. Cruden, S. Gair, and J.R. McDonald, “Dynamic model of a lead acid battery for use in a domestic fuel cell system,” Siencedirecte, Journal of Power Sources, vol. 161, pp. 1400–1411, 2006. [19] M. Coleman, C. K. Lee, C. Zhu, and W. G. Hurley, “State-of-charge determination from EMF voltage estimation: Using impedance, terminal voltage, and current for lead-acid and lithium-ion batteries,” IEEE Trans. on Industrial Electronics, vol. 54, no. 5, pp. 2550–2557, 2007. [20] Yuasa Battery, “SWL Batteries stationnaires étanches au plomb à recombinaison de gas régulées par soupapes,” 2010. [21] R. Mkahl, and A. Nait Sidi Moh, “Modeling of charging station batteries for electric vehicles,” J. of Asian Electric Vehicles, vol. 11, no. 2, pp. 1667–1672. 2013. [22] M. Becherif, M. Y. Ayad, D. Hissel, and R. REFERENCES Jorge, “Design Methodology of Energy Storage Systems for a Small Electric Vehicle,” World Electric Vehicle Journal, vol. 3, pp. 1–12, 2009. He has more than six years’ experiences in participating and working in European and national/regional projects. He is principal investigator of a regional project Com-SloT (Community of logistic services on the Internet of Things, 2013-2016). He was Co-PI (UTBM side) of two European projects ASSET (Advanced Safety and Driver Support in Efficient Road Transport, FP7-SST, 2008-2011, and TELEFOT (Field Operational Tests of Aftermarket and Nomadic Devises in Vehicles, FP7-ICT, 2008-2012. He is also a member (UTBM side) of EU EACEA Erasmus Mundus project TARGET (Transfer of Appropriate Requirements for Global Education and Technology), 2011-2014. [8] A. Nouh, “Contribution au développement d'un simulateur pour les véhicules électriques routiers,” Ph. D. thesis, University of Technologie Belfort-Montbeliard 2008. [9] A. Cherif, M. Jraidi, and A. Dhouib, “A battery ageing model using in stand-alone PV systems,” Journal of Power Sources, vol. 112, no. 1, pp. 49–53, 2002. [10] A. Zegaouia, P. Petita, M. Ailleriea, J-P. Sawickia, and J.P Charlesa, “ Experimental Validation of Photovoltaic Direct and Reverse Mode Model. Influence of Partial Shading,” Energy Procedia, vol. 18, pp. 1247–1253, 2012. His research interests include modeling, analysis and control of discrete event systems, resources and information sharing, decision-making and interoperability for service composition, Internet of Things, Geo-positioning and mobility, decision-making and management of EV charging. [11] J. Surya Kumari, and Ch. Sai Babu, “Mathematical modeling and simulation of photovoltaic cell using Matlab-Simulink environment,” International Journal of Electrical and Computer Engineering, vol. 2, no. 1, pp. 26–34, 2012. Maxime, Wack received his PhD degree in 1981 in computer science from the Université de Technologie de Compiègne (UTC), France. Currently, he is associate professor-HDR (Habilité à diriger les recherches) at the laboratory OPERA/FCLAB at the Université de Technologie de Belfort Montbéliard (UTBM). He is the head of the research group Geopositioning, Embedded systems and mobility (GSEM) at UTBM. [12] E. Mboumboue, and D. Njomo, “Mathematical modeling and digital simulation of PV solar panel using Matlab software,” Inter. Journal of Emerging Technology and Advanced Engineering, vol. 3, pp. 24–23, 2013. [13] S. AGBLI, “Modélisation multiphysique des flux énergétiques d’un couplage photovoltaïque-électrolyseur PEM–Pile à Combustible PEM en vue d’une application stationnaire,” Ph. D. thesis, University of Franche comté 2012. REFERENCES [23] A. Ruzmetov, A Nait-Sidi-Moh, M. Bakhouya, and J Gaber, “A (max - plus)-based approach for charging management of electric vehicles,” In Proc. of the 2nd World Conference on Complex Systems, Agadir, Morocco. Nov. 2014. [1] M. G. Egan, D. L. O’Sullivan, J. G. Hayes, M.J. Willers, and C.P. Henze, “Power-factor-corrected single-stage inductive charger for electric vehicle batteries,” IEEE Trans. on Industrial Electronics, vol. 54, no. 2, pp. 1217–1226, 2007. [2] Z. P. Wang, P. Liu, H. B. Han, C. Lu, and T. Xin, “ Adistribution model of electric vehicle charging station,” Applied Mechanics and Materials, 44–47, pp. 1543–1548, 2011. Rania, Mkahl is a PhD student working at the University of Technology Belfort-Montbéliard (UTBM), Belfort, France. Her PhD thesis is part of research collaboration between the UTBM and the University of Picardie Jules Verne (UPJV) – INSSET, Saint Quentin, France. She received her Master’s degree in Electrical Engineering from the Ecole Centrale of Lyon, France in 2010. Prior to beginning the Master and PhD programs, she worked as assistant professor at the Tishreen University, Syria. Her main research interests are modeling, control and management of energy flows H2V and V2H, charging process of electric vehicles, renewable energy for EV charging, decision-making, simulation and operations research. [3] A. Ruzmetov, A. Nait Sidi Moh, M. Bakhouya, J. Gaber, and M.Wack, “Optimal assignment and charging scheduling of electric vehicles,” IEEE International Renewable and Sustainable Energy Conference (IRSEC'13) Ouarzazate, Morocco, 10.1109/IRSEC.2013.6529691, pp. 537 –541, 2013. [4] J. Song, Amir Toliyat, Dave Tuttle, and Alexis Kwasinski, “A Rapid Charging Station with an Ultracapacitor Energy Storage System for Plug-In Electrical Vehicles,” Electrical Machines and Systems (ICEMS), 2010, International Conference on, pp. 2003-2007, 2010. [5] J. Baker, “New technology and possible advances in energy storage,” Energy Policy, vol. 36, pp. 4368–4373, 2008. Ahmed, Nait-Sidi-Moh received his Ph.D. degree in 2003 in Computer Engineering and Automatic Control from the University of Technology of Belfort Montbéliard (UTBM), France. He is currently an Associate Professor of Computer Engineering at the University of Picardie Jules Verne (UPJV). Prior to joining the UPJV, he was research scientist and lecturer at the UTBM. [6] A. Boucherit, “ Conception d’un convertisseur de puissance pour les véhicules électriques multi-sources,” Ph. D thesis of University Technologie de Belfort –Montbéliard, 2011. g [7] J. P. Trovão, P. G. Pereirinha, and H. M. B. Simulation Results of Battery Lead-Acid 20 Li-Ion batteries charge from Lead-Acid batteries using the combined charge cycle scholar.waset.org/1307-6892/10000396 International Scholarly and Scientific Research & Innovation 9(1) 2015 78 World Academy of Science, Engineering and Technology International Journal of Computer and Information Engineering Vol:9, No:1, 2015 VI. CONCLUSION Mkahl, “Design and sizing of a stand-alone recharging point for battery electrical vehicles using photovoltaic energy,” IEEE Vehicle Power and Propulsion Conference, 10.1109/VPPC.2011.6043075, pp. 1–6, 2011. World Academy of Science, Engineering and Technology International Journal of Computer and Information Engineering Vol:9, No:1, 2015 EACEA Erasmus Mundus project TARGET (Transfer of Appropriate Requirements for Global Education and Technology), 2011-2014. His research interests include information systems, security, digital signature and certification, ubiquitous and pervasive computing, location based services and distributed systems, energy. scholar.waset.org/1307-6892/10000396 International Scholarly and Scientific Research & Innovation 9(1) 2015 REFERENCES He was Co-PI of two European projects ASSET (Advanced Safety and Driver Support in Efficient Road Transport, FP7-SST, 2008-2011, and TELEFOT (Field Operational Tests of Aftermarket and Nomadic Devises in Vehicles, FP7-ICT, 2008-2012. He is also a member (UTBM side) of EU [14] N. Achaibou, M. Haddadi, and A. Malek, “Modelling of lead acid batteries in PV systems,” Siencedirecte, Energy Procedia, Clean Energy scholar.waset.org/1307-6892/10000396 International Scholarly and Scientific Research & Innovation 9(1) 2015 79 International Science Index, Computer and Information Engineering Vol:9, No:1, 2015 waset.org/Publication/10000396 ce Index, Computer and Information Engineering Vol:9, No:1, 2015 waset.org/Publication/10000396 International Science Index, Computer and Information Engineering Vol:9, No:1, 2015 waset.org/Publication/10000396 International Science Index, Computer and Information Engineering Vol:9, No:1, 2015 waset.org/Publica scholar.waset.org/1307-6892/10000396 International Scholarly and Scientific Research & Innovation 9(1) 2015 80
https://openalex.org/W2113199572	https://www.ajol.info/index.php/lex/article/download/49172/35515	English	null	Target Users' Expectations versus the Actual Compilation of a Shona Children's Dictionary	Lexikos	2,009	cc-by	6,784	Target Users' Expectations versus the Actual Compilation of a Shona Children's Dictionary Esau Mangoya, African Languages Research Institute, University of Zimbabwe, Harare, Zimbabwe (emangoya@arts.uz.ac.zw) Esau Mangoya, African Languages Research Institute, University of Zimbabwe, Harare, Zimbabwe (emangoya@arts.uz.ac.zw) Abstract: The article discusses the challenges that confronted the team of compilers working on the monolingual Shona Children's Dictionary (henceforth SCD). It looks at the active involve- ment of the target users in shaping the project and discusses the considerations for the implemen- tation of their recommendations. Matters of concern include issues of headword selection, espe- cially problems of dialect representation in the dictionary. The article also discusses the inclusion of grammatical information such as tone marking. The SCD is one of the many efforts to promote and raise the functions of the indigenous languages in Zimbabwe. Of special interest in this article is therefore the discussion on the inclusion of international words like those denoting weight, dis- tance and mathematical and scientific concepts. The article also considers the treatment of taboo words in the SCD, which takes into account that society has to open up culturally to be able to unequivocally view some of the aspects of life with which present-day children are confronted. The article lastly discusses how a balance was reached for accommodating the target users' proposal for the inclusion of an elaborate back matter in view of the limited available scope and space of the SCD. Introduction A team of three people at the University of Zimbabwe's African Languages Research Institute (ALRI) embarked on a pioneering project to compile a monolingual Shona Children's Dictionary (henceforth SCD) whose main target users are school children at primary level. This team worked closely with teachers and parents who automatically became an interested party in the dic- tionary project, consequently constituting the majority of the target users. The project started in 2002 and the dictionary was supposed to be published in 2006. However, there were technical hitches which saw the draft of the diction- ary being submitted to the publishers only in 2009. y g p y In Zimbabwe, a culture of dictionary compilation and use has not been thoroughly developed. Consequently, most of the lexicographic products in the Zimbabwean context come as pioneering projects. In societies with a long his- tory of lexicography, it is easy to identify the gaps that a newly initiated dic- tionary project is meant to fill within the context of other earlier and previously published dictionary products. In such situations, both the compiler and the target users have a common goal and expectation. The target users' needs are easy to identify and satisfy since the compiler will not be starting a completely new process. However, despite the varying needs of the target users and the difference in the challenges faced by compilers working on different dictionary projects, the target users' needs have to remain a priority. Hartmann (2001: 80) says: "Ultimately all dictionaries are motivated by and judged against the lexi- cal needs of those who consult them." Emphasis is always laid on the need to address the target users' expecta- tions. Gouws (1993: 29) says: "It is an accepted fact that every dictionary has to be compiled in accordance with specific needs and demands of a well-defined target user." Here the task for the compiler is clearly defined. The parameters for the production of a "perfect" dictionary are delineated. The expectations are that a dictionary must have authority, scholarship and precision (Landau 2001: 6). At the same time, it has to be considered that their expectations differ for various reasons, including the linguistic and the socio-cultural nature of the communities for which the dictionaries are compiled. As a result, each diction- ary project may face problems that remain peculiar to it. Keywords: TARGET USERS, DIALECT REPRESENTATION, INTERNATIONAL WORDS, TABOO WORDS, TONE MARKING, BACK MATTER, DICTIONARY, LEXICOGRAPHY Keywords: TARGET USERS, DIALECT REPRESENTATION, INTERNATIONAL WORDS, TABOO WORDS, TONE MARKING, BACK MATTER, DICTIONARY, LEXICOGRAPHY Opsomming: Teikengebruikers se verwagtinge teenoor die werklike same- stelling van 'n Sjonakinderwoordeboek. Die artikel bespreek die uitdagings waarvoor die span samestellers te staan gekom het tydens werk aan die eentalige Sjonakinderwoordeboek (voortaan SKW). Dit kyk na die aktiewe betrokkenheid van die teikengebruikers by die ontwikke- ling van die projek en bespreek die oorwegings vir die uitvoering van hulle aanbevelings. Sake van belang sluit in kwessies van trefwoordkeuse, veral probleme van dialekverteenwoordiging in die woordeboek. Die artikel bespreek ook die insluiting van grammatiese inligting soos toonaandui- ding. Die SKW is een van die baie pogings om die funksies van die inheemse tale in Zimbabwe te bevorder en te vergroot. Van spesiale belang in hierdie artikel is dus die bespreking oor die inslui- ting van internasionale woorde soos dié wat gewig, afstand en wiskundige en wetenskaplike kon- septe benoem. Die artikel beskou ook die behandeling van taboewoorde in die SKW wat in aan- merking neem dat die gemeenskap kultureel oper moet word om in staat te wees om sommige aspekte van die lewe waarvoor hedendaagse kinders te staan kom, onomwonde te beskou. Laas- tens bespreek die artikel hoe 'n balans verkry is om die teikengebruikers se voorstel vir die inslui- ting van 'n uitvoerige agterwerk in te pas in ooreenstemming met die beperkte beskikbare omvang en ruimte van die SKW. Lexikos 19 Supplement (AFRILEX-reeks/series 19: 2009): 120-133 121 The Compilation of a Shona Children's Dictionary Dialectal Representation Shona has five major dialects namely: Karanga, Zezuru, Ndau, Manyika and Korekore. What the research team gathered from the outreach programmes and workshops was that in the past there has been preferential treatment of the Zezuru and Karanga dialects in the Shona resource books used at primary school level. These two dialects have minimal differences compared to the rest of the dialects. This tended to disadvantage pupils who speak other dialects and it was alleged that there was a high failure rate in Shona examinations among them. Zezuru and Karanga which are more central to the whole Shona- speaking community are inclined to dominate and the rest of the dialects play a peripheral role. The feeling was that children particularly from Ndau- and Korekore-speaking areas fail to comprehend the vocabulary from the central dialects. Manyika figured less prominently in the debate as it forms a buffer dialect that is in contact with the central dialects and Ndau and Korekore. Doke (1931: 81) laid down the basic principle that was to be followed in the compilation of Shona dictionaries. He recommended that if a Shona dic- tionary should be compiled, it has to be as inclusive as possible of words from Zezuru, Karanga, Manyika and Ndau, but for the time being Korekore words have to be admitted sparingly. On the contrary, earlier in his report, on page 9, he shows reservations about the inclusion of Ndau words when he says: "Into their vocabulary is quite a noticeable infiltration of Zulu following the conquest of Gungunhana." This gives the impression that from the time of the defeat of the Ndau, their dialect is no longer pure. As a result Ndau and Korekore can- not without reservations be seriously considered as dialects of Shona. Today the speakers of these dialects are fighting to have their dialects recognized as belonging to the Shona language. A new environmental situation has given them reason to clamour for recognition of their dialects. This is in view of the recommendations by the National Language Advisory Panel of 1998. In the report, Chimhundu et al. Introduction The SCD team con- ducted outreach programmes and workshops with the aim of determining the actual needs and expectations of the target users. Primary school teachers, pri- mary school children and interested and knowledgeable parents were con- sulted during these programmes and workshops. This article discusses the challenges that the team compiling the SCD faced in view of the recommenda- tions given by the target users. The problematic areas that the team had to deal 122 Esau Mangoya with are mainly dialectal representation, tone marking for the purpose of pro- nunciation, inclusion of international words, the treatment of words deemed culturally taboo and the setting of the back matter. Dialectal Representation (1998: 36) say: "Use of indigenous/local languages should be made mandatory in schools firstly as subjects and later as the medium of instruction." Although Korekore and Ndau are dialects of Shona, their clamour for recognition is similar to that of some Zimbabwean indigenous languages which ought also, according to the report, to be granted the same right as the other languages. The SCD team had to do strategic research in order to cover the so-called neglected dialects as much as possible. After going through the usual head- word selection process in which student research assistants were involved, the team printed a list of the collected headwords. This also saw a substantial number of headwords from these neglected dialects appearing. The next stage 123 The Compilation of a Shona Children's Dictionary was to engage the target users, comprising teachers, children and parents. The group of teachers that was engaged firstly had to be speakers of one of these neglected dialects, and secondly had to be teaching in schools in areas where these dialects are spoken. These teachers had the advantage of being speakers and practitioners who were knowledgeable about the problems their pupils face when learning Shona as a subject. g j In this way, an additional list of headwords was collected. However, at the same time this strategy also encountered its own problems. As is always the case with groups that feel excluded, there is a tendency to be emotional about their dialects and languages, trying to be as inclusive and exhaustive as possi- ble. Therefore, because of this, a problem surfaced with regard to the many synonymous headwords from the Ndau and Korekore dialects in comparison with the single headwords from the central dialects as shown in (1) and (2) below. Dialectal Representation (1) central dialects headword headwords from Ndau chikuvauro chikwa, chichayo chiroo (wooden tool used for flattening the floor) guvhu chikuvhu, chikumvu, mukuvhu (navel) (2) central dialects headword headwords from Korekore gangamwiwe fengu, topo, mupagada (type of inedible pumpkin) gangemukange madzimba, majochwe (type of ant) (1) central dialects headword headwords from Ndau chikuvauro chikwa, chichayo chiroo (wooden tool used for flattening the floor) guvhu chikuvhu, chikumvu, mukuvhu (navel) (2) central dialects headword headwords from Korekore gangamwiwe fengu, topo, mupagada (type of inedible pumpkin) gangemukange madzimba, majochwe (type of ant) headwords from Ndau chikwa, chichayo chiroo (wooden tool used for flattening the floor) chikuvhu, chikumvu, mukuvhu (navel) headwords from Ndau chikwa, chichayo chiroo (wooden tool used for flattening the floor) chikuvhu, chikumvu, mukuvhu (navel) (2) central dialects headword gangamwiwe This automatically implies that lexical items from those regions are discriminated against when it comes to entering them into the dictionary. This is a feature not peculiar to the Zimbabwean situation only. In general lexicographic works elsewhere, it has also been pointed out that dialects are considered substandard if not plainly incorrect (Landau 2001: 220). The assertion is showing that dialect issues are always problematic. Wherever they feature, they present emotional sentiments. In the Korekore and Ndau situation, their exclusion or selective inclusion leads to protest by speakers who see it as an injustice. y To omit or partly omit some dialect forms such as those from Korekore and Ndau as suggested by Doke, should serve as a warning not to disregard peripheral dialects. However, sidelined dialects will always have their head- words not well recognised in other Shona-speaking areas. As a result, words from these dialects are not quite as familiar to many of the compilers compared to items from the central dialects. The forms of these recognised dialects are always the first to be included in the dictionaries. The central varieties also enjoy being used as the standard, their vocabulary featuring prominently in literary publications which are normally the source for headwords in the dic- tionaries. Consequently, Korekore and Ndau dialects have always been side- lined in Shona publications including dictionaries that are purported to be comprehensive. p There has to be a policy for handling the dialectal items. As one way of standardising Shona, Doke (1931: 81) recommended that a dictionary has to be a collection of headwords gathered from the different dialects. This has how- ever been found to be a too simplistic way of dealing with the persistent ques- tion of unfair treatment of dialects in Shona dictionaries. With every publica- tion there have been allegations by speakers of other dialects that there were no headwords included from their dialects. These groups that maintain that their dialects are not being considered in written Shona also claim that the pass rate in Shona examinations in those areas where Ndau and Korekore are spoken are perpetually low because pupils are always made to study texts representing the central dialects. This is the major reason why target users from those areas were engaged as a reference group. (2) central dialects headword gangamwiwe headwords from Korekore fengu, topo, mupagada (type of inedible pumpkin) madzimba, majochwe (type of ant) headwords from Korekore fengu, topo, mupagada (type of inedible pumpkin) madzimba, majochwe (type of ant) There came plenty of headwords from the excited group of target users who had received the opportunity to show the linguistic ability of their dialects. This resulted in a variety of synonyms and variants to single headwords from the central dialects. This obviously was a strenuous effort by the speakers to try to prove as much as possible that their dialects have plenty of words that are always omit- ted. This great number of words posed yet another problem to the dictionary compilers because not all the headwords that were given would be of complete equivalence to the headwords from the central dialects and even amongst the presumed peripheral dialects themselves. This would result in a situation whereby words from the presumed peripheral dialects dominate at the expense of the central dialects. The other problem was how to determine the level of synonymy the terms from the peripheral dialects have among themselves and to those from the central dialects. It seems as if these dialects have a higher level of term productivity than the central dialects. This touches on the sociolinguistic situation of the people in Zimbabwe. Theirs is a reaction to the colonial history in which the administrative demar- cations have become tribal demarcations as well. Dialects become a feature of speech that serves to identify the speaker as a member of a particular speech community, past or present (Landau 2001: 220). This provides the speakers of 124 Esau Mangoya dialects with arguments as they protest at being deliberately excluded where the rule and policy is that the local and indigenous languages have to be empowered without discrimination. The empowerment of languages, as given in the Advisory Panel Report, also means all the dialects have to enjoy equal status. After independence, the policy has been set as a goal to empower the indigenous languages which have always come second to English. The policy sounds all-embracing, implying that there has to be equal treatment of the indigenous languages and implicitly their dialectal varieties. As pointed out by Landau (2001: 219), forms of expression and pronunciation limited to a par- ticular region have long been regarded as objects of contempt and ridicule. (4) central dialects headword gangamwiwe (4) central dialects headword gangamwiwe gangemukange headwords from Korekore (3) fengu, (2) opo, (1) mupagada (type of inedible pumpkin) (1) madzimba, (2) jochwe (type of ant) gangemukange This ranking was helpful in eliminating headwords from Korekore and Ndau as all those indicated as first were also considered for entry into the dictionary. Tone marking Tone is a fundamental aspect of language. It refers to the distinctive pitch level of a syllable (Crystal 1983: 353). In Shona, the tone contrasts of syllables in words are vital in bringing out the meaning of words. Indicating the tone of words in a dictionary is also important in demonstrating the way words are pronounced. The issue of tone marking became problematic when a recom- mendation against its inclusion was suggested by the prospective target users during the workshops and outreach programmes. As compilers become well acquainted with dictionary making, some of the practices become routine, usu- ally laid down as standard. ALRI's first two monolingual dictionaries Dura- mazwi reChiShona (1996) and the advanced Duramazwi Guru reChiShona (2001) did not only include tone markings but went a step further by coining the Shona terms for high and low tone. These are K from kwidza (raise) for high tone and D from dzikisa (lower) for low tone, as shown in example (5) of the headword akapero (acapella music). 5) akapero DDKD z 9 (2) central dialects headword gangamwiwe However, with the abundance of head- words coming from these target users, some of them had to be eliminated to balance the dialect ratio in the dictionary. The target users were asked to rank 125 The Compilation of a Shona Children's Dictionary the Ndau and Korekore words they had provided in order of preference based on their knowledge of the precise equivalence of these words with the head- words from the central dialects. Eventually the ordering of the above examples was reached as shown under (3) and (4). (3) central dialects headword headwords from Ndau chikuvauro (2) chikwa, (3) chichayo, (1) chiroo (wooden tool used for flattening the floor), guvhu (1) chikuvhu, (3) chikumvu, (2) mukuvhu (navel) (3) central dialects headword chikuvauro guvhu headwords from Ndau (2) chikwa, (3) chichayo, (1) chiroo (wooden tool used for flattening the floor), (1) chikuvhu, (3) chikumvu, (2) mukuvhu (navel) (5) akapero DDKD z 9 (5) (5) The headword has four syllables a+ka+pe+ro. D stands for low tone and K for high tone. So the first two syllables a and ka marked by DD are low tones, the third syllable pe marked by K is high tone and the last one ro marked again by D is low tone. After the tone comes other vital information like z from zita (noun) which is of class 9, then followed by the definition. In Shona lexicogra- phy, tone markings are relatively new grammatical coinages that are being 126 Esau Mangoya standardised not only in the SCD, but also in other ongoing projects that deal with Shona language and grammar. standardised not only in the SCD, but also in other ongoing projects that deal with Shona language and grammar. The target users' position can be understood. The introduction of new elements such as tone marking which gives specialised grammatical informa- tion vital to the pronunciation of words is mostly regarded with reservation. The main question arose whether the SCD team had to include information which did not interest the target users. On the other hand it can also be asked if tone marking should be omitted simply because some target users are not fully acquainted with its use. The problem was complicated by the fact that the speakers of the central dialects needed assistance in the pronunciation of the many new headwords from Ndau and Korekore as much as the speakers of these peripheral dialects needed to acquaint themselves with the tone of the headwords from the central dialects. As a result, the inclusion of tone was considered helpful to users when pronouncing words. It may be argued that children have not yet been exposed to these complicated grammatical aspects such as tone. However, this informa- tion may assist the teacher working with the children. A dictionary that is inclusive has an added advantage to those who might need that extra informa- tion. The teacher can help pupils extract what is relevant to them. At the same time the availability of a dictionary which has extra details leaves the teacher assisting the pupils with additional information although the pupils may not use it themselves. This does not mean that the suggestions by the target users had to be disregarded completely, but the merit of such suggestions had to be reviewed as well. (5) As a reference work, a dictionary has many functions. The SCD team agreed that there was need to have the tone marked in the data base and again recommended the idea of indicating tone in the dictionary to the working groups consisting of target users. The admissibility of tone markings in the dictionary were again assessed in the sample headwords and definitions which were taken to the target users during the outreach and seminar programmes. When the same aspects are again brought up for discussion and viewed in print the target users are normally forthcoming with suggestions for improve- ment. Tone marking was eventually accepted as an essential tool in the diction- ary. International Words What have come to be regarded as international words are those ones that have originated in particular languages but are now used across the world in almost every language. At this stage, they have not been adopted in the Shona lan- guage yet but because of their specialised sphere of use are always mentioned in Shona texts. Examples of these are words denoting weight, length and sci- entific and mathematical terms, as demonstrated in the examples under (6). 127 The Compilation of a Shona Children's Dictionary (6) g g length: centimetre, metre, kilometre These and many others are recognised standard terms in many languages. The initial view on the possible inclusion of these terms in the SCD was purist in nature. As they were considered foreign, they were seen as unsuitable for a dic- tionary meant to strengthen children's communication skills in the Shona lan- guage. However, because the purpose of compiling the dictionary was to empower the Shona language, brought some change in perception. The lan- guage has to be developed so that it would be able to describe scientific con- cepts. The target users did not accept the idea of outright adoption but opted for the coinage of "pure" Shona terms. A precedence has already been set before when coinage was attempted in Hannan's (1981) Standard Shona Diction- ary which yielded words like dutavanhu (bus), hambautare (motorcar) and bhi- zautare (bicycle) in an attempt to avoid using English terms. Naturally, when people speak they say bhazi (bus), motokari (motorcar) and bhasikoro (bicycle) which are all adoptives. People hardly use the once prescribed coinages. p p y p g Being aware of the fate of some coinages in Hannan's dictionary, the SCD team of compilers argued against their inclusion. These coinages would end up as redundant entries in the dictionary and the terminological gap they were meant to fill would remain open. If its natural behaviour is considered, a lan- guage gives preference to adoptives that are close to the source language in spelling and pronunciation. However, there was the difficulty that some of the borrowed headwords had to be adapted to suit the Shona spelling system. Taboo words Another category of words that needed special attention was taboo words. Taboo words are known to speakers but avoided in some, most or all forms or contexts of speech (Matthews 1997: 371). Considerations regarding taboo words can be culture-bound. Culture, however, changes, causing the taboo tag given to some lexical elements to be removed partly or wholly. It is important for dictionary compilers to fully know and understand the feelings of the target users on some important aspects of the dictionary they compile. The issue becomes much more complicated when questions arise on whether some aspects are culturally acceptable to be entered into the dictionary. The diction- ary compiler has to have a full understanding of the users whom the compilers have sometimes ignored so that there was little appreciation for some diction- ary products. The position of target users has to be ascertained in order to avoid criticism of the dictionary product. About the dictionary user, Hartmann (2001: 80) says: "We still know very little about the dictionary user whom sev- eral authorities have called the familiar stranger, presumably because we take him for granted without bothering further." g g Hartmann's assertion assumes that the participation of the target users in making their recommendations towards the contents of a dictionary may help enhance the lexicographer's insights leading to a lexicographic product that answers the user's needs. The question that has to be answered is whether the shape of a dictionary should be completely determined by the target users. Does the compiler have some authority and power to incorporate new chal- lenges that can help change the society's view of the world? Such challenges were encountered in the compilation of the SCD with regard to taboo words. The target users, particularly the teachers and parents, were clear and adamant that human private parts and some physiological terms would not be accept- able in a dictionary meant for children. According to their view, these children are at the critical stage where there has to be a total induction into their culture. Part of the culture forbids having access to knowledge of these taboo words, particularly by primary school children. The refusal to enter headwords deemed to be breaching the ethical code of the society is not peculiar to the Zimbabwean situation. International Words The examples of the international words above were entered as shown under (7): (7) weight: kirogiramu, tani length: sendimita, mita, kiromita scientific and mathematical terms: atomu, ikwezheni, ikweta (7) weight: kirogiramu, tani length: sendimita, mita, kiromita scientific and mathematical terms: atomu, ikwezheni, ikweta The Shona alphabet does not have the letter l but the trend has been that it can be substituted by r, hence the change from the English term kilogramme to kiro- giramu. In Shona, the vowel o cannot be realised as /a/ as in the case of the English word tone in which the first syllable sounds like /ta/. The new adop- tive for tone is now /tani/. Similarly the initial vowel e in the word equator is realised as /i/, therefore the change to /ikweta/. All syllables in Shona are marked by a vowel, hence the change of the word mitre to /mita/ where it now has the syllables /mi/ and /ta/. The adoptives had to be adapted to the Shona writing system. This was found to be an important step towards empowering the Shona language, par- ticularly in a book targeted at a group that is at the foundation level of the edu- cation system. This will give the target users the confidence to use the language in fields where other languages such as English have always dominated. The target users' creative abilities will be enhanced. This will broaden their view of 128 Esau Mangoya the world in which they, using their mother tongue, can be active participants. The belief is that what has already been initiated will continue and eventually the Shona language will not be void of such important terminology. Taboo words All communities at the initial stages of their lexicographic tradition seem to follow the trend of reser- vations on the inclusion of what is deemed taboo (see Landau 2001 : 228). There is the feeling that the exclusion of taboo words contributes towards the up- holding of ethical values. Because the situation of the SCD was found to be rather unique, it brought 129 The Compilation of a Shona Children's Dictionary a number of challenges. Firstly, by aiming primary school children as target users, the dictionary belongs to a type of its own in comparison to the earlier publications Duramazwi reChiShona (1996), targeted at ordinary level students and Duramazwi Guru reChiShona (2001), targeted at college and university level students. Secondly, there are new challenges not only to the Zimbabwean soci- ety but to societies worldwide that need to be addressed to this age group as a matter of urgency. One channel of doing so is through books such as the SCD. Because the world is changing fast, what society once might have strongly con- sidered taboo might need to be reconsidered in today's world. One of the big- gest challenges that have besieged the world is the advent of HIV and AIDS. There is also the problem of the early indulgence in sexual intercourse by today's children and the dangers that go with such activities. There is the ram- pant problem of child abuse and rape. These and other challenges have re- sulted in the need to teach and inform children as a precautionary measure about the dangers of the various sexually transmitted diseases and the eventual consequences. All these problems cannot be fully understood if discussed in unclear and veiled language. In Zimbabwe, the language situation is such that English has been ac- corded the official status and Shona has always come second to English. This has led to a culture in which anything presumed to contain difficult and for- eign concepts should be explained in English. Everything is left to feature in the English texts to which the majority of school-going children eventually have access. However, since English in Zimbabwe is learnt as a second lan- guage, there is little consideration at what time or stage in the children's edu- cational development, they will be able to fully comprehend English. This has consequently led to the exclusion and avoidance of vital terms in Shona text- books. Taboo words The SCD was caught up in a similar dilemma. As a result, the teachers who have become accustomed to this culture over the years oppose the inclu- sion of these taboo words in a children's dictionary, their argument being that it will be antisocial to expose children to such terminology. After serious discussions, it was agreed that some of these presumed taboo words had to be included as long as their role was to give information found to be vital to the children. These are terms specifying the body parts, describing the signs and symptoms of venereal diseases and naming some sex- ual activities which cannot be avoided mentioning when teaching the children. Examples of such headwords are listed under (8). mhata (anus) mheche (vagina) mboro (penis) maronyo (sperm/semen) siki (venereal disease) njovhera (type of venereal disease) -svira (have sexual intercourse) (8) 130 Esau Mangoya These were allowed into the dictionary because of needs and challenges. How- ever, despite their relatedness to the words under (8), there were some reserva- tions on the inclusion of another group of words which were deemed unneces- sary for the level of primary school children. These are common taboo words describing the nature of certain sexual organs or the manner of some sexual ac- tivities. Examples of words about which teachers had reservations are given under (9). (9) beche (big vagina) zimboro (big penis) nzvonyo (circumcised penis) mukodo (sexual intercourse from the back) mufongo (sexual intercourse from the back) (9) beche (big vagina) zimboro (big penis) nzvonyo (circumcised penis) mukodo (sexual intercourse from the back) mufongo (sexual intercourse from the back) (9) beche (big vagina) zimboro (big penis) nzvonyo (circumcised penis) mukodo (sexual intercourse from the back) mufongo (sexual intercourse from the back) (9) beche (big vagina) zimboro (big penis) nzvonyo (circumcised penis) mukodo (sexual intercourse from the back) mufongo (sexual intercourse from the back) (9) It was agreed that specimens of normal use should be given for nouns. It was felt that including words meant to give an obscure or indistinct explanation of the mentioned organs and activities would actually destroy the main aim of teaching, and could create confusion and uncertainty in the children that will in reality be contrary to the aim and purpose of the words' inclusion. So the taboo words would have to be selectively entered into the dictionary. Taboo words Inclusion of some of the vital words will assist in the newly introduced subjects on HIV and AIDS. The children are also being taught issues of human rights in which child sexual abuse would be discussed. Children should know what abuse is and should be able to describe it if the need arises. Because teachers and parents should be able to talk to the children, the vocabulary that was considered to be taboo has to be made normal and familiar by including it in a general diction- ary such as the SCD. Back Matter One of the main concerns of a dictionary is to give comprehensive information. A dictionary of the SCD type has to contain information on issues children might not have had a true experience of and direct exposure to in their lives. For example, a child born and bred in town might not be fully aware of aspects that are predominantly rural and vice versa. As a result the dictionary is expected to play an informative role with many aspects reserved for the back matter. As has been pointed out, the SCD project generated much interest among teachers who played a major role as a reference group advising the compiling team. There was also much interest from the community at large who also felt the dictionary would be a useful resource for their children, par- ticularly when writing the Grade 7 Shona examinations. These are vital exami- nations which need to be passed before children can proceed to secondary level. There have always been complaints from teachers and parents about the 131 The Compilation of a Shona Children's Dictionary lack of resource materials for the Shona examinations at primary school level. The idea was to try as much as possible to provide for the different sections of the examinations usually written at this level. So the SCD project was consid- ered to be a major step in compensating for this lack. This resulted in a number of suggestions on additions to be made to the dictionary over and above the general headwords and their definitions. All the additional information could be placed in the dictionary as back matter. It was suggested that this back mat- ter could include a list of people and their professions, a list of animals and their young ones, illustrations of kitchen utensils, a list of common proverbs and idioms and a list of kinship terms. While this would generally be a recom- mendable idea, it had its own limitations. An example of how this was to be done is shown under (10). Conclusion It is every dictionary compiler's wish to produce a dictionary that fully makes provision for the needs of target users. As a result, it is necessary to consult its prospective target users. The SCD project involved them to a great extent. However, each dictionary project faces challenges of a general nature and at times some which are peculiar to it. This article has discussed those which are peculiar to the SCD, starting with the nature of the target user group which mostly comprised teachers and parents, despite the fact that the real target users are children. Children as a group have to be guided, hence they are mostly represented by the teachers and parents so that their own contribution is minimal. The article has discussed the discrepancies that in the past occurred in the treatment of Shona dialects. It has looked at the pressure exerted by the speak- ers of the neglected dialects, showing how the new scenario has impacted on the SCD project, including the problem of excessive headwords that had to be reduced to bring about a balance with other dialects. Dictionary making is also important in introducing and standardising new aspects for the benefit of tar- get users. As a result, the SCD tried to uphold the new phenomenon of mark- ing the tone, which is vital for the pronunciation of headwords. Although tar- get users are not yet comfortable with tone marking, it is hoped that once introduced and maintained as a lexicographic tradition, target users will soon be familiar with it. This being a pioneering project of a unique nature, it had to confront new challenges. As a result it included some adopted international words. This is an attempt to bring Shona on the same level of communicative ability with other developed languages so that foreign languages need not to be used in discus- sions dealing with foreign concepts. As the world is changing, this also de- mands cultural change. Therefore, the once considered taboo words found their way into the SCD. The aim is to enable communication with the children, so resources such as the SCD must have a high information level. For this rea- son, some additional carefully selected material has been added as back matter to the general dictionary, at the same time taking limitations of space and detail into account. (10) Tete ihanzvadzi yababa (One's aunt is one's father's sister) Tezvara ndibaba vomudzinai (One's father-in-law is one's wife's father) (10) Tete ihanzvadzi yababa (One's aunt is one's father's sister) Tezvara ndibaba vomudzinai (One's father-in-law is one's wife's father) From the first example, the words tete (aunt), hanzvadzi (sister) and baba (father) would have been entered as headwords in the dictionary. Such is also the case with words like tezvara (father-in-law), baba (father) and mudzimai (wife) from the second example. They again appear as repeat entries in the back matter where they, despite being combined in a new context, are in fact a duplication within the same dictionary. Such is also the case with illustrations and all the aspects that were suggested for inclusion in the back matter. A careful selection had to be made to include only those that would bring extra information beyond what could already be found in the headword section. An example of a topographical illustration that was chosen, is a mountain. The illustration would then show gomo (mountain), mawere (cliff), mupata (valley) and bako (cave). The lexicographer is always contending with the issue of space in the dic- tionary. At the same time, it has to be understood that the dictionary has its limitations as a reference book. It cannot be the ultimate solution to everything related to examinations. Moreover, as this is a general and not an encyclopae- dic dictionary, it was found to be difficult to include all the suggested aspects in the back matter limited by size and detail. Eventually it was resolved that the dictionary could include trimmed lists. However, the compilers had to be careful not to outrightly refuse special requests from the reference group which forms a major component of the target users. As a compromise it was agreed that some of the requests would actually be better incorporated in school text books. A dictionary was only meant to ad- dress problems that usually arise out of the subject matter of text books. It was not meant to provide solutions for these problems and at the same time func- tion as a text book which addresses all aspects of Shona as a school subject. It was hoped that the compilation of the SCD would help generate interest by other relevant participants to write Shona books for primary school level. 132 Esau Mangoya 132 Esau Mangoya Conclusion Challenges occur in dictionary making but they can be discussed and overcome in some way or other. Chimhundu, H. et al. 1998. Report on the Formulation of a National Language Policy. Harare: Ministry of Sport, Recreation and Culture. Chimhundu, H. (Ed.). 1996. Duramazwi reChiShona. Harare: College Press. Chimhundu, H. (Ed.). 2001. Duramazwi Guru reChiShona. Harare: College Press. Doke, C. 1931. Report on the Unification of Shona Dialects Carried Out under the Auspices of the Govern- ment of Southern Rhodesia and the Carnegie Corporation. Hertford: Stephen Austin. Crystal, D. 1991. A Dictionary of Linguistics and Phonetics. Third Edition. Cambridge: Basil Black- well. Hartmann, R.R.K. 2001. Teaching and Researching Lexicography. London: Longman. Hannan, M. 1959. Standard Shona Dictionary. Salisbury: The College Press. Landau, S.I. 2001. Dictionaries: The Art and Craft of Lexicography. Second Edition. Cambridge: Cam- bridge University Press. Crystal, D. 1991. A Dictionary of Linguistics and Phonetics. Third Edition. Cambridge: Basil Black- well. Gouws, R.H. 1993. Afrikaans Learner's Dictionaries for a Multilingual South Africa. Lexikos 3: 29- 66. Hannan, M. 1959. Standard Shona Dictionary. Salisbury: The College Press. Hartmann, R.R.K. 2001. Teaching and Researching Lexicography. London: Longman. Landau, S.I. 2001. Dictionaries: The Art and Craft of Lexicography. Second Edition. Cambridge: Cam- bridge University Press. Matthews, P.H. 1997. Oxford Concise Dictionary of Linguistics. Oxford: Oxford University Press. Mpofu, N. et al. (Eds.). 2004. Duramazwi reUrapi neUtano. Gweru: Mambo Press. References 133 The Compilation of a Shona Children's Dictionary Crystal, D. 1991. A Dictionary of Linguistics and Phonetics. Third Edition. Cambridge: Basil Black- well. Gouws, R.H. 1993. Afrikaans Learner's Dictionaries for a Multilingual South Africa. Lexikos 3: 29- 66. Hannan, M. 1959. Standard Shona Dictionary. Salisbury: The College Press. Hartmann, R.R.K. 2001. Teaching and Researching Lexicography. London: Longman. Landau, S.I. 2001. Dictionaries: The Art and Craft of Lexicography. Second Edition. Cambridge: Cam- bridge University Press. Matthews, P.H. 1997. Oxford Concise Dictionary of Linguistics. Oxford: Oxford University Press.
https://openalex.org/W2305302216	https://iris.uniroma1.it/bitstream/11573/904109/1/Midulla_Paediatrics%20messages%20from_2015.pdf	English	null	Paediatrics: messages from Munich	ERJ open research	2,015	cc-by	16,144	MESSAGES FROM MUNICH PAEDIATRICS MESSAGES FROM MUNICH PAEDIATRICS MESSAGES FROM MUNICH PAEDIATRICS @ERSpublications Paediatric highlights from the 2014 ERS International Congress in Munich, Germany http://ow.ly/MaXZv aed a c esp a Forced oscillations Several researchers have investigated how to apply the forced oscillation technique (FOT) clinically and previous publications have provided technical recommendations for adults and children [2, 3]. In a study designed to evaluate lung function using impulse oscillometry (IOS) and spirometry, UYAN et al. [4] followed 41 children with post-infectious bronchiolitis obliterans (mean±SD age 8.8±5.0 years) for a median of 29 months in three different centres in Turkey. Among the 26 subjects who were able to perform spirometry, percentage of predicted respiratory system resistance (Rrs) at 5, 10, 15 and 20 Hz and resonance frequency correlated negatively with forced expiratory volume in 1 s (FEV1), whereas reactance values correlated positively with FEV1 results, suggesting that IOS, which requires only minimal cooperation, is suitable for evaluating lung function in children with bronchiolitis obliterans. IOAN et al. [5] proposed using FOT at a single frequency to detect the paradoxical glottis adduction during inspiration that underlies the clinical manifestations of vocal cord dysfunction (VCD). Six children with VCD and 36 controls with asthma underwent a bronchial challenge and the investigators measured the difference between inspiratory and expiratory Rrs (Rrsi−e). Rrsi−e in children with VCD versus controls was lower at baseline than after the challenge (0.51±2.0 versus −0.37±1.0 hPa·s·L−1 (p<0.03) and 6.0±5.0 versus −0.5±1.0 hPa·s·L−1 (p<0.001)), confirming that FOT can detect vocal cord adduction during inspiration in children with VCD, a pattern that was exacerbated during the bronchial challenge. To assess whether a modified FOT (wave-tube FOT) could be used to measure respiratory mechanics, GRAY et al. [6] studied 219 unsedated infants aged 6–10 weeks enrolled in a birth cohort study in South Africa. A total of 198 (90%) infants had successful measurements. Sex was a determinant of Rrs (higher in males) and compliance (Crs) (lower in males), and Crs was significantly affected by maternal smoking during pregnancy. These data suggest that the modified FOT is a feasible method for measuring respiratory mechanics in healthy unsedated infants and might also be used for assessing lung function early in life. Multiple-breath washout Many researchers presented abstracts on the multiple-breath inert gas washout (MBW), a technique addressed in a recently published technical consensus statement [7]. YAMMINE et al. Paediatrics: messages from Munich Paediatrics: messages from Munich Fabio Midulla1, Enrico Lombardi2, Marielle Pijnenburg3, Ian M. Balfour-Lynn4, Jonathan Grigg5, Kajsa Bohlin6, Franca Rusconi7, Petr Pohunek8 and Ernst Eber9 Affiliations: 1Dept of Paediatrics, Sapienza University of Rome, Rome, Italy. 2Dept of Paediatrics, Anna Meyer Children’s University Hospital, Florence, Italy. 3Dept of Paediatrics, Erasmus MC – Sophia Children’s Hospital, Rotterdam, The Netherlands. 4Dept of Paediatric Respiratory Medicine, Royal Brompton Hospital, London, UK. 5Blizard Institute, Queen Mary University, London, UK. 6Dept of Neonatology, Karolinska University Hospital and Karolinska Institutet, Stockholm, Sweden. 7Unit of Epidemiology, Anna Meyer Children’s University Hospital, Florence, Italy. 8Dept of Paediatrics, 2nd Faculty of Medicine and University Hospital Motol, Charles University, Prague, Czech Republic. 9Respiratory and Allergic Disease Division, Dept of Paediatrics, Medical University of Graz, Graz, Austria. Correspondence: Ernst Eber, Klinische Abteilung für Pulmonologie und Allergologie, Univ.-Klinik für Kinder- und Jugendheilkunde, Medizinische Universität Graz, Auenbruggerplatz 34/2, 8036 Graz, Austria. E-mail: ernst.eber@medunigraz.at ABSTRACT The aim of this article is to describe paediatric highlights from the 2014 European Respiratory Society (ERS) International Congress in Munich, Germany. Abstracts from the seven groups of the ERS Paediatric Assembly (Respiratory Physiology and Sleep, Asthma and Allergy, Cystic Fibrosis, Respiratory Infection and Immunology, Neonatology and Paediatric Intensive Care, Respiratory Epidemiology, and Bronchology) are presented in the context of the current literature. @ERSpublications Paediatric highlights from the 2014 ERS International Congress in Munich, Germany http://ow.ly/MaXZv Copyright ©ERS 2015. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial Licence 4.0. ERJ Open Res 2015; 1: 00016–2015 \| DOI: 10.1183/23120541.00016-2015 PAEDIATRICS \| F. MIDULLA ET AL. PAEDIATRICS \| F. MIDULLA ET AL. Introduction The 2014 International Congress of the European Respiratory Society (ERS) in Munich, Germany, included a substantial paediatric programme, with numerous high-quality scientific communications. As for the 2013 Annual Congress [1], the chairs of the scientific groups of the Paediatric Assembly have selected and discussed the most important abstracts to give a review of the research presented during the meeting. Due to the large number of abstracts, this update cannot be comprehensive but aims to address schemes of research in major areas of paediatric respiratory medicine. Reference values After the Global Lung Initiative (GLI) reference equations for spirometry were published in 2012 (GLI 2012) [12], researchers focussed on validating these equations in different populations and age groups. ARIGLIANI et al. [13] performed spirometry in 399 children (202 boys) aged 6–12 years from two public and two private schools in Ambanja, Madagascar. GLI 2012 equations for black people fitted well with spirometric data for Malagasy children. FEV1 and forced vital capacity (FVC) were reduced in malnourished children with no evidence of airflow limitation, reflecting stunted growth. Conversely, IGLESIAS et al.[14] found that the GLI 2012 equations significantly overestimated FEV1 and FVC values in 318 white, healthy Spanish children aged 7–15 years, suggesting that the reasons for this discrepancy should be investigated before applying the GLI 2012 equations in Spanish school-age children. Similarly, KIRKBY et al. [15] found, in 811 healthy Indian children aged <12 years, that GLI 2012 significantly underestimated FEV1 and FVC values, and calculated a new coefficient for Indian children. Focussing on other lung function techniques, MANDADZHIEVA et al. [16] produced prediction equations for transfer factor of the lung for carbon monoxide (TLCO) in 449 healthy Bulgarian children aged 7–18 years (254 boys). Boys had significantly higher values for single-breath TLCO than girls (7.19±2.47 versus 6.51±1.71 mmol min−1·kPa−1, p<0.001), while alveolar volume (VA) was similar in both sexes. TLCO and VA showed close correlations with height (r2 between 0.82 and 0.90), and with increasing height, all children presented a faster increase in TLCO and VA. Functional monitoring of sleep disordered breathing Functional monitoring of sleep disordered breathing BOUDEWYNS et al. [17] assessed the usefulness of endoscopy under sedation as a tool to detect anatomical factors for upper airway obstruction in 13 obese children with obstructive sleep apnoea (OSA) aged 9– 14 years. In the six surgically naïve children, endoscopy showed upper airway obstruction at the level of the adenoids and tonsils with associated upper airway hypotonia in five, whereas in six out of seven children with previous upper airway surgery, endoscopy documented partial or complete tongue base obstruction, and in three out of four children with previous adenoidectomy, significant tonsillar obstruction was seen. Endoscopy under sedation was reported to be a useful tool for obtaining insights in the pathogenesis of OSA in obese children and making treatment decisions. Seeking ways to reduce the need for polysomnography (PSG), PIETROPAOLI et al. aed a c esp a Forced oscillations [8] compared the repeatability of shortened protocols to measure the lung clearance index (LCI) (calculated at 5% of initial nitrogen washout) and standard LCI (calculated at 2.5% of initial nitrogen washout) in a longitudinal sample of 37 children with CF (age range 5.7–7.2 years) over 35 months. Both indexes yielded similar intra-test, mid-term (<4 months) and long-term (>4 months) repeatability. The authors also showed nonsignificant mean changes after intravenous antibiotics. These data suggest that LCI at 5% of initial nitrogen washout might be useful as a potential study end-point. To explain their previous finding that nitrogen equipment (N2MBW) in younger children induced a higher LCI than sulfur hexafluoride equipment with a mass spectrometer (SF6MBW), BENSELER et al. [9] evaluated the effects of equipment dead space (VD) on LCI measurements. As previously reported, N2LCI was higher than SF6LCI in 10 healthy adults. When SF6MBW VD was increased to be equivalent to N2MBW VD, SF6LCI increased, becoming higher than N2LCI, whereas when VD was added to the N2MBW system in steps approximating 1 mL·kg−1 to mimic the VD spectrum/weight ratios observed in preschool children, N2LCI increased linearly by 0.4 units for each unit increase in weight-adjusted VD. These data suggest that the two methods yield contrasting LCI values because their VD equipment differs and have important implications for interpreting MBW data in young children. To obtain objective physiological assessments in children with acute bronchiolitis, STAFLER et al. [10] measured LCI using SF6 and an ultrasonic flowmeter in 29 infants with acute bronchiolitis (age 2.9±2.1 months) and 23 healthy controls (age 4.2±2.5 months). Valid tidal breathing measurements were obtained in only 19 (66%) out of 29 infants with acute bronchiolitis and 15 of these achieved more than two valid MBW measurements, whereas valid tidal breathing measurements were achieved in 22 (97%) out of 23 of the controls and 19 also achieved more than two valid MBW measurements. LCI was 8.4±0.8 in infants with acute bronchiolitis and 7.3±0.7 in controls (p<0.001). In five infants with acute bronchiolitis who returned after 4 months, LCI decreased from 8.3±0.9 to 7.4±0.7 (p=0.08). The authors concluded that measuring LCI in infants with acute bronchiolitis is feasible, although technically challenging, and LCI can potentially serve as an objective severity measure. In a ERJ Open Res 2015; 1: 00016–2015 \| DOI: 10.1183/23120541.00016-2015 2 PAEDIATRICS \| F. MIDULLA ET AL. similar study, KOBBERNAGEL et al. Reference values [18] investigated whether a sleep clinical record (SCR) and nocturnal pulse oximetry with a McGill Oximetry Score (MOS) could detect OSA. MOS yielded positive results (>2) in 52 out of 285 children with a sensitivity of 20.5% and a specificity of 97.2% in detecting OSA when compared with PSG, whereas 227 (79.6%) children had a positive SCR score (>6.5) with a sensitivity of 81.1% and a specificity of 30.5%. In 48 (16.8%) children, all of whom received a diagnosis of OSA at PSG, SCR and MOS in combination detected the presence of obstructive apnoea with a sensitivity of 52% and a specificity of 100%. These findings suggest that SCR and nocturnal pulse oximetry with MOS may offer a valid alternative to PSG. Finally, ALEXOPOULOS et al. [19] aimed to study whether nocturnal enuresis (defined as urine incontinence on more than one night per week) was associated with an increased risk of moderate to severe OSA (apnoea–hypopnoea index >5 episodes·h−1) among 525 children with snoring (age 7.5±2.2 years) referred for PSG over 12 years. 355 (67.6%) children had nocturnal enuresis and 87 (16.6%) had moderate to severe OSA. Nocturnal enuresis was associated with moderate to severe OSA after adjustment for tonsillar hypertrophy, obesity, sex and age. Nocturnal enuresis had a high sensitivity (78.2%) and a low positive predictive value (19.2%) for detecting moderate to severe OSA, and a low specificity (34.5%) and a high negative predictive value (88.8%) for ruling it out, suggesting that children with snoring and without nocturnal enuresis referred for PSG are less likely to have OSA than those with nocturnal enuresis. aed a c esp a Forced oscillations [11] assessed the long-term variability in N2MBW indices in 26 children (6–18 years) with primary ciliary dyskinesia (PCD) on three occasions during a 1-year follow-up. For LCI, the within-occasion standard deviation (95% CI) was 0.63 (0.55–0.70) and the coefficient of variation was 6.6% (6.0–7.2%), whereas the between-occasion standard deviation was 1.08 (0.83–1.28) and coefficient of variation was 11.4% (10.4–12.5%). The authors also reported within-occasion and between-occasion variability for other MBW indices (Scond and Sacin), thus providing important information for using and interpreting N2MBW indices in the clinical management of PCD in children and as outcome measures in interventional studies. Databases “Big data” found its way into paediatric asthma. Although guidelines heavily rely on results from randomised controlled trials, only few patients treated for asthma meet the eligibility criteria for major trials [20]. MURRAY et al. [21] compared the effectiveness of adding a long-acting β-agonist (LABA) versus increasing the dose of inhaled corticosteroids (ICS) in a real-life population of children whom low-dose ERJ Open Res 2015; 1: 00016–2015 \| DOI: 10.1183/23120541.00016-2015 PAEDIATRICS \| F. MIDULLA ET AL. ICS left uncontrolled. Patients (n=3129) were recruited from two UK databases. Increasing the dose of ICS led to fewer exacerbations with similar asthma control than adding a LABA. These results provide no support for the step 3 recommendations of the 2014 British Thoracic Society/Scottish Intercollegiate Guidelines Network and Global Initiative for Asthma (GINA) guidelines on asthma management, and this gap between guidelines and implementation is exactly what MURRAY et al. [22] showed in a second presentation from the same study. Seeking further information an asthma management, ENGELKES et al. [23] used a large general practitioners’ database to identify children with asthma and studied severe asthma exacerbations in this group of patients. The overall rate of severe exacerbations was 0.02 per year, being highest in children <7 and >15 years of age, and in children on ICS who were treated by a paediatrician. A history of severe exacerbations increased the risk of subsequent severe exacerbations, a finding in line with earlier studies [24]. In a separate study, ENGELKES et al. [25] found that LABA monotherapy was decreasing but, despite the guidelines, the prevalence remains at 1.44 per 100 per year. Although few studies have addressed clinical effectiveness in childhood asthma, research of this kind gives a major insight into the problem and may be complimentary to the results reported in randomised controlled trials. Preschool wheeze Preschool wheeze remains a topic of high clinical and scientific interest. Much effort has focussed on defining the clinical phenotypes that warrant different treatment or monitoring strategies, and on predicting asthma at school age [26]. MUTTI et al. [27] were unable to distinguish preschool children with episodic viral wheeze (EVW) from children with multiple-trigger wheeze (MTW) by clinical and endobronchial biopsy results, suggesting a similar pathophysiology for both entities. They did, however, find thicker basal membranes and more epithelial loss, and higher mucosal eosinophil and mast cell numbers in children with wheeze compared to controls. Databases These results are supported by animal data and data from autopsy studies presented by ANDERSSON et al. [28] showing that increased mast cells in the alveolar parenchyma after respiratory viral infections may affect susceptibility towards allergens and asthma development in later life. The biopsy data provided by MUTTI et al. [27] might imply that asthma is as likely to develop in children with EVW as in those with MTW. In preschool children, others found no difference in reticular basement membrane thickness and mucosal eosinophils or airway smooth muscle mast cell values between children who did or did not have asthma at school age, but they made no distinction between preschool children with MTW and EVW [29]. Yet, biopsy studies, in general, include highly selected populations of children who had a clinical indication for bronchoscopy. GOKSÖR et al. [30] nevertheless showed a higher risk of doctor-diagnosed asthma at 8 years for children with EVW and MTW than for children without preschool wheeze (MTW: OR 48.9, 95% CI 29.3–81.6; EVW: OR 16.2, 95% CI 10.3–25.6). This risk was independent of atopic heredity and allergic sensitisation during preschool years. These data showed that many children with EVW will continue to wheeze and will have asthma, suggesting that EVW is not always a transient, “innocent” disease. In an attempt to define asthmatic and preschool wheezing phenotypes in an unbiased clustering of children, BRINKMAN et al. [31] used an electronic nose (eNose) platform and, with a metabolomics approach, the researchers identified five clinical clusters, which differed in age, atopic state, asthma control and asthma-related quality of life (fig. 1). Whether these five clusters are clinically meaningful and can guide management remains unclear. In a study by VAN DER SCHEE et al. [32], the eNose was also able to discriminate atopic wheeze from nonatopic wheeze in a small cohort of 64 patients, with moderate accuracy (area under the curve 0.74, p=0.008). Another possible source of noninvasive and easily obtainable inflammatory biomarkers in preschool children is urine. CHIU et al. [33], in a birth cohort study, showed that elevated urinary leukotriene E4 had a high specificity and a high positive predictive value for asthma and rhinitis after age Cluster 1 Cluster 2 Cluster 3 Cluster 4 Cluster 5 FIGURE 1 Topology clusters of asthma patients identified using an eNose platform. Reproduced from [31] with permission from the authors. Databases 4 ERJ Open Res 2015; 1: 00016–2015 \| DOI FIGURE 1 Topology clusters of asthma patients identified using an eNose platform. Reproduced from [31] with permission from the authors. FIGURE 1 Topology clusters of asthma patients identified using an eNose platform. Reproduced from [31] with permission from the authors. ERJ Open Res 2015; 1: 00016–2015 \| DOI: 10.1183/23120541.00016-2015 PAEDIATRICS \| F. MIDULLA ET AL. 2 years. These data suggest that biomarkers in the urine may have an added value in diagnosis and monitoring of preschool children. 2 years. These data suggest that biomarkers in the urine may have an added value in diagnosis and monitoring of preschool children. 2 Several groups showed that preschool wheeze and asthma have a multifactorial aetiology that includes viruses (in particular rhinovirus) [34], traffic-related air pollution [35], prenatal exposures to environmental contaminants [36], foods and genetics. LUKKARINEN et al. [34], in a cohort of 64 infants hospitalised for their first wheeze episode, showed that rhinovirus detection and allergic sensitisation were the most important risk factors for atopic asthma at age 7 years, but not for nonatopic asthma. Current evidence therefore confirms that rhinovirus interacts with atopy and with immune system maturity in the development of asthma. The mechanisms underlying rhinovirus-induced wheeze now need unravelling in prospective studies of rhinovirus infections in asymptomatic, healthy preschool children [37]. SHULTZ et al. [35], reporting results from the BAMSE cohort study, showed that exposure to air pollution from road traffic, especially during the first year of life and between years 1 and 8, was associated with impaired small airway function as measured by IOS in adolescents. Their data conflicted with the results reported by OLSSON et al. [38], who failed to find a higher risk of asthma after prenatal exposure or exposure during infancy to air pollution. These discrepancies may depend on differences in how exposure to air pollution was measured and in the outcomes used. Similarly, SMIT et al. [36] found only very limited evidence linking prenatal exposure to contaminants to asthma and eczema in school-age children in Ukraine and Greenland. Obviously, such an association needs assessment in more studies on different exposures and in different populations. Previous studies have shown that preterm birth, low birth weight and high infant weight gain are associated with increased risk of later asthma development. To investigate these possible risks, DUIJTS et al. Databases [39] collected data from 24 birth cohorts to assess the association of early growth and lung function. Preterm birth (<37 weeks) had an adverse effect on FEV1/FVC and forced expiratory flow at 75% of FVC (FEF75%), and low birth weight (<2500 g) on FEV1, FVC and FEF25–75%. The authors concluded that smaller airways might be partly responsible for the associations between early growth characteristics and asthma. Monitoring of asthma During the International Congress, the ERS Task Force on Monitoring of Asthma in Children presented their results. Clinical history, spirometry, and assessment of adherence to therapy, inhaler technique and exposures remain the mainstays of monitoring of asthma [40]. The Task Force members found no evidence supporting the use of composite asthma control scores and asthma-related quality of life or symptom diaries in daily practice. Future directions in monitoring of asthma should include monitoring strategies in specific populations, also called “phenotyped” or “personalised” monitoring. VAN BRAGT et al. [41] in a randomised controlled trial enrolling 93 school children with asthma showed that adding an individualised self-management support programme to usual care resulted in better asthma-related quality of life. Personalised monitoring strategies could usefully include biomarkers in blood, urine, sputum or exhaled breath, genetic markers, and electronic health initiatives. Cystic fibrosis Therapies p Cystic fibrosis transmembrane conductance regulator therapies In the past years, the most exciting new therapies for cystic fibrosis (CF) have focussed on small-molecule therapy, to correct or potentiate the faulty or missing chloride channel on epithelial cell surfaces. The initial studies on the cystic fibrosis transmembrane conductance regulator (CFTR) potentiator ivacaftor in patients with the p.Gly551Asp mutation showed an impressive reduction in sweat chloride, accompanied by a significant improvement in lung function, weight gain and quality of life [42]. Greater clinical experience with the drug has resulted in publication of post-marketing experience in 151 children aged 6 years and above [43]. The hospitalisation rate decreased by 19%, thus improving this expensive drug’s health economic evaluation. Pseudomonas aeruginosa growth decreased, mucociliary clearance doubled and microbial diversity increased. Additionally, intestinal bicarbonate secretion increased (and gut pH increased by 1.5), which may explain the observed improvement in weight gain. A recent publication has extended safety and efficacy data to 144 weeks, and showed maintained improvements and good tolerance [44]. Data have now been published on the use of ivacaftor for the other nine, much rarer (non-p.Gly551Asp) gating mutations that have shown encouraging effects (apart from the p.Gly970Arg mutation) on sweat chloride, lung function, nutrition and quality of life [45]. Ivacaftor has been studied in combination with the CFTR corrector lumacaftor in patients homozygous for the p.Phe508del mutation. The results from the phase 2 studies were published last year [46]. Ataluren is a drug that leads to the production of full-length CFTR protein in patients with nonsense mutations (e.g. p.Gly542X). Last year saw the publication of the phase 3, 48-weeks study in 236 patients ERJ Open Res 2015; 1: 00016–2015 \| DOI: 10.1183/23120541.00016-2015 PAEDIATRICS \| F. MIDULLA ET AL. over 6 years old [47]. The results were disappointing after the drug had shown early promise; however post hoc analysis showed a positive effect in patients not taking inhaled tobramycin. Antimicrobial therapies Some important publications have addressed antibiotic therapy. A phase 2 trial of inhaled Arikace, a liposomal amikacin preparation for nebulisation, showed, in 105 patients after 28 days treatment, a significant rise in FEV1, which was maintained (although to a lesser extent) after a month off therapy [48]. In an open-label extension, lung function also improved with 28 days of therapy followed by 56 days off; this was accompanied by a reduction in sputum P. aeruginosa density [48]. Cystic fibrosis Therapies This should offer another inhaled antibiotic option for treating chronic P. aeruginosa infection. At the ERS International Congress, BILTON et al. [49] presented interim data from the CLEAR-110 extension study in which long-term safety, tolerability and efficacy of once-daily liposomal amikacin (in monthly on–off cycles) is being studied in over 200 patients for 2 years. The treatment was well tolerated and improvements in lung function were maintained during both the on and off treatment months. Another study published last year looked at patients with Burkholderia infection, who are usually excluded from trials [50]. The study investigated the use of inhaled aztreonam in 105 children and adults with chronic Burkholderia infection, and found no significant improvement in lung function, hospitalisation rate or chest exacerbations, with a trend for less antibiotic use. Unfortunately, it was a negative study, but at least it was published, given the definite bias towards publishing only positive results [51]. As well as nebulised antibiotics, some pharmaceutical companies have introduced dry powder forms. Inhaled colistimethate sodium is now being given for chronic P. aeruginosa infection in a dry powder form because it is quicker to administer than the traditional nebulised form and may therefore improve adherence. SU et al. [52] used radioactive scintigraphic imaging to look at lung and oropharyngeal deposition, and found that the dry powder gave a greater lung deposition than the nebuliser (12% versus 6%). Nebulised mannitol is a recently introduced mucoactive osmotic agent [53]. ONG et al. [54] reported that in vitro the drug promotes P. aeruginosa dispersion from the biofilm and enhances the antibacterial effect of ciprofloxacin (fig. 2). This may add to the benefit of this treatment. The study also demonstrated a model for studying bacterial biofilms. Azithromycin has become a standard part of therapy in CF, used both in the long term as well as for shorter antibiotic courses. Respiratory viral infections play an important role in CF lung disease, with few specific therapies available. SCHOEGLER et al. [55], in an in vitro study, showed that azithromycin pre-treatment reduced rhinovirus replication seven-fold in bronchial epithelial cells, although it did not prevent rhinovirus-induced cytokine production. This suggests that azithromycin may have an antiviral role and, in their recent full publication, the authors suggest that studies are necessary to see whether azithromycin might have a role in preventing or managing rhinovirus exacerbations in CF [56]. Local drug deposition on bioﬁlm FIGURE 2 Scanning electron micrographs of a) the nonhomogeneous codeposition of ciprofloxacin and mannitol on the biofilm, b) the site of aerosol deposition and c) areas of no drug deposition. Scale bars=2 μm. Reproduced from [54] with permission from the authors. ERJ Open Res 2015; 1: 00016–2015 \| DOI: 10.1183/23120541.00016-2015 Cystic fibrosis Therapies Influenza immunisation has been recommended for people with CF for many years. For the winter of 2013–2014, the US CF Foundation stated that the new live attenuated influenza vaccine delivered as a nasal spray was contraindicated in CF, due to a lack of data. A safety study has now been published in 168 CF patients aged 2–18 years receiving FluMist (MedImmune, Gaithersburg, MD, USA) [57]. There was no increase in respiratory deteriorations or hospitalisation for 28 days post-immunisation, although 8% of the patients did wheeze in the first week (two thirds on the actual day). This should allow its use in children with CF, which will help those with needle anxiety. Contraindications remain immunosuppression (including oral corticosteroids), acute wheezing on the day and large nasal polyps. Local drug deposition on bioﬁlm No drug deposition FIGURE 2 Scanning electron micrographs of a) the nonhomogeneous codeposition of ciprofloxacin and mannitol on the biofilm, b) the site of aerosol deposition and c) areas of no drug deposition. Scale bars=2 μm. Reproduced from [54] with permission from the authors. Local drug deposition on bioﬁlm No drug deposition Local drug deposition on bioﬁlm ERJ Open Res 2015; 1: 00016–2015 \| DOI: 10.1183/23120541.00016-2015 PAEDIATRICS \| F. MIDULLA ET AL. Other therapies Other therapies p Intravenous aminophylline is sometimes used during chest exacerbations, especially when the patient has a tight chest with severe bronchoconstriction. WADDINGHAM et al. [58] reviewed their use in 30 patients. 60% had subtherapeutic levels; only 0.4% levels were high. Although adverse effects were common (60%), none led to drug cessation. The investigators suggested that high doses are required to reach therapeutic levels. Finally, vitamin D deficiency is common but the levels recommended for optimising bone mineralisation (75–150 nmol·L−1) are hard to achieve in CF. MISTRY et al. [59] reviewed their prescribing practice and found that, initially, many patients were not receiving the correct daily dose. As expected, adherence was a major issue. Vitamin D seems to have a role in many things and is the subject of a profusion of publications. SCHOEGLER et al. [60] in an in vitro study showed that vitamin D lowered rhinovirus 16 replication in bronchial epithelial cells, possibly by inducing the antimicrobial peptide cathelicidin. Assessment of disease The LCI measures ventilation inhomogeneity and has been shown to be a useful marker of early CF lung disease. It is also one of the more sensitive outcome measures available for clinical trials; for example, the LCI decreased by a mean of 2.2 in 21 patients receiving ivacaftor [61]. ELBORN et al. [62] showed an association of a raised LCI with systemic inflammation (C-reactive protein and white-cell count); HATZIAGOROU et al. [63], an association with a positive sputum culture and chest exacerbations; and GROSSE-ONNEBRINK et al. [64], LCI as a predictor of future exacerbations. Although the LCI was also associated with computed tomography (CT) findings of bronchiectasis in children (excluding infants), RAMSEY et al. [65] suggested that it is not a substitute for a chest CT but has a role as a surveillance tool. Chest CT scanning has its advocates, although many believe its role should be restricted to clinical need on an individual basis rather than routine annual surveillance. CT scores for bronchiectasis may be useful in more advanced disease, but in mild cases, scoring is less helpful. The publication from the London CF Collaboration newborn screened cohort illustrated this disadvantage by showing that CT changes in 1-year-olds had poor to fair agreement between two scorers (best for air trapping), probably because the changes were very mild (especially compared with the Australian AREST-CF data) [66, 67]. ERJ Open Res 2015; 1: 00016–2015 \| DOI: 10.1183/23120541.00016-2015 ROSENOW et al. [68] reported on a new imaging technique, PRAGMA (Perth–Rotterdam Annotated Grid Morphometry), which is said to have good inter- and intraobserver agreement in young children. BOON et al. [69] used CT scans of explanted lungs, including micro-CT on lung tissue cores, to study end-stage lung disease. Magnetic resonance imaging The radiation burden of CT scans has aroused increasing interest in the use of magnetic resonance imaging (MRI) for long-term monitoring of lung disease. JUNG et al. [70] reported that typical findings in children with CF can be demonstrated and dynamic contrast-enhanced imaging reveals lung perfusion abnormalities, which may differentiate chronic consolidation from acute atelectasis due to sedation. MARSHALL et al. [71] reported that early lung changes, including those in subclinical disease, can be detected using hyperpolarised helium MRI, and that this technique was more sensitive than LCI, lung function and proton MRI (fig. 3). Inflammatory markers If assessed noninvasively, inflammatory markers could be used routinely and repeatedly. PAREKH et al. [72], in a pilot study, looked at several urinary biomarkers and found higher levels of the protease inhibitors TIMP2 (tissue inhibitor of metalloproteinase) and α1-antitrypsin, and lower levels of fMLP (formyl-methionyl-leucyl-phenylalanine), a neutrophil chemotactic agent, in patients with CF compared to healthy volunteers. More work is needed to determine whether these markers will have any clinical use. Elafin is a serine elastase inhibitor and therefore has anti-inflammatory properties. YACOB et al. [73] measured significantly lower plasma elafin levels in children with CF (who were free from exacerbation) than in controls, whereas elastase levels were comparable in the two groups. The authors concluded that although elafin replacement therapy may be effective, much work remains to be done before this possibility can be considered. Using automated proteomic technology, DEBOER et al. [74] analysed over 1100 proteins in bronchoalveolar lavage fluid, to assess whether they may be biomarkers for bronchiectasis on a CT scan; 82 proteins correlated significantly with the CT airway score but no adjustment was made for multiple comparisons. As many as 43 of these proteins correlated more strongly than neutrophil elastase with the airway score. Miscellaneous With increasing survival and a known increased risk of gastrointestinal malignancy, the adult centre in Munich has instituted screening colonoscopy at 40 years of age (routine screening is recommended a ERJ Open Res 2015; 1: 00016–2015 \| DOI: 10.1183/23120541.00016-2015 7 PAEDIATRICS \| F. MIDULLA ET AL. Paediatric respiratory infection and immunology h f h l d f The major cause of hospital admissions for acute respiratory symptoms during the first years of life is bronchiolitis. To date, the efficacy of inhaled therapies for bronchiolitis remains unclear. This uncertainty may account for the wide variation in approaches to treatment. For example, CONSTANT et al. [84], in a survey including members of the Portuguese Society of Paediatrics (n=514) and general practitioners (n=165), found that inhaled salbutamol was used by 43% of respondents for mild disease and by 78% for moderate disease. 20% of paediatricians routinely used hypertonic saline for moderate disease. The routine use of inhaled therapy was also reported by BON et al. [85], who compared bronchiolitis treatment in a single Italian tertiary unit in 2002 and 2012. At both time-points, most infants received bronchodilator therapy (72.4% in 2002 and 91.3% in 2012). However, a recent Cochrane review that included data from 30 trials representing 1992 infants with bronchiolitis found that bronchodilators do not improve oxygen saturation, do not reduce hospital admission after outpatient treatment and do not shorten the duration of hospitalisation [86]. The Cochrane review’s authors therefore concluded that “bronchodilators are not effective in the routine management of bronchiolitis”. Since there is good evidence to suggest that bronchodilators are ineffective in bronchiolitis, the efficacy of hypertonic saline is of interest. MAGUIRE et al. [87] reported the results of a systematic review of controlled trials of nebulised hypertonic saline for infants hospitalised with primary acute bronchiolitis. They identified 15 trials representing 1722 infants. Overall, hypertonic saline reduced the mean length of stay in the hospital by 0.41 (95% CI −0.56–−0.27) days, but the researchers found a risk of bias and high levels of heterogeneity. Remarkably, no trial reported intervention-related severe adverse events. EVERARD et al. [88] also reported the results of their own trial. This was a parallel-group, pragmatic randomised controlled trial performed in 10 UK hospitals. Infants with acute bronchiolitis were randomised either to standard supportive care with oxygen as required or to supportive care plus 6-hourly 3% nebulised hypertonic saline. The primary outcome was the time to when infants were assessed as being “fit for discharge”, defined as, in air with saturations of >92% for 6 h. 141 infants were allocated to hypertonic saline and 159 to standard care. PAEDIATRICS \| F. MIDULLA ET AL. 55 years in Germany). NAEHRIG et al. [75] found a high rate of abnormalities in 11 patients studied (two adenocarcinomas, three tubulovillous adenomas and one patient with an inflammatory polyp. The CF-ABLE score (based on age, body mass index (BMI), lung function and number of exacerbations) has been developed as a prognostic tool. MEURLING et al. [76] showed that the score predicts death or transplantation over a 4-year period. 3He MRI Patient 1 Patient 3 Patient 4 Patient 5 Patient 10 CT 1H MRI FIGURE 3 Images from five different patients with mild cystic fibrosis: hyperpolarised helium magnetic resonance imaging (MRI), computed tomography (CT) and proton MRI. Arrows indicate common features between MRI and CT. Images courtesy of POLARIS, The University of Sheffield, Sheffield Children’s Hospital and the Cystic Fibrosis Trust. Reproduced from [71] with permission from the authors. 1H MRI 3He MRI Patient 1 Patient 3 Patient 4 Patient 5 Patient 10 FIGURE 3 Images from five different patients with mild cystic fibrosis: hyperpolarised helium magnetic resonance imaging (MRI), computed tomography (CT) and proton MRI. Arrows indicate common features between MRI and CT. Images courtesy of POLARIS, The University of Sheffield, Sheffield Children’s Hospital and the Cystic Fibrosis Trust. Reproduced from [71] with permission from the authors. ERJ Open Res 2015; 1: 00016–2015 \| DOI: 10.1183/23120541.00016-2015 ERJ Open Res 2015; 1: 00016–2015 \| DOI: 10.1183/23120541.00016-2015 PAEDIATRICS \| F. MIDULLA ET AL. High cyanide levels have been found in the sputum of CF patients infected with P. aeruginosa and correlate negatively with lung function. Recently, cyanide has been shown to impair mucociliary function in vitro [77]. GILCHRIST et al. [78] evaluated exhaled breath hydrogen cyanide (HCN) measured by SIFT (selected ion flow tube) mass spectrometry as a marker of P. aeruginosa infection in children. While raised HCN is a marker of Pseudomonas infection, the sensitivity needs to be improved to make this a useful measure. International guidelines and standards of care The US CF Foundation Infection Control guidelines were updated in 2013 (published in 2014). Among many suggestions, the guidelines recommend that staff should all wear gowns and gloves when in contact with CF patients, and the patients themselves should wear facemasks when in the hospital environment [79]. There may be quite an effect on patient morale in terms of stigmatisation and it would be a surprise if many European centres fully adopted this approach. PAEDIATRICS \| F. MIDULLA ET AL. The US CF 2014 guidelines also recommended keeping patients 2 m (6 feet) apart based on how far cough aerosols travel. Since this publication, work from Australia has shown that after intense coughing for 5 min, P. aeruginosa can travel as far as 4 m (13 feet) and viable organisms remain in the air for 45 min [80]. This observation proves, once again, that people with CF should stay apart and not mix socially with each other. The European CF Society Standards of Care were published as three separate documents, a framework for a CF centre that describes what that centre needs in terms of facilities and multidisciplinary staffing [81]; practice guidelines outlining recommendations for clinical care and what CF centres should provide [82]; and quality management, which describes areas for quality improvement moving from an individual to national and international levels [83]. Paediatric respiratory infection and immunology h f h l d f There was no difference between the two arms in the time to being declared fit for discharge (hazard ratio (HR) 0.95, 95% CI 0.75–1.20) or to actual discharge (HR 0.97, 95% CI 0.76–1.23). There was also no difference in adverse events. In a separate trial, LOZA et al. [89] assessed the efficacy of nebulised 3% and 6% hypertonic saline compared with normal saline in children hospitalised with bronchiolitis. In this multicentre, randomised and double-blind controlled trial, 292 infants were randomised to receive either nebulised 3% or 6% hypertonic saline, or normal saline during their hospital stay. The primary end-point was length of hospital stay. Overall, no difference was found between the three treatment groups in median length of hospital stay and adverse effects were similar in the three groups. Since the UK trial conducted by EVERARD et al. [88] suggests that 3% hypertonic saline is ineffective and the Dutch trial by LOZA et al. [89] suggests that the efficacy of 3% hypertonic saline is no ERJ Open Res 2015; 1: 00016–2015 \| DOI: 10.1183/23120541.00016-2015 9 PAEDIATRICS \| F. MIDULLA ET AL. different from either 6% hypertonic saline or normal saline, it is likely that all three inhaled saline therapies are ineffective. Despite recent evidence that inhaled therapies should not be routinely used in bronchiolitis, there may be ways of improving the quality and efficiency of standard supportive care. First, BEKHOF et al. [90], in a prospective cohort study, assessed the effect of cohorting respiratory syncytial virus (RSV)-positive infants separately from RSV-negative infants. Infants shared a two- to four-bed hospital room during the entire admission, irrespective of the virological diagnosis. Of the 65 infants, 94% were RSV positive, 56 (86%) of whom shared a room with another bronchiolitis patient. 18 (28%) of these infants had bronchiolitis related to another respiratory virus. On admission, 10 (15%) infants were already co-infected with another virus and eight (12%) infants became cross-infected with a different virus during their admission. Of these cross-infected infants, only one had shared a room with the virus identified in one of its roommates. Given that there are no significant differences in disease severity between patients infected with single or multiple viruses, nor between RSV-positive and RSV-negative patients, the researchers concluded that room sharing does not seem to play a major role in the transmission of viruses between infants with bronchiolitis sharing a room. Neonatology and paediatric intensive care N l Minimising lung injury and preventing the development of chronic lung disease continues to be a challenge in the care of very preterm infants. Intensive research efforts now focus on seeking reliable markers to predict short- and long-term lung morbidity. BARALDI et al. [92], in a pilot study, collected amniotic fluid samples and performed metabolomic analysis, which revealed the presence of metabolic profiles characterising preterm delivery and bronchopulmonary dysplasia (BPD) development. PRAMANA et al. [93] reported that levels of pro-endothelin-1, a potent pulmonary vasoconstrictor, might serve as a marker for severe lung injury and the development of BPD in very preterm infants. In established BPD, DASSIOS et al. [94] showed that the chest radiographic thoracic area is significantly related to disease severity as quantified by ventilation–perfusion inequality and oxygen requirement. HENCKEL et al. [95] investigated the relationship between telomere length as a marker of previous oxidative stress, chitinase-like protein YKL-40 as a marker of airway remodelling and lung function in school-age children with a history of BPD. They found that BPD was associated with increased levels of YKL-40, which also correlated with significantly shorter telomeres. The clinical progression of BPD and long-term lung health in preterm infants are difficult to predict, as was demonstrated in a recent thorough systematic review and an external validation study [96]. The aforementioned studies all help to advance research for better predictive tools to be used in future intervention studies. Understanding the many and complex causes driving the development of BPD is another challenge still facing neonatologists, and is even more pressing now that survival is increasing even in the most extremely immature gestational age groups [97]. Experimental evidence suggests that the bone morphogenic protein (BMP) pathway participates in regulating pulmonary vasculogenesis, and might thereby intervene in the development of pulmonary hypertension and chronic lung disease. In a mouse model of BPD, KOSCHLIG et al. [98] showed that activating the BMP receptor II signalling pathway improved impaired vasculogenesis. In another mouse model of hypoxia-induced pulmonary hypertension presented by MORENO et al. [99], upregulation of the BMP antagonist Gremlin-1 was shown to contribute to increased vascular tone. Both studies indicate that modulating the BMP signalling pathway may be a promising strategy to prevent pulmonary hypertension in the newborn (PHN). This is also in line with recent experimental findings showing that sildenafil, a treatment option increasingly used in PHN, potentiates BMP signalling [100]. Paediatric respiratory infection and immunology h f h l d f Second, in an uncontrolled study, RUBIO et al. [91] reported that infants with bronchiolitis treated with a high-flow nasal oxygen cannula did at least as well as infants given usual oxygen care. Indeed, preliminary evidence showed a decreased risk of transfer to intensive care and no technical complications. Neonatology and paediatric intensive care N l The maturational changes in the immune system after preterm birth are still incompletely understood. Immunological factors may play a role in the development of BPD during the perinatal period. Two studies extended current knowledge on this relationship. MUSTAFAYEV et al. [101] evaluated cellular immunity in patients with BPD and reported that the acute phase of the disease was characterised by T-cell inhibition. FRANKENBERGER et al. [102] described a strong expansion and aberrant phenotype of intermediate blood monocytes in preterm neonates related to the innate immune system immaturity. Lung function testing in the neonatal period continues to be a rapidly expanding field. The importance of an adequate diaphragm function for extubation success was investigated by BHAT et al. [103] who found that tension time index measurements of the diaphragm may be useful in predicting extubation success in term but not preterm neonates. The mechanism explaining why the prone sleeping position is a risk factor for sudden infant death syndrome was elucidated by ROSSOR et al. [104] in a crossover study in term neonates. The authors found no difference in carbon dioxide sensitivity between the prone and supine ERJ Open Res 2015; 1: 00016–2015 \| DOI: 10.1183/23120541.00016-2015 10 PAEDIATRICS \| F. MIDULLA ET AL. positions, but a significantly longer time constant for the carbon dioxide response in prone sleep, indicating a damped ventilatory response to hypercarbia. BARIKBIN et al. [105] in a study of infants with congenital cystic adenomatoid malformations of the lung found that a breathing pattern consistent with a more severe restrictive ventilation disorder, in particular, reduced respiratory compliance, was associated with the need for surgery during the first 2 years of life, thus showing that lung function testing may be an important tool for monitoring and guiding treatment of infants with this rare condition. positions, but a significantly longer time constant for the carbon dioxide response in prone sleep, indicating a damped ventilatory response to hypercarbia. BARIKBIN et al. [105] in a study of infants with congenital cystic adenomatoid malformations of the lung found that a breathing pattern consistent with a more severe restrictive ventilation disorder, in particular, reduced respiratory compliance, was associated with the need for surgery during the first 2 years of life, thus showing that lung function testing may be an important tool for monitoring and guiding treatment of infants with this rare condition. Neonatology and paediatric intensive care N l Paediatric intensive care There is much interest in noninvasive respiratory support for children with moderate to severe acute onset of respiratory distress. The use of high-flow nasal cannula (HFNC) oxygen therapy has expanded dramatically over the past few years. In a retrospective study, RUBIO et al. [91] reported that HFNC provided a safe and effective means of respiratory support for children with viral bronchiolitis in a secondary hospital, and that the introduction of HFNC was associated with a 58% reduction in transfers to the paediatric intensive care unit over two seasons. HFNC has also been successfully used during emergency transport, as shown by IGLESIAS et al. [106]. HFNC was well tolerated during transport and reduced the need for continuous positive airway pressure. LUCIANI et al. [107], in a crossover study, found that breathing efforts decreased significantly more after HFNC than after low-flow oxygen therapy. FOUZAS et al. [108] presented data from a simulation model they had developed including a new equation to estimate the effective oxygen delivery in relation to flow and respiratory rate more accurately. In summary, accumulating evidence implies that HFNC has physiological benefits but larger randomised trials are warranted [109]. Severe acute asthma continues to be a life-threatening condition in children. In a randomised controlled trial in children 1–12 years of age, TIWARI et al. [110] compared ketamine with aminophylline as second line adjuvant therapy and found both drugs equally effective. THOUVENIN et al. [111] in a retrospective review following the publication of the GINA recommendations in 2011 showed that intravenous magnesium sulfate, if used within the first hour as recommended by GINA, significantly reduced both the total duration of hospitalisation and the length of stay in the paediatric intensive care unit. This is in accordance with a very recent systematic review that recommends the use of intravenous magnesium sulfate as a safe and effective adjunct to conventional bronchodilator therapy in children with acute severe asthma [112]. Some researchers even believe that the accumulating evidence confirming a favourable “cost benefit” profile justifies using magnesium for all children presenting with acute severe asthma [113]. Preterm birth A recent systematic review and meta-analysis showed that preterm-born survivors, even those in whom BPD does not develop, have moderate deficits in FEV1 compared with term-born controls [125]. DUIJTS et al. [39] examined the association between preterm birth and low birth weight with lung function at age 4–18 years, using data from 24715 children in 24 European birth cohorts. Random-effect meta-analyses were used with individual participant data and pooled effect estimates. The strongest independent effects were found for preterm birth (<37 versus ⩾37 weeks of gestation) on FEV1/FVC and FEF75% and for low birth weight (<2500 versus ⩾2500 g) on FEV1, FVC and FEF25–75% (p<0.05). FAINARDI et al. [126], in a small group of 7-year-old children born very preterm (<32 weeks of gestation), assessed lung function by IOS. Compared with preterm children born appropriate for gestational age (AGA), children born small for gestational age or AGA with intrauterine growth restriction showed higher small-airway resistance and lower pulmonary compliance. EDWARDS et al. [127], in a large cross-sectional survey of all surviving preterm-born children in Wales (n=13361) and matched term control children, confirmed previous findings that preterm birth increases the risk of wheezing disorders [128]. Both the <5-year-old and 5–10-year-old preterm born children in the RANOPS had higher reported rates of wheezing ever, wheeze in the last 3 months and use of inhaled medication than term infants. Analysis by gestational age showed a gradient of increasing respiratory symptoms and use of inhaled medication for each week of gestational age prior to 38 weeks. Primary ciliary dyskinesia A number of national and international databases have been developed in order to better define clinical and laboratory data of paediatric PCD. MAURER et al. [129] presented preliminary data from a large multinational dataset (2128 patients in 16 countries, in the framework of the European Union Seventh Framework project BESTCILIA); THOUVENIN et al. [130], data from a French national cohort (298 patients in 23 centres); and SNIJDERS et al. [131], data from an Italian survey (261 patients in 21 centres). The latter group reported that age at diagnosis in patients <18 years was 5.0 years lower in children with situs inversus (4.2 versus 6.0 years, p<0.0127) and higher in children with bronchiectasis (7.0 versus 3.5 years, p<0.0001). GOUTAKI et al. Preterm birth [132] assessed height and BMI in 263 paediatric and adult patients with PCD from six different centres (BESTCILIA project) and found comparable height z-scores and slightly higher BMI z-scores compared with World Health Organization reference data, suggesting that undernutrition might not be a predominant problem in PCD. In a study presenting 5-year data on re-testing in 162 patients diagnosed with PCD across a broad range of phenotypic variants, HIRST et al. [133] showed that inner dynein arm phenotypic variants in the cilia of PCD can in some cases revert to normal on re-testing. They concluded that re-testing for PCD should be considered in patients whose initial biopsy suggested an absence of inner dynein arms. Paediatric respiratory epidemiology Early-life factors for respiratory disorders Longitudinal birth cohort studies have identified a number of early risk factors for respiratory disorders in childhood as well as protective factors [114]. Several studies have reported an increased risk of wheezing and asthma development in children after prenatal exposure to antibiotics [115]. The suggested mechanism is a derangement in neonatal and infant microbial colonisation patterns. Whether the association between maternal antibiotics during pregnancy and wheezing or asthma in the offspring is causal or due to confounding factors has been explored in a cohort study by POPOVIC et al. [116]. and in a crossover and a case–control study by MULDER et al. [117]. Both studies found an increased risk of wheezing or asthma for antibiotic prescription in the third trimester of pregnancy but could not rule out unmeasured confounding as a possible explanation. In particular, in the Italian cohort study [116], the association greatly diminished after a statistical analysis controlling for several maternal factors. A recent study using data from the Danish national register found an association between maternal use of antibiotics and asthma in the offspring regardless of the timing for maternal antibiotic treatment (either during or before or after pregnancy) [118]. These results are also in favour of a confounding effect due to a maternal or offspring propensity to infections. Another topic of interest is vitamin D deficiency in pregnancy and early childhood and the development of infections or wheezing or asthma in the first years of life. Findings from several cohort studies suggest a protective effect of 25-hydroxyvitamin D levels both in pregnancy and in childhood against respiratory infections [119, 120] whereas evidence on protection against wheezing and asthma is inconclusive. In a case–control study, INGHAM et al. [121] reported an association between vitamin D deficiency and severity of acute respiratory infections in New Zealand children. Few data are available on how exposure to air pollution in pregnancy influences respiratory health in offspring. A recent study in the INMA cohort found an association between prenatal exposure to residential traffic-related air pollution and lung function deficits at preschool age [122]. Conversely, OLLSON et al. [38], in a Swedish register-based cohort study, showed no association between nitrogen dioxide levels at the place of residence during pregnancy or infancy and prescribed asthma medication ERJ Open Res 2015; 1: 00016–2015 \| DOI: 10.1183/23120541.00016-2015 11 PAEDIATRICS \| F. MIDULLA ET AL. during the sixth year of life. Paediatric respiratory epidemiology Nitrogen dioxide levels had been evaluated with a dispersion model and used as an indicator of motor vehicle exhaust. The role of allergen exposure in causing asthma remains highly debatable [123]. In a collaborative study of five birth cohorts in Europe, CASAS et al. [124] found a geographical variation in the concentration of house dust mite in infants’ homes, with lower levels in colder areas, such as Scandinavian regions, and higher levels in more humid and warm areas, such as the Mediterranean regions. No association was found with subsequent asthma up to the age of 10 years. Preterm birth Paediatric bronchology h ll b Bronchoscopy is a well-established and standardised technique. Many groups are now looking at the paediatric bronchoscope as an interventional tool, especially in congenital anomalies and airway narrowing. In a retrospective study of 61 children with endoscopic findings of vascular anomalies causing compression of the tracheobronchial tree, SERIO et al. [134] found a good agreement between bronchoscopy and CT angiography. The same group recently published follow-up data for a series of 100 consecutive children who underwent stent insertion for airway narrowing of various aetiologies [135]. They had inserted 235 airway stents with no fatal complications and 80% of the patients had experienced significant improvement. ANTON-PACHECO et al. [136] presented their initial experience with biodegradable polydioxanone stents in two infants (5 and 8 months of age) with severe airway narrowing (fig. 4). Although one child had to undergo multiple stent insertions, no intervention led to stent-related complications, such as stent displacement, bleeding, granulation tissue formation or perforation during the 2-year follow-up. This group has already published information on the use of biodegradable stents in children after slide tracheoplasty, reporting the technique’s good safety profile [137]. Although ERJ Open Res 2015; 1: 00016–2015 \| DOI: 10.1183/23120541.00016-2015 12 PAEDIATRICS \| F. MIDULLA ET AL. FIGURE 4 Bronchoscopic view 15 weeks after polydioxanone tracheal stent insertion. Reproduced from [136] with permission from the authors. FIGURE 4 Bronchoscopic view 15 weeks after polydioxanone tracheal stent insertion. Reproduced from [136] with permission from the authors. FIGURE 4 Bronchoscopic view 15 weeks after polydioxanone tracheal stent insertion. Reproduced from [136] with permission from the authors. FIGURE 4 Bronchoscopic view 15 weeks after polydioxanone tracheal stent insertion. Reproduced from [136] with permission from the authors. biodegradable stents seem to be a very promising alternative to metallic or silicone stents, more experience is needed to define the precise indications in children. MADKOUR et al. presented their experience with the use of a laryngeal mask airway (LMA) for foreign body extraction with a flexible bronchoscope [138]. In a prospective 1-year study, they included 49 children (mean age 5.9 years) suspected of having aspirated a foreign body. They identified 41 mainly organic foreign bodies and successfully removed 34 (82.9%). In six cases they had to use a rigid bronchoscope and ultimately removed one foreign body via open thoracotomy. The authors reported no critical complications related to flexible bronchoscopy. Paediatric bronchology h ll b They concluded that the main advantage of the LMA is that larger (adult size) flexible bronchoscopes and, thus, larger extraction forceps may be used. This study adds to the growing evidence showing that flexible bronchoscopy may be a feasible technique for extracting foreign bodies in children. MORENO et al. [139] addressed the association of protracted bacterial bronchitis (PBB) and airway malacia. They retrospectively analysed data of 165 children who underwent bronchoscopy for persistent cough and received a final diagnosis of PBB or airway malacia. The average age at diagnosis of PBB was 3 years. In 53 children with PBB, only 10 had no symptoms indicating airway malacia. Airway malacia was associated with poor response to treatment and the authors concluded that patients with PBB and malacia should probably receive prolonged antibiotic treatment. Several papers in recent years have looked at bacterial bronchitis and airway malacia [140, 141]. Children with clinically diagnosed PBB should undergo bronchoscopy to document possible airway malacia, a factor that potentially can contribute to disease severity and to poor response to treatment. GOUSSARD et al. [142], in children with tuberculosis (TB), studied the use of GeneXpert MTB/RIF (Cepheid, Sunnyvale, CA, USA) testing on bronchoalveolar lavage (BAL) fluid. Among 40 children (median age 18 months), TB was confirmed in 29 (73%) by Xpert and 23 (58%) by culture. In 21 (53%) children both methods yielded positive results. Positive Xpert testing was associated with lymph nodes ERJ Open Res 2015; 1: 00016–2015 \| DOI: 10.1183/23120541.00016-2015 13 PAEDIATRICS \| F. MIDULLA ET AL. ulcerating into the airway (p=0.03), lung disease on chest X-ray or duration of treatment <14 days. Because median time to culture positivity in this group was 14 days, methods such as Xpert can speed-up the diagnostic workup and increase the diagnostic yield. SVEDBERG et al. [143] looked at BAL fluid return and factors that may influence the amount of BAL fluid recovered from the bronchial tree. In 311 children who underwent bronchoscopy and BAL for diagnostic purposes, they found that younger age and higher percentage of neutrophils were associated with reduced BAL fluid return. HONKOVÁ et al. [144] presented new data on bronchial remodelling. They did bronchial biopsies in 27 children with asthma, 15 with CF and 14 with PCD, and compared basement membrane (BM) thickness and the deposition of structural proteins (tenascin C and collagen IV) in patients and seven controls. References 15 Kirkby J, Lum S, Stocks J, et al. Adaptation of the GLI-2012 spirometry reference equations for use in Indian children. Eur Respir J 2014; 44: Suppl. 58, 191. an I, Clisson R, Bonabel C, et al. Inspiratory vs expiratory resistance in diagnosing vocal cord dysfunction in hildren. Eur Respir J 2014; 44: Suppl. 58, 4533. Gray D, Czövek D, Willemse L, et al. Non-invasive measurement of respiratory impedance in unsedated healthy infants. Eur Respir J 2014; 44: Suppl. 58, 186. Robinson PD, Latzin P, Verbanck S, et al. Consensus statement for inert gas washout measurement using multiple- and single-breath tests. Eur Respir J 2013; 41: 507–522. g p Yammine S, Salzmann S, Singer F, et al. Repeatability of shortened and standard lung clearance index. Eur Respir J 2014; 44: Suppl. 58, 4529. pp Benseler A, Stanojevic S, Jensen R, et al. The effect of equipment dead space on lung clearance index. Eur Respir J 2014; 44: Suppl. 58, 189. pp 9 Benseler A, Stanojevic S, Jensen R, et al. The effect of equipment dead space on lung clearance index. Eur Respir J 2014; 44: Suppl. 58, 189. 10 Stafler P, Weinreb S, Mussaffi H, et al. Lung clearance index – a new parameter for airway obstruction in acute bronchiolitis? Eur Respir J 2014: Suppl. 58, 44: 188. 10 Stafler P, Weinreb S, Mussaffi H, et al. Lung clearance index – a new parameter for airway obstruction in acute bronchiolitis? Eur Respir J 2014: Suppl. 58, 44: 188. p 11 Kobbernagel H, Green K, Madsen A, et al. Prospective longitudinal measurements of N2 multiple breath washout indices in primary ciliary dyskinesia. Eur Respir J 2014; 44: Suppl. 58, 4527. obbernagel H, Green K, Madsen A, et al. Prospective longitudinal measurements of N2 multiple breath washout dices in primary ciliary dyskinesia. Eur Respir J 2014; 44: Suppl. 58, 4527. p y y y p J pp 12 Quanjer PH, Stanojevic S, Cole TJ, et al. Multi-ethnic reference values for spirometry for the 3–95-yr age range the global lung function 2012 equations. Eur Respir J 2012; 40: 1324–1343. p y y y p pp 12 Quanjer PH, Stanojevic S, Cole TJ, et al. Multi-ethnic reference values for spirometry for the 3–95-yr age range: the global lung function 2012 equations. Eur Respir J 2012; 40: 1324–1343. 13 Arigliani M, Canciani MC, Altomare M, et al. p 2 Oostveen E, MacLeod D, Lorino H, et al. The forced oscillation technique in clinical practice: methodo recommendations and future developments. Eur Respir J 2003; 22: 1026–1041. p p 3 Beydon N, Davis SD, Lombardi E, et al. An official American Thoracic Society/European Respiratory Society statement: pulmonary function testing in preschool children. Am J Respir Crit Care Med 2007; 175: 1304–1345. 1 Midulla F, Lombardi E, Rottier B, et al. Paediatrics in Barcelona. Eur Respir J 2014; 44: 457–474. p pp 6 Gray D, Czövek D, Willemse L, et al. Non-invasive measurement of respiratory impedance in unsedated healthy infants. Eur Respir J 2014; 44: Suppl. 58, 186. 5 Ioan I, Clisson R, Bonabel C, et al. Inspiratory vs expiratory resistance in diagnosing vocal cord dysfunction in children. Eur Respir J 2014; 44: Suppl. 58, 4533. p y g p J p ; 4 Uyan ZS, Çakir E, Gökdemir Y, et al. The evaluation of pulmonary function of children with bronchiolitis obliterans by spirometry and impulse oscillometry methods. Eur Respir J 2014; 44: Suppl. 58, 4532. y y p p y statement: pulmonary function testing in preschool children. Am J Respir Crit Care Med 2007; 175: 1304–13 4 Uyan ZS, Çakir E, Gökdemir Y, et al. The evaluation of pulmonary function of children with bronchi obliterans by spirometry and impulse oscillometry methods. Eur Respir J 2014; 44: Suppl. 58, 4532. Paediatric bronchology h ll b In contrast to earlier opinions, this study found similar airway remodelling in all studied chronic respiratory diseases. No difference was found in BM thickness and tenascin layer between children with asthma, CF or PCD. These data add new knowledge about airway remodelling in asthma [145, 146]. KRIVEC et al. [147] studied the role of bronchoscopy in children with neuromuscular diseases (NMDs) and acute hypoxaemic respiratory failure requiring noninvasive ventilation. In eight consecutive children (median age 3.2 years) with NMD, lower respiratory infections and atelectasis, the authors used a specific protocol consisting of flexible bronchoscopy for airway suction (six children), noninvasive ventilation (eight children) and physiotherapy with a cough-assist device (eight children). In all the children who underwent full treatment including bronchoscopy, but not in two who only had noninvasive ventilation and physiotherapy, atelectasis fully resolved. This small series supports the idea of using flexible bronchoscopy for managing respiratory complications in NMD. ARUMUGAM et al. [148] in 53 children with difficult asthma, cystic fibrosis and chronic cough studied the diagnostic value of the lipid-laden macrophage index (LLMI) in BAL fluid. Comparing oesophageal pH or impedance with symptoms and LLMI, they found that neither technique achieved sufficient diagnostic sensitivity and specificity in detecting acidic or nonacidic gastro-oesophageal reflux-related pulmonary aspiration in children with respiratory problems. JEDYNAK-WASOWICZ et al. [149] studied the extraoesophageal reflux by monitoring oropharyngeal reflux in 21 children with chronic cough and observed that in coughing children, reflux events detected by standard multichannel intraluminal impedance/pH measurement and oropharyngeal pH monitoring correlated only when subjects were examined in the supine position. This interesting observation merits further studies because it could influence current diagnostic standards. UMUTOGLU et al. [150], in a randomised, controlled, crossover study of patients aged between 0 and 15 years of age, compared various manoeuvres used for visualising the glottis during flexible bronchoscopy. They found that the most effective manoeuvres were the head tilt, chin lift and triple-airway manoeuvres. References 1 Midulla F, Lombardi E, Rottier B, et al. Paediatrics in Barcelona. Eur Respir J 2014; 44: 457–474. 2 Oostveen E, MacLeod D, Lorino H, et al. The forced oscillation technique in clinical practice: methodology, recommendations and future developments. Eur Respir J 2003; 22: 1026–1041. 3 Beydon N, Davis SD, Lombardi E, et al. An official American Thoracic Society/European Respiratory Society statement: pulmonary function testing in preschool children. Am J Respir Crit Care Med 2007; 175: 1304–1345. 4 Uyan ZS, Çakir E, Gökdemir Y, et al. The evaluation of pulmonary function of children with bronchiolitis obliterans by spirometry and impulse oscillometry methods. Eur Respir J 2014; 44: Suppl. 58, 4532. 5 Ioan I, Clisson R, Bonabel C, et al. Inspiratory vs expiratory resistance in diagnosing vocal cord dysfunction in children. Eur Respir J 2014; 44: Suppl. 58, 4533. 6 Gray D, Czövek D, Willemse L, et al. Non-invasive measurement of respiratory impedance in unsedated healthy infants. Eur Respir J 2014; 44: Suppl. 58, 186. 7 R bi PD L i P V b k S l C f i h i 1 Midulla F, Lombardi E, Rottier B, et al. Paediatrics in Barcelona. Eur Respir J 2014; 44: 457–474. 2 Oostveen E, MacLeod D, Lorino H, et al. The forced oscillation technique in clinical practice: methodology, recommendations and future developments. Eur Respir J 2003; 22: 1026–1041. 3 Beydon N, Davis SD, Lombardi E, et al. An official American Thoracic Society/European Respiratory Society statement: pulmonary function testing in preschool children. Am J Respir Crit Care Med 2007; 175: 1304–1345. 4 Uyan ZS, Çakir E, Gökdemir Y, et al. The evaluation of pulmonary function of children with bronchiolitis obliterans by spirometry and impulse oscillometry methods. Eur Respir J 2014; 44: Suppl. 58, 4532. 5 Ioan I Clisson R Bonabel C et al Inspirator vs e pirator resistance in diagnosing ocal cord d sfunction in References References 1 Midulla F, Lombardi E, Rottier B, et al. Paediatrics in Barcelona. Eur Respir J 2014; 44: 457–474. 2 Oostveen E, MacLeod D, Lorino H, et al. The forced oscillation technique in clinical practice: methodology, recommendations and future developments. Eur Respir J 2003; 22: 1026–1041. 3 Beydon N, Davis SD, Lombardi E, et al. An official American Thoracic Society/European Respiratory Society statement: pulmonary function testing in preschool children. Am J Respir Crit Care Med 2007; 175: 1304–1345. 4 Uyan ZS, Çakir E, Gökdemir Y, et al. The evaluation of pulmonary function of children with bronchiolitis obliterans by spirometry and impulse oscillometry methods. Eur Respir J 2014; 44: Suppl. 58, 4532. 5 Ioan I, Clisson R, Bonabel C, et al. Inspiratory vs expiratory resistance in diagnosing vocal cord dysfunction in children. Eur Respir J 2014; 44: Suppl. 58, 4533. 6 Gray D, Czövek D, Willemse L, et al. Non-invasive measurement of respiratory impedance in unsedated healthy infants. Eur Respir J 2014; 44: Suppl. 58, 186. 7 Robinson PD, Latzin P, Verbanck S, et al. Consensus statement for inert gas washout measurement using multiple- and single-breath tests. Eur Respir J 2013; 41: 507–522. 8 Yammine S, Salzmann S, Singer F, et al. Repeatability of shortened and standard lung clearance index. Eur Respir J 2014; 44: Suppl. 58, 4529. 9 Benseler A, Stanojevic S, Jensen R, et al. The effect of equipment dead space on lung clearance index. Eur Respir J 2014; 44: Suppl. 58, 189. 10 Stafler P, Weinreb S, Mussaffi H, et al. Lung clearance index – a new parameter for airway obstruction in acute bronchiolitis? Eur Respir J 2014: Suppl. 58, 44: 188. 11 Kobbernagel H, Green K, Madsen A, et al. Prospective longitudinal measurements of N2 multiple breath washout indices in primary ciliary dyskinesia. Eur Respir J 2014; 44: Suppl. 58, 4527. 12 Quanjer PH, Stanojevic S, Cole TJ, et al. Multi-ethnic reference values for spirometry for the 3–95-yr age range: the global lung function 2012 equations. Eur Respir J 2012; 40: 1324–1343. 13 Arigliani M, Canciani MC, Altomare M, et al. Lung function and nutritional status in urban Malagasy children. Eur Respir J 2014; 44: Suppl. 58, 192. 14 Iglesias I, Benavides M, Delgado YP, et al. Validation of the global lung function initiative (GLI) spirometry reference equations in school age Spanish children. Eur Respir J 2014; 44: Suppl. 58, 4519. p 3 Beydon N, Davis SD, Lombardi E, et al. An official American Thoracic Society/European Respiratory So statement: pulmonary function testing in preschool children. Am J Respir Crit Care Med 2007; 175: 1304–134 4 Uyan ZS, Çakir E, Gökdemir Y, et al. The evaluation of pulmonary function of children with bronchi obliterans by spirometry and impulse oscillometry methods. Eur Respir J 2014; 44: Suppl. 58, 4532. 5 Ioan I Clisson R Bonabel C et al Inspiratory vs expiratory resistance in diagnosing vocal cord dysfunctio References g gy 30 Goksör E, Loid P, Alm B, et al. Increased risk of school age asthma following multiple trigger wheeze independent of atopy. Eur Respir J 2014; 44: Suppl. 58, 4243. p py p pp 31 Brinkman P, Hashimoto S, Fleming LJ, et al. Unbiased clustering of children with asthma or pre-school wheeze using the U-BIOPRED electronic nose platform. Eur Respir J 2014; 44: Suppl. 58, 431. g p p pp 32 Van der Schee MP, Hashimoto S, Van den Ouweland M, et al. Identification of atopy amongst wheezing infant via electronic nose. Eur Respir J 2014; 44: Suppl. 58, 4202. via electronic nose. Eur Respir J 2014; 44: Suppl. 58, 420 p pp 33 Chiu CY, Yeh KW, Huang YL, et al. Urinary LTE4 level as a diagnostic marker for atopic diseases in preschoo children: A birth cohort study. Eur Respir J 2014; 44: Suppl. 58, 4200. p 33 Chiu CY, Yeh KW, Huang YL, et al. Urinary LTE4 level as a diagno children: A birth cohort study. Eur Respir J 2014; 44: Suppl. 58, 4200. 34 Lukkarinen M, Vuorinen T, Lehtinen P, et al. Rhinovirus aetiology at the first wheeze predicts atopic but no non-atopic asthma at 7 years. Eur Respir J 2014; 44: Suppl. 58, 430. 34 Lukkarinen M, Vuorinen T, Lehtinen P, et al. Rhinovirus aetiology non-atopic asthma at 7 years. Eur Respir J 2014; 44: Suppl. 58, 430. 35 Schultz ES, Hallberg J, Bellander T, et al. Preschool exposure to traffic-related air pollution and small airway involvement in adolescence. Eur Respir J 2014; 44: Suppl. 58, 2991. 35 Schultz ES, Hallberg J, Bellander T, et al. Preschool exposure to t involvement in adolescence. Eur Respir J 2014; 44: Suppl. 58, 2991. 36 Smit LAM, Lenters V, Høyer BB, et al. Prenatal exposure to environmental contaminants and asthma and eczema in school-age children. Eur Respir J 2014; 44: Suppl. 58, 2918. 37 Kieninger E, Fuchs O, Latzin P, et al. Rhinovirus infections in infancy and early childhood. Eur Respir J 2013; 41 443–452. 38 Olsson D, Bråbäck L, Forsberg B. Air pollution exposure during pregnancy and infancy and childhood asthma Eur Respir J 2014; 44: Suppl. 58, 4237. p 39 Duijts L, Sonnenschein-van der Voort AMM, De Jongste JC, et al. Preterm birth and early growth characteristic are associated with lower lung function in childhood: a meta-analysis of 24,000 European children. References Lung function and nutritional status in urban Malagasy children. Eur Respir J 2014; 44: Suppl. 58, 192. 13 Arigliani M, Canciani MC, Altomare M, et al. Lung function and nutritional status in urban Malagasy children Eur Respir J 2014; 44: Suppl. 58, 192. p pp 14 Iglesias I, Benavides M, Delgado YP, et al. Validation of the global lung function initiative (GLI) spirometry reference equations in school age Spanish children. Eur Respir J 2014; 44: Suppl. 58, 4519. q g p p pp 15 Kirkby J, Lum S, Stocks J, et al. Adaptation of the GLI-2012 spirometry reference equations for use in Indian children. Eur Respir J 2014; 44: Suppl. 58, 191. ERJ Open Res 2015; 1: 00016–2015 \| DOI: 10.1183/23120541.00016-2015 ERJ Open Res 2015; 1: 00016–2015 \| DOI: 10.1183/23120541.00016-2015 14 PAEDIATRICS \| F. MIDULLA ET AL. 16 Mandadzhieva S, Marinov B, Kostianev S. Prediction equations for carbon monoxide diffusing capacity of the lung (TL,CO) in healthy Bulgarian children aged 7 to 18 years. Eur Respir J 2014; 44: Suppl. 58, 193. 17 Boudewyns A, Verhulst S, Saldien V, et al. Drug-induced sedation endoscopy (DISE) as a tool for upper airway l ti i b hild ith b t ti l E R i J 2014 44 S l 58 235 17 Boudewyns A, Verhulst S, Saldien V, et al. Drug-induced sedation endoscopy (DISE) as a tool for upper airway evaluation in obese children with obstructive sleep apnoea. Eur Respir J 2014; 44: Suppl. 58, 235. 17 Boudewyns A, Verhulst S, Saldien V, et al. Drug-induced sedation endoscopy (DISE) as a tool for evaluation in obese children with obstructive sleep apnoea. Eur Respir J 2014; 44: Suppl. 58, 235. 18 Pie tropaoli N, Supino MC, Rabasco J, et al. The role of sleep clinical record and nocturnal pulse oximetry in predicting childhood obstructive sleep apnea. Eur Respir J 2014; 44: Suppl. 58, 3271. 18 Pie tropaoli N, Supino MC, Rabasco J, et al. The role of sleep clinical record and nocturnal pulse o di ti hildh d b t ti l E R i J 2014 44 S l 58 3271 18 Pie tropaoli N, Supino MC, Rabasco J, et al. The role of sleep clinical record and nocturnal pulse oximetry in predicting childhood obstructive sleep apnea. Eur Respir J 2014; 44: Suppl. 58, 3271. p g p p p pp 19 Alexopoulos E, Malakasioti G, Varlami V, et al. References Nocturnal enuresis as risk factor for moderate-to-severe OSA in children with snoring. Eur Respir J 2014; 44: Suppl. 58, 4912. children with snoring. Eur Respir J 2014; 44: Suppl. 58, 4912 g p pp 20 Price D, Hillyer EV, van der Molen T. Efficacy versus effectiven management. Curr Opin Allergy Clin Immunol 2013; 13: 50–57. g p pp 20 Price D, Hillyer EV, van der Molen T. Efficacy versus effectiveness trials: informing guidelines for asthma management. Curr Opin Allergy Clin Immunol 2013; 13: 50–57. 21 Murray CS, Turner S, Thomas M, et al. Comparative effectiveness of paediatric asthma step-up options Increasing ICS dose vs adding separate LABA. Eur Respir J 2014; 44: Suppl. 58, 434. 22 Murray CS, Turner S, Thomas K, et al. Predictors of BTS Step 3 paediatric asthma prescribing. Eur Respir J 2014 44: Suppl. 58, 1171. 23 Engelkes M, Janssens H, De Ridder M, et al. Epidemiology and characteristics of severe asthma exacerbations in children. Eur Respir J 2014; 44: Suppl. 58, 435. p pp 24 Forno E, Celedón JC. Predicting asthma exacerbations in children. Curr Opin Pulm Med 2012; 18: 63–69. lk l h h l d 25 Engelkes M, Janssens H, De Jongste J, et al. Long-acting β2 agonist without concomitant inhaled cortic i hild ith th i i E R pi J 2014 44 S l 58 1166 25 Engelkes M, Janssens H, De Jongste J, et al. Long-acting β2 agonist without concomitant inhaled corticosteroids in children with asthma in primary care. Eur Respir J 2014; 44: Suppl. 58, 1166. asthma in primary care. Eur Respir J 2014; 44: Suppl. 58, 1166 26 Brand PL, Caudri D, Eber E, et al. Classification and pharmacological treatment of preschool wheezing: change since 2008. Eur Respir J 2014; 43: 1172–1177. p 27 Mutti E, Baraldo S, Bazzan E, et al. Defining phenotypes of wheeze at preschool age: Comparison of episodic v multitrigger wheeze. Eur Respir J 2014; 44: Suppl. 58, 429. gg p pp 28 Andersson C, Shikhagaie M, Al-Garawi A, et al. Mast cells infiltrate the alveolar parenchyma in young children with respiratory viral infections Eur Respir J 2014; 44: Suppl 58, 3225 29 O’Reilly R, Ullmann N, Irving S, et al. Increased airway smooth muscle in preschool wheezers who have asthma at school age. J Allergy Clin Immunol 2013; 131: 1024–1032. school age. J Allergy Clin Immunol 2013; 131: 1024–1032. References Eur Respir 2014; 44: Suppl. 58, 442. pp 40 Pijnenburg MW, Baraldi E, Brand PLP, et al. Monitoring asthma in children. Eur Respir J 2015; 45: 906–925. S d b A l ff f d d l d lf 41 Van Bragt S, Van den Bemt L, Vaessen-Verberne A, et al. Effectiveness of individualized self managemen support for children with asthma in Dutch outpatient clinics, preliminary results of a randomized controlled trial. Eur Respir J 2014; 44: Suppl. 58, 1159. p pp 42 Ramsey BW, Davies J, McElvaney NG, et al. A CFTR potentiator in patients with cystic fibrosis and the G551D mutation. N Engl J Med 2011; 365: 1663–1672. 43 Rowe SM, Heltshe SL, Gonska T, et al. Clinical mechanism of the cystic fibrosis transmembrane conductance regulator potentiator ivacaftor in G551D-mediated cystic fibrosis. Am J Respir Crit Care Med 2014; 190: 175–184. 44 McKone EF, Borowitz D, Drevinek P, et al. Long-term safety and efficacy of ivacaftor in patients with cystic fibrosis who have the Gly551Asp-CFTR mutation: a phase 3, open-label extension study (PERSIST). Lancet Respir Med 2014; 2: 902–910. k k A lk S l ff d f f f h f b d p 45 De Boeck K, Munck A, Walker S, et al. Efficacy and safety of ivacaftor in patients with cystic fibrosis and a non-G551D gating mutation. J Cyst Fibros 2014; 13: 674–680. g g y 46 Boyle MP, Bell SC, Konstan MW, et al. A CFTR corrector (lumacaftor) and a CFTR potentiator (ivacaftor) fo treatment of patients with cystic fibrosis who have a phe508del CFTR mutation: a phase 2 randomised controlled trial. Lancet Respir Med 2014; 2: 527–538. p 47 Kerem E, Konstan MW, De Boeck K, et al. Ataluren for the treatment of nonsense-mutation cystic fibrosis: a randomised, double-blind, placebo-controlled phase 3 trial. Lancet Respir Med 2014; 2: 539–547. 48 Clancy JP, Dupont L, Konstan MW, et al. Phase II studies of nebulised Arikace in CF patients with Pseudomona aeruginosa infection. Thorax 2013; 68: 818–825. 49 Bilton D, Pressler T, Fajac I, et al. Interim data from a long-term safety, tolerability, and efficacy study of liposomal amikacin for inhalation in cystic fibrosis patients with chronic Pseudomonas aeruginosa infection. Eur Resp J 2014; 44: Suppl. 58, 3445. p pp 50 Tullis DE, Burns JL, Retsch-Bogart GZ, et al. Inhaled aztreonam for chronic Burkholderia infection in cystic fibrosis: a placebo-controlled trial. PAEDIATRICS \| F. MIDULLA ET AL. 51 Balfour-Lynn IM. At last, Burkholderia spp. is one of the inclusion criteria – a negative (but published) randomised controlled trial. J Cyst Fibros 2014; 13: 241–242. 52 Su S, Riccobene T, Scott C. Lung deposition of inhaled colistimethate sodium in cystic fibrosis patients. Eur Respir J 2014; 44: Suppl. 58, 1975. p pp 53 Bilton D, Bellon G, Charlton B, et al. Pooled analysis of two large randomised phase III inhaled mannitol studies in cystic fibrosis. J Cyst Fibros 2013; 12: 367–376. y y 54 Ong HX, Loo CY, Lee WH, et al. Sweetening antibiotic treatment for eradication of bacteria biofilm. Eur Respir J 2014; 44: Suppl. 58, 3443. 55 Schoegler A, Kopf BS, Muster RJ, et al. Antiviral activity of azithromycin in cystic fibrosis airway epithelial cells Eur Respir J 2014; 44: Suppl. 58, 3450. p 56 Schögler A, Kopf BS, Edwards MR, et al. Novel antiviral properties of azithromycin in cystic fibrosis airway epithelial cells. Eur Respir J 2015; 45: 428–439. p p 57 Boikos C, De Serres G, Lands LC, et al. Safety of live-attenuated influenza vaccination in cystic fibrosis. Pediatrics 2014; 134: e983–e991. 58 Waddingham P, Simmonds N, Bilton D. Safety, tolerability and efficacy of intravenous aminophylline in adult patients with pulmonary exacerbations of cystic fibrosis. Eur Respir J 2014; 44: Suppl. 58, 1969. 59 Mistry A, Huggins R, Ward A, et al. Difficulties in optimizing vitamin D levels in paediatric cystic fibrosis patients. Eur Respir J 2014; 44: Suppl. 58, 1223. 60 Schoegler A, Kopf BS, Muster RJ, et al. Novel antiviral properties of vitamin D in cystic fibrosis airway epithelial cells. Eur Respir J 2014; 44: Suppl. 58, 3449. p pp 61 Davies J, Sheridan H, Bell N, et al. Assessment of clinical response to ivacaftor with lung clearance index in cystic fibrosis patients with a G551D-CFTR mutation and preserved spirometry: a randomised controlled trial Lancet Respir Med 2013; 1: 630–638. p 62 Elborn JS, O’Neill K, Johnston E, et al. Increased systemic inflammation in cystic fibrosis (CF) is associated with deterioration in lung clearance index (LCI). Eur Respir J 2014; 44: Suppl. 58, 1206. 63 Hatziagorou E, Avramidou V, Kampouras A, et al. Clinical value of lung clearance index (LCI) among patients with cystic fibrosis. Eur Respir J 2014; 44: Suppl. 58, 1218. 64 Grosse-Onnebrink J, Stehling F, Mellies U. Lung clearance index predicts pulmonary exacerbations in cystic fibrosis. PAEDIATRICS \| F. MIDULLA ET AL. Eur Respir J 2014; 44: Suppl. 58, 1219. 64 Grosse-Onnebrink J, Stehling F, Mellies U. Lung clearance index predicts pulmonary exacerbations in cystic fibrosis. Eur Respir J 2014; 44: Suppl. 58, 1219. 65 Ramsey K, Rosenow T, Skoric B, et al. Agreement between the lung clearance index and bronchiectasis detected 65 Ramsey K, Rosenow T, Skoric B, et al. Agreement between the lung clearance index and bronchiectasis detected via chest computed tomography in infants and children with cystic fibrosis (CF). Eur Respir J 2014; 44: Suppl 58, 1974. 66 Thia LP, Calder A, Stocks J, et al. Is chest CT useful in newborn screened infants with cystic fibrosis at 1 year of age? Thorax 2014; 69: 320–327. g 67 Mott LS, Park J, Murray CP, et al. Progression of early structural lung disease in young children with cystic fibrosis assessed using CT. Thorax 2012; 67: 509–516. g 68 Rosenow T, Kuo W, de Bruijne M, et al. A new gold standard for assessing CT in early CF lung disease? Eur Respir J 2014; 44: Suppl. 58, 3446. 69 Boon M, Bosch B, Lammertyn E, et al. Quantitative evaluation of CF airways using HRCT, µCT and histology Eur Respir J 2014; 44: Suppl. 58, 3447. 70 Jung A, Amaxopoulou C, Gnannt R, et al. Lung MRI findings in children with cystic fibrosis. Eur Respir J 2014 44: Suppl. 58, 1215. pp 71 Marshall H, Hughes D, Horsley A, et al. Detection of early-stage lung disease in cystic fibrosis; comparing the sensitivity of 3He and 1H MRI, CT and LCI. Eur Respir J 2014; 44: Suppl. 58, 3448. 72 Parekh G, Tunney M, Vallières E, et al. Inflammatory biomarkers in urine of cystic fibrosis patients. Eur Respir J 2014; 44: Suppl. 58, 1208. pp 73 Yacob A, Zirbes J, Dunn C, et al. A relative plasma Elafin deficiency in children with cystic fibrosis (CF) is associated with pulmonary disease. Eur Respir J 2014; 44: Suppl. 58, 1200. 74 DeBoer E, Kroehl M, Wagner B, et al. Novel protein biomarkers of bronchiectasis in children with cystic fibrosis Eur Respir J 2014; 44: Suppl. 58, 1960. p pp 75 Naehrig S, Huber RM, Gülberg V, et al. Screening colonoscopy in adults with cystic fibrosis. Eur Respir J 2014 44: Suppl. 58, 1967. 76 Meurling J, McCarthy C, Gunaratnam C, et al. The CF-ABLE score: A 2-year evaluation of a 4-year prognostic tool. References J Cyst Fibros 2014; 13: 296–305. 15 ERJ Open Res 2015; 1: 00016–2015 \| DOI: 10.1183/23120541.00016-2015 PAEDIATRICS \| F. MIDULLA ET AL. PAEDIATRICS \| F. MIDULLA ET AL. 87 Maguire C, Cantrill H, Hind D, et al. Hypertonic saline (HS) for acute bronchiolitis: systematic review and meta-analysis. Eur Respir J 2014; 44: Suppl. 58, 1226. 88 Everard ML, Hind D, Freeman JV, et al. Hypertonic saline in acute bronchiolitis: randomised controlled trial (RCT) and economic evaluation (the SABRE study). Eur Respir J 2014; 44: Suppl. 58, 3451. 89 Loza B, Teunissen J, Hochs A, et al. Effect of 3 and 6% hypertonic saline in viral bronchiolitis: a RCT. Eur Respir 2014; 44: Suppl. 58, 389. 90 Bekhof J, Wessels M, Reimink R, et al. Roomsharing in hospitalized children with bronchiolitis. Eur Respir 2014; 44: Suppl. 58, 390. pp 91 Rubio MTR, Sendra RL, Pérez JM, et al. High-flow nasal oxygen cannula: a useful non-invasive therapy fo mild-severe bronchiolitis in a secondary hospital. Eur Respir J 2014; 44: Suppl. 58, 3294. 92 Baraldi E, Guiseppe G, Stocchero M, et al. Metabolomic profiling of the amniotic fluid predicts the risk o preterm delivery and BPD development. Eur Respir J 2014; 44: Suppl. 58, 274. 93 Pramana I, Baumann P, Grass B, et al. Plasma pro-endothelin-1 as marker of severe lung injury in very preterm infants. Eur Respir J 2014; 44: Suppl. 58, 275. 94 Dassios T, Curley A, Molnar Z, et al. Chest radiographic thoracic area and severity of bronchopulmonary dysplasia. Eur Respir J 2014; 44: Suppl. 58, 279. y p p pp 95 Henckel E, James A, Konradsson J, et al. A novel association between YKL-40, a marker for structural lung disease, and short telomere length in 10-year old children with BPD. Eur Respir J 2014; 44: Suppl. 58, 3304. 96 Onland W, Debray TP, Laughon MM, et al. Clinical prediction models of bronchopulmonary dysplasia: a systematic review and external validation study. BMC Paediatrics 2013; 13: Suppl. 58, 207. y y pp 97 Fellman V, Hellstrom-Westas L, Norman M, et al. One-year survival of extremely preterm infants after active perinatal care in Sweden. JAMA 2009; 301: 2225–2233. p 98 Koschlig M, Tian X, Sudheendra D, et al. Reduced BMP signaling in lungs of mechanically ventilated newborn mice as a potential target for neonatal chronic lung disease. Eur Respir J 2014; 44: Suppl. 58, 278. p g g p pp 99 Moreno L, Menéndez C, Barreira B, et al. A potential role for Gremlin1 in pulmonary maturation and neonata hypoxia-induced pulmonary hypertension. Eur Respir J 2014; 44: Suppl. 58, 3296. PAEDIATRICS \| F. MIDULLA ET AL. 111 Thouvenin G, Monhounso H, Busson PF, et al. M g py p pp 112 Albuali WH. The use of intravenous and inhaled magnesium sulphate in management of children with bronchia asthma. J Matern Fetal Neonatal Med 2014; 27: 1809–1815. 113 Ohn M, Jacobe S. Magnesium should be given to all children presenting to hospital with acute severe asthma Paediatr Respir Rev 2014; 15: 319–321. p 114 Duijts L. Fetal and infant origins of asthma. Eur J Epidemiol 2012; 27: 5–14. 115 Murk W, Risnes KR, Bracken MB. Prenatal or early-life exposure to antibiotics and risk of childhood asthma: a systematic review. Pediatrics 2011; 127: 1125–1138. y 116 Popovic M, Zugna D, Galassi C, et al. Prenatal exposure to antibiotics and wheezing: An internet-based birth cohort study. Eur Respir J 2014; 44: Suppl. 58, 441. y p pp 117 Mulder B, Pouwels K, Schuiling-Veninga C, et al. Antibiotic use among Dutch pregnant woman and the development of toddler asthma: the influence of confounding. Eur Respir J 2014; 44: Suppl. 58, 440. 118 Stokholm J, Sevelsted A, Bønnelykke K, et al. Maternal propensity for infections and risk of childhood asthma: a registry-based cohort study. Lancet Respir Med 2014; 2: 631–637. 118 Stokholm J, Sevelsted A, Bønnelykke K, et al. Maternal propensity fo registry-based cohort study. Lancet Respir Med 2014; 2: 631–637. 119 Camargo CAJr, Ingham T, Wickens K, et al. Cord-blood 25-hydroxyvitamin D levels and risk of respiratory infection, wheezing, and asthma. Pediatrics 2011; 127: e180–e187. 120 Morales E, Romieu I, Guerra S, et al. Maternal vitamin D status in pregnancy and risk of lower respiratory trac infections, wheezing, and asthma in offspring. Epidemiology 2012; 23: 64–71. 120 Morales E, Romieu I, Guerra S, et al. Maternal vitamin D status in pregnancy infections, wheezing, and asthma in offspring. Epidemiology 2012; 23: 64–71. g p g p gy 121 Ingham TR, Jones B, Camargo CAJr, et al. Association of vitamin D deficiency with severity of acute respiratory infection: A case-control study in New Zealand children. Eur Respir J 2014; 44: Suppl. 58, 439. 122 Morales E, Garcia-Esteban R, de la Cruz OA, et al. Intrauterine and early postnatal exposure to outdoor ai pollution and lung function at preschool age. Thorax 2015; 70: 64–73. g g 123 Fuchs O, von Mutius E. Genetic and environmental factors in bronchial asthma and wheezing disorders. In: Eber E, Midulla F, eds. PAEDIATRICS \| F. MIDULLA ET AL. Eur Respir J 2014; 44: Suppl. 58, 1964. 77 Nair C, Shoemark A, Chan M, et al. Cyanide levels found in infected cystic fibrosis sputum inhibit airway ciliary function. Eur Respir J 2014; 44: 1253–1261. p 78 Gilchrist F, Jones A, Webb K, et al. The sensitivity and specificity of Pseudomonas aeruginosa detection using hydrogen cyanide concentration in exhaled breath – the SPACE study. Eur Respir J 2014; 44: Suppl. 58, 3444. 79 Saiman L, Siegel JD, LiPuma JJ, et al. Infection prevention and control guideline for cystic fibrosis: 2013 update Infect Control Hosp Epidemiol 2014; 35: Suppl. 1, S1–S67. f p p 80 Knibbs LD, Johnson GR, Kidd TJ, et al. Viability of Pseudomonas aeruginosa in cough aerosols generated by persons with cystic fibrosis. Thorax 2014; 69: 740–745. p y 81 Conway S, Balfour-Lynn IM, De Rijcke K, et al. European Cystic Fibrosis Society Standards of Care: framework for the cystic fibrosis centre. J Cyst Fibros 2014; 13: Suppl. 1, S3–22. y y pp 82 Smyth AR, Bell SC, Bojcin S, et al. European Cystic Fibrosis Society Standards of Care: best practice guidelines J Cyst Fibros 2014; 13: Suppl. 1, S23–S42. 83 Stern M, Bertrand DP, Bignamini E, et al. European Cystic Fibrosis Society Standards of Care: quality management in cystic fibrosis. J Cyst Fibros 2014; 13: Suppl. 1, S43–S59. g y y 84 Constant C, Magalhães M, Andrade MG, et al. Perspectives of pediatricians and general practitioners on diagnosis and treatment of bronchiolitis. Eur Respir J 2014; 44: Suppl. 58, 1237. g p pp 85 Bon A, Mauro M, Manfredi A, et al. Bronchiolitis in the last ten years in a tertiary paediatric department Evaluation of evidence-based guidelines. Eur Respir J 2014; 44: Suppl. 58, 3459. d k b h d l f b h l h b 85 Bon A, Mauro M, Manfredi A, et al. Bronchiolitis in the last ten years in Evaluation of evidence-based guidelines. Eur Respir J 2014; 44: Suppl. 58, 3459. 86 Gadomski AM, Scribani MB. Bronchodilators for bronchiolitis. Cochrane Datab 86 Gadomski AM, Scribani MB. Bronchodilators for bronchiolitis. Cochrane Database Syst Rev 2014; 6: CD001266. ERJ Open Res 2015; 1: 00016–2015 \| DOI: 10.1183/23120541.00016-2015 ERJ Open Res 2015; 1: 00016–2015 \| DOI: 10.1183/23120541.00016-2015 16 PAEDIATRICS \| F. MIDULLA ET AL. PAEDIATRICS \| F. MIDULLA ET AL. 100 Yang J, Li X, Al-Lamki RS, et al. Sildenafil potentiates bone morphogenetic protein signaling in pulmonary arterial smooth muscle cells and in experimental pulmonary hypertension. Arterioscler Thromb Vasc Biol 2013 33: 34–42. arterial smooth muscle cells and in experimental pulmonary hypertension. Arterioscler Thromb Vasc Biol 2013; 33: 34–42. 101 Mustafayev I, Allahverdiyeva L, Bogdanova A. Immune status in bronchopulmonary dysplasia. Eur Respir J 2014; 44 Suppl 58 3306 33: 34–42. 101 Mustafayev I, Allahverdiyeva L, Bogdanova A. Immune status in bronchopulmonary dysplasia. Eur Respir J 2014; 44: Suppl 58, 3306 101 Mustafayev I, Allahverdiyeva L, Bogdanova A. Immune status in bronchopulmonary dysplasia. Eur Respir J 2014 44: Suppl. 58, 3306. 102 Frankenberger M, Kaßner G, Oak P, et al. Intermediate CD14++CD16+ blood monocytes are elevated in preterm neonates. Eur Respir J 2014; 44: Suppl. 58, 3307. p 103 Bhat P, Rafferty G, Hannam S, et al. Prediction of extubation outcome in neonates born prematurely or at term Eur Respir J 2014; 44: Suppl. 58, 277. 104 Rossor T, Ali K, Trenear R, et al. The effects of sleeping position on the ventilatory response to hypercarbia. Eu Respir J 2014; 44: Suppl. 58, 276. p pp 105 Barikbin P, Roehr CC, Wilitzki S, et al. Congenital cystic adenomatoid malformation: Initial lung function tes predicts respiratory impairment. Eur Respir J 2014; 44: Suppl. 58, 3302. p p y p p pp 106 Iglesias I, Rajmil R, Dominguez P. High flow nasal cannulae for bronchiolitis in pediatric interhospital critica care transport. Eur Respir J 2014; 44: Suppl. 58, 3300. p p pp 107 Luciani S, Caresta E, Cicchetti R, et al. Effort of breathing indices in infants with acute respiratory insufficiency receiving different noninvasive respiratory support. Eur Respir J 2014; 44: Suppl. 58, 3295. 108 Fouzas S, Tzifas S, Pelekouda P, et al. Effective concentrations of oxygen delivered to infants via nasal cannula: Back to the model. Eur Respir J 2014; 44: Suppl. 58, 3298. p pp 109 Phillips B. Towards evidence based medicine for paediatricians. Arch Dis Child 2013; 98: 1018. 110 Tiwari A, Guglani V, Jat KR. Ketamine versus aminophylline for status asthmatic in children: A randomized controlled trial. Eur Respir J 2014; 44: Suppl. 58, 281. 111 Thouvenin G, Monhounso H, Busson PF, et al. Management of severe acute asthma in childhood: impact of intravenous magnesium therapy. Eur Respir J 2014; 44: Suppl. 58, 280. PAEDIATRICS \| F. MIDULLA ET AL. Treatment strategies in the management of severe complications following slide tracheoplasty in children. Eur J Cardiothorac Surg 2014; 46: 280–285. g p y g 138 Madkour A, Elmansoury A, Sharkawy S. The efficacy of fiberoptic bronchoscopy via laryngeal mask airway in pediatric foreign bodies extraction. Eur Respir J 2014; 44: Suppl. 58, 4652. 139 Moreno M, Castillo S, Castaño C, et al. Protracted bacterial bronchitis and malacias: which came first, the chicken or the egg? Eur Respir J 2014; 44: Suppl. 58, 4654. 140 Kompare M, Weinberger M. Protracted bacterial bronchitis in young children: association with airway malacia J Pediatr 2012; 160: 88–92. 141 Zgherea D, Pagala S, Mendiratta M, et al. Bronchoscopic findings in children with chronic wet cough. Pediatrics 2012; 129: e364–e369. 142 Goussard P, Walters E, Bosch C, et al. Factors that predict for positive GeneXpert MTB/RIF on bronchoalveolar lavage samples in children with suspected tuberculosis. Eur Respir J 2014; 44: Suppl. 58, 4653. 143 Svedberg M, Kötz K, Hesselmar B. Bronchoalveolar lavage fluid recovery in children correlates with age and neutrophils. Eur Respir J 2014; 44: Suppl. 58, 4657. p p pp 144 Honková L, Beránková K, Uhlík J, et al. Deposition of tenascin C and collagen IV in bronchial basement membrane of children with chronic respiratory diseases. Eur Respir J 2014; 44: Suppl. 58, 4655. 145 Berankova K, Uhlik J, Honkova L, et al. Structural changes in the bronchial mucosa of young children at risk of developing asthma. Pediatr Allergy Immunol 2014; 25: 136–142. developing asthma. Pediatr Allergy Immunol 2014; 25: 136– 146 Saglani S, Malmström K, Pelkonen AS, et al. Airway remodeling and inflammation in symptomatic infants with reversible airflow obstruction. Am J Respir Crit Care Med 2005; 171: 722–727. p 147 Krivec U, Praprotnik M, Lepej D, et al. Flexible bronchoscopy in children with neuromuscular disease and acute hypoxemic respiratory failure requiring noninvasive ventilation. Eur Respir J 2014; 44: Suppl. 58, 4656. 148 Arumugam JM, Hartt C, Vadamalayan B, et al. Lipid laden macrophages in bronchoalveolar lavage as a marker of pulmonary aspiration secondary to acid and non-acid gastroesophageal reflux. Eur Respir J 2014; 44: Suppl 58, 4468. 149 Jedynak-Wasowicz U, Cichocka-Jarosz E, Sladek M, et al. Evaluation of gastroesophageal reflux in children with persistent cough: correlation between multichannel intraluminal impedance/pH-metry and oropharyngeal pH monitoring. Eur Respir J 2014; 44: Suppl. 58, 4469. PAEDIATRICS \| F. MIDULLA ET AL. 124 Casas L, García-Esteban R, Tischer C, et al. Early life mite allergens and respiratory and allergic health Eur Respir J 2014; 44: Suppl. 58, 437. p pp 125 Kotecha SJ, Edwards MO, Watkins WJ, et al. Effect of preterm birth on later FEV1: a systematic review and meta-analysis. Thorax 2013; 68: 760–766. y 126 Fainardi V, Sapienza E, Magnani C, et al. IOS in school-aged children born preterm: is small for gestational age a risk factor for small airway dysfunction? Eur Respir J 2014; 44: Suppl. 58, 1255. y y p pp 127 Edwards M, Kotecha S, Lowe J, et al. Respiratory and neurological outcomes in children born preterm study (RANOPs): Preterm outcomes. Eur Respir J 2014; 44: Suppl. 58, 1253. 128 Been JV, Lugtenberg MJ, Smets E, et al. Preterm birth and childhood wheezing disorders: a systematic review and meta-analysis. PLoS Med 2014; 11: e1001596. y 129 Maurer E, Goutaki M, Behan L, et al. Creating a large multinational dataset of patients with primary ciliary dyskinesia (PCD). Eur Respir J 2014; 44: Suppl. 58, 1244. y p pp 130 Thouvenin G, de Blic J, Epaud R, et al. The French national cohort of children with primary ciliary dyskinesia Eur Respir J 2014; 44: Suppl. 58, 1242. 131 Snijders D, Quartesan S, di Cicco ME, et al. A national survey on primary ciliary dyskinesia in Italy. Eur Respir J 2014; 44: Suppl. 58, 1243. pp 132 Goutaki M, Maurer E, Boon M, et al. Growth in patients with primary ciliary dyskinesia (PCD): a multinational study. Eur Respir J 2014; 44: Suppl. 58, 1246. y p pp 133 Hirst R, Rutman A, Williams G, et al. Re-testing for primary ciliary dyskinesia (PCD) is only indicated for inner dynein arm defects. Eur Respir J 2014; 44: Suppl. 58, 1247. y p pp 134 Serio P, Fainardi V, Leone R, et al. Role of bronchoscopy in diagnosis, treatment and follow-up of vascular anomalies: A multidisciplinary approach. Eur Respir J 2014; 44: Suppl. 58, 4650. 135 Serio P, Fainardi V, Leone R, et al. Tracheobronchial obstruction: follow-up study of 100 children treated with airway stenting. Eur J Cardiothorac Surg 2014; 45: e100–e109. y g g 136 Anton-Pacheco JL, Luna-Paredes C, García-Hernández G, et al. Initial experience with biodegradable tracheal stents in children. Eur Respir J 2014; 44: Suppl. 58, 4651. p pp 137 Antón-Pacheco JL, Comas JV, Luna C, et al. PAEDIATRICS \| F. MIDULLA ET AL. g p pp 150 Umutoglu T, Gedik AH, Bakan M, et al. The influence of airway supporting maneuvers on glottis view in paediatric patients undergoing diagnostic flexible fiberoptic bronchoscopy. Eur Respir J 2014; 44: Suppl. 58, 4476. ERJ Open Res 2015; 1: 00016–2015 \| DOI: 10.1183/23120541.00016-2015 PAEDIATRICS \| F. MIDULLA ET AL. ERS Handbook of Paediatric Respiratory Medicine. Sheffield, European Respiratory Society, 2013; pp. 298–304. 17 ERJ Open Res 2015; 1: 00016–2015 \| DOI: 10.1183/23120541.00016-2015 PAEDIATRICS \| F. MIDULLA ET AL. ERJ Open Res 2015; 1: 00016–2015 \| DOI: 10.1183/23120541.00016-2015 18
https://openalex.org/W4229044803	https://www.nature.com/articles/s42003-022-03372-2.pdf	English	null	Succession comprises a sequence of threshold-induced community assembly processes towards multidiversity	Communications biology	2,022	cc-by	9,057	1 Department of Environment and Biodiversity, Paris Lodron University Salzburg, 5020 Salzburg, Austria. 2 Evolutionary Ecology of Plants, Department of Biology, Philipps-University Marburg, 35043 Marburg, Germany. ✉email: robert.junker@uni-marburg.de COMMUNICATIONS BIOLOGY \| (2022) 5:424 \| https://doi.org/10.1038/s42003-022-03372-2 \| www.nature.com/commsbio ARTICLE ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-022-03372-2 S Yet, it is an ongoing debate whether ecological communities generally converge towards a core set of species over the course of succession21,22, and studies on successional convergence have mostly been focused on singular taxa neglecting potential inter- actions and co-occurrences across organismal groups23–25. To speciﬁcally test the extent of species turnover across several taxa, we introduce multi-betadiversity, an aggregated measure of taxonomic community dissimilarity. This index allows to test whether the expected increase in biotic interactions is reﬂected in a reduced compositional variation (i.e., reduced multi- betadiversity regarding several taxa) of the multidiverse com- munity, i.e., a community that comprises a number of taxonomic groups, once niche-based and interaction-mediated assembly processes dominate over stochastic dispersal as main mechanisms shaping multidiversity. S uccession and community assembly research are neigh- boring ﬁelds, each with a long history of constructive debates and a large body of ecological literature. Both research lines are founded on overlapping concepts considering processes such as dispersal, environmental ﬁltering, biotic inter- actions, and stochasticity as structuring elements of local com- munities and ecosystems. Nevertheless, succession and community assembly research focus on different spatial and temporal scales, which may have hindered a mutual exchange of ideas1. Succession research relates to the initial development of ecosystems and communities over time. Community assembly studies, on the other hand, try to elucidate the past drivers of local and recent diversity patterns regardless of temporal components. Attempts to synthesize the two ﬁelds led to the development of an integrated conceptual framework of succession and community assembly dynamics. Here, drivers of community structure are considered from an explicitly temporal perspective, assuming a temporal sequence of assembly processes shifting from stochastic (likely dispersal) to rather deterministic (niche-based and inter- action-mediated) processes along the course of succession1. The proposed framework also emphasizes the importance of threshold dynamics, predicting that gradual changes in the abiotic and biotic environment are followed by rapid shifts in the pre- dominant processes that determine community assembly2,3. These shifts in processes are expected to be associated with var- ious community-level responses such as changes in diversity, species composition, and functionality which ultimately leads to a community state shift1,2,4. Recent research suggests that sudden ecosystem shifts are not restricted to changes in species compo- sition of single organismal groups but that local diversity patterns emerge from on-site interactions between multiple taxa5,6. ARTICLE Mul- tidiversity, an aggregate measure of biodiversity that integrates the standardized diversities of multiple taxa7, has been shown to reﬂect ecosystem functionality8, species composition9, and mul- titrophic interactions10 more accurately than diversity measures considering only one taxon. Additionally, the responses of dif- ferent organismal groups to successional gradients may differ either due to stochastic or deterministic drivers11–15. For instance, the assembly of belowground bacterial and fungal communities follows individual trajectories during primary succession that differ from the assembly of vascular plant communities16,17. Thus, the concept of multidiversity is a well-suited approach that can reﬂect various community-level responses and allows more concise conclusions about different ecosystem states along suc- cessions, as well as underlying assembly processes than con- sidering a single or few taxa only16. Assuming that multidiversity more precisely reﬂects ecosystem responses and functions, we tested predictions deduced from contemporary succession hypotheses using a dataset on multi- diversity comprising inventories of plants, animals, and micro- organisms along a successional gradient. Most studies on multidiversity have been undertaken in well-developed ecosys- tems that have undergone severe anthropogenic alterations in the past and focused on the drivers of biodiversity decline7,9,10. The mechanisms behind the successional emergence of multidiversity and ecosystem complexity under natural conditions, however, are yet poorly understood. Thus, considering multidiversity within a successional framework will help to gain insights into the tem- poral dynamics of the processes that shape the initial emergence of biodiversity in natural ecosystems that comprise multiple organismal groups with unique and complementary ecological functions. We adopted the concept of multidiversity to empiri- cally test predictions of the integrated framework of succession and community assembly dynamics on an ecological gradient of primary succession following glacial retreat in the Austrian Alps26. We assessed the multidiversity and multi-betadiversity of ﬁve organismal groups, including vascular plants, bryophytes, invertebrates, and soil-inhabiting fungi and bacteria on 110 plots spanning 170 years of primary succession. We estimated multi- diversity by the mean of ranked normalized Shannon-diversities and calculated multi-betadiversity as the mean Bray-Curtis dis- similarities between plots regarding the composition of individual taxa. According to the conceptual framework by Chang and HilleRisLambers (2016)1, we predicted that the multidiverse community will undergo a rapid shift that is induced by a sudden change of the biotic and abiotic environment along the succes- sional gradient. ARTICLE We expect this shift to be associated with increased biotic interactions and stronger environmental inﬂu- ences that result in a reduced compositional variation between plots of the multidiverse community. g g y The assumed changes of assembly processes along successions, a decrease in the importance of stochasticity, and a simultaneous increase in niche-based and interaction-based processes, have consequences for the taxonomic, functional, and phylogenetic composition of communities and thus beta diversity1,17. The framework by Chang and HilleRisLambers (2016)1 predicts an increase in functional and phylogenetic diversity of local single- taxon communities in later successions due to niche differentia- tion and intensiﬁed biotic interactions. In a multidiversity context where multiple taxa are considered, increased interactions and thus potentially strong dependencies between taxa may result in aggregated co-occurrence patterns18, which may have con- sequences for the beta-diversity between local communities—and not necessarily for the functional and phylogenetic diversity within a community. For instance, interactions between plants and soil-inhabiting microorganisms intensify with successional age19 and regulate plant-animal interactions20, which is reﬂected in the co-occurrence of the partners involved in these interac- tions. Such interactions may thus result in reduced species turnover across local assemblages in older successional stages. Accordingly, we identiﬁed a threshold in multidiversity development (i.e., a shift from an increase of multidiversity with time to a rather stationary phase) and estimated the relative importance of stochasticity, as well as environmental and biotic drivers on the multidiverse community before and after the threshold using path analysis. We further highlighted the more structured character of community assembly after the shift by comparing multi-betadiversity estimates of the early and late- successional communities. Succession comprises a sequence of threshold- induced community assembly processes towards multidiversity Maximilian Hanusch 1, Xie He 1, Victoria Ruiz-Hernández1 & Robert R. Junker 1, Research on successions and community assembly both address the same processes such as dispersal, species sorting, and biotic interactions but lack unifying concepts. Recent theore- tical advances integrated both research lines proposing a sequence of stochastic and deterministic processes along successional gradients. Shifts in ecosystem states along suc- cessional gradients are predicted to occur abruptly once abiotic and biotic factors dominate over dispersal as main driver. Considering the multidiversity composed of ﬁve organismal groups including plants, animals, and microbes, our results imply that stochastic, likely dis- persal-dominated, processes are replaced by rather deterministic processes such as envir- onmental ﬁltering and biotic interactions after around 60 years of succession in a glacier foreﬁeld. The niche-based character of later successional processes is further supported by a decline in multi-beta-diversity. Our results may update concepts of community assembly by considering multiple taxa, help to bridge the gap between research on successions and community assembly, and provide insights into the emergence of multidiverse and complex ecosystems. 1 Results 1 Multidiversity along the successional gradient. Multidiversity increases during the early successional stage and remains stationary as Fig. 1 Multidiversity along the successional gradient. Multidiversity increases during the early successional stage and remains stationary as succession continues. The vertical dashed line indicates the estimated threshold in multidiversity development, which is the threshold in community assembly as predicted by the framework of Chang and HilleRisLambers (2016)1. The grey regression line indicates the linear correlation between multidiversity and time since deglaciation using a broken-stick model. Blue dashed lines indicate the 2.5% and 97.5% quantiles of ﬁtted values. The yellow curve on the x-axis resembles the posterior distribution for the estimate of the breaking point, i.e., higher density of the frequency indicates a higher probability for the breaking point to be located at a given value. whereas temperature did not affect multidiversity (β = 0.01, p = 0.93, Fig. 3a). Furthermore, diversities of the ﬁve organismal groups varied independently of each other suggesting little or no mutual inﬂuences. In late succession, mean growth-season soil temperature had a signiﬁcant positive direct effect on the diversity of bryophytes (β = 0.35, p = 0.01) and a clear positive indirect effect on multidiversity mediated through all organismal groups (β = 0.30, p = 0.01). Plot age had a negative effect on fungal diversity (β = −0.35, p < 0.01) and a positive effect on bryophyte diversity (β = 0.30, p = 0.01) but no effect on multidiversity (β = −0.01, p = 0.89). We further detected positive residual covariances between vascular plant (β = 0.26, p = 0.04), inverte- brate (β = 0.25, p = 0.05) and bacterial (β = 0.29, p = 0.03) diversities with fungal diversity, suggesting mutual inﬂuences between taxa (Fig. 3b). Drivers of multidiversity. To quantify the putative relative importance of stochastic dispersal, species interactions, and environmental drivers before and after the threshold, we speciﬁed a path model in which time since deglaciation and mean growth- season soil temperature act as exogenous variables and directly affect diversities of single organismal groups. Soil temperature is an environmental factor that is important for the diversity of several trophic levels in alpine environments29,30. We also tested the effects of other environmental variables, such as soil nutrient content and soil-pH value but could not detect any signiﬁcant effects and thus removed these variables from the ﬁnal model (see “Methods” and Supplementary Data 1–4). Results After accounting for environmental variation, we consider the effect of time since deglaciation as a proxy for stochastic events, such as successful dispersal and colonization events31,32, leading to cumulating multidiversity over time. The total effects of time since degla- ciation (proxy for stochastic dispersal) and environmental drivers on multidiversity were estimated as indirect effects mediated through the organismal groups. Biotic interactions were modeled as the residual covariance among the diversity values among all organismal groups that remained after accounting for stochasti- city and environmental variation. If corrected for such underlying common causes, strong positive covariances within communities can be interpreted as biotic interactions33,34. We further tested whether the residuals of the estimated models show signiﬁcant spatial autocorrelation but could not detect any unexplained variance that could be attributed to geographic variation (see Methods and Supplementary Table 2). Multi-betadiversity. We predicted that the shift from dispersal dominated stochastic events in early successional stages to more deterministic niche-based processes that are induced through environmental ﬁltering and biotic interactions in late successional stages is associated with lower beta-diversity among communities after the threshold. To test this prediction, we calculated a mea- sure of total community dissimilarity, multi-betadiversity (mbD) for both successional stages by the mean Bray-Curtis dissim- ilarities of the multidiverse community (see “Methods”). Of n = 5 organismal groups surveyed, n = 4 showed a decrease in betadi- versity and in total, multi-betadiversity was signiﬁcantly lower in the late successional stage (mean ± SD; mbDearly = 0.75 ± 0.09; mbDlate = 0.70 ± 0.08; Fig. 4). y The path analysis provided good model ﬁt (Model ﬁt: pchi-square > 0.05; CFI > 0.95; TLI ≥0.9; SRMR < 0.09; RMSEAearly < 0.05; RMSEAlate = 0.066 with lower bound of conﬁdence interval = 0) and revealed different inﬂuences of the exogenous variables between the early and late successional environments (Fig. 3, Supplementary Data 4). During early succession, time since deglaciation had a strong positive effect on the diversity of bryophytes (β = 0.66, p < 0.01), arthropods (β = 0.62, p < 0.01), and bacteria (β = 0.32, p = 0.04), whereas there was no signiﬁcant effect of temperature on any organismal group. Time since deglaciation also had a signiﬁcant positive indirect effect on total multidiversity (β = 0.74, p < 0.01) during early succession, Results Breaking point in multidiversity. Multidiversity follows a non- linear trajectory along the successional gradient. We screened the gradient for the predicted shift from a developing (characterized by an increase in multidiversity over time) to a more stationary ecosystem (characterized by non-monotonic variation in COMMUNICATIONS BIOLOGY \| (2022) 5:424 \| https://doi.org/10.1038/s42003-022-03372-2 \| www.nature.com/commsbio 2 ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-022-03372-2 Fig. 1 Multidiversity along the successional gradient. Multidiversity increases during the early successional stage and remains stationary as succession continues. The vertical dashed line indicates the estimated threshold in multidiversity development, which is the threshold in community assembly as predicted by the framework of Chang and HilleRisLambers (2016)1. The grey regression line indicates the linear correlation between multidiversity and time since deglaciation using a broken-stick model. Blue dashed lines indicate the 2.5% and 97.5% quantiles of ﬁtted values. The yellow curve on the x-axis resembles the posterior distribution for the estimate of the breaking point, i.e., higher density of the frequency indicates a higher probability for the breaking point to be located at a given value. multidiversity over time) using breaking point analysis, which detects changes in the slope of associations. According to Groffman (2006)27 breaking points represent ecological thresh- olds, which is characterized by a rapid change of the slope in the association between time since deglaciation as explanatory vari- able and multidiversity as dependent variable in our study. We found such a breaking point by cross-validating a broken-stick model to alternative models that either did not include a breaking point or included a disjunct breaking point using Bayesian inference28. We then used Bayes Factors analysis to validate the exact location of the breaking point after about 60 years of suc- cession, i.e., plot index 44 along the gradient (lower-upper bounds: 40–74 years after deglaciation; see “Methods”, Fig. 1, Supplementary Table 1). Resulting piecewise linear models showed a signiﬁcant linear increase in multidiversity during the early successional stage and a stationary multidiversity (non- signiﬁcant relationship with time) for the late successional stage (early: t42 = 5.77, p < 0.001, r2 = 0.44; late: t64 = −0.55, p = 0.58, r2 = 0.004; Fig. 1). To assess whether the delineated threshold is also reﬂected in the composition of the multidiverse community, we calculated the relative abundance of each taxon along the successional gradient and visualized it in a temporally ordered multi-taxa community table of species occurrences (see “Meth- ods”, Fig. 2). Fig. Discussion Multidiversity is indicated as light pink density plot on top of the community composition plot. Grey density plot on the left side indicates the range size of the taxa, i.e., whether their occurrence is restricted to a narrow range of the successional gradient or whether taxa occur along the whole gradient. Black bars on the right represent the organismal group of the taxa in each row, symbols of organismal groups are ordered by decreasing frequency; white dashed horizontal line marks the threshold. Fig. 3 Path analysis regarding the effects of stochastic and deterministic drivers on the multidiverse community for the early and late successional stages. Time since deglaciation and mean growth-season soil temperature act as exogenous variables with directed effects on the diversities of vascular plants, bryophytes, invertebrates, and soil-inhabiting fungi and bacteria. Indirect effects of exogenous variables on multidiversity were calculated by mediation analysis through the direct effects of the organismal groups on multidiversity. Biotic interactions are modeled as covariances between organismal groups. Numbers represent standardized path coefﬁcients and are given for signiﬁcant paths only. The path model was calculated for (a) the early and (b) the late successional stage separately. Silhouette images were obtained from PhyloPic (http://phylopic.org) under a public domain license (CC0 1.0 license). Fig. 3 Path analysis regarding the effects of stochastic and deterministic drivers on the multidiverse community for the early and late successional g. 3 Path analysis regarding the effects of stochastic and deterministic drivers on the multidiverse community f Fig. 3 Path analysis regarding the effects of stochastic and deterministic drivers on the multidiverse community for the early and late successional stages. Time since deglaciation and mean growth-season soil temperature act as exogenous variables with directed effects on the diversities of vascular plants, bryophytes, invertebrates, and soil-inhabiting fungi and bacteria. Indirect effects of exogenous variables on multidiversity were calculated by garding the effects of stochastic and deterministic drivers on the multidiverse community for the early and late s g y g g y y stages. Time since deglaciation and mean growth-season soil temperature act as exogenous variables with directed effects on the diversities of vascular plants, bryophytes, invertebrates, and soil-inhabiting fungi and bacteria. Indirect effects of exogenous variables on multidiversity were calculated by mediation analysis through the direct effects of the organismal groups on multidiversity. Biotic interactions are modeled as covariances between organismal groups. Discussion Our study is an empirical test of the conceptual framework integrating succession and community assembly dynamics pro- posed by Chang and HilleRisLambers (2016)1 and modiﬁed here in the context of multidiverse communities. We showed that threshold dynamics play an important role in the generation of multidiversity under natural conditions and that succession comprises a sequence of different ecosystem states that can be detected using multidiversity. These threshold-mediated shifts in ecosystem states are associated with substantial changes in the COMMUNICATIONS BIOLOGY \| (2022) 5:424 \| https://doi.org/10.1038/s42003-022-03372-2 \| www.nature.com/commsbio 3 ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-022-03372-2 Fig. 2 Single taxa occurrence along the successional gradient for each taxon that occurs on at least three plots along the gradient. Tiles indicate the relative abundance of each taxon at a given plot relative to the mean abundance of the taxon along the gradient. Taxa are ordered by the weighted average plot of their occurrence, i.e., taxa that reach their abundance optima early in succession appear in the top left and late successional specialist in the bottom right. Relative taxon abundance is color-coded and reﬂects z-scores with the mean abundance as 0 and one standard deviation as +1 or −1, rescaled between −1 and 1. Multidiversity is indicated as light pink density plot on top of the community composition plot. Grey density plot on the left side indicates the range size of the taxa, i.e., whether their occurrence is restricted to a narrow range of the successional gradient or whether taxa occur along the whole gradient. Black bars on the right represent the organismal group of the taxa in each row, symbols of organismal groups are ordered by decreasing frequency; white dashed horizontal line marks the threshold. Fig. 2 Single taxa occurrence along the successional gradient for each taxon that occurs on at least three plots along the gradient. Tiles indicate the relative abundance of each taxon at a given plot relative to the mean abundance of the taxon along the gradient. Taxa are ordered by the weighted average plot of their occurrence, i.e., taxa that reach their abundance optima early in succession appear in the top left and late successional specialist in the bottom right. Relative taxon abundance is color-coded and reﬂects z-scores with the mean abundance as 0 and one standard deviation as +1 or −1, rescaled between −1 and 1. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-022-03372-2 Fig. 4 Betadiversity values for each organismal group and community-wide multi-betadiversity values for the early and late successional stages. Mean Bray-Curtis dissimilarities were used to calculate betadiversity and multi-betadiversity values. Green violin plots resemble the dissimilarity values of the early successional stage (n = 44 plots), rose violin plots indicate the late successional stage (n = 66 plots). Boxplots indicate the upper and lower quartile of the estimates, and the size of the notch represents the conﬁdence interval of the median. a mean of all organismal groups, i.e., multi-betadiversity (b) bacteria, c fungi, d vascular plants, e bryopyhtes, and (f) invertebrates. Silhouette images were obtained from PhyloPic (http://phylopic.org) under a public domain license (CC0 1.0 license). Fig. 4 Betadiversity values for each organismal group and community-wide multi-betadiversity values for the early and late successional stages. Mean Bray-Curtis dissimilarities were used to calculate betadiversity and multi-betadiversity values. Green violin plots resemble the dissimilarity values of the early successional stage (n = 44 plots), rose violin plots indicate the late successional stage (n = 66 plots). Boxplots indicate the upper and lower quartile of the estimates, and the size of the notch represents the conﬁdence interval of the median. a mean of all organismal groups, i.e., multi-betadiversity (b) bacteria, c fungi, d vascular plants, e bryopyhtes, and (f) invertebrates. Silhouette images were obtained from PhyloPic (http://phylopic.org) under a public domain license (CC0 1.0 license). Our data do not allow to directly test for mutual inﬂuences between organismal groups on the taxon level, but we ﬁnd a strong pattern of positive covariances of the diversities of various taxonomic groups after accounting for environmental variation that is indicative of biotic dependencies within the community. Community covariances have initially been applied to evaluate the relative importance of biotic interactions in multitrophic communities by estimating synchronized ﬂuctuations in popu- lation abundances where positive covariances have been inter- preted as a result of facilitative interactions within a community33,34,39. On the diversity level as analyzed in this study, however, positive covariances among organismal groups do not necessarily reﬂect only facilitative interactions, because diversity in one group is likely to be positively linked to diversity of another trophic group through specialized interactions, either positive or negative40. For instance, in host-pathogen relation- ships, more diverse host communities harbor a higher diversity of obligate pathogens41 and below-ground fungal diversity is pro- moted by diverse bacterial assemblages through complex feed- back loops42. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-022-03372-2 A previous study in the same glacier foreﬁeld showed that after more than 50 years of ecosystem development, the soil microbial community is mainly supported by carbon from recent plant production, whereas during the initial stage of ecosystem development, the microbial community is mainly sustained by ancient carbon sources43. The time point of the shift in the main carbon source largely coincides with our delineated threshold in multidiversity development. The increased inter- dependence of the soil microbial and plant community during the later successional stage is also reﬂected in the positive covariances of vascular plant, fungal and bacterial diversities in our path model. Previous studies have also shown strong mutual depen- dencies of invertebrate and fungal communities in developing alpine environments44,45. Invertebrates increase the diversity of soil-inhabiting microbial taxa by propagule dispersal and create a variability of suitable habitats through modiﬁcations of the phy- sical environment46. Some fungal-feeding taxa show preferences community assembly processes. So far, the importance of threshold dynamics has been recognized for anthropogenically altered ecosystems either as catastrophic shifts with respect to climate change4,6 or for restoration efforts of anthropogenically altered landscapes2,3. Here, we revealed a threshold after about 60 years of ecosystem development when multidiversity became stationary. Signiﬁcant thresholds that are marked by a steep increase followed by stationarity after 40–60 years of succession appear to be a generalizable pattern that occurs in various aspects of ecosystem development, such as plant, invertebrate, and microbial diversities and functionality in glacier foreﬁelds in Europe and Northern America11,35–38. The threshold became also evident in the change of community composition with different sets of taxa before and after the breaking point. Prior to the threshold, communities mainly consist of species that reach their abundance optima early in succession. These pioneering species were soon accompanied by taxa with no clear preference of early or late successional stages leading to an increase in multidiversity over time. After the threshold, specialists for early successions are replaced by specialists for late successional stages, consequently multidiversity remained stationary over time (Fig. 2). Thus, acknowledging the effects of threshold dynamics during the development of natural multidiverse ecosystems, and their uni- versality in ecological systems provides valuable insights into the patterns and processes of initial ecosystem development. Discussion Numbers represent standardized path coefﬁcients and are given for signiﬁcant paths only. The path model was calculated for (a) the early and (b) the late successional stage separately. Silhouette images were obtained from PhyloPic (http://phylopic.org) under a public domain license (CC0 1.0 license). COMMUNICATIONS BIOLOGY \| (2022) 5:424 \| https://doi.org/10.1038/s42003-022-03372-2 \| www.nature.com/commsbio 4 ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-022-03372-2 COMMUNICATIONS BIOLOGY \| (2022) 5:424 \| https://doi.org/10.1038/s42003-022-03372-2 \| www.nature.com/commsbio Methods S d d i Although co-occurrence patterns must not necessarily reﬂect biotic interactions54, reduced beta-diversity between plots in later successional stages still may be indicative for stronger biotic interactions, which ﬁnds support in studies on plant community assembly55,56. The literature suggests the increasing importance of interactions in community development over time. One explanation put forward is that with increasing niche differentiation, energy and nutrient ﬂows in an ecosystem follow more complex biochemical pathways that necessitate biotic interactions to retain nutrients in the ecosystem through closed mineral cycles and complex food-webs1,17. Our results support the increasing importance of biotic interactions as in the late successional stage, our path model revealed positive community covariances between the soil microbial, vascular plant, and arthropod communities that may represent such complex nutritional cycles comprising primary producers, het- erotrophic organisms, and decomposers For the organismal groups forming these putative cycles, we ﬁnd a reduced betadi- versity between the plots that indicates a strong interrelatedness of individual taxa after the threshold, which may lead to re- occurring core assemblages of species of several taxa. These assumptions and the threshold suggested by our data after 60 years are further supported by the study of Bardgett et al. (2007)43 that revealed a high dependence of heterotrophic microorganisms on plant communities in the Ödenwinkel fore- ﬁeld after a similar number of years. We thus recommend extending the integrative framework of succession and commu- nity assembly for multiple interacting taxa that mutually shape their diversity and composition and thus cause a reduction of betadiversity. Sampling. In 2019, we identiﬁed all vascular plant and bryophyte species present on the plots and estimated their cover with a resolution of 0.1%. We sampled above-ground arthropod diversity by installing two pitfall traps on each plot. Traps were set active for a total of n = 7 days. The abundance of all arthropods, excluding Collembola and Acari, larger than 3 mm was counted. The abundance of Col- lembola and Acari and of animals smaller than 3 mm was estimated based on random samples of aliquots of the total sample. All arthropods and other animals are identiﬁed to the order level. Soil-inhabiting bacteria and fungi were sampled from soil cores from an approximate depth of 3 cm, as soil development in the proglacial study area was limited to the top layers of the pedosphere. Methods S d d i Study design. The study was conducted in the long-term ecological research platform Ödenwinkel which was established in 2019 in the Hohe Tauern National Park, Austria (Dynamic Ecological Information Management System—site and dataset registry: https://deims.org/activity/fefd07db-2f16-46eb-8883-f10fbc9d13a3, last access: March 2021). A total of n = 135 permanent plots was established within the glacier foreﬁeld of the Ödenwinkelkees which was covered by ice at the latest glacial maximum in the Little Ice Age around 1850. For this study, we used a subset of n = 110 plots with complete datasets with all biotic and abiotic variables available. The plots represent a successional gradient spanning over 1.7 km in length and were distributed within the glacier foreﬁeld to reﬂect the gradient of glacial retreat at an altitude ranging from 2070 to 2170 m a.s.l (distance between neighboring plots, median ± SD: 19 m ± 6.3 m). Each plot was given a unique index number according to the location along the gradient: the plot with index 1 is located closest to the glacier, plot index 135 is the farthest away from the glacier. Plot age was estimated based on data of historical glacial extent and as glacial loss did not occur at a constant rate, plot locations were pre-selected to represent a linear gradient of time since deglaciation rather than a spatial gradient with equally spaced intervals between plots. To ensure independence of plots in areas of slow glacial retreat, we chose a minimum distance of 5 m between two neighboring plots. Plots were deﬁned as squares with an area of 1 m² and were all oriented in the same cardinal direction. Further details on the design of the research platform, exact plot positions and details on the surrounding environment, as well as on the historical glacial extent can be found in Junker et al. (2020)26. litter). The threshold-mediated character of successional processes is further supported by a pronounced decline in multi-betadiversity that is indicative of an increased dependence structure among the organismal groups. In line with that assumption, we detected a lower beta-diversity in all those organismal groups that also show positive community covariances in the path model. Out of the ﬁve organismal groups we studied, only bryophytes were char- acterized by a higher betadiversity in the late successional stage that might be attributable to increasing variability in micro- habitats as the communities mature52,53. ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-022-03372-2 for certain types of fungal hyphae that frequently have distinct associations with selected plant species as pathogens47, mycor- rhizal partners48, or litter decomposers49, which well explains the pattern of covariance among these groups in our analysis. Fur- ther, the importance of fungi-animal interactions for the devel- opment of ecological complexity was predicted in foundational works on succession theory by Connell and Slatyer (1977)50, as heterotrophic fungi were expected to feed on the carcasses and dung of animals and in turn, certain arthropods were hypothe- sized to be reliant on decomposed substrate for the develop- mental stages of their life cycles. Although bryophytes have been shown to closely interact with epiphytic microorganisms after glacial retreat51, potential interactions of bryophytes with microorganisms may not be detectable in our path model as we sampled soil microbial communities and not all substrate types colonized by bryophytes in our study site (e.g., rocks, scree, and litter). natural ecosystems. These results are not only a proof of concept but also (re-)emphasize that succession is a multi-faceted rather than a linear process that comprises a sequence of assembly processes towards multidiversity and ecosystem complexity. Methods S d d i Soil- microbiome samples were analyzed by next-generation sequencing, and micro- biome proﬁling of isolated DNA was performed by Euroﬁns Genomics (Ebersberg, Germany). Prior to the statistical analysis of microbial communities, we performed a cumulative sum scaling (CSS) normalization (R-package “metagenomeSeq” v1.28.2)57 on the count data to account for differences in sequencing depth among samples. Detailed information on the sampling strategies of all organismal groups can be found in Junker et al. (2020)26. Soil temperature measurements were done by installing temperature loggers (MF1921G iButton, Fuchs Elektronik, Weinheim, Germany) 10 cm north of each plot center, at the same depth of 3 cm at which the microbial samples were taken. Mean growth-season soil temperature was calculated based on the recordings ranging from 26th of June to 16th of September repre- senting the period in which the plots were free of permanent snow cover before and after the winter 2019/2020. In 2020, soil samples were taken and soil nutrients (Ca, P, K, Mg, and total N2) as well as soil pH were measured on all plots by AGROLAB Agrar und Umwelt GmbH (Sarstedt, Germany). Soil nutrient analysis was per- formed according to the Ö-Norm Boden: L 1087: 2012-12 (K and P—mg/1000 g), L 1093: 2010-12 (Mg—mg/1000 g), and L 1083: 2006-04 (pH). Total N2 (%) was determined according to the DIN EN 16168: 2012-11. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-022-03372-2 Our results indicate an ecosystem state shift associated with different assembly processes: the ﬁrst 60 years of succession are characterized by stochastic species additions, afterwards rather deterministic processes such as niche-ﬁltering and biotic inter- actions dominate. Although different organismal groups follow individual trajectories during primary succession, multidiversity is mainly promoted by stochastic drivers during the initial phase of ecosystem development, most assumably by heterogenous dispersal events. Over the course of succession, stochasticity is replaced by environmental ﬁltering and biotic interactions as the structuring mechanism of multidiversity. 5 5 COMMUNICATIONS BIOLOGY \| (2022) 5:424 \| https://doi.org/10.1038/s42003-022-03372-2 \| www.nature.com/commsbio COMMUNICATIONS BIOLOGY \| (2022) 5:424 \| https://doi.org/10.1038/s42003-022-03372-2 \| www.nature.com/commsbio Statistics and reproducibility models by calculating spatial neighbor matrices for distance classes of 5 m and 10 m between plots (i.e., the potential to include a maximum of three or ﬁve neighboring plots). For both distance classes, we estimated Moran´s I for the residuals of the piece wise linear models of the breaking point analysis and the case-wise residuals of the exogenous variables of the path models using the R-Package “spdep” v1.1-865. Breaking point analysis. According to the hypothesis of community assembly dynamics1 and by visual inspection of the relationship between multidiversity and successional age of the plots, we expected two different stages of community establishment along the successional gradient. These stages are separatable by the transition from a developing (characterized by an increase in multidiversity over time) to a more stationary ecosystem (characterized by non-monotonic variation in multidiversity over time). Such non-linear associations are suggestive of regime- shifts of the ecosystem and can be interpreted as ecological thresholds27. Piecewise regression models have been shown to be the most suitable tool for the detection of ecological thresholds in natural systems, as they are able to correctly estimate the probability, as well as the number and position of ecological thresholds58. We screened the successional gradient for the existence of a threshold by comparing four different models with mD as the dependent and plot age as the independent variable using the R-package “mcp” v0.3.0.928. The mcp-method ﬁts piecewise regression models with a pre-deﬁned number of breaking points and is based on Bayesian inference. We speciﬁed a base model m1 (mD-values remain constant along the gradient), and compared it to three alternative models (m2 = constant linear increase of mD-values without a breaking point, m3 = one breaking point in mD-values with a segregated slope (abrupt-threshold model), m4 = one breaking point in mD-values with a joined slope (broken-stick or smooth-threshold model)). For each model, we separately ran three Markov Chain Monte Carlo estimators with a uniform prior for a total of n = 11,000 generations while discarding the ﬁrst n = 1000 generations as burn-in. Model convergence was estimated by visually inspecting the trace plots and checking that all model parameters reached statio- narity. We then compared the predictive performance of the four models using leave-one-out-cross-validation and conﬁrmed the exact position of the breaking point using Bayes Factors. References 1. Chang, C. & HilleRisLambers, J. Integrating succession and community assembly perspectives. F1000Research 5, 1–10 (2016). 1. Chang, C. & HilleRisLambers, J. Integrating succession and community assembly perspectives. F1000Research 5, 1–10 (2016). 1. Chang, C. & HilleRisLambers, J. Integrating succession and community assembly perspectives. F1000Research 5, 1–10 (2016). 2. Suding, K. N. & Hobbs, R. J. Threshold models in restoration and conservation: a developing framework. Trends Ecol. Evol. 24, 271–279 (2009) 2. Suding, K. N. & Hobbs, R. J. Threshold models in restoration and conservation: a developing framework. Trends Ecol. Evol. 24, 271–279 (2009 g, , J conservation: a developing framework. Trends Ecol. Evol. 24, 271–279 (2009). 3. Kadowaki, K., Nishijima, S., Kéﬁ, S., Kameda, K. O. & Sasaki, T. Merging community assembly into the regime-shift approach for informing ecological restoration. Ecol. Indic. 85, 991–998 (2018). Path analysis. We used path analysis (i) to model the inﬂuence of the abiotic environment on the diversities of all taxonomic groups individually, (ii) to estimate the strength of covariance between the diversities of those groups, (iii) to infer the effect sizes of the diversity of each group on total mD, and (iv) estimate the strength of indirect effects of the exogenous variables that are mediated through the organismal groups on mD. We built separate models with identical structure for the early (n = 44 plots) and late (n = 66 plots) successional stages. The stages were delineated by the threshold identiﬁed in the breakpoint analysis. Exogenous vari- ables in the model were time since deglaciation and mean growth-season soil temperature. Time since deglaciation reﬂects the plot age and can be seen as a proxy for the increasing chance of heterogeneous dispersal events that occur over time. The mean temperature of the growing season is an estimate for environ- mental heterogeneity and has been shown to affect diversity directly and indirectly on various trophic levels29. All variables were scaled by subtracting the mean and dividing through the standard deviation. We ﬁrst ran a full model that included soil pH and soil nutrient content as additional exogenous variables. None of the two variables showed a signiﬁcant direct effect on any organismal group or a signiﬁcant indirect effect on multidiversity during either the young or late successional stage. References Accordingly, we stepwise removed the two variables from the model while cross- checking whether the effect strength of one variable became signiﬁcant in the absence of the other variable. As no signiﬁcant effects occurred or the model ﬁt signiﬁcantly decreased, we decided to remove both variables from the ﬁnal model. Within the ﬁnal model, the strength of residual covariance between the diversities of all taxonomic groups was estimated while accounting for inﬂuences of time since deglaciation and temperature. If corrected for an underlying common cause, such as environmental autocorrelation, strong covariances between members of a community can be interpreted as biotic interactions and especially positive com- munity covariances are indicative of facilitative effects within the community33,34. All path models were estimated using the R-package “lavaan” v0.6-762. 4. Scheffer, M., Carpenter, S., Foley, J. A., Folke, C. & Walker, B. Catastrophic shifts in ecosystems. Nature 413, 591–596 (2001). 5. Lu, M. & Hedin, L. O. Global plant–symbiont organization and emergence of biogeochemical cycles resolved by evolution-based trait modelling. Nat. Ecol. Evol. 3, 239–250 (2019). 6. Berdugo, M. et al. Global ecosystem thresholds driven by aridity. Science 367, 787–790 (2020). 7. Allan, E. et al. Interannual variation in land-use intensity enhances grassland multidiversity. Proc. Natl Acad. Sci. USA 111, 308–313 (2014). y ( ) 8. Delgado-Baquerizo, M. et al. Multiple elements of soil biodiversity drive t f ti bi N t E l E l 4 210 220 (2020) y 8. Delgado-Baquerizo, M. et al. Multiple elements of soil biodiversity drive ecosystem functions across biomes. Nat. Ecol. Evol. 4, 210–220 (2020). y 9. Felipe-Lucia, M. R. et al. Land-use intensity alters networks between y 9. Felipe-Lucia, M. R. et al. Land-use intensity alters networks between biodiversity, ecosystem functions, and services. Proc. Natl Acad. Sci. USA 117, 28140–28149 (2020). biodiversity, ecosystem functions, and services. Proc. Natl Acad. Sci. USA 117, 28140–28149 (2020). 10. Manning, P. et al. Grassland management intensiﬁcation weakens the associations among the diversities of multiple plant and animal taxa. Ecology 96, 1492–1501 (2015). 11. Kaufmann, R. Invertebrate succession on an alpine glacier foreland. Ecology 82, 2261–2278 (2001). 12. Blaalid, R. et al. Changes in the root-associated fungal communities along a primary succession gradient analysed by 454 pyrosequencing. Mol. Ecol. 21, 1897–1908 (2012). 13. Fenton, N. J. & Bergeron, Y. Stochastic processes dominate during boreal bryophyte community assembly. Ecology 94, 1993–2006 (2013). 14. Dini-Andreote, F., Stegen, J. C., Van Elsas, J. Data availability R f Raw sequences of next-generation 16 S rRNA gene amplicon sequencing are available at the NCBI Sequence Read Archive (SRA) under the BioProject accession PRJNA701884 and PRJNA701890. Raw ﬂoristic, microbial, and zoological community composition data are available in a Mendeley Data repository66. https://doi.org/10.17632/xkv89tbftc.1. Code availability R i il bl i R scripts are available in a Mendeley Data repository67. https://doi.org/10.17632/ dr6d3728xb.1. Community taxa occurrence. To visualize the distribution of individual taxa and thus the community-wide taxonomic turnover along the successional gradient, we estimated the abundance optimum of each taxon that occurred on at least three plots. Abundance optima were estimated by calculating the weighted mean plot of occurrence with abundance of each taxon as weighting factor (i.e., cover for vas- cular plants and bryophytes, individual count for invertebrates, and CSS- normalized read number for microorganisms). We further calculated the relative abundance of each taxon per plot by using z-scores of abundances. The mean abundance was set as 0 with one standard deviation as +1 or −1. To allow a comparison across taxa, z-scores were then rescaled between −1 and 1. Range size was estimated by calculating the variance of occurrence plots (i.e., the span of plots on which a taxon was found along the gradient) weighted by the abundance of the taxon on the respective plots. Received: 3 November 2021; Accepted: 14 April 2022; Statistics and reproducibility Both validation methods can be applied for a robust and accurate testing of competing hypotheses in ecological datasets59–61. Multi-betadiversity. We deﬁned Multi-betadiversity (mbD) as the cumulative averaged pairwise-dissimilarity across a number of organismal groups. First, we split the total community composition data table (sites x species, n = 110 plots) for each organismal group into two tables containing the plots before (n = 44 plots) and after (n = 66 plots) the threshold. Second, we calculated pairwise Bray-Curtis dissimilarities for each of the organismal groups individually. Third, we calculated mbD as the mean of the n = 5 dissimilarity estimates of each plot pair. Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article. Statistics and reproducibility Calculation of multidiversity. Multidiversity is deﬁned as the cumulative diversity of a number of taxonomic groups7. The multidiversity of the n = 110 plots composed of the diversities of vascular plants, bryophytes, invertebrates, fungi, and bacteria present in each plot and was deﬁned as follows: First, we calculated the Shannon diversities of each of the taxonomic groups in each plot. Second, we ranked the plots by increasing diversity for each of the ﬁve taxonomic groups individually, i.e., for each plot we received n = 5 ranks. The mean rank of each plot was deﬁned as multidiversity mD. For better interpretability of the index, we then normalized the mean ranks as Understanding the relative importance and temporal dynamics of deterministic and stochastic processes is a key challenge in community ecology, especially in natural systems and has been in the center of broad debates among ecologists. Using the multi- diversity and multi-betadiversity approach allows us to compre- hensively understand the processes leading to stable, resilient, and complex ecosystems, which may remain vague in single-taxon approaches. Thus, our study contributes to a synthesis of com- munity ecological theories into succession research, acknowl- edging the fundamental importance of abrupt state shifts in x0 ¼ ðx minðxÞÞ=ðmaxðxÞ minðxÞÞ ð1Þ ð1Þ to scale them between zero and one. Ranks were used to give the same weighting to each taxonomic group despite deviations in the absolute values of Shannon diversity and to reduce the impact of outliers in mD values. To account for dif- ferences in the sequencing depth of the microbial raw dataset and sampling effort of the invertebrate trapping, we performed multiple rarefactioning prior to the calculation of Shannon diversity by averaging the results of n = 999 iterations 6 ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-022-03372-2 (R-package “rtk” v0.2.5.7) and used original read numbers instead of using the CSS-normalized dataset for the microbial dataset. models by calculating spatial neighbor matrices for distance classes of 5 m and 10 m between plots (i.e., the potential to include a maximum of three or ﬁve neighboring plots). For both distance classes, we estimated Moran´s I for the residuals of the piece wise linear models of the breaking point analysis and the case-wise residuals of the exogenous variables of the path models using the R-Package “spdep” v1.1-865. COMMUNICATIONS BIOLOGY \| (2022) 5:424 \| https://doi.org/10.1038/s42003-022-03372-2 \| www.nature.com/commsbio COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-022-03372-2 Contrasting primary successional trajectories of fungi and bacteria in retreating glacier soils. Mol. Ecol. 23, 481–497 (2014). 54. Blanchet, F. G., Cazelles, K. & Gravel, D. Co‐occurrence is not evidence of ecological interactions. Ecol. Lett. 23, 1050–1063 (2020). 24. Castle, S. C. et al. Biogeochemical drivers of microbial community convergence across actively retreating glaciers. Soil Biol. Biochem. 101, 74–84 (2016). 55. Fukami, T. & Nakajima, M. Complex plant-soil interactions enhance plant species diversity by delaying community convergence. J. Ecol. 101, 316–324 (2013). 25. Chang, C. C. et al. Testing conceptual models of early plant succession across a disturbance gradient. J. Ecol. 107, 517–530 (2019). 56. Martínez-García, L. B., Richardson, S. J., Tylianakis, J. M., Peltzer, D. A. & Dickie, I. A. Host identity is a dominant driver of mycorrhizal fungal community composition during ecosystem development. N. Phytol. 205, 1565–1576 (2015). 26. Junker, R. R. et al. Ödenwinkel: an Alpine platform for observational and experimental research on the emergence of multidiversity and ecosystem complexity. Web Ecol. 20, 95–106 (2020). p y 27. Groffman, P. M. et al. Ecological thresholds: the key to successful environmental management or an important concept with no practical application? Ecosystems 9, 1–13 (2006). 57. Paulson, J. metagenomeSeq: statistical analysis for sparse high-throughput sequencing. Bioconductor.Jp https://github.com/nosson/metagenomeSeq/ (2014). pp y 28. Lindeløv, J. K. mcp: an R package for regression With multiple change points. J. Stat. Softw. https://doi.org/10.31219/osf.io/fzqxv (2020). 58. Francesco Ficetola, G. & Denoël, M. Ecological thresholds: an assessment of methods to identify abrupt changes in species-habitat relationships. Ecography 32, 1075–1084 (2009). 29. Ohler, L. M., Lechleitner, M. & Junker, R. R. Microclimatic effects on alpine plant communities and ﬂower-visitor interactions. Sci. Rep. 10, 1–9 (2020). 59. Aho, K., Derryberry, D. & Peterson, T. Model selection for ecologists: the worldviews of AIC and BIC. Ecology 95, 631–636 (2014). 30. Cavieres, L. A. et al. Facilitative plant interactions and climate simultaneously drive alpine plant diversity. Ecol. Lett. 17, 193–202 (2014). 60. Conn, P. B., Johnson, D. S., Williams, P. J., Melin, S. R. & Hooten, M. B. A guide to Bayesian model checking for ecologists. Ecol. Monogr. 88, 526–542 (2018). 31. Lowe, W. H. & McPeek, M. A. Is dispersal neutral? Trends Ecol. Evol. 29, 444–450 (2014). 61. Hanusch, M., Ortiz, E. M., Patiño, J. & Schaefer, H. Biogeography and integrative taxonomy of epipterygium (Mniaceae, Bryophyta). Taxon 69, 1150–1171 (2020). ( ) 32. Legendre, P., Borcard, D. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-022-03372-2 COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-022-03372-2 16. Pulsford, S. A., Lindenmayer, D. B. & Driscoll, D. A. A succession of theories: purging redundancy from disturbance theory. Biol. Rev. 91, 148–167 (2016). 17. Odum, E. P. The strategy of ecosystem development. Science 164, 262–270 (1969). 16. Pulsford, S. A., Lindenmayer, D. B. & Driscoll, D. A. A succession of theories: purging redundancy from disturbance theory. Biol. Rev. 91, 148–167 (2016). 48. Klironomos, J. N. & Kendrick, W. B. Palatability of microfungi to soil arthropods in relation to the functioning of arbuscular mycorrhizae. Biol. Fertil. Soils 21, 43–52 (1996). p g g y y 17. Odum, E. P. The strategy of ecosystem development. Science 164, 262–270 (1969). 49. Klironomos, J. N., Widden, P. & Deslandes, I. Feeding preferences of the collembolan Folsomia candida in relation to microfungal successions on decaying litter. Soil Biol. Biochem. 24, 685–692 (1992). 18. Ohlmann, M. et al. Mapping the imprint of biotic interactions on β-diversity. Ecol. Lett. 21, 1660–1669 (2018). 19. Chen, C., Chen, H. Y. H., Chen, X. & Huang, Z. Meta-analysis shows positive effects of plant diversity on microbial biomass and respiration. Nat. Commun. 10, 1–10 (2019). y g 50. Connell, J. H. & Slatyer, R. O. Mechanisms of succession in natural communities and their role in community stability and organization. Am. Nat 111, 1119–1144 (1977). 20. Howard, M. M., Kao-Knifﬁn, J. & Kessler, A. Shifts in plant–microbe interactions over community succession and their effects on plant resistance to herbivores. N. Phytol. 226, 1144–1157 (2020). 51. Arróniz-Crespo, M. et al. Bryophyte-cyanobacteria associations during primary succession in recently deglaciated areas of Tierra del Fuego (Chile). PLoS One 9, 15–17 (2014). y 21. Lepš, J., Rejmánek, M., Leps, J. & Rejmanek, M. Convergence or divergence: what should we expect from vegetation succession? Oikos 62, 261–264 (1991). 52. Jean, M. et al. Experimental assessment of tree canopy and leaf litter controls on the microbiome and nitrogen ﬁxation rates of two boreal mosses. N. Phytol. 227, 1335–1349 (2020). 22. Fukami, T., Bezemer, T. M., Mortimer, S. R. & Van Der Putten, W. H. Species divergence and trait convergence in experimental plant community assembly. Ecol. Lett. 8, 1283–1290 (2005). 53. Jean, M., Alexander, H. D., Mack, M. C. & Johnstone, J. F. Patterns of bryophyte succession in a 160-year chronosequence in deciduous and coniferous forests of boreal Alaska. Can. J. Res. 47, 1021–1032 (2017). 23. Brown, S. P. & Jumpponen, A. Acknowledgements 40. Vellend, M. Effects of diversity on diversity: consequences of competition and facilitation. Oikos 117, 1075–1085 (2008). We thank the Hohe Tauern National Park Salzburg administration and the Rudolfshütte for organizational and logistic support, the governing authority Land Salzburg for the permit to conduct our research (permit no. 20507-96/45/7-2019), Jan-Christoph Otto, Tobias Seifert, and Anna Vojtkó for help in the ﬁeld. Hamed Azarbad, Lisa-Maria Ohler and Verena Zieschank provided valuable comments to improve the study. This research has been supported by the Austrian Science Fund (FWF), which provided funding to R.R.J. (grant no. Y1102). 41. Rottstock, T., Joshi, J., Kummer, V. & Fischer, M. Higher plant diversity promotes higher diversity of fungal pathogens, while it decreases pathogen infection per plant. Ecology 95, 1907–1917 (2014). p p gy 42. Baudy, P. et al. Fungal–fungal and fungal–bacterial interactions in aquatic decomposer communities: bacteria promote fungal diversity. Ecology 102, 1–16 (2021). 43. Bardgett, R. D. et al. Heterotrophic microbial communities use ancient carbon following glacial retreat. Biol. Lett. 3, 487–490 (2007). Author contributions 44. Bokhorst, S. & Wardle, D. A. Snow fungi as a food source for micro- arthropods. Eur. J. Soil Biol. 60, 77–80 (2014). R.R.J. conceived and initiated the study. M.H., X.H., V.R.-H., and R.R.J. designed the study and conducted ﬁeldwork. M.H. performed the processing and analysis of the data with main inputs from R.R.J. and X.H. M.H. and R.R.J. drafted the initial version of the manuscript. All authors contributed critically to the interpretation of the results, revising, and approving the ﬁnal version of the manuscript. p 45. Hågvar, S. et al. Ecosystem birth near melting glaciers: a review on the pioneer role of ground-dwelling arthropods. Insects 11, 1–35 (2020). role of ground-dwelling arthropods. Insects 11, 1–35 (2020). 46. Wardle, D. A. The inﬂuence of biotic interactions on soil biodiversity. Ecol. Lett. 9, 870–886 (2006). 47. Sabatini, M. A. & Innocenti, G. Functional relationships between Collembola and plant pathogenic fungi of agricultural soils. Pedobiologia (Jena.) 44, 467–475 (2000). COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-022-03372-2 & Peres-Neto, P. R. Analyzing beta diversity: partitioning the spatial variation of community composition data. Ecol. Monogr. 75, 435–450 (2005). 62. Oksanen, J. et al. vegan: community ecology package. Github https://github. com/vegandevs/vegan (2019). g 33. Ranta, E. et al. Detecting compensatory dynamics in competitive communities under environmental forcing. Oikos 117, 1907–1911 (2008). 63. Baker, S. C. & Barmuta, L. A. Evaluating spatial autocorrelation and depletion in pitfall-trap studies of environmental gradients. J. Insect Conserv. 10, 269–276 (2006). 34. Houlahan, J. E. et al. The utility of covariances: a response to Ranta et al. Oikos 117, 1912–1913 (2008). 64. Pickett, S. T. A. in Long-Term Studies in Ecology (ed. Likens, G. E.) 110–135 (Springer, 1989). 35. Tscherko, D., Rustemeier, J., Richter, A., Wanek, W. & Kandeler, E. Functional diversity of the soil microﬂora in primary succession across two glacier forelands in the Central Alps. Eur. J. Soil Sci. 54, 685–696 (2003). 65. Bivand, R. S. & Wong, D. W. S. Comparing implementations of global and local indicators of spatial association. Test 27, 716–748 (2018). 36. Rafﬂ, C., Mallaun, M., Mayer, R. & Erschbamer, B. Vegetation succession pattern and diversity changes in a Glacier Valley, Central Alps, Austria. Arct. Antarct. Alp. Res. 38, 421–428 (2006). 66. Hanusch, M., He, X., Ruiz-Hernández, V. & Junker, R. R. Successional generation of functional multidiversity and ecosystem complexity—a dataset from the Ödenwinkel research platform. Mendeley Data. https://doi.org/10. 17632/xkv89tbftc.1 (2022). p 37. Schütte, U. M. E. et al. Bacterial diversity in a glacier foreland of the high Arctic. Mol. Ecol. 19, 54–66 (2010). 67. Hanusch, M., He, X., Ruiz-Hernández, V. & Junker, R. R. Supporting dataset and code: succession comprises a sequence of threshold-induced community assembly processes towards multidiversity. Mendeley Data. https://doi.org/10. 17632/dr6d3728xb.1 (2022). 38. Dong, K. et al. Soil fungal community development in a high Arctic glacier foreland follows a directional replacement model, with a mid-successional diversity maximum. Sci. Rep. 6, 1–9 (2016). y p 39. Houlahan, J. E. et al. Compensatory dynamics are rare in natural ecological communities. Proc. Natl Acad. Sci. USA 104, 3273–3277 (2007). References D. & Salles, J. F. Disentangling mechanisms that mediate the balance between stochastic and deterministic processes in microbial succession. Proc. Natl Acad. Sci. USA 112, E1326–E1332 (2015). Test for spatial autocorrelation. Studies taking advantage of space-for-time sub- stitution generally are prone to erroneous conclusions caused by spatial auto- correlation, especially when sampling points are located at varying distances63,64. We tested for the presence of residual spatial autocorrelation in the estimated path 15. Måren, I. E., Kapfer, J., Aarrestad, P. A., Grytnes, J. A. & Vandvik, V. Changing contributions of stochastic and deterministic processes in community assembly over a successional gradient. Ecology 99, 148–157 (2018). 7 COMMUNICATIONS BIOLOGY \| (2022) 5:424 \| https://doi.org/10.1038/s42003-022-03372-2 \| www.nature.com/commsbio © The Author(s) 2022 Competing interests The authors declare no competing interests. 8 COMMUNICATIONS BIOLOGY \| (2022) 5:424 \| https://doi.org/10.1038/s42003-022-03372-2 \| www.nature.com/commsbio Additional information Open Access This article is licensed under a Creative Commons Attri- bution 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/W3050064217	https://www.frontiersin.org/articles/10.3389/fchem.2020.00715/pdf	English	null	High Power Energy Storage via Electrochemically Expanded and Hydrated Manganese-Rich Oxides	Frontiers in chemistry	2,020	cc-by	10,381	Edited by: Jennifer L. Schaefer, University of Notre Dame, United States Reviewed by: Yanliang Liang, University of Houston, United States Liqiang Mai, Wuhan University of Technology, China Reviewed by: Yanliang Liang, University of Houston, United States Liqiang Mai, Wuhan University of Technology, China Keywords: aqueous electrolyte, manganese oxide, interlayer, conﬁned water, electrochemical capacitor Specialty section: This article was submitted to Electrochemistry, a section of the journal Frontiers in Chemistry Received: 14 May 2020 Accepted: 09 July 2020 Published: 18 August 2020 Citation: Boyd S, Geise NR, Toney MF and Augustyn V (2020) High Power Energy Storage via Electrochemically Expanded and Hydrated Manganese-Rich Oxides. Front. Chem. 8:715. doi: 10.3389/fchem.2020.00715 Specialty section: This article was submitted to Electrochemistry, a section of the journal Frontiers in Chemistry ORIGINAL RESEARCH published: 18 August 2020 doi: 10.3389/fchem.2020.00715 High Power Energy Storage via Electrochemically Expanded and Hydrated Manganese-Rich Oxides Shelby Boyd 1, Natalie R. Geise 2,3, Michael F. Toney 3 and Veronica Augustyn 1* 1 Department of Materials Science and Engineering, North Carolina State University, Raleigh, NC, United States, 2 Departme of Chemistry, Stanford University, Stanford, CA, United States, 3 Stanford Synchrotron Radiation Lightsource, SLAC Nationa Accelerator Laboratory, Menlo Park, CA, United States 1 Department of Materials Science and Engineering, North Carolina State University, Raleigh, NC, United States, 2 Department of Chemistry, Stanford University, Stanford, CA, United States, 3 Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, CA, United States Understanding the materials design features that lead to high power electrochemical energy storage is important for applications from electric vehicles to smart grids. Electrochemical capacitors offer a highly attractive solution for these applications, with energy and power densities between those of batteries and dielectric capacitors. To date, the most common approach to increase the capacitance of electrochemical capacitor materials is to increase their surface area by nanostructuring. However, nanostructured materials have several drawbacks including lower volumetric capacitance. In this work, we present a scalable “top-down” strategy for the synthesis of EC electrode materials by electrochemically expanding micron-scale high temperature-derived layered sodium manganese-rich oxides. We hypothesize that the electrochemical expansion induces two changes to the oxide that result in a promising electrochemical capacitor material: (1) interlayer hydration, which improves the interlayer diffusion kinetics and buffers intercalation-induced structural changes, and (2) particle expansion, which signiﬁcantly improves electrode integrity and volumetric capacitance. When compared with a commercially available activated carbon for electrochemical capacitors, the expanded materials have higher volumetric capacitance at charge/discharge timescales of up to 40 s. This shows that expanded and hydrated manganese-rich oxide powders are viable candidates for electrochemical capacitor electrodes. Keywords: aqueous electrolyte, manganese oxide, interlayer, conﬁned water, electrochemical capacitor INTRODUCTION Electrochemical energy storage plays ever-increasing roles in our daily lives by powering everything from portable electronics to electric vehicles. Electrochemical capacitors (ECs) are a class of energy storage devices with intermediate energy and power between those of batteries and dielectric capacitors. Commercialized ECs are highly attractive for diverse applications requiring reliability, large currents, and high energy eﬃciency. They exhibit speciﬁc energy of up to 10 Wh/kg, speciﬁc power of up to 30 kW/kg, typical charge/discharge timescales on the order of seconds, and lifetimes of up to ∼1,000,000 cycles (Conway, 1999; Nybeck et al., 2019). Most commercialized ECs store energy by the rapid (within seconds) formation of the electric double layer at the electrode/electrolyte interface. This mechanism can give rise to speciﬁc capacitance values of up to 150 F/g when the electrolyte ion size matches the average pore size of the electrode material (Largeot et al., 2008). Another capacitive energy storage mechanism, termed pseudocapacitance, Received: 14 May 2020 Accepted: 09 July 2020 Published: 18 August 2020 Citation: Boyd S, Geise NR, Toney MF and Augustyn V (2020) High Power Energy Storage via Electrochemically Expanded and Hydrated Manganese-Rich Oxides. Front. Chem. 8:715. doi: 10.3389/fchem.2020.00715 August 2020 \| Volume 8 \| Article 715 Frontiers in Chemistry \| www.frontiersin.org 1 Electrochemically Expanded Hydrated Manganese-Rich Oxides Boyd et al. poor electronic and ionic conductivity (Clément et al., 2015; Clement et al., 2016; Nam et al., 2015; Liu C. et al., 2019). However, these materials could be promising as EC electrode materials due to their tendency to form hydrated phases under ambient conditions and upon electrochemical cycling in aqueous electrolytes (Lu and Dahn, 2001; Buchholz et al., 2014; Boyd et al., 2018). In our previous work, we established that this water intercalation causes a 25% c-axis expansion and a phase transformation to a birnessite-like structure, which subsequently exhibits capacitive electrochemical behavior (Boyd et al., 2018). While these electrodes experience a severe capacity decline from active material detachment due to the large structural change, the capacitive response of the remaining expanded, hydrated oxide led us to consider whether it was possible to perform the expansion prior to electrode assembly. The resulting expanded micron-sized particles of manganese-rich oxide with hydrated interlayers could then be used for high rate capability and high volumetric energy density EC electrodes with improved stability upon extended cycling. uses fast and reversible redox reactions in materials such as transition metal oxides and can lead to capacitances >150 F/g (Fleischmann et al., 2020). There is a need to increase the energy density and reduce the cost of ECs to enable their use for broader applications. However, this requires the development of new electrode materials that exhibit high energy storage performance and are made from abundant elements via scalable synthesis processes. Thus far, the search for new EC materials has primarily focused on the synthesis of nanostructured materials. This improves the power density by increasing the interfacial area between the electrode and electrolyte, and reducing the solid-state diﬀusion distance within the particles (Okubo et al., 2007; Rauda et al., 2014; DeBlock et al., 2019; Li et al., 2020). However, the high surface area of nanostructured particles can lead to undesirably low volumetric capacitance electrodes and increased parasitic side reactions with the electrolyte (Palacín et al., 2016; Augustyn and Gogotsi, 2017). Citation: Additionally, the decrease of tap density with particle size lowers the volumetric capacity of electrodes made with small particles (Jouanneau et al., 2003; Li et al., 2019). Therefore, there is a need for high-power energy storage materials that match the rate performance of high surface area materials but exhibit higher volumetric capacitance, as well as scalable strategies to synthesize such materials. In this work, we present a scalable “top-down” strategy for the synthesis of EC electrode materials by electrochemically expanding micron-scale high temperature-derived layered manganese-rich oxides. Inspired by the electrochemical exfoliation methods developed for large sheet graphite and MoS2, this batch process is signiﬁcantly quicker than traditional chemical exfoliations and produces expanded bulk particles (Ma et al., 2008; Liu et al., 2014; Achee et al., 2018; Ambrosi and Pumera, 2018). We then show that assembly of the micron-sized expanded materials into electrodes leads to high power capacitive energy storage. Comparison with commercial activated carbon shows that the oxides exhibit similar rate capability but higher volumetric capacitance. We hypothesize that the electrochemical expansion enables high power and high volumetric capacitance via two important changes in the oxide: (1) interlayer hydration, which improves interlayer diﬀusion kinetics while also buﬀering intercalation-induced structural changes, and (2) particle expansion, which signiﬁcantly improves electrode integrity and volumetric capacitance. Commonly studied materials for ECs include transition metal oxides with hydrated interlayers. Oxides such as V2O5·xH2O, WO3·2H2O, and birnessite (typical formula KxMnO2·nH2O) show pseudocapacitive behavior in aqueous electrolytes due to reversible cation intercalation (Nam et al., 2015; Sai Gautam et al., 2016; Mitchell et al., 2017; Xiong et al., 2017; Wang et al., 2018; Wu et al., 2019). The interlayer water in these materials is hypothesized to enable fast energy storage by buﬀering structural changes during cycling, reducing transition metal dissolution into the electrolyte, and/or improving the interlayer diﬀusion kinetics of intercalating cations (Zhu et al., 2017; Wang et al., 2018; Liu Z. et al., 2019). In particular, birnessite manganese oxides are promising EC materials due to the abundance and safety of manganese. However, their low crystallinity and frequently nanoscale morphology result in low material utilization and packing density, and therefore low volumetric capacitance in thick electrodes (Wu et al., 2015; Wang et al., 2017; Liu et al., 2018). Citation: An attractive alternative is developing material synthesis techniques to produce micron- scale transition metal oxide particles with hydrated interlayers, which could lead to high volumetric capacitance EC electrodes made via traditional slurry electrode manufacturing techniques. Frontiers in Chemistry \| www.frontiersin.org Materials Synthesis: Bulk Powders Materials Synthesis: Bulk Powders A coprecipitation and calcination method was used to prepare the micron-scale layered oxide powders with a P2 structure (Boyd et al., 2018). Brieﬂy, a 2:1 ratio of Mn(CH2CHOO)2·4H2O and Cu(CH2CHOO)2 (Alfa Aesar) were dissolved in deionized water, and added at ∼1 drop/s into a stirred aqueous NaOH (Fisher Scientiﬁc) solution of pH ∼12. After aging for ∼1 h, the Mn0.62Cu0.31(OH)2 precipitate was washed until pH neutral, ﬁltered, and dried overnight at 120◦C. The hydroxide precursor was ball milled in ethanol for 24 h. After drying the material again, Na+ was added in a 0.74:1 molar ratio of Na+:MCu(OH)2 by mixing the precursor and NaOH in DI water for 1 h at room temperature and then heating it at 100◦C on a stir plate until dry. The dried powder was ground with a mortar and pestle to break up any large chunks, and annealed at 500◦C for 5 h. After cooling to room temperature, the powder was ground To address this, we propose the use of electrochemically expanded and hydrated manganese oxides based on P2 oxides of the formula Na0.67MO2. Here, M is a combination of manganese and other transition metals and P2 indicates the prismatic (P) coordination of Na+ and ...ABBA... stacking of the oxygen layers. The high temperature (∼900◦C) synthesis of the P2 oxides leads to micron-scale primary particles with surface areas of ∼1 m2/g (Yabuuchi et al., 2014). P2 Na0.67MO2 are widely studied for non-aqueous sodium ion battery cathodes, but often exhibit poor cyclability due to the Jahn-Teller distortion and dissolution of the Mn atoms during electrochemical cycling, and August 2020 \| Volume 8 \| Article 715 2 Electrochemically Expanded Hydrated Manganese-Rich Oxides Boyd et al. for 15 min before calcination at 850◦C for 6 h and subsequent transfer to an inert-atmosphere glovebox (<1 ppm H2O and O2) while at ∼70◦C. The calcined powder was ground with a mortar and pestle for 30 min in the glovebox. The composition of the as-synthesized material was Na0.64Mn0.62Cu0.31O2, abbreviated NaMCu. A Ni-containing material, Na0.64Ni0.22Mn0.66Cu0.12O2 (NaNMCu), was synthesized in the same manner. electrochemical cycling. XRD was performed with standard Bragg-Brentano geometry and Cu-Ka radiation (PANalytical Empyrean). The powder samples were rotated at 7.5 RPM, while the electrodes remained stationary. Scanning electron microscopy (SEM, FEI Verios 460L) was used to determine the morphology of the P2 powders before and after expansion, as well as after electrochemical cycling. Materials Synthesis: Bulk Powders The water content of the expanded samples was measured with thermogravimetric analysis (TGA; SII EXSTAR6000 TG/DTA6200), using aluminum pans to hold ∼12 mg of powder during heating in air at 5◦C /min from 25 to 300◦C. Methods: Electrochemical Characterization Electrode Preparation The electrochemical behavior of the expanded particles was characterized using slurry electrodes cast onto either Ti mesh or foil (Alfa Aesar). The metal substrates were cleaned by sonication in ethanol, after which the mesh electrodes were dried and coated with slurry. The working face of the Ti foil was etched with sandpaper to improve slurry adhesion while the back of the foil electrode was covered with Kapton tape to minimize its current contribution during electrochemical cycling. The electrode was then plasma cleaned (Harrick Plasma PDC-32G) for 3 min also to improve slurry adhesion. p y The slurry composition varied depending on the type of current collector (mesh or foil). We found that for the expanded oxide on Ti mesh, a slurry composition of 80 wt% active material, 17.5 wt% acetylene black (Alfa Aesar), and 2.5 wt% polyvinylidene ﬂuoride (PVDF, Arkema Kynar KV 900) gave good adhesion to the mesh and the best rate capability. The acetylene black slurries, used to understand the current contribution from the conductive carbon additive, consisted of 67 wt% acetylene black and 33 wt% PVDF. To compare the electrochemically expanded NaMCu to commercial activated carbon on a volumetric basis, we used foil current collectors. For the activated carbon slurry on Ti foil, 90 wt% YP50 activated carbon (Kuraray Co., Ltd.) was combined with 10 wt% PVDF. The expanded oxide slurry on Ti foil consisted of 80 wt% active material, 15 wt% acetylene black, and 5 wt% PVDF. The dry components of each slurry were ground for 5 min before adding n-methyl pyrrolidone (NMP; Sigma Aldrich) and stirring overnight on a magnetic stir plate at 990 RPM. After painting the slurry onto the current collector, the electrodes dried for several hours at room temperature in the fume hood. At this point, the foil electrodes were placed between a folded weigh paper and calendared to the approximate thickness of the electrode-weigh paper assembly with a rolling mill (Durston) before transfer to an oven at 120◦C. We transferred the mesh electrodes directly from the fume hood to the oven at 120◦C, where they dried for several hours before calendaring to 125 µm between the sheets of a folded weigh paper. The ﬁnal thickness of the mesh electrodes was ∼90 µm. Methods: Synchrotron X-Ray Diffraction Methods: Synchrotron X-Ray Diffraction Synchrotron X-ray diﬀraction (XRD) was performed at the Stanford Synchrotron Radiation Lightsource (SSRL) on beamline 7–2 with a 14.013 keV beam. The operando diﬀraction patterns were collected with a Pilatus 300 K area detector (DECTRIS Ltd.) in portrait mode, in a Bragg-Brentano (θ-2θ) reﬂection geometry at a sample-to-detector distance of 750 mm. XRD patterns were taken with the middle of the detector at 11.4 and 23.0◦2θ with a 3 s exposure every 20 s while cycling the electrode at 0.5 mV/s. After calibrating the detector geometry (tilts and distance) with LaB6, the area diﬀraction patterns were reduced to one dimensional intensity vs. 2θ patterns with the pyFAI library (Ashiotis et al., 2015). The electrochemical cell consisted of a 1 M Na2SO4 (Fisher Scientiﬁc) electrolyte, a Pt counter electrode, and a miniature leakless Ag/AgCl reference electrode (eDAQ ET072-1) using a cell developed for in situ electrochemical X- ray scattering (Cao et al., 2016; Mitchell et al., 2019). The preparation of the working electrode is discussed in more detail in section Methods: Electrochemical Characterization. Brieﬂy, an 8:1:1 slurry of the P2 oxide, acetylene black, and polyvinylidene ﬂuoride was prepared in n-methyl pyrrolidone, cast onto a plasma-cleaned platinized silicon substrate (MTI Corp.), and dried at 120◦C overnight. Methods: Physical Characterization XRD was used to determine the structure of the as-synthesized P2 and expanded powders, and the electrodes before and after Expansion To electrochemically expand and hydrate the P2 powders, ∼0.5 g of the material was placed into an electrolyte-permeable and electronically-connected pouch acting as the positive electrode in a two electrode cell, vs. a Ni foam negative electrode. The pouch was made by folding double-layer dialysis tubing (Thermo Scientiﬁc SnakeSkin R⃝, 10,000 MWCO, 22 mm diameter) around a coiled Pt wire electrode (Pine Instruments, 99.99% pure) and sealing it with paraﬁlm (Figure S1). The dialysis tubing held the NaMCu powder near (and optimally in contact with) the Pt wire. After ensuring that the 0.5 M K2SO4 (Fisher Scientiﬁc) electrolyte fully wet the dialysis tubing, +4 V was applied to the cell for 24 h. After expansion, the pouch was rinsed and cut open. The expanded material was washed out of the pouch using DI water, and centrifuged at 4,500 RPM for 3– 5 min to remove excess water. The powders were rinsed at least twice more to remove all electrolyte salt, and centrifuged after each rinse. Finally, the expanded powder was air dried at room temperature. RESULTS AND DISCUSSION Mechanism of Electrochemical Expansion To determine the mechanism of electrochemical expansion and interlayer hydration of P2-type layered sodium manganese- rich oxides in aqueous electrolytes, we performed operando XRD during cyclic voltammetry of NaMCu and NaNMCu. The crystal structure of a P2-type layered oxide (Figure S2A) consists of layers of edge-sharing MO6 octahedra, and ∼67% of the interlayer prismatic sites are ﬁlled with Na+ (Delmas et al., 1980). Figure 1A shows the cyclic voltammogram (CV) of P2 NaMCu at 0.5 mV/s in 1 M Na2SO4. During the ﬁrst anodic cycle, the CV of NaMCu shows current peaks around 0.2 and 0.9 V vs. Ag/AgCl. On the subsequent cathodic scan, the overall current decreases and is relatively constant as a function of potential. These electrochemical changes suggest a change in the structure and energy storage mechanism in the material, which we attribute to expansion (vide infra) and interlayer water insertion as shown in Figure 1B and described in our previous work (Boyd et al., 2018). Well-deﬁned current peaks correlate to a well-deﬁned Gibbs free energy of reaction, which in an intercalation-type material indicates little dispersion in the intercalation site energy. On the other hand, a constant current as a function of potential, such as observed after the ﬁrst anodic sweep, is indicative of a capacitive mechanism exhibited by many layered manganese oxides in neutral pH electrolytes (Brousse et al., 2006; Ghodbane et al., 2012; Leong and Yang, 2019). The capacitive charge storage mechanism of manganese oxides under these conditions is a In addition to this expected behavior, the appearance of a new peak at 7.19◦2θ at ∼0.9 V during the ﬁrst anodic cycle indicates the formation of a new phase. According to prior results, this peak corresponds to the (001) plane of a birnessite-like hydrated phase with an interlayer spacing of 7.05 Å (Lu and Dahn, 2001; Abou-El-Sherbini et al., 2002; Buchholz et al., 2014; Boyd et al., 2018; Yao et al., 2019). The full XRD patterns of the material before and after the phase transformation are shown in Figure S2B. The large interlayer spacing of the new phase indicates the insertion of a single layer of water molecules from the aqueous electrolyte into the interlayer of NaMCu (Nam et al., 2015; Li et al., 2016). Electrochemistry XRD was used to determine the structure of the as-synthesized P2 and expanded powders, and the electrodes before and after All electrodes were tested with a three-electrode set up in glass 50 mL round bottom ﬂasks with an aqueous 0.5 M K2SO4 (Fisher August 2020 \| Volume 8 \| Article 715 Frontiers in Chemistry \| www.frontiersin.org 3 Electrochemically Expanded Hydrated Manganese-Rich Oxides Boyd et al. FIGURE 1 \| Mechanism of water intercalation into P2-type NaMCu upon electrochemical cycling in 1 M Na2SO4. (A) Cyclic voltammetry at 0.5 mV/s. A.B. indicates “acetylene black,” the electrochemical response of the conductive carbon in the electrode. (B) Cartoon showing the insertion of water molecules (red) from the electrolyte into the material interlayer to compensate for the electrochemical de-intercalation of Na+ (dark blue). (C) Operando synchrotron XRD during the ﬁrst two CV cycles at 0.5 mV/s. The right-most panel shows the applied potential (dashed line) and the current response (solid line). During the ﬁrst anodic cycle, the interlayer peak of the hydrated birnessite-like phase (∼7◦2θ) emerges around 0.9 V. Both the P2 and the hydrated peak show reversible, continuous changes in the interlayer spacing as a function of potential attributed to Na+ de/intercalation. The peak at 8.1◦2θ is due to the electrochemical cell. FIGURE 1 \| Mechanism of water intercalation into P2-type NaMCu upon electrochemical cycling in 1 M Na2SO4. (A) Cyclic voltammetry at 0.5 mV/s. A.B. indicates “acetylene black,” the electrochemical response of the conductive carbon in the electrode. (B) Cartoon showing the insertion of water molecules (red) from the electrolyte into the material interlayer to compensate for the electrochemical de-intercalation of Na+ (dark blue). (C) Operando synchrotron XRD during the ﬁrst two CV cycles at 0.5 mV/s. The right-most panel shows the applied potential (dashed line) and the current response (solid line). During the ﬁrst anodic cycle, the interlayer peak of the hydrated birnessite-like phase (∼7◦2θ) emerges around 0.9 V. Both the P2 and the hydrated peak show reversible, continuous changes in the interlayer spacing as a function of potential attributed to Na+ de/intercalation. The peak at 8.1◦2θ is due to the electrochemical cell. Scientiﬁc) electrolyte, using an Ag/AgCl in 3.5 M KCl reference electrode (Pine Instruments) and a platinum wire counter electrode (99.997%, Alfa Aesar). All tests used Ni-plated stainless steel alligator clips to hold the working and counter electrodes in the electrolyte. Electrochemistry The cyclic voltammetry was performed with a Biologic MPG2 potentiostat. subject of debate, but has been attributed to the correlated intercalation of cations and water molecules (Bélanger et al., 2008; Arias et al., 2014; Costentin et al., 2017). The structural changes of NaMCu during the ﬁrst two CV cycles were measured with operando synchrotron XRD (Figure 1C). Two changes are apparent: the appearance of a new peak during the ﬁrst anodic cycle (discussed below) and shift of the NaMCu diﬀraction peak. The interlayer spacing of the P2 phase, indicated by the (002) peak at ∼9◦2θ, increases from 5.59 Å (9.07◦2θ) at 0 V to 5.65 Å (8.97◦2θ) at 1.1 V. Upon reduction to 0 V, the spacing returns to nearly its original value, 5.61 Å (9.04◦2θ). This shows that the interlayer spacing reversibly increases during oxidation and decreases during reduction. The continuous change of the NaMCu interlayer spacing as a function of potential indicates a solid-solution intercalation mechanism, similar to its structural behavior in non-aqueous Na+ electrolytes (Kang et al., 2015; Li et al., 2015; Wang L. et al., 2017). The faint splitting of the P2 (001) peak near the end of the cathodic cycle may indicate that the remaining P2 material forms two phases with slightly diﬀerent Na+ content. Frontiers in Chemistry \| www.frontiersin.org Batch Expansion Process During the second cycle it increases to 7.07 Å (7.17◦2θ) at 1.1V and shifts back to 6.97 Å (7.27◦2θ) at 0 V. This change of ∼0.1 Å is about half of that typically observed in birnessite in aqueous electrolytes (Ghodbane et al., 2012; Xiong et al., 2017). The reversible peak shifts of the hydrated phase indicate that cation (de)intercalation into the hydrated interlayer at least partially contributes to the capacitive energy storage mechanism. These changes would not appear if the capacitive behavior was simply due to the formation of the double layer at the outer surface of the particles. The intensity increase of the hydrated phase peak and corresponding decrease in the P2 (002) peak during the ﬁrst anodic sweep show the partial phase transformation of the electrode material. However, all peak intensities decrease after ∼0.5 V during the second cathodic sweep, as the change in particle volume during electrochemical expansion causes the electrode to delaminate from the current collector. During the second cycle it increases to 7.07 Å (7.17◦2θ) at 1.1V and shifts back to 6.97 Å (7.27◦2θ) at 0 V. This change of ∼0.1 Å is about half of that typically observed in birnessite in aqueous electrolytes (Ghodbane et al., 2012; Xiong et al., 2017). The reversible peak shifts of the hydrated phase indicate that cation (de)intercalation into the hydrated interlayer at least partially contributes to the capacitive energy storage mechanism. These changes would not appear if the capacitive behavior was simply due to the formation of the double layer at the outer surface of the particles. The intensity increase of the hydrated phase peak and corresponding decrease in the P2 (002) peak during the ﬁrst anodic sweep show the partial phase transformation of the electrode material. However, all peak intensities decrease after ∼0.5 V during the second cathodic sweep, as the change in particle volume during electrochemical expansion causes the electrode to delaminate from the current collector. p To circumvent the electrical contact and slurry delamination problems resulting from in situ P2 particle expansion on electrodes, we developed a batch expansion process for the P2 oxide powder, as described in the Experimental section. Figure 2 shows the expansion cell, along with scanning electron microscopy (SEM) images of a P2 particle (Figure 2A) and an expanded particle (Figure 2C). Batch Expansion Process The resulting expanded and hydrated particles can be assembled into electrodes, thus bypassing the issue of electrode delamination after direct electrochemical expansion of the P2 particles. The batch process uses a neutral pH electrolyte, a voltage source, permeable membrane, and a Ni foam counter electrode, rendering it environmentally friendly and scalable. Figure S4 shows representative chronoamperometry for the process. We observed that a smoother current response is a good indicator of expansion quality—noisier curves resulted in less expansion of the P2 oxide, as indicated by XRD (Figure 3A). This is likely due to the variability of the electrical contact between the powders and the Pt coil in the pouch cell, which could be improved with more advanced pouch designs. This synthesis method oﬀers a new, scalable method to produce materials for EC electrodes that can provide high volumetric capacitance. Figure S3 shows similar behavior in a P2 oxide with a diﬀerent composition, NaNMCu, where the inclusion of nickel led to Na+ deintercalation and the subsequent formation of the birnessite- like phase at a lower potential (Kang et al., 2015; Wang L. et al., 2017; Chen et al., 2018). Overall, the operando results show that when these P2 oxides are cycled in an aqueous electrolyte, water incorporation occurs during Na+ deintercalation, likely to oﬀset the increasing electrostatic repulsion between the transition metal and oxygen layers. Within an individual particle, the formation of this birnessite-like phase may lead to the capacitive CV observed after the ﬁrst cycle. However, the large expansion of the P2 particles (25% c-axis increase) in the slurry electrode leads to loss of electronic connection to the current collector, or even delamination, when cast onto the electrode before expansion. If the electrochemical expansion is better controlled, the formation of micron-scale particles with hydrated interlayers with a capacitive electrochemical response indicates that these particles could be viable for high volumetric capacitance EC materials. RESULTS AND DISCUSSION Moreover, reversible peak shifts indicate that this new hydrated phase is electrochemically active: upon reduction from 1.1 to 0 V, the interlayer spacing decreases to 6.97 Å (7.27◦2θ). August 2020 \| Volume 8 \| Article 715 4 Electrochemically Expanded Hydrated Manganese-Rich Oxides Boyd et al. FIGURE 2 \| Effect of batch expansion on particle morphology: (A) a pristine P2 NaMCu particle has a compact structure, which expands after electrochemical water insertion. (B) A schematic of the electrochemical expansion setup, where the P2 material is contained in a dialysis tubing pouch around a coil of Pt wire, and the counter electrode is Ni foam. (C) A typical fully expanded NaMCu particle, which retains its ab-plane morphology but expands vertically due to water insertion. Scale bar 2 µm. FIGURE 2 \| Effect of batch expansion on particle morphology: (A) a pristine P2 NaMCu particle has a compact structure, which expands after electrochemical water insertion. (B) A schematic of the electrochemical expansion setup, where the P2 material is contained in a dialysis tubing pouch around a coil of Pt wire, and the counter electrode is Ni foam. (C) A typical fully expanded NaMCu particle, which retains its ab-plane morphology but expands vertically due to water insertion. Scale bar 2 µm. Structural Characterization of the Expanded Materials To determine the extent to which the batch expansion process aﬀects material structure and electrochemical performance, we compared three structures: as-synthesized NaMCu, fully expanded (F.E.) NaMCu, and partially expanded (P.E.) NaMCu. Figure 3 shows the XRD, TGA, and SEM of these materials. The pristine NaMCu has the same structure as reported previously (Boyd et al., 2018): a primary P2-type phase (∗) and a small amount of CuO (+). The ﬁrst peak at 15.92◦2θ (5.57 Å) corresponds to the (002) peak of the P2 phase. The F.E. NaMCu August 2020 \| Volume 8 \| Article 715 Frontiers in Chemistry \| www.frontiersin.org 5 Electrochemically Expanded Hydrated Manganese-Rich Oxides Boyd et al. FIGURE 3 \| Structural characterization of expanded NaMCu materials. (A) Powder XRD shows the complete transformation of the F.E. material into the hydrated phase, while the P.E. material contains both the hydrated phase and residual P2 phase. (B) TGA shows that both F.E. and P.E. NaMCu materials lose a signiﬁcant amount of water with increasing temperature. SEM of the (C) pristine NaMCu powder and (D) F.E. NaMCu powder shows that the expanded particles retain their initial hexagonal morphology and particle size but are now expanded along the layers. Scale bar 3 µm. FIGURE 3 \| Structural characterization of expanded NaMCu materials. (A) Powder XRD shows the complete transformation of the F.E. material into the hydrated phase, while the P.E. material contains both the hydrated phase and residual P2 phase. (B) TGA shows that both F.E. and P.E. NaMCu materials lose a signiﬁcant amount of water with increasing temperature. SEM of the (C) pristine NaMCu powder and (D) F.E. NaMCu powder shows that the expanded particles retain their initial hexagonal morphology and particle size but are now expanded along the layers. Scale bar 3 µm. shows a completely expanded and hydrated structure: the P2- phase is no longer present and the interlayer peak at 12.99◦2θ (6.99 Å) indicates a 25% expansion, the same as the operando XRD experiments. As discussed above, the large increase in interlayer spacing is due to the transformation to a new hydrated phase. Both the P2 and F.E. NaMCu materials exhibit a peak at 35.8◦2θ (2.5 Å). This peak corresponds to the (100) plane of the P2 structure, and its presence in both the pristine and F.E. materials indicates that the lattice spacing along the transition metal oxide sheets is unaﬀected by electrochemical expansion. Structural Characterization of the Expanded Materials The larger c-axis in the expanded materials leads to slightly decreased angles of the (101) and (102) peaks at 36.5 and 38.1◦2θ. The two-phase P.E. structure indicates that the expansion proceeds via a direct phase transformation and agrees with the operando XRD, which shows the emergence of the hydrated phase at a distinct potential as opposed to a gradual structural transition over a broad potential range. varying degrees of partial exfoliation. Figure 2 shows higher magniﬁcation SEM images of pristine and expanded NaMCu particles. Before expansion, the material consists of dense, roughly hexagonal, micron-scale primary particles. Based on the crystal structure, the interlayer spacing is parallel to the surface of hexagon-like primary particles. After electrochemical oxidation, there is clear partial exfoliation of the particles along the c axis that gives rise to large pores hundreds of nanometers in dimension. We hypothesize that the large pores and interlayer hydration formed by the expansion process enable high power energy storage by increasing access of electrolyte ions to the interlayer and buﬀering the structural changes during ion (de)intercalation. Electrochemical Behavior of Expanded Materials Electrochemical characterization shows that the capacitive character of the material increases with amount of interlayer hydration and expansion. We characterized the electrochemical behavior of pristine, P.E., and F.E. NaMCu in a 0.5 M K2SO4 electrolyte. This electrolyte was selected over Na2SO4 because K+ has a smaller hydrated ion radius compared to Na+, which is hypothesized to allow higher rate capability and cyclability in aqueous electrolytes (Shao et al., 2013; Xiong et al., 2017; Dupont et al., 2018; Phadke et al., 2020). Figure 4 shows the CVs of the pristine, P.E., and F.E. NaMCu in 0.5 M K2SO4 at 1, 10, and 100 mV/s. The response of the acetylene black conductive additive is also shown for reference. It contributes only minimal double-layer current. The F.E. NaMCu exhibits a capacitive CV at 1 and 10 mV/s, with a semi-rectangular shape and broad peaks similar to those often observed in birnessite (Xiong et al., 2017; Yao et al., 2019). The P.E. NaMCu also has a semi-rectangular CV, albeit with a lower speciﬁc current and lacking the broad peaks present in the F.E. material. In contrast, the pristine P2 NaMCu particles expand upon intercalating water during the ﬁrst anodic cycle, and the electrode capacity drops p g TGA of each material indicates decreasing mass loss between room temperature (∼25◦C) and 300◦C in the order F.E. NaMCu > P.E. NaMCu > NaMCu. In this temperature range, mass losses are most likely due to the removal of water molecules from the oxide surface and interlayer. The composition of the F.E. material for TGA was Mn0.62Cu0.31O2·xH2O, assuming the removal of all Na+ during electrochemical expansion. This means that the 6.2 wt% lost from F.E. NaMCu between 50 and 120◦C corresponds to 0.33 mol H2O per MCu (x = 0.33). Many birnessites also have a oxide:water content ratio near 1:0.33, supporting the XRD data that electrochemical expansion leads to insertion of a single layer of water molecules into the interlayer of the P2 oxides. The P.E. NaMCu lost 2.4 wt%, which suggests only ∼38% of the powder expanded based on the mass lost from F.E. NaMCu. Lastly, NaMCu exhibits only a minor 1.2 wt% loss, likely due to the desorption of surface water. Frontiers in Chemistry \| www.frontiersin.org Electrochemical Behavior of Expanded Materials The SEM images in Figures 3C,D show that the crystallographic change aﬀects the morphology as well: the ab plane particle size and shape are retained after expansion, while the c-axis dimensions increase as the layers experience August 2020 \| Volume 8 \| Article 715 6 Electrochemically Expanded Hydrated Manganese-Rich Oxides Boyd et al. FIGURE 4 \| CVs of the pristine P2, P.E., and F.E. NaMCu materials in 0.5 M K2SO4 electrolyte at (A) 1 mV/s, (B) 10 mV/s, and (C) 100 mV/s. The speciﬁc current from just the acetylene black (A.B.) conductive additive is shown for reference. FIGURE 5 \| Electrochemical energy storage rate capability and post-electrochemical cycling material structure. (A) Cathodic capacitance and (B) rate capability (cathodic capacitance normalized to the capacitance at 0.1 mV/s), from 0.1 to 100 mV/s. The low capacity and rate capability of the acetylene black (A.B.) conductive additive shows that the capacitive electrochemical response is due to the oxide active materials. (C) Ex situ XRD of electrodes after electrochemical cycling shows that the F.E. and P.E. retain their initial structures. FIGURE 4 \| CVs of the pristine P2, P.E., and F.E. NaMCu materials in 0.5 M K2SO4 electrolyte at (A) 1 mV/s, (B) 10 mV/s, and (C) 100 mV/s. The speciﬁc current from just the acetylene black (A.B.) conductive additive is shown for reference. FIGURE 4 \| CVs of the pristine P2, P.E., and F.E. NaMCu materials in 0.5 M K2SO4 electrolyte at (A) 1 mV/s, (B) 10 mV/s, and (C) 100 mV/s. The speciﬁc current f j h l bl k (A B ) d i ddi i i h f f FIGURE 4 \| CVs of the pristine P2, P.E., and F.E. NaMCu materials in 0.5 M K2SO4 electrolyte at (A) 1 mV/s, (B) 10 mV/s, and (C) 100 mV/s. The speciﬁc current from just the acetylene black (A.B.) conductive additive is shown for reference. FIGURE 4 \| CVs of the pristine P2, P.E., and F.E. NaMCu materials in 0.5 M K2SO4 electrolyte at (A) 1 mV/s, (B) 10 mV/s, and (C) 100 mV/s. The speciﬁc current from just the acetylene black (A.B.) conductive additive is shown for reference. FIGURE 5 \| Electrochemical energy storage rate capability and post-electrochemical cycling material structure. (A) Cathodic capacitance and (B) rate capability (cathodic capacitance normalized to the capacitance at 0.1 mV/s), from 0.1 to 100 mV/s. Electrochemical Behavior of Expanded Materials The low capacity and rate capability of the acetylene black (A.B.) conductive additive shows that the capacitive electrochemical response is due to the oxide active materials. (C) Ex situ XRD of electrodes after electrochemical cycling shows that the F.E. and P.E. retain their initial structures. FIGURE 5 \| Electrochemical energy storage rate capability and post-electrochemical cycling material structure. (A) Cathodic capacitance and (B) rate capability (cathodic capacitance normalized to the capacitance at 0.1 mV/s), from 0.1 to 100 mV/s. The low capacity and rate capability of the acetylene black (A.B.) conductive additive shows that the capacitive electrochemical response is due to the oxide active materials. (C) Ex situ XRD of electrodes after electrochemical cycling shows that the F.E. and P.E. retain their initial structures. of activated carbon materials, which is surprising given the much smaller surface area of the oxide materials. When cycled from 0.1 to 50 mV/s, the F.E. NaMCu retains 34% of its initial capacitance. The maximum capacitance of the P.E. NaMCu is lower, 62.4 F/g (14 mAh/g), but its rate capability is slightly better than that of F.E. NaMCu. The pristine P2 NaMCu undergoes electrochemical expansion and hydration directly on the electrode, and its second-cycle capacitance of 57 F/g (13 mAh/g) drops oﬀsigniﬁcantly as the material delaminates from the electrode. Ex situ XRD (Figure 5C) indicates that F.E. and P.E. NaMCu retain their initial structure after electrochemical cycling. There is no ex situ XRD of the P2 electrodes after electrochemistry because the slurries delaminated from the current collectors during cycling. Together, these results show that the F.E. and P.E. NaMCu exhibit relatively high capacitance and rate capability for aqueous energy storage due to the hydrated interlayer and particle expansion. The batch electrochemical expansion of P2 NaMCu and subsequent electrode assembly leads to electrodes signiﬁcantly as the 25% increase along the c-axis causes particles to delaminate. P2 NaMCu electrodes do not show clear redox peaks in K2SO4 (Figure S5), possibly because the interlayer Na+ partially exchanges with K+ and H2O immediately upon immersion into the electrolyte. The semi-rectangular CV, with the lowest speciﬁc current of the three electrodes, is likely from the response of the remaining, partially-hydrated material that is still in electrical contact with the current collector. By 100 mV/s, all three materials exhibit signiﬁcant polarization arising from increased Ohmic resistance from the large measured currents. Frontiers in Chemistry \| www.frontiersin.org Electrochemical Behavior of Expanded Materials Overall, these results show that the degree of expansion directly determines the material’s electrochemical behavior, and that electrochemical expansion before electrode assembly leads to stable, capacitive energy storage. Figure 5 shows the cathodic capacitance and rate capability of the three materials. The highest capacitance of 102 F/g (23 mAh/g) is obtained with the F.E. materials, indicating that the interlayer expansion enabled access of electrolyte ions to the interlayer. This value is comparable to capacitances August 2020 \| Volume 8 \| Article 715 7 Electrochemically Expanded Hydrated Manganese-Rich Oxides Boyd et al. FIGURE 6 \| (A) Cycling stability of F.E., P.E., and P2 NaMCu over 600 cycles at 10 mV/s. (B) Ex situ XRD after 600 cycles shows that the F.E. NaMCu retains the hydrated phase, while some of the remaining P2 material in the P.E. NaMCu electrode expanded after prolonged cycling. FIGURE 6 \| (A) Cycling stability of F.E., P.E., and P2 NaMCu over 600 cycles at 10 mV/s. (B) Ex situ XRD after 600 cycles shows that the F.E. NaMCu retains the hydrated phase, while some of the remaining P2 material in the P.E. NaMCu electrode expanded after prolonged cycling. showed that after 50 cycles, NaMCu partially transformed to the hydrated phase (Boyd et al., 2018). with high rate capability and is promising for high-power energy storage. Post-electrochemical SEM (Figure 7) shows that while most particles in the F.E. and P.E. electrodes retained their original morphology, some exhibit a nanostructured surface coating. After 600 cycles, the morphology of the F.E. NaMCu primary particles appears similar as before cycling. However, the surface of these primary particles now has a nano–structured, semi- porous coating. One possible mechanism of this surface coating could be restructuring due to the dissolution of Mn2+ during reduction, and subsequent re-deposition during oxidation (Liang et al., 2019). The variability of this surface coating from particle to particle in F.E. NaMCu may highlight how the electrode architecture aﬀects the relevant electrochemical reaction. A well- connected particle may experience more overall charge transfer, leading to surface restructuring. However, there was little surface coating in the P.E. NaMCu, suggesting that more of the current may have gone into expanding the remaining P2 particles. Figure 7D shows one of the few P.E. NaMCu particles with partial surface coating. Finally, Figures 7E,F show the pristine P2 NaMCu after 600 cycles. Electrochemical Behavior of Expanded Materials While the P2 particles expanded somewhat, their morphology is much smoother than F.E. or P.E. electrode particles. The presence of only F.E. and Ti substrate peaks in the XRD results conﬁrm that this surface ﬁlm is either the same hydrated birnessite-like phase or it is just a minor weight fraction of the total electrode mass. Overall, the electrochemistry, XRD, and SEM results highlight that electrodes assembled from pre-expanded F.E. or P.E. NaMCu possess high rate capability and structure stability that make them suitable as EC materials. Evaluation of the cyclability of the three oxide materials shows that the F.E. NaMCu electrode retained its structure and the most capacity after 600 cycles at 10 mV/s (Figure 6), while the P.E. NaMCu converted to the F.E. structure and the P2 NaMCu delaminated from the electrode. The initial capacitances were 58 F/g (13 mAh/g) for the F.E. electrode, 34 F/g (8 mAh/g) for the P.E. electrode, and 37 F/g (8 mAh/g) for the P2 electrode. After 600 cycles at 10 mV/s, the F.E. electrode retained 86.3% capacity, the P.E. retained 85.7%, and the P2 retained 40.4%. Figure 6 shows that the F.E. electrode experienced an initial capacity drop, but after 60 cycles the capacity increased close to its initial value. The P.E. electrode experienced a steady capacity increase over the ﬁrst 50 cycles, and then a steady decline during the subsequent 550. The P2 NaMCu lost most of its capacity within the ﬁrst 50 cycles, as water insertion into the P2 NaMCu partially expanded the oxide particles, causing them to fall oﬀthe electrode due to the change in volume. The materials with an expanded and hydrated interlayer exhibit a signiﬁcant improvement over the as-synthesized P2 materials, likely due to the improved access of the electrolyte to the material. However, further investigation is required to understand the reaction mechanisms and develop their electrode architecture so they can withstand the ∼1,000,000 cycles required of ECs (Conway, 1999; Nybeck et al., 2019). XRD of the F.E. and P.E. NaMCu after the cyclability testing (Figure 6B) shows that the F.E. phase is stable under these electrochemical conditions, as the only visible change is a slight broadening of the peaks after cycling. This suggests that the particle expansion and interlayer hydration limit further structural changes in the material, leading to better stability. Ex situ XRD of the P.E. material conﬁrms that an increasing fraction of the F.E. Electrochemical Behavior of Expanded Materials material develops after extended cycling. It appears that the initial fraction of expanded particles was enough to maintain electronic connectivity with the particles expanded in situ. Slurry delamination during cycling prevented ex situ post- cycling XRD of the pristine NaMCu, but our previous work Frontiers in Chemistry \| www.frontiersin.org Comparison of Expanded NaMCu to Commercial Activated Carbon To test our hypothesis that the hydrated and expanded NaMCu would allow an increase in the volumetric capacitance of EC electrodes by increasing access of electrolyte ions to the interlayer and buﬀering structural changes associated with August 2020 \| Volume 8 \| Article 715 Frontiers in Chemistry \| www.frontiersin.org 8 Electrochemically Expanded Hydrated Manganese-Rich Oxides Boyd et al. than F.E. NaMCu. To enable comparison of the volumetric capacity/capacitance, the materials were cast onto foil (vs. mesh) current collectors. Figure 8 compares the CVs for both materials at 1, 10, and 100 mV/s in 0.5 M K2SO4 while Figure 9 shows the speciﬁc capacity/capacitance and rate capability. Both P.E. NaMCu and activated carbon exhibit nearly rectangular, ideally capacitive CVs. At sweep rates up to 20 mV/s (corresponding to a charge/discharge time of 40 s), P.E. NaMCu shows a higher speciﬁc current than activated carbon, indicating that the expanded oxide particles give better volumetric performance than the high surface area activated carbon. At 0.1 mV/s, the volumetric capacitances of P.E. NaMCu and activated carbon are 57.2 F/cm3 (12.7 mAh/cm3) and 20.9 F/cm3 (4.64 mAh/cm3), respectively. However, the response of the two materials becomes similar at faster sweep rates; by 100 mV/s the CVs essentially overlap and their diagonality indicates both are more Ohmically resistive. The similarity of the material response at faster sweep rates may be due to similar issues of maintaining adequate ionic and electronic conductivity in the electrodes. These results show that an oxide material with expanded and hydrated micron- size particles can increase EC energy density over traditionally used high surface area activated carbon at timescales of up to 40 s. While further studies are required to determine the exact mechanism, we propose that the expanded and hydrated interlayer facilitates access of electrolyte ions for charge storage, enabling better material utilization at high rates. ion (de)intercalation, we compared the P.E. NaMCu with a commercially available activated carbon used for EC electrodes. P.E. NaMCu was selected due to its better rate capability FIGURE 7 \| SEM images of (A,B) F.E., (C,D) P.E., and (E,F) P2 NaMCu electrodes after 600 cycles in 0.5 M K2SO4. While most F.E. particles show expansion or a surface coating, few P.E. particles show surface coating, and few P2 particles even show expansion. Scale bar 500 nm. CONCLUSIONS This work describes a promising new material for EC electrodes by detailing how electrochemically expanding micron-sized layered oxides and hydrating the interlayer leads to EC electrodes with high volumetric capacitance and cyclability in aqueous electrolytes. We propose a scalable “top-down” synthesis to electrochemically expand particles, changing their electrochemical characteristics from battery-like for the initial Na+ deintercalation to capacitive upon subsequent cycling. Speciﬁcally, after interlayer hydration and subsequent particle expansion, micron-sized P2 oxide particles exhibit capacitive FIGURE 7 \| SEM images of (A,B) F.E., (C,D) P.E., and (E,F) P2 NaMCu electrodes after 600 cycles in 0.5 M K2SO4. While most F.E. particles show expansion or a surface coating, few P.E. particles show surface coating, and few P2 particles even show expansion. Scale bar 500 nm. FIGURE 8 \| CV comparison of P.E. NaMCu and activated carbon (A.C.) on the basis of their speciﬁc current at (A) 1 mV/s, (B) 10 mV/s, and (C) 100 mV/s. At sweep rates up to ∼20 mV/s, the P.E. NaMCu out-performs the activated carbon, although both electrodes appear relatively resistive at 100 mV/s. FIGURE 8 \| CV comparison of P.E. NaMCu and activated carbon (A.C.) on the basis of their speciﬁc current at (A) 1 mV/s, (B) 10 mV/s, and (C) 100 mV/s. At sweep rates up to ∼20 mV/s, the P.E. NaMCu out-performs the activated carbon, although both electrodes appear relatively resistive at 100 mV/s. August 2020 \| Volume 8 \| Article 715 Frontiers in Chemistry \| www.frontiersin.org 9 Electrochemically Expanded Hydrated Manganese-Rich Oxides Boyd et al. FIGURE 9 \| (A) Volumetric capacity/capacitance and (B) rate capability of P.E. NaMCu and activated carbon (A.C.) electrodes. FIGURE 9 \| (A) Volumetric capacity/capacitance and (B) rate capability of P.E. NaMCu and activated carbon (A.C.) electrodes. behavior that is at least in part pseudocapacitive, as evidenced by reversible interlayer spacing changes during operando XRD. The obvious reduction in redox peaks in CVs after expansion suggests that the material deformation is further buﬀered by the interlayer water. In addition, the surface area of the bulk expanded particles is only slightly greater than that of the pristine materials. Therefore, we hypothesize that the interlayer expansion and hydration eﬀectively increase the surface area by extending the electrolyte into the bulk of the particles in two ways. DATA AVAILABILITY STATEMENT The authors thank Cynthia Koehler and Prof. Thom LaBean (NC State University) for providing dialysis tubing. The authors thank Cynthia Koehler and Prof. Thom LaBean (NC State University) for providing dialysis tubing. The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. FUNDING SB was supported by the National Science Foundation Graduate Research Fellowship Program under Grant No. 571800. NG was supported by the Department of Defense (DoD) through the National Defense Science and Engineering Graduate Fellowship (NDSEG) Program. XRD and SEM studies were supported as part of the Fluid Interface Reactions, Structures, and Transport (FIRST) Center, an Energy Frontier Research Center funded by the U.S. Department of Energy, Oﬃce of Science, Oﬃce of Basic Energy Sciences. This work was performed in part at the Analytical Instrumentation Facility (AIF) at North Carolina State University, which was supported by the State of North Carolina and the National Science Foundation (award number ECCS-1542015). The AIF was a member of the North Carolina Research Triangle Nanotechnology Network (RTNN), a site in the National Nanotechnology Coordinated Infrastructure (NNCI). Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, was supported by the U.S. Department of Energy, Oﬃce of Science, Oﬃce of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. CONCLUSIONS First, hydrating the material interlayer improves cation transfer from the bulk electrolyte to the particles, and second, the large (∼100 nm) pores between expanded sections of the material allow easier access of the bulk electrolyte to the particles. The hydrated interlayers may also cushion the structural eﬀects of ion intercalation. All of these increase the rate at which electrolyte cations can approach transition metal oxide storage sites. These results show that hydrating the material interlayer allowed the micron-scale particles to maintain their structure during extended cycling, making them compatible for capacitive energy storage in aqueous electrolytes. By expanding the interlayer of P2 NaMCu oxide, we show that particles with a large solid-state cation diﬀusion distance can be competitive with the state-of- the-art activated carbon for EC electrodes in aqueous electrolytes. While this synthesis is conducted on P2 oxides, it can be applied to other layered materials that possess an electrochemically labile ion. Overall, this shows that electrochemically-driven expansion of oxide materials can tune both the interlayer chemistry and particle size, leading to a new scalable synthesis strategy for EC materials with high volumetric capacitance. NG extracted and analyzed the synchrotron data. SB performed all other experiments and performed the data analysis. All authors contributed to writing and revising the manuscript, and read and approved the submitted version. REFERENCES Quantifying the volumetric performance metrics of supercapacitors. Adv. Energy Mater. 9, 1–11. doi: 10.1002/aenm.201900079 Bélanger, D., Brousse, T., and Long, J. W. (2008). Manganese oxides: battery materials make the leap to electrochemical capacitors. Electrochem. Soc. Interface 17, 47–52. Available online at: https://www.electrochem.org/dl/ interface/spr/spr08/spr08_p49-52.pdf Li, X., Xiang, L., Xie, X., Zhang, C., Liu, S., Li, Z., et al. (2020). Impact of the electrode thickness and crystal water to pseudocapacitive performances of layered birnessite MnO2. Nanotechnology 31:215406. doi: 10.1088/1361-6528/ab73bf Boyd, S., Dhall, R., LeBeau, J. M., and Augustyn, V. (2018). Charge storage mechanism and degradation of P2-type sodium transition metal oxides in aqueous electrolytes. J. Mater. Chem. A 6, 22266–22276. doi: 10.1039/C8TA08367C Li, Y., Feng, X., Cui, S., Shi, Q., Mi, L., and Chen, W. (2016). From α-NaMnO 2 to crystal water containing Na-birnessite: enhanced cycling stability for sodium- ion batteries. CrystEngComm 18, 3136–3141. doi: 10.1039/C6CE00191B Brousse, T., Toupin, M., Dugas, R., Athouël, L., Crosnier, O., and Bélanger, D. (2006). Crystalline MnO2 as possible alternatives to amorphous compounds in electrochemical supercapacitors. J. Electrochem. Soc. 153, A2171–A2180. doi: 10.1149/1.2352197 Li, Y., Yang, Z., Xu, S., Mu, L., Gu, L., Hu, Y., et al. (2015). Air-stable copper- based P2-Na7/9Cu2/9Fe1/9Mn2/3O2 as a new positive electrode material for sodium-ion batteries. Adv. Sci. 2:1500031. doi: 10.1002/advs.201500031 Buchholz, D., Chagas, L. G., Vaalma, C., Wu, L., and Passerini, S. (2014). Water sensitivity of layered P2/P3-NaxNi0.22Co0.11Mn0.66O2 cathode material. J. Mater. Chem. A 2:13415. doi: 10.1039/C4TA02627F Liang, G., Mo, F., Li, H., Tang, Z., Liu, Z., Wang, D., et al. (2019). A universal principle to design reversible aqueous batteries based on deposition–dissolution mechanism. Adv. Energy Mater. 9:1901838. doi: 10.1002/aenm.201901838 Cao, C., Steinrück, H.-G., Shyam, B., Stone, K. H., and Toney, M. F. (2016). In situ study of silicon electrode lithiation with x-ray reﬂectivity. Nano Lett. 16, 7394–7401. doi: 10.1021/acs.nanolett.6b02926 Liu, C., Luo, S., Huang, H., Zhai, Y., and Wang, Z. (2019). Layered potassium- deﬁcient P2- and P3-type cathode materials KxMnO2 for K-ion batteries. Chem. Eng. J. 356, 53–59. doi: 10.1016/j.cej.2018.09.012 Chen, T., Liu, W., Liu, F., Luo, Y., Zhuo, Y., Hu, H., et al. (2018). Beneﬁts of copper and magnesium Co-substitution in Na0.5Mn0.6Ni0.4O2 as a superior cathode for sodium-ion batteries. ACS Appl. Energy Mater. 2, 844–851. doi: 10.1021/acsaem.8b01909 Liu, H., Wang, J. G., Hua, W., Wang, J., Nan, D., and Wei, C. (2018). Scale-up production of high-tap-density carbon/MnOx/carbon nanotube microcomposites for Li-ion batteries with ultrahigh volumetric capacity. Chem. Eng. J. REFERENCES dioxide for electrochemical capacitors. Electrochim. Acta 271, 337–350. doi: 10.1016/j.electacta.2018.03.141 Abou-El-Sherbini, K. S., Askar, M. H., and Schöllhorn, R. (2002). Hydrated layered manganese dioxide: part I. Synthesis and characterization of some hydrated layered manganese dioxides from??-NaMnO2. Solid State Ionics 150, 407–415. doi: 10.1016/S0167-2738(02)00258-8 Fleischmann, S., Mitchell, J. B., Wang, R., Zhan, C., Jiang, D., Presser, V., et al. (2020). Pseudocapacitance: from fundamental understanding to high power energy storage materials. Chem. Rev. 120, 6738–6782. doi: 10.1021/acs.chemrev.0c00170 Achee, T. C., Sun, W., Hope, J. T., Quitzau, S. G., Sweeney, C. B., Shah, S. A., et al. (2018). High-yield scalable graphene nanosheet production from compressed graphite using electrochemical exfoliation. Sci. Rep. 8, 1–8. doi: 10.1038/s41598-018-32741-3 Ghodbane, O., Ataherian, F., Wu, N.-L. L., and Favier, F. (2012). In situ crystallographic investigations of charge storage mechanisms in MnO2-based electrochemical capacitors. J. Power Sources 206, 454–462. doi: 10.1016/j.jpowsour.2012.01.103 Ambrosi, A., and Pumera, M. (2018). Electrochemical exfoliation of MoS2 crystal for hydrogen electrogeneration. Chem. Eur. J. 24, 18551–18555. doi: 10.1002/chem.201804821 Jouanneau, S., Eberman, K. W., Krause, L. J., and Dahn, J. R. (2003). Synthesis, characterization, and electrochemical behavior of improved Li[NixCo1–2xMnx]O2 (0.1 ≤x ≤0.5). J. Electrochem. Soc. 150:A1637. doi: 10.1149/1.1622956 Arias, C. R., Debiemme-Chouvy, C., Gabrielli, C., Laberty-Robert, C., Pailleret, A., Perrot, H., et al. (2014). New insights into pseudocapacitive charge- storage mechanisms in Li-birnessite type MnO2 monitored by fast quartz crystal microbalance methods. J. Phys. Chem. C 118, 26551–26559. doi: 10.1021/jp508543h Kang, W., Zhang, Z., Lee, P. K., Ng, T. W., Li, W., Tang, Y., et al. (2015). Copper substituted P2-type Na0.67CuxMn1−xO2: a stable high-power sodium-ion battery cathode. J. Mater. Chem. A 3, 22846–22852. doi: 10.1039/C5TA06371J Largeot, C., Portet, C., Chmiola, J., Taberna, P., Gogotsi, Y., and Simon, P. (2008). Relation between the ion size and pore size for an electric double-layer capacitor. J. Am. Chem. Soc. 130, 2730–2731. doi: 10.1021/ja7106178 jp Ashiotis, G., Deschildre, A., Nawaz, Z., Wright, J. P., Karkoulis, D., Picca, F. E., et al. (2015). The fast azimuthal integration python library: pyFAI. J. Appl. Crystallogr. 48, 510–519. doi: 10.1107/S1600576715004306 Leong, Z. Y., and Yang, H. Y. (2019). A study of MnO2 with diﬀerent crystalline forms for pseudocapacitive desalination. ACS Appl. Mater. Interfaces 11, 13176–13184. doi: 10.1021/acsami.8b20880 Augustyn, V., and Gogotsi, Y. (2017). 2D materials with nanoconﬁned ﬂuids for electrochemical energy storage. Joule 1, 443–452. doi: 10.1016/j.joule.2017.09.008 Li, H., Qi, C., Tao, Y., Liu, H., Wang, D. W., Li, F., et al. (2019). AUTHOR CONTRIBUTIONS The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fchem. 2020.00715/full#supplementary-material SB and VA contributed to the study conception and design. All authors designed the synchrotron experiments. August 2020 \| Volume 8 \| Article 715 Frontiers in Chemistry \| www.frontiersin.org 10 Electrochemically Expanded Hydrated Manganese-Rich Oxides Boyd et al. REFERENCES 354, 220–227. doi: 10.1016/j.cej.2018.08.031 Clement, R. J., Billaud, J., Armstrong, A. R., Singh, G., Filo Rojo, T., Bruce, P. G., et al. (2016). Structurally stable Mg-doped P2-Na2/3Mn1−yMgyO2 sodium-ion battery cathodes with high rate performance: insights from electrochemical, NMR and diﬀraction studies. Energy Environ. Sci. 9, 3240–3251. doi: 10.1039/C6EE01750A Liu, N., Kim, P., Kim, J. H., Ye, J. H., Kim, S., and Lee, C. J. (2014). Large-area atomically thin MoS2 nanosheets prepared using electrochemical exfoliation. ACS Nano 8, 6902–6910. doi: 10.1021/nn5016242 Liu, Z., Tian, R., Mushtaq, M., Guo, W., Yao, M., and Feng, J. (2019). Performance modulation through synergetic eﬀect of interstitial water with Ti-substitution for sodium ion battery cathode. Chem. Lett. 48, 670–673. doi: 10.1246/cl.1 90137 Clément, R. J., Bruce, P. G., and Grey, C. P. (2015). Review—manganese-based P2-type transition metal oxides as sodium-ion battery cathode materials. J. Electrochem. Soc. 162, A2589–A2604. doi: 10.1149/2.0201514jes Lu, Z., and Dahn, J. R. (2001). Intercalation of water in P2, T2 and O2 Structure Az[CoxNi1/3−xMn2/3]O2. Chem. Mater. 13, 1252–1257. doi: 10.1021/cm000721x Conway, B. E. (1999). Electrochemical Supercapacitors: Scientiﬁc Fundamentals and Technological Applications. New York, NY: Kluwer-Academic. Costentin, C., Porter, T. R., Save, J., and Savéant, J. M. (2017). How do pseudocapacitors store energy? Theoretical analysis and experimental illustration. ACS Appl. Mater. Interfaces 9, 8649–8658. doi: 10.1021/acsami.6b14100 Ma, B., Hou, W., Han, Y., Sun, R., and Liu, Z. H. (2008). Exfoliation reaction of birnessite-type manganese oxide by a host-guest electrostatic repulsion in aqueous solution. Solid State Sci. 10, 141–147. doi: 10.1016/j.solidstatesciences.2007.09.003 DeBlock, R. H., Choi, C., Ashby, D. S., Butts, D. M., DeBlock, R. H., Wei, Q., et al. (2019). Achieving high energy density and high power density with pseudocapacitive materials. Nat. Rev. Mater. 5, 5–19. doi: 10.1038/s41578-019-0142-z Mitchell, J. B., Geise, N. R., Paterson, A. R., Osti, N. C., Sun, Y., Fleischmann, S., et al. (2019). Conﬁned interlayer water promotes structural stability for high-rate electrochemical proton intercalation in tungsten oxide hydrates. ACS Energy Lett. 4, 2805–2812. doi: 10.1021/acsenergylett.9b02040 Delmas, C., Fouassier, C., and Hagenmuller, P. (1980). Structural classiﬁcation and properties of the layered oxides. Phys. B 99, 81–85. doi: 10.1016/0378-4363(80)90214-4 Mitchell, J. B., Lo, W. C., Genc, A., LeBeau, J., and Augustyn, V. (2017). Transition from battery to pseudocapacitor behavior via structural water in tungsten oxide. Chem. Mater. 29, 3928–3937. doi: 10.1021/acs.chemmater.6b 05485 Dupont, M. F., Forghani, M., Cameron, A. P., and Donne, S. W. (2018). REFERENCES Eﬀect of electrolyte cation on the charge storage mechanism of manganese August 2020 \| Volume 8 \| Article 715 Frontiers in Chemistry \| www.frontiersin.org 11 Electrochemically Expanded Hydrated Manganese-Rich Oxides Boyd et al. Nam, K. W., Kim, S., Yang, E., Jung, Y., Levi, E., Aurbach, D., et al. (2015). Critical role of crystal water for a layered cathode material in sodium ion batteries. Chem. Mater. 27, 3721–3725. doi: 10.1021/acs.chemmater.5b00869 water driven battery-to-pseudocapacitor transition. ACS Nano 12, 6032–6039. doi: 10.1021/acsnano.8b02273 Wu, T., Zhu, K., Qin, C., and Huang, K. (2019). Unraveling the role of structural water in bilayer V2O5 during Zn2+-intercalation: insights from DFT calculations. J. Mater. Chem. A 7, 5612–5620. doi: 10.1039/C8TA12014E Nybeck, C. N., Dodson, D. A., Wetz, D. A., and Heinzel, J. M. (2019). Characterization of ultracapacitors for transient load applications. IEEE Trans. Plasma Sci. 47, 2493–2499. doi: 10.1109/TPS.2019.2904562 Wu, T. H., Hesp, D., Dhanak, V., Collins, C., Braga, F., Hardwick, L. J., et al. (2015). Charge storage mechanism of activated manganese oxide composites for pseudocapacitors. J. Mater. Chem. A 3, 12786–12795. doi: 10.1039/C5TA03334A Okubo, M., Hosono, E., Kim, J., Enomoto, M., Kojima, N., Kudo, T., et al. (2007). Nanosize eﬀect on high-rate Li-ion intercalation in LiCoO2 electrode. J. Am. Chem. Soc. 129, 7444–7452. doi: 10.1021/ja0681927 Palacín, M. R., Simon, P., and Tarascon, J. M. (2016). Nanomaterials for electrochemical energy storage: the good and the bad. Acta Chim. Slov. 63, 417–423. doi: 10.17344/acsi.2016.2314 Xiong, P., Ma, R., Sakai, N., Bai, X., Li, S., and Sasaki, T. (2017). Redox active cation intercalation/deintercalation in two-dimensional layered MnO2 nanostructures for high-rate electrochemical energy storage. ACS Appl. Mater. Interfaces 9, 6282–6291. doi: 10.1021/acsami.6b14612 Phadke, S., Mysyk, R., and Anouti, M. (2020). Eﬀect of cation (Li+, Na+, K+, Rb+, Cs+) in aqueous electrolyte on the electrochemical redox of prussian blue analogue (PBA) cathodes. J. Energy Chem. 40, 31–38. doi: 10.1016/j.jechem.2019.01.025 Yabuuchi, N., Hara, R., Kajiyama, M., Kubota, K., Ishigaki, T., Hoshikawa, A., et al. (2014). New O2/P2-type Li-excess layered manganese oxides as promising multi-functional electrode materials for rechargeable Li/Na batteries. Adv. Energy Mater. 4:1301453. doi: 10.1002/aenm.201301453 Rauda, I. E., Augustyn, V., Saldarriaga-Lopez, L. C., Chen, X., Schelhas, L. T., Rubloﬀ, G. W., et al. (2014). Nanostructured pseudocapacitors based on atomic layer deposition of V2O5 onto conductive nanocrystal-based mesoporous ITO scaﬀolds. Adv. Funct. Mater. 24, 6717–6728. doi: 10.1002/adfm.201401284 Yao, M., Ji, X., Chou, T. F., Cheng, S., Yang, L., Wu, P., et al. (2019). Frontiers in Chemistry \| www.frontiersin.org August 2020 \| Volume 8 \| Article 715 REFERENCES Simple and cost-eﬀective approach to dramatically enhance the durability and capability of a layered δ-MnO2 based electrode for pseudocapacitors: a practical electrochemical test and mechanistic revealing. ACS Appl. Energy Mater. 2, 2743–2750. doi: 10.1021/acsaem.9b00075 aﬀolds. Adv. Funct. Mater. 24, 6717–6728. doi: 10.1002/adfm.2014 Sai Gautam, G., Canepa, P., Richards, W. D., Malik, R., and Ceder, G. (2016). Role of structural H2O in intercalation electrodes: the case of Mg in nanocrystalline xerogel-V2O5. Nano Lett. 16, 2426–2431. doi: 10.1021/acs.nanolett. 5b05273 Zhu, K., Guo, S., Li, Q., Wei, Y., Chen, G., and Zhou, H. (2017). Tunable electrochemistry via controlling lattice water in layered oxides of sodium-ion batteries. ACS Appl. Mater. Interfaces 9, 34909–34914. doi: 10.1021/acsami.7b09658 Shao, J., Li, X., Qu, Q., and Wu, Y. (2013). Study on diﬀerent power and cycling performance of crystalline KxMnO2·nH2O as cathode material for supercapacitors in Li2SO4, Na2SO4, and K2SO4 aqueous electrolytes. J. Power Sources 223, 56–61. doi: 10.1016/j.jpowsour.2012.09.046 Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Wang, J.-G., Liu, H., Liu, H., Fu, Z., and Nan, D. (2017). Facile synthesis of microsized MnO/C composites with high tap density as high performance anodes for Li-ion batteries. Chem. Eng. J. 328, 591–598. doi: 10.1016/j.cej.2017.07.039 Copyright © 2020 Boyd, Geise, Toney and Augustyn. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Wang, L., Sun, Y.-G., Hu, L.-L., Piao, J.-Y., Guo, J., Manthiram, A., et al. (2017). Copper-substituted Na0.67Ni0.3−xCuxMn0.7O2 cathode materials for sodium- ion batteries with suppressed P2-O2 phase transition. J. Mater. Chem. A 5, 8752–8761. doi: 10.1039/C7TA00880E Wang, R., Mitchell, J. B., Gao, Q., Tsai, W., Boyd, S., Pharr, M., et al. (2018). Operando atomic force microscopy reveals mechanics of structural August 2020 \| Volume 8 \| Article 715 Frontiers in Chemistry \| www.frontiersin.org 12
https://openalex.org/W4310363527	https://pure.eur.nl/files/79456829/The_impact_of_pancreatic_cancer_screening_on_life_expectancy.pdf	English	null	The impact of pancreatic cancer screening on life expectancy: A systematic review of modeling studies	International journal of cancer	2,022	cc-by	8,504	The impact of pancreatic cancer screening on life expectancy: A systematic review of modeling studies Brechtje D. M. Koopmann1,2 \| Amir-Houshang Omidvari1 \| Iris Lansdorp-Vogelaar1 \| Djuna L. Cahen2 \| Marco J. Bruno2 \| Inge M. C. M. de Kok1 1Department of Public Health, Erasmus MC University Medical Center, Rotterdam, The Netherlands 2Department of Gastroenterology & Hepatology, Erasmus MC University Medical Center, Rotterdam, The Netherlands Correspondence Brechtje D. M. Koopmann, Department of Public Health, Erasmus MC University Medical Center, Rotterdam, The Netherlands. Email: b.koopmann@erasmusmc.nl Funding information Maag Lever Darm Stichting, Grant/Award Number: SK 17-52 1Department of Public Health, Erasmus MC University Medical Center, Rotterdam, The Netherlands Abstract Evidence supporting the effectiveness of pancreatic cancer (PC) screening is scant. Most clinical studies concern small populations with short follow-up durations. Math- ematical models are useful to estimate long-term effects of PC screening using short- term indicators. This systematic review aims to evaluate the impact of PC screening on life expectancy (LE) in model-based studies. Therefore, we searched four data- bases (Embase, Medline, Web-of-science, Cochrane) until 30 May 2022 to identify model-based studies evaluating the impact of PC screening on LE in different risk populations. Two authors independently screened identified papers, extracted data and assessed the methodological quality of studies. A descriptive analysis was per- formed and the impact of screening strategies on LE of different risk groups was reported. Our search resulted in 419 studies, of which eight met the eligibility criteria (mathematical model, PC screening, LE). Reported relative risks (RR) for PC varied from 1 to 70. In higher risk individuals (RR > 5), annual screening (by imaging with 56% sensitivity for HGD/early stage PC) predicted to increase LE of screened individ- uals by 20 to 260 days. In the general population, one-time PC screening was esti- mated to decrease LE (2-110 days), depending on the test characteristics and treatment mortality risk. In conclusion, although the models use different and some- times outdated or unrealistic assumptions, it seems that PC screening in high-risk populations improves LE, and that this gain increases with a higher PC risk. Updated model studies, with data from large clinical trials are necessary to predict the long- term effect of PC screening more accurately. Funding information Maag Lever Darm Stichting, Grant/Award Number: SK 17-52 Received: 7 March 2022 Revised: 9 October 2022 Accepted: 7 November 2022 Received: 7 March 2022 Revised: 9 October 2022 Accepted: 7 November 2022 DOI: 10.1002/ijc.34379 C A N C E R E P I D E M I O L O G Y Int. J. Cancer. 2022;1–11. 2.1 These models all have a similar principle of decision processes. These processes relate to potential events that can occur. For exam- ple, an individual can be at a certain risk to develop a precursor lesion. Based on this risk, a precursor lesion will or will not develop during his or her lifetime. If a precursor lesion develops, the associated cancer risk will determine the chance that it will progress to a malignancy. Decision processes can be relatively simple, such as a decision tree, or more complex, such as a microsimulation model. In a decision tree the risk of events and states of nature is diagramed over a fixed time hori- zon. Markov models simulate a hypothetical cohort of individuals through health states over time. A microsimulation model simulates individual life histories and tracks the past health states and future events for each individual.2 These decision-analytic models can inform policy makers on optimal screening strategies for several cancer types. Also, they can estimate the cost-effectiveness and harms and benefits of a screening program.3,4 In the past, these models have been used to quantify the effects of screening for cancer such as breast, colon, cervix, and lung cancer in a population.3-6 Mathematical modeling is, for example, used to translate colorectal cancer screening trial results (that showed screening effectiveness in a controlled setting) to real world health outcomes for the total population and a longer time period.7,8 We searched for studies that used a mathematical model to evaluate the effect of PC screening on LE in different PC risk groups. Mathe- matical models were defined as models using given input to simulate a decision process with the help of algorithms (eg, simulation, Markov, microsimulation or decision tree models). Our search was limited to papers written in English and involving human subjects. We excluded studies that evaluated the effect of PC treatment. We also excluded reviews, in vitro studies, case reports and letters. What's new? Robust evidence on the effectiveness of pancreatic cancer screening is lacking, as published studies are mostly cohort studies, limited in size and duration. Here, the authors performed a systematic review to evaluate the impact of pancreatic cancer screening on life expectancy as determined in model-based studies. The results show that, while it could be detrimental in the general population, pancreatic cancer screening in high-risk populations may increase life expec- tancy. Updated model studies, with data from large clinical trials, are necessary to predict the long-term effect of pancreatic cancer screening more accurately. 2 \| METHODS This systematic review was executed according to the Preferred Report- ing Items for Systematic reviews and Meta-Analyses (PRISMA) guideline. The review protocol is available in PROSPERO (CRD42020168804). We searched four electronic databases (Embase, Ovid Medline, Web of Science, Cochrane central) for studies published before 30 May 2022. The search strategies used for each database are presented in the Supplementary Materials (Data S1). Mathematical models, also known as decision-analytic models, can simulate health outcomes of individual patients or a population under a variety of scenarios. They can be used to estimate the conse- quences of distinct screening scenarios under varying circumstances. Several types of mathematical models exist, such as decision trees, Markov models and microsimulation models. 1 \| INTRODUCTION objective of the current systematic review is to gain insight into the esti- mated effects of these mathematical models pertaining life expec- tancy (LE). Pancreatic cancer (PC) is mostly diagnosed at an incurable stage.1 Early detection by screening might improve survival, but robust evi- dence on the effectiveness of PC screening is lacking, as published studies are cohort studies, limited in size and duration. Ideally, large clinical trials with decades of follow-up would provide solid evidence on the efficacy of screening. Since such studies are unlikely to be concluded promptly, mathematical models can be used to predict long-term outcomes, using short-term indicators from observational studies. K E Y W O R D S high-risk, mathematical model, pancreatic cancer, screening Abbreviations: AGA, American Gastroenterological Association; CI, confidence interval; CT, computed tomography; EUS, endoscopic ultrasound; FPC, familial pancreatic cancer; ICER, incremental cost effectiveness ratio; LE, life expectancy; LYG, life years gained; MRI, magnetic resonance imaging; NA, not applicable; NNS, number needed to screen; NNT, number needed to treat; PanIN, pancreatic intraepithelial neoplasia; PC, pancreatic cancer; PRISMA, Preferred Reporting Items for Systematic reviews and Meta-Analyses; PROSPERO, Prospective Register of Systematic Reviews; RR, relative risk; SB-IPMN, Side Branch—Intraductal Papillary Mucinous Neoplasm; Sens., sensitivity; Spec., specificity. Systematic review registration number: CRD42020168804. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. g © 2022 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC. wileyonlinelibrary.com/journal/ijc 1 2 KOOPMANN ET AL. 2.3 \| Quality assessment We adapted the ISPOR-AMCP-NPC Questionnaire14 to assess the relevance and credibility of each modeling study according to the following criteria: (I) Validation; (II) Bias due to the study design; (III) Limitations in data sources; (IV) Appropriateness of the model analy- sis; (V) Reporting bias; (VI) Interpretation bias; and (VII) Conflict of interest. The risk of bias for each domain was rated as low, high, or unclear. The checklist is available in the Supplementary Materials (Data S1). This assessment provided us with information on the qual- ity of the included studies, which is important when results are trans- lated to clinical practice. FIGURE 1 PRISMA flow chart (CT), a combination of imaging tests or a hypothetical test. Two papers assumed a 100% sensitive test for PanIN3 and PC detec- tion.11,17 Although unrealistic, this model provides insight in the maxi- mal gain of screening in a hypothetical optimal scenario. (CT), a combination of imaging tests or a hypothetical test. Two papers assumed a 100% sensitive test for PanIN3 and PC detec- tion.11,17 Although unrealistic, this model provides insight in the maxi- mal gain of screening in a hypothetical optimal scenario. 3.1 \| Models Rulyak et al used an EUS sensitivity of 90% for the detection of dysplasia based on expert opinion.12 An abnormal EUS was defined as two or more of the following abnormalities: heteroge- nous parenchyma with echogenic foci, hypoechoic nodules, hyper- echoic main duct wall or discrete masses. A positive test led to a diagnostic Endoscopic Retrograde Cholangiopancreatography (ERCP) for confirmation. Koopmann et al used preliminary data from a large surveillance cohort to assess a stage dependent sensi- tivity (60%-99%) for a combined EUS/MRI test.13 An overview of the extracted data from the included models is presented in Table 2. Our search resulted in 514 articles, of which 11 underwent full text review after title and abstract screening (Figure 1). Four papers were excluded based on study type (review) and/or model outcome (LE was not reported). One paper was found by evaluation of reference lists. Altogether, eight articles met the inclusion criteria, published between 2003 and 2021. Table 1 provides an overview of the included studies. Six were performed in the United States, one in Italy and one in the Netherlands. The simulated populations varied from general popula- tions, to high-risk groups (such as BRCA2 gene mutation carriers or kindreds of familial pancreatic cancer patients [FPC] or individuals with pancreatic cystic lesions), to PC patients. In this latter group the effect of detection in an earlier stage on LE was evaluated. The models encompass five Markov models, one Monte Carlo model, one Microsimulation model and one decision tree.9-13,15-17 Three studies from a single research group used the same base model, which was adjusted for different research questions. 2.2 After removing duplicates, two reviewers (B.K. and A.O) indepen- dently screened titles, abstracts and full texts for potentially eligible studies. In case of disagreement, discrepancies between the two reviewers were discussed, and if no consensus was reached, a third reviewer (IdK) joined to settle. The reference lists of included studies were scanned to identify potential additional studies. The following information was extracted (if available): authors, publication year, model type, model input (source), simulated population (PC risk level, gender), screening method (diagnostic method, test characteristics, screening interval, start-, stop age, complication risk), treatment (type, effect, complication risk, morbidity), screening effect (LE), Life years gained (LYG), number needed to screen (NNS), number needed to In the last decade, several mathematical models on PC screening have been developed to evaluate the efficacy of screening with a variety of screening modalities, screening strategies, and risk populations.9-13 The Electronic Database searches: PubMed, Embase, Medline Ovid, Web of Science and Cochrane central (n = 514 records) Titles/abstracts screened (n = 514) Excluded (n = 503) Full-text articles assessed for eligibility (n = 11) Excluded (n = 4): - 2 Reviews - 2 Measured outcome (biomarker availability) Full-text articles assessed for inclusion (n = 8) n oit a cifit n e d I g nin e e r c S ytilibigil E d e d ulc n I Included (n = 1): - From reference list FIGURE 1 PRISMA flow chart 3 KOOPMANN ET AL. treat (NNT). If detailed results of a certain analysis were not available, we contacted the corresponding author to get access to the results. treat (NNT). If detailed results of a certain analysis were not available, we contacted the corresponding author to get access to the results. 2.4 \| Data synthesis and analysis We performed a descriptive analysis and summarized the effect of PC screening on LE. We compared the screening effect on LE for the dif- ferent risk groups in the included studies (general population, high-risk group, different risk levels/mutation carriers). A really low sensitivity of 56% for detection of HGD/early stage PC with MRI was described by Pandharipande et al9,10 used a sensitiv- ity of 56% for detection of HGD/early stage PC with MRI. This sensi- tivity was based on pooled data from six clinical studies dating from 2009 to 2012. Cases (resected and/or clinical PC) within these studies were classified into true-positive (resectable PC), false-negative (advanced PC), true-negative and false-positive (surgery + no PC). Because of a paucity of available data on cystic lesions, similar test characteristics were assumed for high risk cysts. 3.2 \| Screening in a high-risk population Four studies reported on the effect of screening in different high-risk populations.9,10,12,13 The effect of PC screening on LE depended on the relative risk (RR) for PC of the screened population and the test characteristics (Table 3). Pandharipande et al reported that one-time MRI screening (sensitivity 56%, specificity 97% for pancreatic cyst and early stage cancer) in 50-year-old individuals with a 2.4 to 4.5 RR for PC (eg, BRCA2) led to a negligible gain in LE of 3.9 to 5.8 days per simulated individual. Annual MRI screening even reduced LE by 1.3 to 12.9 days in this risk group. When higher risk individuals (RR 6.4% or The study aims of all included articles differed. Some focused on efficacy of screening in different risk groups, while others focused on the natural disease course of PC and its precursor lesions. Besides variations in screen frequency and starting age, models used different surveillance tests; varying between magnetic resonance imaging (MRI), Endoscopic Ultrasound (EUS), Computed tomography KOOPMANN ET AL. TABLE 1 Overview included studies Study Modeling aim Modeling method Simulated population Outcome Rulyak, 200312 Cost-effectiveness of EUS screening in FPC kindreds Decision tree Members of familial pancreatic cancer kindreds Endoscopic screening was cost-effective, with an incremental cost-effectiveness ratio of 16 885/life-year saved. Screening was more cost-effective as the probability of dysplasia increased and as the sensitivity of the test increased. Expected gain in LE was 38 years for a cohort of 100 people Weinberg, 201015 Assist patients and physicians with understanding how their decisions concerning pancreatic cysts affect overall survival/QoL Markov model Pancreatic cyst (SB-IPMN located in head) Initial pancreaticoduodenectomy was the dominant strategy to maximize overall survival for any cyst >2 cm, regardless of age or comorbidities. “Do nothing” maximized quality of life for all cysts <3 cm in patients aged <75, when measuring quality adjusted survival Pandharipande, 20159 Identify when, from the standpoint of RR, one time screening is effective in high-risk individuals for PC Markov model RR for PC from 1 to 70 One time MR imaging screening in average risk group of 100 000 men at age 50 identifies 2375 low risk cysts, 159 high-risk cysts and 56 cancers. Resulting in 39 cancer deaths averted and a net LE loss of 3 days). 3.2 \| Screening in a high-risk population If the PC risk exceeds 2.4 (men) or 2.7 (women) there was a gain in LE Pandharipande, 201510 Compare effectiveness of different PC screening strategies in BRCA2 mutation carriers, from standpoint of life expectancy Markov model BRCA2 mutation carriers (with or without FDR with PC) One time screening at age 50 resulted in a LE gain of 3.9 days for the BRCA2 cohort. The gain was higher with more FDRs with PC. Annual screening resulted in a LE loss of 12.9 days for BRCA2 mutation carriers. BRCA2 carriers with 2 FDRs gained 20.6 days with annual screening Cucchetti, 201616 Verify survival benefit obtained from a hypothetical screening where a 20,30 or 50% reduction of PC stage was obtained Monte Carlo Simulation Pancreatic cancer patients Mean expected LE for PC patients was 13 months. When a hypothetical screening reduced stage III/IV with 30-50% this LE was: 14-15.9 months Peters, 201811 Gain insight into the natural history of PanIN and to assess the potential of screening Markov model General population Lifetime probability for PanIN1 to progress to PDAC (1.5%). Duration of this progression: 33.6 years. A hypothetical perfect test for PanIN 3 detection and treatment could provide a maximum, average LE gain of 40 days Raphel, 201817 Determine the effect of patient age and comorbidity on LE benefits associated with SB-IPMN follow- up Markov model Pancreatic cyst (SB-IPMN) The LE benefit of SB-IPMN follow-up is 5.3-6.4 months for healthy 60 year old individuals. The effect on LE was limited in case of 80 year old individuals and coexistence of severe comorbidity Koopmann, 202113 Analyze the impact of relevant uncertainties on the effect of PC screening in high-risk individuals Markov model High-risk individuals Screening reduced PC mortality in all modeled scenarios. The reduction depended strongly on natural disease course (progressive vs indolent and faster progressive lesions). The impact of test sensitivity was much smaller. The NNS was impacted most by PC risk 7.5% life time risk) were screened annually, a gain in LE of up to 158 days was predicted, depending on test performance.13 PC life-time risk in the general population, which can be compared to a RR of 12. Rulyak et al reported a gain of 139 days per simulated individual when EUS (sensitivity 90% for pancreatic dysplasia) was performed once at age 50 in family members of familial pancreatic cancer kin- dreds. 3.2 \| Screening in a high-risk population They assumed individuals in this risk group had a 20% risk of having dysplasia and a 90% risk of progression to PC when having dysplasia. This resulted in an estimated 18% risk of PC, given a 1.5% A higher PC risk (ie, RR of 30) also led to a higher LE gain (31.5 for one time and 260 days for annual screening). The effect of one- time MRI screening was highest in individuals with a RR of 70. Their LE increased by 160 days for men and 188 days for women.9 Cucchetti et al evaluated the effect on LE in case of an earlier PC diagnosis after hypothetical screening.16 They evaluated the impact of 10970215, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/ijc.34379 by Erasmus University Rotterdam Universiteitsbibliotheek, Wiley KOOPMANN ET AL. 1 2 3 4 5 6 7 8 hor + publication ar Rulyak et al,12 2003 Weinberg et al,15 2010 Pandharipande et al,9 2015 Pandharipande et al,10 2015 Cucchetti et al,16 2016 Peters et al,11 2018 Raphel et al,17 2018 Koopmann et al,13 2021 al Gastrointestinal endoscopy Gastroenterology Radiology Ebiomedicine Pancreas Pancreatology Radiology International journal of cancer try USA USA USA USA Italy USA USA The Netherlands el type Decision Tree Markov based clinical Nomogram Markov model Markov model Monte Carlo Simulation Markov model Markov model Markov model ation Members of FPC kindreds Pancreatic cystic lesions, male and female (cyst: 0.5 to >3 cm) General population to high risk group (both male and female) BRCA2 mutation carriers (with/ without FDRs) 1000 PC patients General population Pancreatic cyst (SB-IPMN), male and female, different comorbidities High risk group (7.5% life time risk) sk 18% lifetime riska 1% RR 0-70 for PC RR 3.5 or greater 100%b RR 0 Low risk incidental findings 7.5% lifetime risk n method EUS (on indication + ERCP) CT or EUS (± FNA) MRI scan MRI (combined with EUS) Hypothetical test Hypothetical test Hypothetical imaging test EUS and MRI n sens. (sens. alysis) 90% for detection of pancreatic dysplasia 80%-86% 56% (0.25-1.0) For detection of cyst and early stage cancer. No PanIN. 56% (0.5-1.0) For detection of cyst and early stage cancer. No PanIN. NA 100% for detection of PanIN3 100% for malignant lesions Disease stage dependent: 60-99% (±10%) n spec. (sens. 3.2 \| Screening in a high-risk population alysis) 90% 99% 97% (0.5-1.0) 97% (0.9-1.0) NA 100% NA 99% (±5 and 10%) n test mplication Pancreatitis: 5.1% (0.3-8.2) Death from EUS- FNA: 0.01% NA NA NA NA NA NA ning interval One time One time Annual and one time One time One time and continuous Annual Annual and 5 yearly /stop age (sens. alysis) 50 65, 75, 85 50 (40, 60, 70) 50-80 NA 50, 60 or 70 60,80 ment Surgery (total pancreatectomy) Surgery Surgery Surgery Surgery Surgery Surgery Surgery ment mplication risk ns. analysis) Mortality risk: 3% (1%-5%) Mortality risk: 2%-6.4% Chronic compl: 19.5% Mortality risk: 2% (0%-10%) Mortality risk: 1% (0%-2.5%) NA Mortality risk: 0 and 2% Mortality risk: 0% (2%-4%) Mortality risk: 3% (5%) ence of dysplasia (0.20) x progression probability to PC given the presence of dysplasia (0.90) = 0.18. is performed on individuals already diagnoses with PC. Consequences of earlier detection were evaluated. OOPMANN ET AL. 5 0970215, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/ijc.34379 by Erasmus University Rotterdam Universiteitsbibliotheek, Wiley Online Library on [27/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable TABLE 3 Effect of different PC screening strategies on life expectancy Risk group Screening method Screening interval Age Treatment mortality (%) Effect on LE (days) per simulated individual Reference General population MRI (sens. 56%, spec. 97%, for detection of cyst and early stage PC. No PanIN) One time 40 2 5.1 to 5.8a 9 50 2 3.3 to 4.0a 10 27 to 31a 1 2 to 3a 60 2 2.2 to 2.9a 70 2 1.5 to 1.8a MRI (sens. 25%, spec. 97%, for detection of cyst and early stage PC. No PanIN) One time 50 2 4 to 5a MRI (sens. 56%, spec. 50%, for detection of cyst and early stage PC. No PanIN) One time 50 2 94 to 110a MRI (sens. 56%, spec. 100%, for detection of cyst early stage PC. No PanIN) One time 50 2 2 MRI (sens. 100%, spec. 97%, for detection of cyst and early stage PC. No PanIN. One time 50 2 2 Hypothetical perfect test for PanIN3 Continuous 50 0 40 11 2 37 One time 50 0 7.1 2 6.7 60 0 10 2 9.3 70 0 7.9 2 7 Inherited increased risk for PC RR 2.4-4.5 MRI (sens. 56%, spec. 97%, for detection of cyst and early stage PC. TABLE 3 (Continued) stage reduction on LE for different stages. The mean LE without screening was 13 months (395.2 days) from diagnosis. The 3- and 5-year survival rates were 14.1% and 3.8%, respectively. When a hypothetical screening program reduced stage III/IV PC by 20%, 30% or 50%, the mean LE increased to 450, 477 and 547 days, respec- tively.16 After adjustment for lead-time, LE was 426, 444 and 483 days. 3.4 \| Screening in the general population Two studies evaluated the effect of PC screening in the general popu- lation.9,11 The first reported that one-time screening either at age 40, 50, 60 or 70 with MRI (sensitivity 56%, specificity 97% for HGD pancreatic cyst and early stage cancer) resulted in a LE loss of 1.5 to 5.8 days per screened individual. A lower test specificity of 50% resulted in an even higher loss of LE (94-100 days). An increase in test sensitivity to 100% hardly effected LE (LE loss of 2 vs 4 days with 56%-sensitivity). TABLE 3 (Continued) Note: The effect on LE is visible in days lost or gained per simulated individual entering screening. Morality risk is either caused by screentest (EUS) or by treatment (surgery after positive screentest). Abbreviations: CT, computed tomography; EUS, endoscopic ultrasound; HGD, high grade dysplasia; MRI, magnetic resonance imaging; Neg., negative; PanIN, Pancreatic Intraepithelial Neoplasia; PC, pancreatic cancer; Pos., positive; RR, relative risk; SB-IPMN, Side Branch—Intraductal Papillary Mucinous Neoplasm; Sens., sensitivity; Spec., specificity. aEffect on life expectancy for men and women. bIn this model two pathways are simulated, one with only progressive lesions and one with indolent and faster progressive lesions. A larger effect on LE was seen in the progressive-only pathway. cLYG are presented in years per patient in ranges correlated with cyst size (1 cm less gain 3 cm more gain in LE) Note: The effect on LE is visible in days lost or gained per simulated individual entering screening. Morality risk is either caused by screentest (EUS) or by treatment (surgery after positive screentest). Abbreviations: CT, computed tomography; EUS, endoscopic ultrasound; HGD, high grade dysplasia; MRI, magnetic resonance imaging; Neg., negative; PanIN, Pancreatic Intraepithelial Neoplasia; PC, pancreatic cancer; Pos., positive; RR, relative risk; SB-IPMN, Side Branch—Intraductal Papillary Mucinous Neoplasm; Sens., sensitivity; Spec., specificity. aEffect on life expectancy for men and women. Note: The effect on LE is visible in days lost or gained per simulated individual entering screening. Morality risk is either caused by screentest (EUS) or by treatment (surgery after positive screentest). Abbreviations: CT, computed tomography; EUS, endoscopic ultrasound; HGD, high grade dysplasia; MRI, magnetic resonance imaging; Neg., negative; PanIN, Pancreatic Intraepithelial Neoplasia; PC, pancreatic cancer; Pos., positive; RR, relative risk; SB-IPMN, Side Branch—Intraductal Papillary Mucinous Note: The effect on LE is visible in days lost or gained per simulated individual entering screening. Morality risk is either caused by screentest (EUS) or by treatment (surgery after positive screentest). Note: The effect on LE is visible in days lost or gained per simulated individual entering screening. Morality risk is either caused by screentest (EUS) or by treatment (surgery after positive screentest) cLYG are presented in years per patient in ranges correlated with cyst size (1 cm less gain, 3 cm, more gain in LE). comorbidity levels.17 For healthy men and women, the LE benefit was 161 and 195 days, respectively. In 80-year-old individuals with severe comorbidity, surveillance had less effect on LE (36 days). 3.2 \| Screening in a high-risk population No PanIN) One time 50 1 3.9 to 5.8 10 Annual 50 1 12.9 to 1.3 RR 6.4/life time risk 7.5% MRI (sens. 56%, spec. 97%, for detection of cyst and early stage PC. No PanIN) One time 50 1 9.1 10 Annual (50– 80) 50 1 20.6 MRI + EUS (sens. per disease stage [60–99%], spec. 90%) Annual 50 3 120.4-158.4b 13 MRI + EUS (sens. per disease stage [60-99%], spec. 90%) 5-yearly 50 3 48.6-90.4b RR 12 (PC life time risk 18%) EUS (sens. 90%, for detection of pancreatic dysplasia) One time 50 3 138.7 12 RR 30-32 MRI (sens. 56%, spec. 97%, for detection of cyst and early stage PC. No PanIN) One time 50 1 31.5 10 2 65 to 71a 9 Annual 50 1 260 10 RR 70 MRI (sens. 56%, spec. 97%, for detection of early stage PC) One time 50 2 160 to 181a 9 PC Hypothetical screening reducing stage III/IV cancer with 20% NA NA NA 54.7 16 Hypothetical screening reducing stage III/IV cancer with 30% NA NA NA 82.1 Hypothetical screening reducing stage III/IV cancer with 50% NA NA NA 152 6 KOOPMANN ET AL. 10970215, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/ijc.34379 by Erasmus University Rotterdam Universiteitsbibliotheek, Wiley Online Library on [27/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Com 10970215, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/ijc.34379 by Erasmus University Rotterdam Universiteitsbibliotheek, Wiley KOOPMANN ET AL. TABLE 3 Effect of different PC screening strategies on life expectancy /onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License TABLE 3 (Continued) Pancreatic cyst Test mortality (%) Cyst (1-3 cmc) CT/MRI (Detecting HGD + PC. Probability true pos. = 0.80, true neg. = 0.99) Annual 65 0 17 to 439 15 75 0 9 to 50 85 0 3 to 54 EUS ± FNA (Detecting HGD + PC. Probability true pos. = 0.86, true neg. = 0.99) Annual 65 0.01 18 to 445 75 0.01 10 to 55 85 0.01 4 to 59 SB-IPMN Imaging Annual 60 0 161 to 195 17 80 0 69 Note: The effect on LE is visible in days lost or gained per simulated individual entering screening. Morality risk is either caused by screentest (EUS) or by treatment (surgery after positive screentest). 3.3 \| lesions Two studies evaluated the effect of screening in individuals with pan- creatic cysts.15,17 Weinberg et al evaluated the effect of annual screening with computed tomography (CT)/MRI or endoscopic ultra- sound (EUS) in patients with different size cystic lesions suggestive of Side Branch—Intraductal Papillary Mucinous Neoplasm (SB-IPMN), specifically in the pancreatic head. Their results show that the effect on LE was larger when screening started at age 65, as compared to starting at 75 or 85, both for CT/MRI and EUS screening. The effect on LE increased with cyst size. In case of annual screening with CT starting at age 65, a gain of 17 days was achieved for cystic lesions of 1 cm. If the cystic lesion was 3 cm, the LE gain for the same screening scenario was 439 days per screened individual. When individuals aged 85 received annual screening with CT, the average LE gain was 3 days in case of a 1 cm cystic lesion and 54 days in case of a 3-cm cyst. Sim- ilar results were seen for screening with EUS (Table 3). The second study evaluated the maximum effect of surveillance in the general population with a hypothetical perfect test for pancre- atic intraepithelial neoplasia (PanIN).11 When a 2% treatment mortal- ity was considered, one time screening improved LE by a mere 6.7 to 9.3 days per screened individual. In the hypothetical optimal scenario where screening with this perfect test was continuous (eg, daily), a 40-day gain in LE was found for each simulated individual. 3.2 \| Screening in a high-risk population Abbreviations: CT, computed tomography; EUS, endoscopic ultrasound; HGD, high grade dysplasia; MRI, magnetic resonance imaging; Neg., negative; PanIN, Pancreatic Intraepithelial Neoplasia; PC, pancreatic cancer; Pos., positive; RR, relative risk; SB-IPMN, Side Branch—Intraductal Papillary Mucinous Neoplasm; Sens., sensitivity; Spec., specificity. aEffect on life expectancy for men and women. bIn this model two pathways are simulated, one with only progressive lesions and one with indolent and faster progressive lesions. A larger effect on LE was seen in the progressive-only pathway. cLYG are presented in years per patient in ranges correlated with cyst size (1 cm less gain, 3 cm, more gain in LE). KOOPMANN ET AL. 7 TABLE 3 (Continued) Pancreatic cyst Test mortality (%) Cyst (1-3 cmc) CT/MRI (Detecting HGD + PC. Probability true pos. = 0.80, true neg. = 0.99) Annual 65 0 17 to 439 15 75 0 9 to 50 85 0 3 to 54 EUS ± FNA (Detecting HGD + PC. Probability true pos. = 0.86, true neg. = 0.99) Annual 65 0.01 18 to 445 75 0.01 10 to 55 85 0.01 4 to 59 SB-IPMN Imaging Annual 60 0 161 to 195 17 80 0 69 Note: The effect on LE is visible in days lost or gained per simulated individual entering screening. Morality risk is either caused by screentest (EUS) or by t t t ( ft iti t t) KOOPMANN ET AL. 7 KOOPMANN ET AL. \| Screening individuals with pancreatic cystic s 3.3 \| lesions 4 \| DISCUSSION This systematic review evaluates model-based analyses on the effect of PC screening on LE. PC screening in the general population led to decreased LE in most analyses, as screening benefits were out- weighed by false positive tests and surgical mortality risks. For higher- risk individuals, screening was more beneficial, but this conclusion strongly depends on model assumptions such as test characteristics. The effect of surveillance on LE seemed small in the slightly increased risk group (RR < 4.5). However, in this analysis, an unrealistic low test sensitivity (56%) for HGD/early stage PC were used in this analysis. As a result, these predictions likely underestimate the potential effect of screening. In individuals at higher risk (RR 6.4-60, eg, CDKN2A), both annual and one-time screening improved LE, even with a poor screening test. Although most papers were based on outdated and unrealistic assumptions, they do show that PC screening can be worthwhile. In the future, predictions from these models will improve when they are updated with reliable input data from ongoing long- term studies. Also, the use of a composite sensitivity for the detection of both HGD and early stage PDAC is a limitation. Overbeek et al published results of PC screening in high risk individuals, and showed that none of the 14 resected lesions harbored HGD. This shows how difficult detection of HGD is and that using the sensitivity for PDAC likely overestimates test performance for HGD. Ideally, each detected lesion on imaging is pathologically con- firmed. However, a lesion without worrisome features on imaging is not resected, and thus no pathological confirmation is available. In surveillance studies, only resected lesions can be compared head-to- head, causing selection bias. In higher-risk individuals (RR: 30), the expected median LE gain of 260 days for annual PC screening seems worthwhile. In comparison, colorectal cancer screening with an annual FIT test in the general pop- ulation is estimated to increase LE by 89 days per screened individ- ual.18 For lung cancer, 30 days are gained by annual screening with a low dose CT of 50 to 75-year old males who smoke.19 One model evaluated the effect of a hypothetical test that detects all PanIN3 lesions. Under this ideal (but for now unrealistic) assumption, one-time PC screening led to a LE gain, even in the general population, although that gain was still low compared to other cancer screening programs. \| Risk of bias Especially in the field of PC, where data are scarce and evidence is based on small studies, the risk of bias regarding input or target data is relatively high. Figure 2 provides an overview of the risk of bias of Raphel et al evaluated the effect of surveillance in patients with SB-IPMN and analyzed the effect on LE for individuals with different the included articles per item. Bias assessment per study is available in the Supplementary Materials (Data S1). 0% 20% 40% 60% 80% 100% Validation Design Data Analysis Reporting Interpretation Conflict of interest Risk of Bias Low risk Moderate risk High risk FIGURE 2 Risk of bias 8 KOOPMANN ET AL. 8 0% 20% 40% 60% 80% 100% Validation Design Data Analysis Reporting Interpretation Conflict of interest Risk of Bias Low risk Moderate risk High risk adenomas.12,15 Distinct precursor types such as IPMN and PanIN, that can progress from LGD to HGD into cancer, were not defined. The models that did simulate both precursors in separate pathways assumed that 90% of PC developed from PanIN and 10% from cystic lesions.9-11 If the proportion of cyst derived cancers is actually higher, as some studies indicate,20-22 these models would also underestimate the efficacy of screening. Input on cyst prevalence is needed to simulate a population at (increased) risk for pancreatic precursor lesions and PC.9-11,13 The reported cyst prevalence in the general population varies from 2.6% to 55%. This range is caused by differences in imaging techniques, populations and cyst definition (size and type). Pandharipande et al used an imaging based cyst prevalence of 4.6% at age 50 as a calibra- tion target. An increased cyst prevalence was assumed in the models that simulated a higher risk group, as is consistent with literature.23,24 the included articles per item. Bias assessment per study is available in the Supplementary Materials (Data S1). the included articles per item. Bias assessment per study is available in the Supplementary Materials (Data S1). Next to differences in natural history, the models incorporated different screen scenarios and assumed different screen tests charac- teristics. Test sensitivity for cystic lesions and PC varied from 56% to 99%. Also, most models used the same sensitivity for all disease stages, which is unrealistic. Pandharipande et al assumed a sensitivity of 56% for both cystic lesions and early stage cancer. \| Risk of bias When we evalu- ate more recent publications on PC screening in high risk individuals, a sensitivity of 70% to 75% for HGD/early stage cancer seems more realistic.23,25,26 A lower test sensitivity leads to an underestimation of the screening effect. However, with frequent screening, the effect of test sensitivity on life years saved is relatively small.13 4 \| DISCUSSION As the majority of LGD cysts will never progress to PC (given the high cyst prevalence and low PC incidence), the impact of a resected LGD cyst on life expectancy will be low and possibly negative because of treatment complications. Trials also show that patients with early detected and resected PC still have a limited life expectancy.23 The 5 year survival rate of T1 patients was reported to be 30.6%.32 For future research, focus should therefore lay on the detection and treatment of HGD precursor lesions. The detection of early stage cancer, even with annual screening, seems challenging and screen detected early stage PC remains associated with a decreased LE. The simulated screening strategies vary from one time to annual, to 5-yearly screening. Multiple studies evaluate the effect of one time screening at age 50. The relevance of this strategy is questionable given the PC peak incidence at age 60 to 70 years. However, the rele- vance also depends on duration of disease stages and test characteris- tics. One time screening at age 60 or 65 would be an interesting alternative scenario to study. Unfortunately, current screen tests are underperforming in the detection of high-grade dysplastic precursor lesions. Despite the exis- tence of multiple risk prediction tools, it remains difficult to classify cysts and determine their grade of dysplasia correctly, based on imag- ing.27 Also, test specificity for PanIN lesions is nowhere near 97%, while this lesion is believed to account for 50% to 90% of all PC. Thus, it is important to search for new and better screen tests. In this regard biomarkers hold promise, not only to detect early cancer, but also to search for timely identification of (high grade dysplastic) precursor lesions.33 Assumed treatment mortality varied from 0% to 10%. Recent lit- erature shows a mortality risk of 1% to 3% for a Whipple procedure and 1% to 2% for pancreatic tail resections.28,29 Thus, the study that assumed a 10% treatment mortality may have overestimated the harms of screening, resulting in an even larger loss in LE. Finally, of the models that evaluated PC screening in high-risk individuals, only one accounted for the increased risk to die from other prevalent malignancies in mutation carriers.10 The assumption that high-risk individuals have an average probability to die of other causes in the other models seems inappropriate and leads to an over- estimation of life-years gained. 4 \| DISCUSSION However, in clinical practice, we fail to detect PanIN lesions with current imaging modalities, let alone determine their dysplastic grade. However, this unrealistic scenario provides useful information on the upper limit of effective- ness of screening, and the maximal benefit in life expectancy it can provide. However, LE gain is not the only parameter to determine effec- tiveness of a screening program. Other factors, such as impact on quality of life, but also harms and costs should be considered. More- over, morbidity associated with the screening methodology or the sur- gical treatment (pancreatic fistula, exocrine pancreatic insufficiency and diabetes) was not taken into account in most studies, although this is highly relevant. It is very difficult to distinguish low from high grade dysplasia, based on imaging. Although cyst characteristics can give some guid- ance, a discrepancy between the imaging based diagnosis, and the true pathological state is observed.27 In the model of Pandharipande et al,9 the proportion of high-risk cystic lesions was estimated using a proxy as a calibration target: the proportion of patients with cysts The effect of screening on LE varies within the models because of different model assumptions, for instance on the natural history. In two models, PC could only evolve from “pancreatic dysplasia” or 10970215, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/ijc.34379 by Erasmus University Rotterdam Universiteitsbibliothe 9 KOOPMANN ET AL. who underwent subsequent pancreatic surgery was considered “high- risk” (5/84). However, as we know from surgical series, not nearly all individuals with high-risk features harbor actual high-grade dysplasia or PC. A large meta-analysis showed a pooled sensitivity of 0.67 (95% Confidence Interval [CI] 0.64-0.70) and a specificity of 0.64 (95% CI 0.62-0.66) for the Fukuoka guideline to detect advanced neoplasia (HGD or PC). Similar results were seen for the American Gastroenter- ological Association (AGA) guideline (pooled sensitivity of 0.59 [95% CI 0.52-0.65] and specificity of 0.77 [95% CI 0.74-0.80]).27 The effect of PC screening in ongoing clinical trials is mainly based on early detection of cancer rather than resection of HGD cyst, as published data shows that the number of resected HGD lesions is low (n = 0,31 n = 0,23 n = 1025). Precursor lesions that have been resected were mainly LGD. 4 \| DISCUSSION Importantly, mathematical models use observational data to simulate disease progression and evaluate effects of screening. Compared to other cancer models, observational PC data on the natural history and test characteristics are scant. This is a source for bias and may hamper the reliability of decision-analytic models. In conclusion, even though most included papers used out- dated and unrealistic assumptions, our study shows that screening can be effective in high-risk individuals. The extend of this effect depends on PC risk, test characteristics and screen frequency. Long lasting clinical trials are needed to gain more insight in the natural history of PC and the true test sensitivity by disease stage. Such input data will improve the predictive performance of PC screening models. Despite these limitations, the results of this systematic review on PC screening are important for clinical practice and future research. They show the potential and limitations of screening on LE in different PC risk groups and emphasize the importance of selecting the right pop- ulation for screening and the need for better screening tests to correctly identify and classify pancreatic precursor lesions. Especially for PanIN lesions, there is a lot to gain. For a screening program to be successful, given the current screening tests, the focus should be on individuals from the highest risk groups. Consensus guidelines advice screening for individuals with a 5% life time risk for PC.30 This PC risk greatly depends on test characteristics. Future model updates, based on more reliable input data from large and long-term studies should focus on establishing this. ACKNOWLEDGEMENTS Screening is always associated with lead and length-time bias. In this review, only one study reported on this.16 They estimated a lead- time bias of 23 to 64 days, which has considerable impact, given the short survival. They corrected for this bias by using the tumor volume doubling time, after which the NNS encompassed the harmful thresh- old. Other studies evaluating the effect of PC screening did not account for this bias. Thus, the true effect of surveillance in these groups is even smaller. The authors wish to thank Wichor Bramer from the Erasmus MC Medical Library for developing and updating the search strategies. AUTHOR CONTRIBUTIONS Brechtje D. M. Koopmann: Conceptualization, data curation, formal analysis, methodology, writing original draft. Amir-Houshang Omid- vari: Data curation, formal analysis, methodology, writing original draft. Iris Lansdorp-Vogelaar: Supervision, review & editing. Djuna L. Cahen: Funding, supervision, review & editing. Marco J. Bruno: Funding, supervision, review & editing. Inge M. C. M. de Kok: Fund- ing, Methodology, supervision, review & editing. The work reported in the paper has been performed by the authors, unless clearly specified in the text. REFERENCES 19. Du YSG, Heuvelmans MA, Groen HJM, Vermeulen KM, Greuter MJW, de Bock GH. Cost-effectiveness of lung cancer screen- ing with low-dose computed tomography in heavy smokers: a micro- simulation modelling study. Eur J Cancer. 2020;135:121-129. 1. Huang LJL, Balavarca Y, Molina-Montes E, et al. Resection of pancre- atic cancer in Europe and USA: an international large-scale study highlighting large variations. Gut. 2020;69(1):7-17. 2019 Jan;68(1): 130-139. 1. Huang LJL, Balavarca Y, Molina-Montes E, et al. Resection of pancre- atic cancer in Europe and USA: an international large-scale study highlighting large variations. Gut. 2020;69(1):7-17. 2019 Jan;68(1): 130-139. 20. Poultsides GARS, Cameron JL, Hruban RH, et al. Histopathologic basis for the favorable survival after resection of intraductal papillary mucinous neoplasm-associated invasive adenocarcinoma of the pan- creas. Ann Surg. 2010;251(3):470-476. 2. Kuntz K, Sainfort F, Butler M, et al. Decision and Simulation Modeling in Systematic Reviews [Internet]. Rockville, MD: Agency for Healthcare Research and Quality (US); 2013. 21. Makohon-Moore APMK, Zhang M, Reiter JG, et al. Precancerous neoplastic cells can move through the pancreatic ductal system. Nature. 2018;561(7722):201-205. 3. Geuzinge HA, Obdeijn IM, Rutgers EJT, et al. Cost-effectiveness of breast cancer screening with magnetic resonance imaging for women at familial risk. JAMA Oncol. 2020;6(9):1381-1389. 22. McGinnis TBL, Madan R, Dandawate P, et al. Survival outcomes of pancreatic intraepithelial neoplasm (PanIN) versus intraductal papil- lary mucinous neoplasm (IPMN) associated pancreatic adenocarci- noma. J Clin Med. 2020;9(10):3102. 4. de Kok IM, van Rosmalen J, Dillner J, et al. Primary screening for human papillomavirus compared with cytology screening for cervical cancer in European settings: cost effectiveness analysis based on a Dutch microsimulation model. BMJ. 2012;344:e670. 23. Overbeek KALI, Koopmann BDM, Harinck F, et al. Long-term yield of pancreatic cancer surveillance in high-risk individuals. Gut. 2022; 71(6):1152-1160. 5. Ten Haaf K, van Rosmalen J, de Koning HJ. Lung cancer detectability by test, histology, stage, and gender: estimates from the NLST and the PLCO trials. Cancer Epidemiol Biomarkers Prev. 2015;24(1): 154-161. 5. Ten Haaf K, van Rosmalen J, de Koning HJ. Lung cancer detectability by test, histology, stage, and gender: estimates from the NLST and the PLCO trials. Cancer Epidemiol Biomarkers Prev. 2015;24(1): 154-161. 24. Canto MIHR, Fishman EK, Kamel IR, et al. Frequent detection of pan- creatic lesions in asymptomatic high-risk individuals. Gastroenterology. 2012;142(4):796-804. 6. Lansdorp-Vogelaar I. Effect of Rising Chemotherapy Costs on the Cost Savings of Colorectal Cancer Screening; 2009. http://academic.oup. com. Accessed September 24, 2009. ORCID Brechtje D. M. Koopmann https://orcid.org/0000-0001-5025-5050 Amir-Houshang Omidvari https://orcid.org/0000-0002-5005-8038 Iris Lansdorp-Vogelaar https://orcid.org/0000-0002-9438-2753 Djuna L. Cahen https://orcid.org/0000-0001-6753-4857 Marco J. Bruno https://orcid.org/0000-0001-9181-5499 Inge M. C. M. de Kok https://orcid.org/0000-0002-9419-0452 Brechtje D. M. Koopmann https://orcid.org/0000-0001-5025-5050 Amir-Houshang Omidvari https://orcid.org/0000-0002-5005-8038 Iris Lansdorp-Vogelaar https://orcid.org/0000-0002-9438-2753 Djuna L. Cahen https://orcid.org/0000-0001-6753-4857 Marco J. Bruno https://orcid.org/0000-0001-9181-5499 Inge M. C. M. de Kok https://orcid.org/0000-0002-9419-0452 17. Raphel TJWD, Berland LL, Herts BR, Megibow AJ, Knudsen AB, Pandharipande PV. Imaging follow-up of low-risk incidental pancreas and kidney findings: effects of patient age and comorbidity on pro- jected life expectancy. Radiology. 2018;287(2):504-514. 18. Knudsen ABZA, Rutter CM, Naber SK, et al. Estimation of benefits, burden, and harms of colorectal cancer screening strategies: modeling study for the US Preventive Services Task Force. JAMA. 2016; 315(23):2595-2609. doi:10.1001/jama.2016.6828 FUNDING INFORMATION Brechtje D. M. Koopmann, Djuna L. Cahen, Marco J. Bruno and Inge M. C. M. de Kok received funding from the MLDS (nongovernmental organization, national gastrointestinal disease association, nonprofit). 10 KOOPMANN ET AL. Brechtje D. M. Koopmann, Amir-Houshang Omidvari, Iris Lansdorp- Vogelaar, Djuna L. Cahen, Marco J. Bruno and Inge M. C. M. de Kok have nothing to disclose. 10. Pandharipande PV, Jeon A, Heberle CR, et al. Screening for pancreatic adenocarcinoma in BRCA2 mutation carriers: results of a disease sim- ulation model. EBioMedicine. 2015;2(12):1980-1986. 11. Peters MLB, Eckel A, Mueller PP, et al. Progression to pancreatic duc- tal adenocarcinoma from pancreatic intraepithelial neoplasia: results of a simulation model. Pancreatology. 2018;18(8):928-934. CONFLICT OF INTEREST 12. Rulyak SJ, Kimmey MB, Veenstra DL. Cost-Effectiveness of Pancreatic Cancer Screening in Familial Pancreatic Cancer Kindreds. Elsevier; 2003;57(1):23-9. doi:10.1067/mge.2003.28. The authors declare no potential conflict of interests. The authors declare no potential conflict of interests. DATA AVAILABILITY STATEMENT 13. Koopmann BDM, Harinck F, Kroep S, et al. Identifying key factors for the effectiveness of pancreatic cancer screening: a model-based anal- ysis. Int J Cancer. 2021;149(2):337-346. All data used in this systematic review is available in the original papers that were included. 14. Jaime Caro JED, Kan H, Kaltz C, et al. Questionnaire to assess rele- vance and credibility of modeling studies for informing health care decision making: an ISPOR-AMCP-NPC good practice task force report. Value Health. 2014;17(2):174-182. ETHICS STATEMENT No ethical approval was needed for this systematic review because data from previous published modeling studies with simulated individ- uals was retrieved and analyzed. 15. Weinberg BM, Spiegel BM, Tomlinson JS, Farrell JJ. Asymptomatic pancre- atic cystic neoplasms: maximizing survival and quality of life using Markov- based clinical nomograms. Gastroenterology. 2010;138(2):531-540. 16. Cucchetti A, Ercolani G, Cescon M, et al. Estimation of the survival benefit obtainable from screening for the early detection of pancre- atic cancer. Pancreas. 2016;45(5):714-719. REFERENCES 25. Canto MIKT, Yeo CJ, Hruban RH, et al. Surgical outcomes after pan- creatic resection of screening-detected lesions in individuals at high risk for developing pancreatic cancer. J Gastrointest Surg. 2020;24(5): 1101-1110. 7. Toes-Zoutendijk E vLM, Dekker E, van Hees F, et al. Real-time monitoring of results during first year of Dutch colorectal cancer screening program and optimization by altering fecal imm- unochemical test cut-off levels. Gastroenterology. 2017;152(4): 767-775.e2. 26. Vasen H II, Ponce CG, Slater EP, et al. Benefit of surveillance for pan- creatic cancer in high-risk individuals: outcome of long-term prospec- tive follow-up studies from three European expert centers. J Clin Oncol. 2016;34(17):2010-2019. 8. Kooyker AIT-ZE, Opstal-van Winden AWJ, Spaander MCW, et al. The second round of the Dutch colorectal cancer screening program: impact of an increased fecal immunochemical test cut-off level on yield of screening. Int J Cancer. 2020;147(4): 1098-1106. 27. Wu JWY, Li Z, Miao H. Accuracy of Fukuoka and American Gastroen- terological Association guidelines for predicting advanced neoplasia in pancreatic cyst neoplasm: a meta-analysis. Ann Surg Oncol. 2019; 26(13):4522-4536. 9. Pandharipande PV, Heberle C, Dowling EC, et al. Targeted screening of individuals at high risk for pancreatic cancer: results of a simulation model. Radiology. 2015;275(1):177-187. 28. Peluso H, Abougergi M, Mrad C, Jones W. The incidence, risk factors and impact of 30-day readmission after Whipple procedure KOOPMANN ET AL. for pancreatic cancer on mortality and healthcare resource utilization: a nationwide analysis. Am J Gastroenterol. 2017;112- (October):S22. 33. Tanaka MHM, Liu B, Heger U, Hackert T, Michalski CW. Cytologic analysis of pancreatic juice increases specificity of detection of malignant IPMN—a systematic review. Clin Gastroenterol Hepatol. 2019;17(11):2199-2211.e21. 29. Cardini B, Primavesi F, Maglione M, et al. Outcomes following pancre- atic resections—results and challenges of an Austrian university hos- pital compared to nationwide data and international centres. Eur Surg. 2019;51:81-89. SUPPORTING INFORMATION 30. Goggins MOK, Brand R, Syngal S, et al. International cancer of the pancreas screening (CAPS) consortium. Management of patients with increased risk for familial pancreatic cancer: updated recommenda- tions from the international cancer of the pancreas screening (CAPS) Consortium. Gut. 2020;69(1):7-17. Additional supporting information can be found online in the Support- ing Information section at the end of this article. How to cite this article: Koopmann BDM, Omidvari A-H, Lansdorp-Vogelaar I, Cahen DL, Bruno MJ, de Kok IMCM. The impact of pancreatic cancer screening on life expectancy: A systematic review of modeling studies. Int J Cancer. 2022; 1‐11. doi:10.1002/ijc.34379 31. Klatte DCFBB, Wasser MNJM, Feshtali Shahbazi S, et al. Pancreatic cancer surveillance in carriers of a germline CDKN2A pathogenic variant: yield and outcomes of a 20-year prospective follow-up. J Clin Oncol. 2022;40:3267-3277. 32. Kwon WPT, He J, Higuchi R, et al. Is the new T1 category as defined in the eighth edition of the AJCC pancreatic cancer staging system an improvement? J Gastrointest Surg. 2020; Feb;24(2):262-269.
https://openalex.org/W4294397970	https://www.nature.com/articles/s41598-022-19430-y.pdf	English	null	Miniaturized antenna verified with diffuse optical measurements for native and boiled adipose tissue differentiation	Scientific reports	2,022	cc-by	6,395	Ashraf S. Abdel Halim1, Mohanad Mostafa1 & Omnia Hamdy2* Ashraf S. Abdel Halim1, Mohanad Mostafa1 & Omnia Hamdy2* Medical industries are continuously working towards the development of wearable theragnostic devices which enable monitoring various ailments in the body and then transmitting them to the base-station. The antenna design is of prime importance where the suitable design guarantees proper communication between the antenna and the base-station. In this paper, a co-planar wave-guide antenna is proposed for the use in the medical implant communication service (MICS) band for data transmission. The proposed antenna is studied for ex-vivo applications where the antenna is simulated for bovine intramuscular fat (adipose tissue). The preliminary results showed that the antenna radiates in MICS band. Two types of samples are tested; namely, native fat and boiled fat. The boiled fat is used in order to represent the infected fat tissue. Hence, the antenna was implanted into the fat samples and the results revealed noticeable variations in the radiation characteristics between native and boiled fat. Different parameters of the proposed antenna including the reflection coefficient (S11), radiation patterns, gain, efficiency, and front-to-back ratio are investigated. The simulations showed that S11 parameter was − 12.4 dB in MICS band for the normal fat. On the other hand, the measured S11 values were − 12.3 dB for the native samples and − 9.9 dB for the boiled fat samples. To assert the variation in the biological characteristics of the boiled fat as compared to those of the native fat, diffuse optical measurements of the examined samples were investigated. Such variation in the light scattering and absorbance by the tissue is responsible for varying the S11 parameter for each case. The results have shown that the proposed design is a good candidate for detecting the change in biological tissue. Biomedical antenna has been developed in 1960s by Medtronic, USA1 as a battery powered device. Nowadays, antenna in medical applications provides a virtual environment that has the ability to stimulate and monitor the function of various body organs2. The antenna wirelessly transfers information about glucose level, body tem- perature, pH, and cardiac pressure to a certain receiver (e.g., base-stations in medical centers) via links named “bio-telemetry”3–5. The design of the antenna depends on the industrial, scientific and medical (ISM) band for medical telemetry operations. www.nature.com/scientificreports www.nature.com/scientificreports Ashraf S. Abdel Halim1, Mohanad Mostafa1 & Omnia Hamdy2* Nonetheless, a recent medical implant communication service (MICS) band (402–405 MHz) has been allocated which is regulated by the United States federal communications commission and the European radio communications committee for bi-directional bio-telemetry operations6.h p y p The MICS band permits low noise propagation through human body that makes it suitable for low power implanted device circuits7. The wavelength for free space is 74 cm in the med radio whereas it is around 12 cm in the ISM bands8. Accordingly, there is a need to miniaturize the antenna to be implanted inside the human body considering the biocompatibility properties and other related parameters such as gain, SAR, bandwidth, and directivity. Studying the miniaturized antenna and its radiation characteristics has started since decades. The size of the antenna was directly proportional to the radiation power factor, the total efficiency, and the antenna bandwidth7. Later on, different miniaturization techniques has been proposed including the use of high permittivity dielectric substrate, lengthening the current flow path on the patch surface, and inserting shorted pins between the ground and the patch plane. The latter results in developing antennas of relatively small effec- tive size9. 1Department of Communication, Faculty of Engineering, Canadian International College (CIC), Cairo, Egypt. 2Department of Engineering Applications of Lasers, National Institute of Laser Enhanced Sciences, Cairo University, Giza, Egypt. email: omnia@niles.cu.edu.eg Scientific Reports \| (2022) 12:15035 \| https://doi.org/10.1038/s41598-022-19430-y www.nature.com/scientificreports/ Table 1. Performance comparison between the proposed antenna and the relevant literature. Ref Antenna dimension (mm3) Resonant Freq. (GHz) BW (MHz) Gain (dBi) SAR W/Kg Test tissue 35 57 0.402 38.6 − 30.8 289 Minced pork 38 10 × 10 × 2.54 0.402 115 − 7 341 Pork 39 20.5 × 30 × 0.05 0.402 3.73 − 32 513.7 Deep tissue 40 7 × 6.5 × 0.377 0.402 36.8 − 30.5 588 Skin layer 41 22 × 23 × 1.27 0.402 30 − 36.7 832 Skin-mimicking material 42 14 × 7.5 × 0.5 0.405 64 − 40.85 665.35 Scalp (realistic human phantom) This work 14 × 14 × 0.76 0.454 0.23 − 10 50 Fat Table 1. Performance comparison between the proposed antenna and the relevant literature. Since the signal is highly attenuated within the human body, the near-field effect of antenna causes an increase in the temperature of neighboring tissues. Higher frequency operation also causes similar effect. Ashraf S. Abdel Halim1, Mohanad Mostafa1 & Omnia Hamdy2 This may be useful for some therapeutic applications yet shows a negative impact on communication7. Utilizing meandered antennas has solved such problem. The meandered structure can miniaturize the size of the antenna and keep the same radiation properties for an implantable antenna8. p p p Micro-strip (or patch) antennas have the advantages of being low cost, light weight, compact size and easy fabrication. However, a typical micro-strip antenna has a relatively narrow bandwidth10. Therefore, various techniques to enhance the bandwidth of patch antennas have been proposed including substrate-integrated sus- pended-line technique11, slotted patch12,13, adding stubs14,15, proximity coupled technique16, and cutting edges17. Also, the use of rectangular-shaped coplanar waveguide (CPW) fed printed antennas provides wider bandwidths necessary for wireless and wearable applications18–21. Additionally, to increase the gain of the antenna, several advancements are used such as artificial magnetic conductors (AMCs)22, metamaterials23 or a horn reflector24. i gl Various designs of wireless antennas were developed based on the use of rigid substrates such as FR4 as reported in25–27. Nevertheless, for implanting purpose, the design of efficient antennas requires the use of flex- ible substrates which have the ability to be incorporated with shapes of irregular surfaces28. Example of these flexible substrate are kapton29, liquid crystal polymer (LCP)30, Rogers RT/duroid31, cotton layer32, polyethylene terephthalate (PET) film33, and paper34. A flexible material such as Rogers ULTRALAM has been utilized for multiband conformal implantable antenna fabrication which is utilized in ingestible capsule endoscope and biotelemetry applications35. Moreover, antennas can be designed in different shapes including flat and capsule shapes. The capsule antennas are suitable for deep tissue implantation such as intestine, stomach and heart, while the flat type is used for skin (e.g., scalp) implantation36,37. Table 1 presents a comparison between the proposed antenna performance and the state-of-the-art designs.f p g In the present paper, a micro-strip antenna is designed for monitoring adipose tissue (fat) at two different conditions (native and boiled). The antenna is designed in a simple double layered meandered structure. The different antenna parameters are evaluated at each tissue condition. The proposed antenna radiates near the MICS band for data transmission related to patient monitoring. Additionally, diffuse reflectance and transmittance of the examined samples were measured at four laser wavelengths (532, 660, 780 and 980 nm) using integrating sphere-based optical setup to emphasize the difference in tissue properties at each condition, which in turn results in varying the reflection coefficient (S11). Antenna design Design process. g Design process. A compact antenna of dimensions of 0.116 λ0 × 0.116 λ0 × 0.006 λ0 with respect to the reso- nant frequency is proposed in the current study. The simulations are performed using a commercial simula- tion software (High-Frequency Structure Simulator Technology (HFSS)). The design is applied on a dielectric substrate Rogers 3210 (dielectric constant = 10.5) to achieve the required characteristics. This substrate has a thickness of 0.76 mm and a loss tangent of 0.0027. It should be noted that the design is primarily based on the meander patch antenna. Figure 1 presents the meander patch antenna. Moreover, the dimensions of the pro- posed antenna are listed in Table 2.hih p The first step in the design process is to identify the medium. The proposed antenna is designed to test fat tissue which has dispersive electromagnetic characteristics. The antenna design achieved the 50-Ω excitation in both cases. Figure 2 shows the simulation model in case of fat as a medium. As shown in Fig. 2, the antenna is sandwiched between two layers of fat. The preliminary simulation results for the native fat tissue shows that the resonance frequency achieved by our proposed antenna is 513 MHz. Design approach. The proposed antenna consists of three layers. The first layer is a ground sheet connected with the patch through the pin. The second layer is the substrate which is divided into two layers that sandwiches the patch (the third layer) between them (see Fig. 3).hh p y g The patch layer of the antenna is attached at the top right corner with SMA connector pin. This layer also includes meander tailored resonators and a capacitive coupler gap43. The proposed design aims to elongate the current path. The meander structure is bent to decrease the length and increase stiffness of the antenna. A superstrate is usually added to avoid the short circuit between patch and substrate. Therefore, this eliminates the patch to function as a radiating element. Nevertheless, in our proposed design, there is no need to use a https://doi.org/10.1038/s41598-022-19430-y Scientific Reports \| (2022) 12:15035 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 1. The proposed antenna configuration and dimensions. Table 2. Dimensions of the proposed antenna (in mm). Antenna design Design process. A B C D E F G H i J K L M h W P Q R S 12 12 1.7 6.6 1 1.7 1 3.2 1 1 1 14 1.7 0.76 14 0.8 0.5 1.2 5 Figure 2. The simulation of the measurement setup in fat. Figure 1. The proposed antenna configuration and dimensions. Figure 1. The proposed antenna configuration and dimensions. Table 2. Dimensions of the proposed antenna (in mm). Figure 2. The simulation of the measurement setup in fat. Figure 2. The simulation of the measurement setup in fat. Figure 2. The simulation of the measurement setup in fat. superstrate due to the poor conductivity of fat material (0.102 S/m). Table 3 represents fat parameters in the resonance frequency. The manufacturing and measurements were performed in the National Telecommunica- tion Institute (NTI), Cairo, Egypt.f superstrate due to the poor conductivity of fat material (0.102 S/m). Table 3 represents fat parameters in the resonance frequency. The manufacturing and measurements were performed in the National Telecommunica- tion Institute (NTI), Cairo, Egypt.f Indeed, the microstructural and macroscopic properties of boiled fat are relatively different from the native one44. Once the fat is heated, adipose tissue (fat) is liberated from the adipocytes (a cell in connective tissue that is specialized for the fat storage) and its oxidation increases45. Accordingly, the electrical conductivity of the fat tissue increases as the temperature increases while decreases if its structure changes46. The current distribution. To illustrate the mechanism of operation, the surface current distribution for the proposed antenna is presented in Fig. 4. The current distribution demonstrates that the antenna structure is responsible for producing l dip at 513 MHz as the current concentration along the antenna length. The cur- rent concentrates along the antenna width, patch, and pin. The intensive current indicated by red and a null https://doi.org/10.1038/s41598-022-19430-y Scientific Reports \| (2022) 12:15035 \| www.nature.com/scientificreports/ Figure 3. The common design approach of meander antenna. Table 3. Fats parameters in the resonance frequency. Tissue Conductivity Relative permittivity Loss tangent Wavelength Penetration depth Fat 0.10235 S/m 5.2853 0.14503 0.054193 m 0.11956 m Figure 4. Current distribution of the proposed antenna at MICS band. Fi 3 Th d i h f d t Figure 3. The common design approach of meander antenna. Figure 4. Current distribution of the proposed antenna at MICS band. Figure 4. Current distribution of the proposed antenna at MICS band. Figure 4. Antenna design Design process. Current distribution of the proposed antenna at MICS band. https://doi.org/10.1038/s41598-022-19430-y Scientific Reports \| (2022) 12:15035 \| www.nature.com/scientificreports/ Figure 5. (a) The implemented optical setup using integrating sphere, (b) schematic of the transmission mode, and (c) schematic of the reflection mode. Figure 5. (a) The implemented optical setup using integrating sphere, (b) schematic of the transmission mode, and (c) schematic of the reflection mode. represented by blue. The figure also illustrates the effective area at which the proposed antenna resonates at the certain frequency. Diffuse optical measurementshl f The optical transmission and reflection resulting from light interaction with biological tissue is named as diffuse because of the multiple scattering events affected the light propagation in tissues47. The tissue diffuse reflectance/ transmittance is a representative parameter for each tissue, where they vary according to the tissue condition. Therefore, they can be used in tissue differentiation processes48,49. Integrating sphere is a very common device that is used for the ex-vivo measurement of the tissue diffuse reflectance and transmittance50,51. It is a hollow spherical cavity coated with highly reflective material to ensure reflection of the whole entering light. In our experiments, Barium sulfate coated mcPHERSON integrating sphere has been used to measure diffuse reflectance/transmit- tance of the native and boiled fat samples. A photo of the experimental setup is presented in Fig. 5a. A typical integrating sphere has multiple ports and locations for placing the sample under investigation. Furthermore, a light detector is used where its location is dependent on the required measurement (tissue transmittance or reflectance) as demonstrated in Fig. 5b,c, respectively. The tissue’s detected diffuse light is then delivered to a compact spectrometer that is connected to a computer for data analysis and processing. In the present work, a digital fiber spectrometer (STDFSM, Touptek Photonics Co. Ltd, Zhejiang China) integrated with a detector (Toshiba TCD1304AP linear CCD array) has been used. Collecting fat samples. Ex-vivo bovine intramuscular fat (adipose tissue) samples have been collected from different butcher shops. There is no direct contact with alive animals in the present study. Therefore, no specific ethical approval is required. The sources of the utilized fat samples (bovines) were already slaughtered for commercial food production and the fat tissues can be considered as disposals or remains. Scientific Reports \| (2022) 12:15035 \| https://doi.org/10.1038/s41598-022-19430-y www.nature.com/scientificreports/ Figure 6. The measured reflection coefficient (S11) of the three fabricated antennas for native and boiled fat tissues. Figure 6. The measured reflection coefficient (S11) of the three fabricated antennas for native and boiled fat tissues. Results and discussion To evaluate the proposed design, a Rohde&Schwarz ZVB 20 (Vector Network Analyzer, 10 MHz–20 GHz) is used to test the antenna. As previously mentioned, the fat is examined in this study in two cases; normal and infected (the infected was represented by the boiled tissue). For better profess, three similar antennas with the same design and material were fabricated and measured. Figure 6 shows the measured reflection coefficient of each antenna at the two examined tissue conditions.t Photographs of the fabricated antennas before and after immersed in the fat sample is presented in Fig. 7a,b respectively. Moreover, a comparison between the simulated and average measured reflection coefficient (S11) for the three antennas is presented in Fig. 7c. Figure 7c illustrates the S11 parameter for the three cases; simulations, native tissue and infected tissue (boiled). In the simulations, S11 was − 12.4 dB at 0.513 GHz. However, in the case of native fat the S11 was − 12.3 dB. In case of infected fat S11 changed to − 9.9 dB. In the measurements, both native and infected fat exhibit a frequency shift to the left with respect to that of the simulated value (i.e., 0.454 GHz for the native fat and 0.425 GHz for the infected fat). The observed frequency shift in the three cases is attributed to the non-uniform thickness (structure) of the examined fat tissue samples at the different conditions (native and boiled samples). In addition, the pos- sible fabrication tolerance may lead to the same effect. Table 4 summarizes the results of the proposed antenna. The radiation patterns. The E-plane of the radiation pattern of the proposed antenna (Eθ at φ = 0°) and the H-plane radiation pattern (Eθ at φ = 90°) is presented in Fig. 8.h The E-plane radiation patterns show that the proposed antenna radiates omnidirectional in case of simulated and boiled tissue and bi-directional in case of normal fat. However, the H-plane patterns reveal that the antenna radiates bi-directional in the three cases. It is worth noting that the model does not take into account the probe location. Results and discussion It is also assumes that the dielectric material of the substrate is truncated and does not cover the ground plane beyond the edges of the patch which may be the cause of the discrepancies between the two patterns.hi y g y The 3-D radiation patterns for the proposed antenna at the resonance frequency for the three configurations; simulated, normal and boiled fat are illustrated in Fig. 9a–c, respectively. The gain. Figure 10 shows the gain of the proposed antenna. The maximum obtained gain in the MICS band was about − 10 dB. This is due to limitations in the measurement process in addition to the degenerative effect of the SMA connector due to small size of the antenna. SAR. The electromagnetic parameters of the human tissue are given in Table 5. The calculation formula of SAR value is presented as follows: (1) SAR = σE2 ρ SAR = σE2 ρ (1) where σ is conductivity, E is the electric field intensity, and ρ is the mass density52 where σ is conductivity, E is the electric field intensity, and ρ is the mass density52.h i The average SAR value of the proposed antenna when implanted into the fat tissue is presented in Fig. 11. The maximum SAR of the 10 g is 50 W/Kg. Due to the reflection characteristics, the obtained SAR enhances the main lobe but it also reduces the back lobe. This means that only a small amount of electromagnetic wave energy is radiated to the fat tissue. Therefore, the antenna has good forward radiation and reasonable SAR value. Total efficiency and front to back ratio. The simulated total efficiency and front to back ratio “FBR” versus frequency of the proposed antenna are presented in Fig. 12a,b, respectively.fi As shown in Fig. 12a, the total efficiency of the antenna is almost − 10 dB at the operational frequency. In addition, a relatively high FBR is obtained at the resonance frequency of the antenna (see Fig. 12b). Scientific Reports \| (2022) 12:15035 \| https://doi.org/10.1038/s41598-022-19430-y www.nature.com/scientificreports/ Figure 7. (a) A photograph of the fabricated antennas, (b) a photograph of the antenna with studied fat sample, (c) the S11 parameters for simulation, normal and boiled fat. Validation Operating frequency (GHz) Reflection coefficient S11 (dB) 3 dB Bandwidth (MHz) Figure 7. Results and discussion (a) A photograph of the fabricated antennas, (b) a photograph of the antenna with studied fat sample, (c) the S11 parameters for simulation, normal and boiled fat. Figure 7. (a) A photograph of the fabricated antennas, (b) a photograph of the antenna with studied fat sample, (c) the S11 parameters for simulation, normal and boiled fat. Table 4. Summary of the simulated and measured antenna results. Validation Operating frequency (GHz) Reflection coefficient, S11 (dB) − 3- dB Bandwidth (MHz) Simulated Native fat 0.513 − 12.4 0.20 Measured Native fat 0.454 − 12.3 ± 0.3 0.19 ± 0.02 Boiled fat 0.425 − 9.9 ± 0.2 0.17 ± 0.01 Table 4. Summary of the simulated and measured antenna results. Table 4. Summary of the simulated and measured antenna results. Diffuse optical measurements. Tissue reflectance is the ratio between the reflected light intensity and the incident light intensity, while the transmittance is defined as the ratio between the transmitted light intensity and the incident light intensity. Accordingly, the variations in the reflected light intensity and transmitted light intensity of the fat samples (native and boiled) at each wavelength are presented in Figs. 13 and 14, respectively. For each sample, the experimental measurements have been obtained five times and the average values are plot- ted.fl As shown in Fig. 13, the diffuse reflectance of the boiled fat tissues is less than the native tissues at the four studied wavelengths. On the other hand, the transmittance measurements show the opposite behavior (Fig. 14). Scientific Reports \| (2022) 12:15035 \| https://doi.org/10.1038/s41598-022-19430-y www.nature.com/scientificreports/ Figure 8. The Radiation pattern (a) E-plane at φ = 0 and (b) H-plane at φ = 90°. Figure 9. The 3-D radiation patterns for the proposed antenna (a) simulation, (b) normal fat and (c) boiled fat. Figure 8. The Radiation pattern (a) E-plane at φ = 0 and (b) H-plane at φ = 90°. Figure 8. The Radiation pattern (a) E-plane at φ = 0 and (b) H-plane at φ = 90°. Figure 9. The 3-D radiation patterns for the proposed antenna (a) simulation, (b) normal fat and (c) boiled fat. Figure 9. The 3-D radiation patterns for the proposed antenna (a) simulation, (b) normal fat and (c) boiled fat. igure 9. The 3-D radiation patterns for the proposed antenna (a) simulation, (b) normal fat and (c) boiled fat. Figure 10. The gain of the proposed antenna. Figure 10. Results and discussion The gain of the proposed antenna. Figure 10. The gain of the proposed antenna. https://doi.org/10.1038/s41598-022-19430-y https://doi.org/10.1038/s41598-022-19430-y Scientific Reports \| (2022) 12:15035 \| www.nature.com/scientificreports/ Tissue εr Conductivity (S/m) Density (kg/m3) Thickness (mm) Skin 37.95 1.49 1001 2 Fat 5.27 0.10235 900 5 Muscle 52.67 1.77 1006 20 Bone 18.49 0.82 1008 13 Table 5. The electromagnetic parameters of the human tissue. Skin 37.95 1.49 1001 2 Fat 5.27 0.10235 900 5 Muscle 52.67 1.77 1006 20 Bone 18.49 0.82 1008 13 Figure 11. Simulated SAR of the proposed antenna. Figure 12. The simulated (a) total efficiency, and (b) front to back ratio of the proposed antenna. Table 5. The electromagnetic parameters of the human tissue. Table 5. The electromagnetic parameters of the human tissue. Figure 11. Simulated SAR of the proposed antenna. Figure 11. Simulated SAR of the proposed antenna. Figure 12 The simulated (a) total efficiency and (b) front to back ratio of the proposed antenna Figure 12. The simulated (a) total efficiency, and (b) front to back ratio of the proposed antenna. Figure 12. The simulated (a) total efficiency, and (b) front to back ratio of the proposed antenna. https://doi.org/10.1038/s41598-022-19430-y Scientific Reports \| (2022) 12:15035 \| www.nature.com/scientificreports/ Figure 13. Diffuse reflected light of fat samples at (a) 532 nm, (b) 660 nm, (c) 780 nm, and (d) 980 nm. Figure 13. Diffuse reflected light of fat samples at (a) 532 nm, (b) 660 nm, (c) 780 nm, and (d) 980 nm. Figure 13. Diffuse reflected light of fat samples at (a) 532 nm, (b) 660 nm, (c) 780 nm, and (d) 980 nm. Such results are reasonable as some cellular and intracellular characteristics (such as protein denaturation, hyalinization of collagen, and cell membrane rupture) of fat tissues are supposed to be altered after boiling53. These characteristics have a great effect on tissue scattering properties and hence affect light distribution and propagation54. Conclusions A miniaturized multilayer antenna was designed as a diagnostic tool for differentiating native and infected adi- pose tissue (ex-vivo bovine fat). An additional substrate was added to the design to enable the antenna to test high conductive tissues. The antenna resonated at 454 MHz (near to the MICS band), which makes it applicable for the short range medical applications. Although the resonant frequency of the proposed antenna does not lay exactly in the MICS band (402–405 MHz), the measured reflection coefficient (S11) showed a considerable change at the different conditions of the tissue. Additionally, the measured results of the proposed antenna showed an acceptable agreement with the simulation results. Moreover, the E- and H-field radiation patterns asserted the same conclusions. The antenna gain was − 10 dB in the three cases. Furthermore, the diffuse reflectance and trans- mittance of the studied tissue samples were measured to confirm the variation in the optical properties between native and infected samples which was also demonstrated in the obtained S11 parameter at each case. Compared with the previous literature, the proposed antenna has relatively small dimensions. For future investigations, the proposed antenna will be tested on other tissues with high conductivity. Additionally, multiple antennas with different designs using various Rogers will be considered. https://doi.org/10.1038/s41598-022-19430-y Scientific Reports \| (2022) 12:15035 \| www.nature.com/scientificreports/ Figure 14. Total transmitted light of fat samples at (a) 532 nm, (b) 660 nm, (c) 780 nm, and (d) 980 nm. Figure 14. Total transmitted light of fat samples at (a) 532 nm, (b) 660 nm, (c) 780 nm, and (d) 980 nm. Data availabilityh Data availability The datasets used and/or analyzed during the current study available from the corresponding author on reas able request. Data availability The datasets used and/or analyzed during the current study available from the corresponding author on reason- able request. Received: 15 March 2022; Accepted: 29 August 2022 Received: 15 March 2022; Accepted: 29 August 2022 References 1. Bhattacharjee, S., Maity, S., Metya, S. K. & Bhunia, C. T. Performance enhancement of implantable medical antenna using dif- ferential feed technique. Eng. Sci. Technol. Int. J. 19, 642–650 (2016). q g 2. Cosoli, G., Scalise, L., Poli, A. & Spinsante, S. Wearable devices as a valid support for diagnostic excellence: lessons from a pandemic going forward. Health Technol. (Berl) 11, 673–675 (2021). g g ( ) ( ) 3. Taylor, W. et al. A review of the state of the art in non-contact sensing for covid-19. Sensors 20, 1–19 (2020). g g 3. Taylor, W. et al. A review of the state of the art in non-contact sensing for covid-19. Sensors 20, 1–19 (2020 3. Taylor, W. et al. A review of the state of the art in non-cont y g 4. Yun, S. M. et al. Recent advances in wearable devices for non-invasive sensing. Appl. Sci. 11, 1–28 (2021). y g 4. Yun, S. M. et al. Recent advances in wearable devices for non-invasive sensing. Appl. Sci. 11, 1–28 (2021). 5. Liao, Y., Leeson, M. S. & Higgins, M. D. A communication link analysis based on biological implant wireless body area networks Appl. Comput. Electromagn. Soc. J. 31, 619–628 (2016). pp p g J ( ) 6. Kiourti, A., Costa, J. R., Fernandes, C. A., Santiago, A. G. & Nikita, K. S. Miniature implantable antennas for biomedical telemetry: From simulation to realization. IEEE Trans. Biomed. Eng. 59, 3140–3147 (2012). 6. Kiourti, A., Costa, J. R., Fernandes, C. A., Santiago, A. G. & Nikita, K. S. Miniature implantable antennas for biomedical telemetry From simulation to realization. IEEE Trans. Biomed. Eng. 59, 3140–3147 (2012). g , ( ) 7. Kiourti, A., Costa, J. R., Fernandes, C. A. & Nikita, K. S. A broadband implantable and a dual-band on-body repeater antenna: Design and transmission performance. IEEE Trans. Antennas Propag. 62, 2899–2908 (2014).hfi g ( ) 7. Kiourti, A., Costa, J. R., Fernandes, C. A. & Nikita, K. S. A broadband implantable and a dual-band on-body repeater antenna Design and transmission performance. IEEE Trans. Antennas Propag. 62, 2899–2908 (2014).hfi g p p g 8. Skrivervik, A. K. Implantable antennas: The challenge of efficiency. in 7th European Conference on Antennas and Propagation (EuCAP) 3627–3631 (2013). 9. Kiourti, A. & Nikita, K. S. A review of implantable patch antennas for biomedical telemetry: Challenges and solutions. IEEE Antennas Propag. Mag. 54, 210–228 (2012). 9. www.nature.com/scientificreports/ A low-profile high-gain substrate-integrated waveguide-slot anten zation using metamaterial. IEEE Antennas Wirel. Propag. Lett. 16, 1614–1617 (2017).i y p p g g 23. Pandit, S., Mohan, A. & Ray, P. A low-profile high-gain substrate-integrated waveguide-slot antenna with suppressed cross po i i i l IEEE A Wi l P L 16 1614 1617 (2017) 3. Pandit, S., Mohan, A. & Ray, P. A low-profile high-gain substrate-integrated waveguide-slot antenna with suppressed cross polari zation using metamaterial. IEEE Antennas Wirel. Propag. Lett. 16, 1614–1617 (2017).i 24. Ge, Y., Sun, Z., Chen, Z. & Chen, Y. Y. A high-gain wideband low-profile fabry-perot resonator antenna with a conical short horn. IEEE Antennas Wirel. Propag. Lett. 15, 1889–1892 (2016). 25. Wu, M. T. & Chuang, M. L. Multibroadband slotted bow-tie monopole antenna. IEEE Antennas Wirel. Propag. Lett. 14, 887–890 (2015). 6. Supriya, A. S. & Rajendran, J. A low cost tri-band microstrip patch antenna for GPS application. Prog. Electromagn. Res. Symp 60–65 (2017). https://doi.org/10.1109/PIERS-FALL.2017.8293111. p g 7. Ahmad, A., Arshad, F., Naqvi, S. I., Amin, Y. & Tenhunen, H. Design, fabrication, and measurements of extended l-shaped multi band antenna for wireless applications. Appl. Comput. Electromagn. Soc. J. 33, 388–393 (2018).ll 28. Kavitha, A. & Swaminathan, J. N. Design of flexible textile antenna using FR4, jeans cotton and teflon substrates. Microsyst. Technol. 25, 1311–1320 (2019).l 9. Ahmed, S., Tahir, F. A., Shamim, A. & Cheema, H. M. A compact kapton-based inkjet-printed multiband antenna for flexible wireless devices. IEEE Antennas Wirel. Propag. Lett. 14, 1802–1805 (2015).l g 30. Haque, E., Mahmuda, S. & Ahmed, F. A capsule-like flexible multiband antenna for WBAN applications. IEEE Reg. 10 Annu. Int. Conf. Proceedings/TENCON 1614–1619 (2017). https://doi.org/10.1109/TENCON.2017.8228116. 1. Liu, H. et al. Quad-band CPW-fed monopole antenna based on flexible pentangle-loop radiator. IEEE Antennas Wirel. Propag Lett. 14, 1373–1377 (2015). 2. Hosseini Varkiani, S. M. & Afsahi, M. Grounded CPW multi-band wearable antenna for MBAN and WLAN applications. Microw Opt. Technol. Lett. 60, 561–568 (2018).il p 33. Castro, A. T. & Sharma, S. K. Inkjet-printed wideband circularly polarized microstrip patch array antenna on a PET film flexible substrate material. IEEE Antennas Wirel. Propag. Lett. 17, 176–179 (2018). p g 4. Abutarboush, H. F., Farooqui, M. F. & Shamim, A. Inkjet-printed wideband antenna on resin-coated paper substrate for curved wireless devices. IEEE Antennas Wirel. Propag. Lett. 15, 20–23 (2016). 5. Yousaf, M. et al. www.nature.com/scientificreports/ Compacted conformal implantable antenna with multitasking capabilities for ingestible capsule endoscope. IEEE Access 8, 157617–157627 (2020). 6. Zada, M. & Yoo, H. A miniaturized triple-band implantable antenna system for bio-telemetry applications. IEEE Trans. Antennas Propag. 66, 7378–7382 (2018). 7. Zada, M., Shah, I. A., Basir, A. & Yoo, H. Ultra-compact implantable antenna with enhanced performance for leadless cardiac pacemaker system. IEEE Trans. Antennas Propag. 69, 1152–1157 (2021). 37. Zada, M., Shah, I. A., Basir, A. & Yoo, H. Ultra-compact implantable antenna with enh pacemaker system. IEEE Trans. Antennas Propag. 69, 1152–1157 (2021). 8. Huang, F. J. et al. Rectenna application of miniaturized implantable antenna design for triple-band biotelemetry communication IEEE Trans. Antennas Propag. 59, 2646–2653 (2011). IEEE Trans. Antennas Propag. 59, 2646–2653 (2011). p g 9. Das, R. & Yoo, H. Biotelemetry and wireless powering for leadless pacemaker systems. IEEE Microw. Wirel. Components Lett. 25 262–264 (2015). ( ) 0. Shah, I. A., Zada, M. & Yoo, H. Design and analysis of a compact-sized multi-band spiral-shaped implantable antenna for scalp implantable and leadless pacemaker systems. IEEE Trans. Antennas Propag. 67, 4230–4234 (2019).f 41. Liu, Y., Chen, Y., Lin, H. & Juwono, F. H. A novel differentially fed compact dual-band implantable antenna for biotelemetry applications. IEEE Antennas Wirel. Propag. Lett. 15, 1791–1794 (2016). 42. Gani, I. & Yoo, H. Multi-band antenna system for skin implant. IEEE Microw. Wirel. Components Lett. 26, 294–296 (2016) 43 Abb i M A B Sh hid S Ri M T M A & Abb S M O b d i d h d i h l d 43. Abbasi, M. A. B., Shahid, S., Rizwan, M., Tarar, M. A. & Abbas, S. M. On-board printed handset antenna with coupled mon 8th Eur. Conf. Antennas Propagation, EuCAP 2014 2889–2892 (2014). https://doi.org/10.1109/EuCAP.2014.6902430.f 44. Glorieux, S. et al. Effect of meat type, animal fat type, and cooking temperature on microstructural and macroscopic pro of cooked sausages. Food Bioprocess Technol. 12, 16–26 (2019).f g p ( ) 45. El-Zayat, S. R., Sibaii, H. & El-Shamy, K. A. Physiological effects of fat loss. Bull. Natl. Res. Cent. 43, 1–15 (2019). 46. Bozkurt, H. & Icier, F. Electrical conductivity changes of minced beef-fat blends during ohmic cooking. J. Food Eng. 96, 8 (2010). 47. Wang, L. V., Wu, H. Biomedical Optics: Principles and Imaging. (Wiley-Interscience, 2007).i 48. Hamdy, O. & Mohammed, H. S. References Kiourti, A. & Nikita, K. S. A review of implantable patch antennas for biomedical telemetry: Challenges and solutions. IEEE Antennas Propag. Mag. 54, 210–228 (2012). p g g 10. Liu, Y. et al. Some recent developments of microstrip antenna. Int. J. Antennas Propag. https://doi.org/10.1155/2012/428284 (2012). 11. He, Y., Ma, K., Yan, N. & Zhang, H. Dual-band monopole antenna using substrate-integrated suspended line technology for WLAN application. IEEE Antennas Wirel. Propag. Lett. 16, 2776–2779 (2017). 10. Liu, Y. et al. Some recent developments of microstrip antenna. Int. J. Antennas Propag. https://doi.org/10.1155/2012/428284 (2012). 11. He, Y., Ma, K., Yan, N. & Zhang, H. Dual-band monopole antenna using substrate-integrated suspended line technology for WLAN application. IEEE Antennas Wirel. Propag. Lett. 16, 2776–2779 (2017). Scientific Reports \| (2022) 12:15035 \| https://doi.org/10.1038/s41598-022-19430-y www.nature.com/scientificreports/ www.nature.com/scientificreports/ 12. Boukarkar, A., Lin, X. Q., Jiang, Y. & Yu, Y. Q. Miniaturized Single-Feed Multiband Patch Antennas. IEEE Trans. Antennas Propag. 65, 850–854 (2017). 3. Araghi, A. A compact planar ultra wideband antenna with triple-notched bands using capacitive coupled and parallel LC elements Appl. Comput. Electromagn. Soc. J. 31, 1416–1420 (2016). pp p g J , ( ) 14. Cao, Y. F., Cheung, S. W. & Yuk, T. I. A multiband slot antenna for GPS/WiMAX/WLAN systems. IEEE Trans. Antennas Propag. 63, 952–958 (2015). 5. Gad, N. H. & Vidmar, M. Design of a Microstrip-fed printed-slot antenna using defected ground structures for Multiband applica- tions. Appl. Comput. Electromagn. Soc. J. 33, 854–860 (2018). tions. Appl. Comput. Electromagn. Soc. J. 33, 854–860 (2018). 16. Majidi, N. et al. Design and comparison of 4 types of dual resonance proximity coupled microstrip patch antennas. Appl. Comput. Electromagn Soc J 33 1135–1139 (2018) pp p g 6. Majidi, N. et al. Design and comparison of 4 types of dual resonance proximity coupled microstrip patch antennas. Appl. Comput Electromagn. Soc. J. 33, 1135–1139 (2018). g ( ) 7. Hsu, C. K. & Chung, S. J. Compact multiband antenna for handsets with a conducting edge. IEEE Trans. Antennas Propag. 63 5102–5107 (2015). 8. Prabakaran, N. et al. A coplanar waveguide (CPW) fed circular microstripantenna for UWB applications. Int. J. Innov. Technol Explor. Eng. 8, 531–534 (2019). p g 9. Shanmuganantham, T., Balamanikandan, K. & Raghavan, S. CPW-fed slot antenna for wideband applications. Int. J. Antennas Propag. 1–4 (2008). https://doi.org/10.1155/2008/379247.l Propag. 1–4 (2008). https://doi.org/10.1155/2008/379247. p g ( ) p g 20. Kadry, M., Atrash, M. El & Abdalla, M. A. Design of an ultra-thin compact flexible dual-band antenna for wearable applications. in 2018 IEEE International Symposium on Antennas and Propagation & USNC/URSI National Radio Science Meeting 1949–1950 (2018). https://doi.org/10.1109/APUSNCURSINRSM.2018.8609247.l p g 21. Abdalgalil, O. F., Atrash, M. El & Abdalla, M. A. a flexible high gain wide-band antenna for wireless and wearable applications. in 2018 IEEE International Symposium on Antennas and Propagation & USNC/URSI National Radio Science Meeting 1279–1280 (2018). https://doi.org/10.1109/APUSNCURSINRSM.2018.8608830.i p g 22. Bean, D. & Venkataraman, J. Gain Enhancement of on-chip antenna at 60 GHz using an artificial magnetic conductor. in 2020 IEEE International Symposium on Antennas and Propagation and North American Radio Science Meeting 1423–1424 (2020).i y p p g 23. Pandit, S., Mohan, A. & Ray, P. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 3. Larin, K. V., Larina, I. V. & Esenaliev, R. O. Monitoring of tissue coagulation during thermotherapy using optoacoustic technique J. Phys. D. Appl. Phys. 38, 2645–2653 (2005).l 53. Larin, K. V., Larina, I. V. & Esenaliev, R. O. Monitoring of tissue coagulation during thermotherapy using optoacoustic technique. J. Phys. D. Appl. Phys. 38, 2645–2653 (2005).l y pp y 4. Hamdy, O., El-Azab, J., Al-Saeed, T. A., Hassan, M. F. & Solouma, N. H. A method for medical diagnosis based on optical fluence rate distribution at tissue surface. Materials (Basel). 10, 1–13 (2017). Acknowledgementsh g The authors would like to thank their colleague Dr. Mahmoud M. A. Ahmed for his diligent proofreading of this paper. g The authors would like to thank their colleague Dr. Mahmoud M. A. Ahmed for his diligent proofreading of this paper. Author contributions Study design and conceptualization: A.S.A.H. and O.H. Writing original draft: O.H. and A.S.A.H. Analyzing results: M.M., A.S.A.H., and O.H. All authors reviewed the final manuscript. Competing interests h Competing interests The authors declare no competing interests. p g The authors declare no competing interests. www.nature.com/scientificreports/ Investigating the transmission profiles of 808 nm laser through different regions of the rat ’ s head. Lasers Med. Sci. 36, 803–810 (2021).l 9. Hamdy, O., Fathy, M., Al-Saeed, T. A., El-Azab, J. & Solouma, N. H. Estimation of optical parameters and fluence rate distribution in biological tissues via a single integrating sphere optical setup. Optik (Stuttg). 140, 1004–1009 (2017). 50. Hamdy, O. & Solouma, N. H. Distant-detector versus integrating sphere measurements for estimating tissue optical parameters: A comparative experimental study. Optik (Stuttg). 247, 167981 (2021). 51. Hamdy, O. & Mohammed, H. S. Variations in tissue optical parameters with the incident power of an infrared laser. PLoS ONE 17, e0263164 (2022).li 2. Saeed, S. M., Balanis, C. A., Birtcher, C. R., Durgun, A. C. & Shaman, H. N. Wearable flexible reconfigurable antenna integrated with artificial magnetic conductor. IEEE Antennas Wirel. Propag. Lett. 16, 2396–2399 (2017). https://doi.org/10.1038/s41598-022-19430-y Scientific Reports \| (2022) 12:15035 \| Funding Funding Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in coopera- tion with The Egyptian Knowledge Bank (EKB). Additional information Correspondence and requests for materials should be addressed to O.H. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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https://openalex.org/W4380675504	https://ejde-ojs-txstate.tdl.org/ejde/article/download/506/441	English	null	Stability of initial-boundary value problem for quasilinear viscoelastic equations	Electronic journal of differential equations	2,020	cc-by	7,186	Electronic Journal of Diﬀerential Equations, Vol. 2020 (2020), No. 85, pp. 1–15. ISSN: 1072-6691. URL: http://ejde.math.txstate.edu or http://ejde.math.unt.edu Electronic Journal of Diﬀerential Equations, Vol. 2020 (2020), No. 85, pp. 1–15. ISSN: 1072-6691. URL: http://ejde.math.txstate.edu or http://ejde.math.unt.edu Electronic Journal of Diﬀerential Equations, Vol. 2020 (2020), No. 85, pp. 1–15. ISSN: 1072-6691. URL: http://ejde.math.txstate.edu or http://ejde.math.unt.edu c⃝2020 Texas State University. Submitted November 11, 2019. Published July 30, 2020. KUN-PENG JIN, JIN LIANG, TI-JUN XIAO Abstract. We investigate the stability of the initial-boundary value problem for the quasilinear viscoelastic equation \|ut\|ρutt −∆utt −∆u + Z t 0 g(t −s)∆u(s)ds = 0, in Ω× (0, +∞), u = 0, in ∂Ω× (0, +∞), u(·, 0) = u0(x), ut(·, 0) = u1(x), in Ω, where Ωis a bounded domain of Rn (n ≥1) with smooth boundary ∂Ω, ρ is a positive real number, and g(t) is the relaxation function. We present a general polynomial decay result under some weak conditions on g, which generalizes and improves the existing related results. Moreover, under the condition g′(t) ≤−ξ(t)gp(t), we obtain uniform exponential and polynomial decay rates for 1 ≤p < 2, while in the previous literature only the case 1 ≤p < 3/2 was studied. Finally, under a general condition g′(t) ≤−H(g(t)), we establish a ﬁne decay estimate, which is stronger than the previous results. 1. Introduction 2010 Mathematics Subject Classiﬁcation. 35Q74, 35B35, 74H55, 74H40, 93D15. Key words and phrases. Quasilinear viscoelastic equation; polynomial and exponential decay; relaxation function; uniform decay. c⃝2020 Texas State University. Submitted November 11, 2019. Published July 30, 2020. Key words and phrases. Quasilinear viscoelastic equation; polynomial and exponential decay; relaxation function; uniform decay. c⃝2020 Texas State University ⃝ y Submitted November 11, 2019. Published July 30, 2020 2010 Mathematics Subject Classiﬁcation. 35Q74, 35B35, 74H55, 74H40, 93D15. c⃝2020 Texas State University. 1. Introduction In this article, we consider the stability of the initial-boundary value problem for quasilinear viscoelastic equations, \|ut\|ρutt −∆utt −∆u + Z t 0 g(t −s)∆u(s)ds = 0, in Ω× (0, +∞), u = 0, in ∂Ω× (0, +∞), u(·, 0) = u0(x), ut(·, 0) = u1(x), in Ω, (1.1) (1.1) where Ωis a bounded domain of Rn(n ≥1) with smooth boundary ∂Ω, ρ is a positive real number, and g(t) the relaxation function. where Ωis a bounded domain of Rn(n ≥1) with smooth boundary ∂Ω, ρ is a positive real number, and g(t) the relaxation function. p , g( ) In [16], under the assumption that the bounded C1-function g : R+ →R+ satisﬁes 1 − Z +∞ 0 g(t)ds > 0, g′(t) ≤−ξgp(t), 1 ≤p < 3 2, (1.2) (1.2) where ξ > 0 is a constant, Messaoudi and Tatar obtained decay rates in [16, Theo- rem 3.1]. where ξ > 0 is a constant, Messaoudi and Tatar obtained decay rates in [16, Theo- rem 3.1]. 1 2 K P JIN J LIANG T J XIAO EJDE 2020/85 K. P. JIN, J. LIANG, T.-J. XIAO 2 EJDE-2020/85 EJDE-2020/85 More recently, Messaoudi and Al-Khulaiﬁ[13] improved this result [16, Theorems 3.1] by using the assumption that the non-increasing diﬀerentiable function g : R+ →R+ satisﬁes 1 − Z +∞ 0 g(t)ds > 0, g′(t) ≤−ξ(t)gp(t), 1 ≤p < 3 2, (1.3) (1.3) here ξ(t) : R+ →R+ is a non-increasing diﬀerentiable function with ξ(0) > 0. here ξ(t) : R+ →R+ is a non-increasing diﬀerentiable function with ξ(0) > 0. Messaoudi and Mustafa [14] also studied problem (1.1) and the corresponding decay results were obtained for the following condition on g(t), g′(t) ≤−H(g(t)), t ≥0, (1.4) (1.4) where H is a positive function and satisﬁes some conditions (see details in [14, hypotheses (A2) and (A3)]). where H is a positive function and satisﬁes some conditions (see details in [14, hypotheses (A2) and (A3)]). For more related information on the stability of problem (1.1) and some related equations or systems, we refer the reader to [1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 18, 19, 20, 21, 22, 23, 24] and references therein. In this article, we investigate the stability for problem (1.1) by using more general (weaker) assumptions on the relaxation functions g(t). 1. Introduction We establish ideal stability theorems with exact uniform polynomial decay rates t−1 for the solutions to this problem, under some basic conditions (see Theorem 3.2). Furthermore, in Theorems 3.4 and 3.6, our results hold for all 1 ≤p < 2, while in the previous literature only the case: 1 ≤p < 3 2 was studied. Therefore, all of our results, with much weaker conditions on the relaxation function g(t), are optimal so far. In the next section, we prove some estimates (lemmas) which will be used in Section 3. Finally, we will state and prove our main results in Section 3. 2. Basic estimates In this article we use the following assumptions: (A1) 0 < ρ, if n = 1, 2; and In this article we use the following assumptions: (A1) 0 < ρ, if n = 1, 2; and (A1) 0 < ρ, if n = 1, 2; and 0 < ρ ≤ 2 n −2, if n ≥3; (A2) g(t) : [0, +∞) →[0, +∞) is a non-increasing diﬀerentiable function with meas(J0) = 0, g(0) > 0, g′(t) ≤0, µ0 > 0, (A2) g(t) : [0, +∞) →[0, +∞) is a non-increasing diﬀerentiable function with meas(J0) = 0, g(0) > 0, g′(t) ≤0, µ0 > 0, (A2) g(t) : [0, +∞) →[0, +∞) is a non-increasing diﬀerentiable function with meas(J0) = 0, g(0) > 0, g′(t) ≤0, µ0 > 0, where J0 := {s ≥0; g(s) > 0, g′(s) = 0} = 0, µ0 := 1 − Z +∞ 0 g(t) dt. J0 := {s ≥0; g(s) > 0, g′(s) = 0} = 0, µ0 := 1 − Z +∞ 0 In the sequel, C, Ci > 0, i = 1, 2, . . . represent positive constants which are possibly diﬀerent in diﬀerent places. We denote In the sequel, C, Ci > 0, i = 1, 2, . . . represent positive constants which are possibly diﬀerent in diﬀerent places. We denote G(t) := Z +∞ t g(s)ds, for t ≥0; M(δ) := Z +∞ 0 g(s) Kδ(s)ds, Kδ(s) := −g′(s) g(s) + δ, where δ ∈(0, 1) is a constant. We deﬁne where δ ∈(0, 1) is a constant. We deﬁne I1(t) := Z Ω Z t 0 G(t −s)\|∇u(s)\|2 ds dx, STABILITY OF VISCOELASTIC EQUATIONS EJDE-2020/85 3 I2(t) := M(δ) δ Z Ω Z t 0 G(t −s)\|∇u(s)\|2 ds dx + E(t) . d dtI1(t) ≤−1 2 Z Ω Z t 0 g(t −s)\|∇u(t) −∇u(s)\|2 ds dx + 2G(0) Z Ω \|∇u(t)\|2dx, (2.1) and d dtI2(t) ≤−1 2M(δ) Z Ω Z t 0 Kδ(t −s)g(t −s)\|∇u(t) −∇u(s)\|2 ds dx + 2δM(δ)G(0) Z Ω \|∇u(t)\|2dx. (2.2) d dtI1(t) ≤−1 2 Z Ω Z t 0 g(t −s)\|∇u(t) −∇u(s)\|2 ds dx + 2G(0) Z Ω \|∇u(t)\|2dx, (2.1) and and d dtI2(t) ≤−1 2M(δ) Z Ω Z t 0 Kδ(t −s)g(t −s)\|∇u(t) −∇u(s)\|2 ds dx + 2δM(δ)G(0) Z Ω \|∇u(t)\|2dx. (2.2) (2.2) Moreover, δM(δ) →0, as δ →0. (2.3) δM(δ) →0, as δ →0. , δM(δ) →0, as δ →0. 2. Basic estimates (2.3) (2.3) Proof. Noting that Proof. Noting that −(a ± b)2 ≤−1 2a2 + b2, we see by a direct calculation that, for t ≥0, d dtI1(t) = − Z Ω Z t 0 g(t −s)\|∇u(s)\|2 ds dx + G(0) Z Ω \|∇u(t)\|2dx = − Z Ω Z t 0 g(t −s)\|∇u(t) −∇u(s) −∇u(t)\|2 ds dx + G(0) Z Ω \|∇u(t)\|2dx ≤−1 2 Z Ω Z t 0 g(t −s)\|∇u(t) −∇u(s)\|2 ds dx + Z Ω Z t 0 g(t −s)\|∇u(t)\|2 ds dx + G(0) Z Ω \|∇u(t)\|2dx ≤−1 2 Z Ω Z t 0 g(t −s)\|∇u(t) −∇u(s)\|2 ds dx + 2G(0) Z Ω \|∇ This means that (2.1) holds. This means that (2.1) holds. F th d ﬁiti f K ( ) (2 1) d (3 2) it f ll th t ( ) From the deﬁnition of Kδ(s), (2.1) and (3.2), it follows that ( ) From the deﬁnition of Kδ(s), (2.1) and (3.2), it follows that ( ) From the deﬁnition of Kδ(s), (2.1) and (3.2), it follows that From the deﬁnition of Kδ(s d dtI2(t) ≤−1 2M(δ) Z Ω Z t 0 (δg(t −s) + g′(t −s)) \|∇u(t) −∇u(s)\|2 ds dx + 2δM(δ)G(0) Z Ω \|∇u(t)\|2dx ≤−1 2M(δ) Z Ω Z t 0 Kδ(t −s)g(t −s)\|∇u(t) −∇u(s)\|2 ds dx + 2δM(δ)G(0) Z Ω \|∇u(t)\|2dx. According to [8, P. 1525, lines 8-10], we know that (2.3) is true. Thus, we completed the proof. □ According to [8, P. 1525, lines 8-10], we know that (2.3) is true. Thus, we completed the proof. □ K. P. JIN, J. LIANG, T.-J. XIAO EJDE-2020/85 K. P. JIN, J. LIANG, T.-J. XIAO K. P. JIN, J. LIANG, T.-J. XIAO EJDE-2020/85 4 EJDE-2020/85 We deﬁne F1(t) := 1 ρ + 1 Z Ω \|ut\|ρutu dx + Z Ω ∇u · ∇ut dx, Lemma 2.2. For t ≥0, Lemma 2.2. For t ≥0, d dtF1(t) ≤−µ0 2 Z Ω \|∇u\|2dx + 1 2µ0 Z Ω Z t 0 g(t −s)\|∇u(t) −∇u(s)\|ds 2 dx + Z Ω \|∇ut\|2dx + 1 ρ + 1 Z Ω \|ut\|ρ+2dx. (2.4) (2.4) Proof. Clearly, we can rewrite the ﬁrst equation in (1.1) as Proof. Clearly, we can rewrite the ﬁrst equation in (1.1) as \|ut\|ρutt −∆utt − 1 − Z t 0 g(s)ds ∆u − Z t 0 g(t −s) (∆u(t) −∆u(s)) ds = 0. 2. Basic estimates (2.5) It follows from (2.5) that It follows from (2.5) that tF1(t) = 1 − Z t 0 g(s)ds Z Ω u∆udx + Z Ω u(t) Z t 0 g(t −s) (∆u(t) −∆u(s)) ds + Z Ω \|∇ut\|2dx + 1 ρ + 1 Z Ω \|ut\|ρ+2dx = − 1 − Z t 0 g(s)ds Z Ω \|∇u\|2dx − Z Ω ∇u(t) · Z t 0 g(t −s) (∇u(t) −∇u(s)) ds dx + Z Ω \|∇ut\|2dx + 1 ρ + 1 Z Ω \|ut\|ρ+2dx ≤−µ0 Z Ω \|∇u\|2dx + µ0 2 Z Ω \|∇u\|2dx + 1 2µ0 Z Ω Z t 0 g(t −s)\|∇u(t) −∇u(s)\|ds 2 dx + Z Ω \|∇ut\|2dx + 1 ρ + 1 Z Ω \|ut\|ρ+2dx ≤−µ0 2 Z Ω \|∇u\|2dx + 1 2µ0 Z Ω Z t 0 g(t −s)\|∇u(t) −∇u(s)\|ds 2 dx + Z Ω \|∇ut\|2dx + 1 ρ + 1 Z Ω \|ut\|ρ+2dx. d dtF1(t) = 1 − Z t 0 g(s)ds Z Ω u∆udx + Z Ω u(t) Z t 0 g(t −s) (∆u(t) −∆u(s)) ds dx + Z Ω \|∇ut\|2dx + 1 ρ + 1 Z Ω \|ut\|ρ+2dx = − 1 − Z t 0 g(s)ds Z Ω \|∇u\|2dx − Z Ω ∇u(t) · Z t 0 g(t −s) (∇u(t) −∇u(s)) ds dx + Z Ω \|∇ut\|2dx + 1 ρ + 1 Z Ω \|ut\|ρ+2dx ≤−µ0 Z Ω \|∇u\|2dx + µ0 2 Z Ω \|∇u\|2dx 2 Z Ω Z Ω + 1 2µ0 Z Ω Z t 0 g(t −s)\|∇u(t) −∇u(s)\|ds 2 dx + Z Ω \|∇ut\|2dx + 1 ρ + 1 Z Ω \|ut\|ρ+2dx ≤−µ0 2 Z Ω \|∇u\|2dx + 1 2µ0 Z Ω Z t 0 g(t −s)\|∇u(t) −∇u(s)\|ds 2 dx + Z Ω \|∇ut\|2dx + 1 ρ + 1 Z Ω \|ut\|ρ+2dx. □ This completes the proof. his completes the proof. □ Now, we deﬁne Now, we deﬁne F2(t) := Z Ω ∆ut − 1 ρ + 1\|ut\|ρut Z t 0 g(t −s)(u(t) −u(s)) ds dx. STABILITY OF VISCOELASTIC EQUATIONS EJDE-2020/85 5 Lemma 2.3. There is a constant C1 > 0 such that, for t ≥t0, Lemma 2.3. 2. Basic estimates There is a constant C1 > 0 such that, for t ≥t0, dt ≤− G(0) 2(ρ + 1) Z Ω \|ut(t)\|ρ+2dx −G(0) 2 Z Ω \|∇ut(t)\|2dx + µ0G(0) 16 Z Ω \|∇u(t)\|2dx + C1 Z Ω Z t 0 g(t −s)\|∇u(t) −∇u(s)\|ds 2 dx −C1 Z Ω Z t 0 g′(t −s)\|∇u(t) −∇u(s)\|2 ds dx, (2.6) (2.6) where t0 a positive large number so that where t0 a positive large number so that Z t0 0 g(s)ds = 3G(0) 4 . Proof. By (2.5), we obtain Proof. By (2.5), we obtain d dtF2(t) = − 1 ρ + 1 Z t 0 g(s)ds Z Ω \|ut(t)\|ρ+2dx + Z t 0 g(s)ds Z Ω ut(t)∆ut(t)dx + Z Ω ut Z t 0 g′(t −s)(∆u(t) −∆u(s)) ds dx − 1 ρ + 1 Z Ω \|ut\|ρut Z t 0 g′(t −s)(u(t) −u(s)) ds dx − 1 − Z t 0 g(s)ds Z Ω ∆u(t) · Z t 0 g(t −s)(u(t) −u(s)) ds dx − Z Ω Z t 0 g(t −s)(∆u(t) −∆u(s))ds Z t 0 g(t −s)(u(t) −u(s)) ds dx = − 1 ρ + 1 Z t 0 g(s)ds Z Ω \|ut(t)\|ρ+2dx − Z t 0 g(s)ds Z Ω \|∇ut(t)\|2dx − Z Ω ∇ut · Z t 0 g′(t −s)(∇u(t) −∇u(s)) ds dx − 1 ρ + 1 Z Ω \|ut\|ρut Z t 0 g′(t −s)(u(t) −u(s)) ds dx + 1 − Z t 0 g(s)ds Z Ω ∇u(t) · Z t 0 g(t −s)(∇u(t) −∇u(s)) ds dx + Z Ω Z t 0 g(t −s)\|∇u(t) −∇u(s)\|ds 2 dx. (2.7) (2.7) − 1 ρ + 1 Z Ω \|ut\|ρut Z 0 g′(t −s)(u(t) −u(s)) ds dx + 1 − Z t 0 g(s)ds Z Ω ∇u(t) · Z t 0 g(t −s)(∇u(t) −∇u(s)) ds dx + Z Ω Z t 0 g(t −s)\|∇u(t) −∇u(s)\|ds 2 dx. Next, let us to estimate the third, fourth and ﬁfth terms on the right of (2.7). First we estimate the fourth term. By Young’s and Holder’s inequality, for any ζ1 > 0, we have − 1 ρ + 1 Z Ω \|ut\|ρut Z t 0 g′(t −s)(u(t) −u(s)) ds dx K. P. JIN, J. LIANG, T.-J. XIAO EJDE-2020/85 6 ≤ 1 ρ + 1ζ1 Z Ω \|ut\|2ρ+2dx − g(0) 4ζ1(ρ + 1) Z Ω Z t 0 g′(t −s)\|u(t) −u(s)\|2 ds dx. 2. Basic estimates By (A1), (A2) and the Sobolev embedding inequality, we obtain By (A1), (A2) and the Sobolev embedding inequality, we obtain Z Ω \|ut\|2ρ+2dx ≤Cs(2E(0))ρ Z Ω \|∇ut\|2dx . By Poincar´e’s inequality, we have By Poincar´e’s inequality, we have − Z Ω Z t 0 g′(t −s)\|u(t) −u(s)\|2 ds dx ≤−Cp Z Ω Z t 0 g′(t −s)\|∇u(t) −∇u(s)\|2 ds dx, where Cp is the Poincar´e’s constant and Cs the Sobolev embedding constant. There- fore, where Cp is the Poincar´e’s constant and Cs the Sobolev embedding constant. There- fore, − 1 ρ + 1 Z Ω \|ut\|ρut Z t 0 g′(t −s)(u(t) −u(s)) ds dx ≤ Cs ρ + 1(2E(0))ρζ1 Z Ω \|∇ut\|2dx − g(0)Cp 4ζ1(ρ + 1) Z Ω Z t 0 g′(t −s)\|∇u(t) −∇u(s)\|2 ds dx. (2.8) (2.8) Now, we estimate the third and ﬁfth terms. It is not hard to see that, for any ζ2, ζ3 > 0, − Z Ω ∇ut · Z t 0 g′(t −s)(∇u(t) −∇u(s)) ds dx ≤ζ2 Z Ω \|∇ut\|2dx −g(0) 4ζ2 Z Ω Z t 0 g′(t −s)\|∇u(t) −∇u(s)\|2 ds dx, (2.9) (2.9) and 1 − Z t 0 g(s)ds Z Ω ∇u(t) · Z t 0 g(t −s)(∇u(t) −∇u(s)) ds dx ≤ζ3 Z Ω \|∇u(t)\|2dx + 1 4ζ3 Z Ω Z t 0 g(t −s)\|∇u(t) −∇u(s)\|ds 2 dx. (2.10) (2.10) Thus, combining (2.8), (2.9), (2.10) with (2.7), we know that Thus, combining (2.8), (2.9), (2.10) with (2.7), we know that d dtF2(t) d dtF2(t) ≤− 1 ρ + 1 Z t 0 g(s)ds Z Ω \|ut(t)\|ρ+2dx − Z t 0 g(s)ds −ζ2 − Cs ρ + 1(2E(0))ρζ1 Z Ω \|∇ut(t)\|2dx − g(0) 4ζ2 + g(0)Cp 4ζ1(ρ + 1) Z Ω Z t 0 g′(t −s)\|∇u(t) −∇u(s)\|2 ds dx + ζ3 Z Ω \|∇u(t)\|2dx + 1 + 1 4ζ3 Z Ω Z t 0 g(t −s)\|∇u(t) −∇u(s)\|ds 2 dx. Setting ζ1 = (ρ + 1)G(0) 8Cs(2E(0))ρ , ζ2 = G(0) 8 , ζ3 = µ0G(0) 16 , ζ1 = (ρ + 1)G(0) 8Cs(2E(0))ρ , □ we obtain the estimate (2.6). This completes the proof. we obtain the estimate (2.6). This completes the proof. we obtain the estimate (2.6). This completes the proof. □ EJDE-2020/85 EJDE-2020/85 STABILITY OF VISCOELASTIC EQUATIONS 7 3. Main results and their proofs We ﬁrstly state an existence and uniqueness result for problem (1.1), which can be proved by using similar arguments as in [4, 15] so we omit it here. Theorem 3.1. Let (A1) and (A2) hold. Then for any u0 ∈H1 0(Ω), u1 ∈H1 0(Ω), the problem (1.1) has a unique global solution on [0, ∞) with the regularity u ∈C1 R+; H1 0(Ω) . We introduce the energy functional E(t) := 1 ρ + 2 Z Ω \|ut\|ρ+2dx + 1 2 Z Ω \|∇ut\|2dx + 1 2 1 − Z t 0 g(s)ds Z Ω \|∇u\|2dx + 1 2 Z Ω Z t 0 g(t −s)\|∇u(t) −∇u(s)\|2 ds dx. (3.1) E(t) := 1 ρ + 2 Z Ω \|ut\|ρ+2dx + 1 2 Z Ω \|∇ut\|2dx + 1 2 1 − Z t 0 g(s)ds Z Ω \|∇u\|2dx t (3.1 (3.1) + 1 2 Z Ω Z t 0 g(t −s)\|∇u(t) −∇u(s)\|2 ds dx. ( ) + 1 2 Z Ω Z t 0 g(t −s)\|∇u(t) −∇u(s)\|2 ds dx. ( ) Then, for t ≥0, Then, for t ≥0, d dtE(t) = −1 2g(t) Z Ω \|∇u\|2dx + 1 2 Z Ω Z t 0 g′(t −s)\|∇u(t) −∇u(s)\|2 ds dx ≤1 2 Z Ω Z t 0 g′(t −s)\|∇u(t) −∇u(s)\|2 ds dx, (3.2) nd E(t) ∼ Z Ω \|ut\|ρ+2 + \|∇ut\|2 + \|∇u\|2 dx + Z Ω Z t 0 g(t −s)\|∇u(t) −∇u(s)\|2 ds dx. (3.3) h f ll i i l if d th f th l ti f b d dtE(t) = −1 2g(t) Z Ω \|∇u\|2dx + 1 2 Z Ω Z t 0 g′(t −s)\|∇u(t) −∇u(s)\|2 ds dx 1 Z Z t (3.2) d dtE(t) = −1 2g(t) Z Ω \|∇u\|2dx + 1 2 Z Ω Z t 0 g′(t −s)\|∇u(t) −∇u(s)\|2 ds dx ≤1 2 Z Ω Z t 0 g′(t −s)\|∇u(t) −∇u(s)\|2 ds dx, (3.2) nd (3.2) and and E(t) ∼ Z Ω \|ut\|ρ+2 + \|∇ut\|2 + \|∇u\|2 dx + Z Ω Z t 0 g(t −s)\|∇u(t) −∇u(s)\|2 ds dx. (3.3) (3.3) Z Ω Z 0 The following is our general uniform decay theorem for the solution energy of prob- lem (1.1). Theorem 3.2. Let (A1) and (A2) hold. 3. Main results and their proofs Then, for u0, u1 ∈H1 0(Ω), the solution energy E(t) of the problem (1.1) satisﬁes Z +∞ 0 E(t) ≤CE(0), t ≥0, E(t) ≤CE(0)(t + 1)−1, t ≥0, where C > 0 is a constant. where C > 0 is a constant. Proof. The proof is mainly based on the construction of an auxiliary function L(t) satisfying L(t0) ≤CE(0), L(t) ≥0, t ≥0, and and d dtL(t) ≤−ϵ0E(t), t ≥t0. (3.4) d dtL(t) ≤−ϵ0E(t), t ≥t0. (3.4) Clearly, integrating (3.4) we obtain the desired estimate. Now, we apply the lemmas obtained in the previous section to construct this auxiliary function L(t). We deﬁne J(t) := NE(t) + F1(t) + 4 G(0)F2(t). By the deﬁnitions of F1(t) and F2(t) and a simple calculation, we see that, there is a constant c0 > 0 such that, for t ≥0, \|F1(t)\|, \|F1(t)\| ≤c0E(t). \|F1(t)\|, \|F1(t)\| ≤c0E(t). K. P. JIN, J. LIANG, T.-J. XIAO EJDE-2020/85 8 Taking N > 8C1/G(0) large enough, we obtain c1E(t) ≤J(t) ≤c2E(t), t ≥0, Taking N > 8C1/G(0) large enough, we obtain Taking N > 8C1/G(0) large enough, we obtain c1E(t) ≤J(t) ≤c2E(t), t ≥0, where c1, c2 > 0 are constants. , Thus, by (2.4), (2.6) and (3.2), for t ≥t0, we have d dtJ(t) ≤−µ0 4 Z Ω \|∇u(t)\|2dx − 1 ρ + 1 Z Ω \|ut(t)\|ρ+2dx − Z Ω \|∇ut\|2dx + 4C1 G(0) + 1 2µ0 Z Ω Z t 0 g(t −s)\|∇u(t) −∇u(s)\|ds 2 dx. (3.5) (3.5) Moreover, Z Ω Z t 0 g(t −s)\|∇u(t) −∇u(s)\|ds 2 dx ≤ Z Ω Z t 0 g(s) Kδ(s)ds Z t 0 Kδ(t −s)g(t −s)\|∇u(t) −∇u(s)\|2 ds dx ≤M(δ) Z Ω Z t 0 Kδ(t −s)g(t −s)\|∇u(t) −∇u(s)\|2 ds dx. Hence, by (3.5), for t ≥t0, we see that Hence, by (3.5), for t ≥t0, we see that d dtJ(t) ≤−µ0 4 Z Ω \|∇u(t)\|2dx − 1 ρ + 1 Z Ω \|ut(t)\|ρ+2dx − Z Ω \|∇ut\|2dx + 4C1 G(0) + 1 2µ0 M(δ) Z Ω Z t 0 Kδ(t −s)g(t −s)\|∇u(t) −∇u(s)\|2 ds dx. (3.6) dt ≤−µ0 4 Z Ω \|∇u(t)\|2dx − 1 ρ + 1 Z Ω \|ut(t)\|ρ+2dx − Z Ω \|∇ut\|2dx + 4C1 G(0) + 1 2µ0 M(δ) Z Ω Z t 0 Kδ(t −s)g(t −s)\|∇u(t) −∇u(s)\|2 ds dx. 3. Main results and their proofs (3.6) (3.6) Now we deﬁne Now we deﬁne L(t) := J(t) + µ0 32G(0)I1(t) + 2 4C1 G(0) + 1 2µ0 I2(t). Then, by (2.1), (2.2) and (3.6), for t ≥t0, we obtain d dtL(t) ≤− 3µ0 16 −4G(0) 4C1 G(0) + 1 2µ0 δM(δ) Z Ω \|∇u(t)\|2dx − 1 ρ + 1 Z Ω \|ut(t)\|ρ+2dx − Z Ω \|∇ut\|2dx − µ0 64G(0) Z Ω Z t 0 g(t −s)\|∇u(t) −∇u(s)\|2 ds dx. (3.7) (3.7) − µ0 64G(0) Z Ω Z t 0 g(t −s)\|∇u(t) −∇u(s)\|2 ds dx. Convergence (2.3) shows that there exists δ0 > 0 such that, for any 0 < δ < δ0, Convergence (2.3) shows that there exists δ0 > 0 such that, for any 0 < δ < δ0, δM(δ) ≤ µ0 64G(0) 4C1 G(0) + 1 2µ0 . Thus, by (3.7) and (3.3), we deduce that, for 0 < δ < δ0, there exists a constant ϵ0 > 0 such that, for t ≥t0, d dtL(t) ≤−ϵ0E(t). (3.8) (3.8) STABILITY OF VISCOELASTIC EQUATIONS EJDE-2020/85 9 Since L(t) ≥0 for t ≥0, and L(t0) ≤CE(0), it follows by integrating (3.8) over [t0, τ) that for any τ > t0, Since L(t) ≥0 for t ≥0, and L(t0) ≤CE(0), it follows by integrating (3.8) over [t0, τ) that for any τ > t0, Z τ t0 E(t)dt ≤CE(0). Z τ t0 E(t)dt ≤CE(0). So, Z +∞ 0 E(t)dt ≤CE(0). (3.9) (3.9) Noting that E′(t) ≤0, by(3.9), we obtain E(t) ≤CE(0)(t + 1)−1, t ≥0. This completes the proof. □ □ This completes the proof. Remark 3.3. (1) As showed in Theorem 3.2, the polynomial decay rates can be obtained without the control conditions on g′(t) used previously. There are many functions g(t) satisfying the assumptions (A2) without satisfying the previous restriction that g(t) controls g′(t) as in (1.2), (1.3) and (1.4). For example, if g(t) = √ 2 + sin t e−t, t ≥0, then g′(t) = − √ 2 −cos t + sin t e−t = − √ 2 1 −cos(t + π 4 ) e−t, t ≥0. Clearly, g′(t) ≤0, for t ≥0; g′(t) = 0, for t = 2kπ −π 4 , k = 1, 2, . . . . Hence, g(t) satisﬁes (A2), while g(t) does not satisfy (1.2), (1.3) or (1.4). That is, g′(t) is not controlled by g(t). 3. Main results and their proofs Hence, g(t) satisﬁes (A2), while g(t) does not satisfy (1.2), (1.3) or (1.4). That is, g′(t) is not controlled by g(t). Functions g(t) as above have not been studied in the literature. However, we can treat the problem (1.1) with these general relaxation functions, and according to Theorem 3.2 here, we know the energy E(t) of problem (1.1) decays at least at the rate (t + 1)−1. ( ) (2) The decay rates given in Theorem 3.2 are optimal in a sense according to [13, Example 3.1, Remark 3.2] and [8, Remark 3.3(ii)]. When the derivative g′(s) is controlled by the relaxation function g(t), we can prove the following results. Theorem 3.4. Let (A1) and (A2) hold, and g′(t) ≤−ξ(t)gp(t), t ≥0, (3.10) (3.10) g′(t) ≤−ξ(t)gp(t), t ≥0, (3.10) where ξ(t) : R+ →R+ is a non-increasing diﬀerentiable function with ξ(0) > 0 and 1 ≤p < 2 is a constant. Then there are constants C, η > 0 such that for t ≥0, where ξ(t) : R+ →R+ is a non-increasing diﬀerentiable function with ξ(0) > 0 and 1 ≤p < 2 is a constant. Then there are constants C, η > 0 such that for t ≥0, E(t) ≤    CE(0)e−η R t 0 ξ(s)ds, p = 1, CE(0) 1 1+ R t 0 ξ(s)ds 1 p−1 1 < p < 2. (3.11) (3.11) / K. P. JIN, J. LIANG, T.-J. XIAO 10 EJDE-2020/85 EJDE-2020/85 Proof. A key idea in the proof is to construct a Lyapunov function satisfying R(t) ∼ E(t) and Proof. A key idea in the proof is to construct a Lyapunov function satisfying R(t) ∼ E(t) and d dtR(t) ≤−ϵ2ξ(t)Rp(t). dt To ﬁnd this function, we will use the results of Theorem 3.2 and J(t) deﬁned above. Clearly, Z Ω Z t 0 g(t −s)\|∇u(t) −∇u(s)\|ds 2 dx ≤G(0) Z Ω Z t 0 g(t −s)\|∇u(t) −∇u(s)\|2 ds dx. Thus, by (3.5) and (3.3), for t ≥t0, we have d dtJ(t) ≤−ϵ1E(t) + C2 Z Ω Z t 0 g(t −s)\|∇u(t) −∇u(s)\|2 ds dx, (3.12) 0 i t t (3.12) where ϵ1 > 0 is a constant. where ϵ1 > 0 is a constant. On the other hand, by Theorem 3.2, we know that Z +∞ 0 E(t)dt ≤CE(0), and E(t) ≤CE(0)(t + 1)−1. ∞ E(t)dt ≤CE(0), and E(t) ≤CE(0)(t + 1)−1. 3. Main results and their proofs Since Since Z Ω Z t 0 g(t −s)\|∇u(t) −∇u(s)\|2 ds dx ≤ Z t 0 Z Ω \|∇u(t) −∇u(s)\|2dxds 1−1 p Z Ω Z t 0 gp(t −s)\|∇u(t) −∇u(s)\|2 ds dx 1/p ≤C Z t 0 (E(t) + E(s))ds 1−1 p Z Ω Z t 0 gp(t −s)\|∇u(t) −∇u(s)\|2 ds dx 1/p ≤CE1−1 p (0) Z Ω Z t 0 gp(t −s)\|∇u(t) −∇u(s)\|2 ds dx 1/p , Z t 0 g(t −s)\|∇u(t) −∇u(s)\|2 ds dx by (3.12) it follows that for t ≥t0, by (3.12) it follows that for t ≥t0, d dtJ(t) ≤−ϵ1E(t)+C3E1−1 p (0) Z Ω Z t 0 gp(t−s)\|∇u(t)−∇u(s)\|2 ds dx 1/p . (3.13) Multiplying (3.13) by ξ(t)Ep−1(t), for t ≥t0, we obtain Multiplying (3.13) by ξ(t)Ep−1(t), for t ≥t0, we obtain ξ(t)Ep−1(t) d dtJ(t) ≤−ϵ1ξ(t)Ep(t) ξ(t)Ep−1(t) d dtJ(t) ≤−ϵ1ξ(t)Ep(t) + C3E1−1 p (0)ξ(t)Ep−1(t) Z Ω Z t 0 gp(t −s)\|∇u(t) −∇u(s)\|2 ds dx 1/p ≤−ϵ1 2 ξ(t)Ep(t) + C4ξ(t) Z Ω Z t 0 gp(t −s)\|∇u(t) −∇u(s)\|2 ds dx. (3.14) + C3E1−1 p (0)ξ(t)Ep−1(t) Z Ω Z t 0 gp(t −s)\|∇u(t) −∇u(s)\|2 ds dx 1/p ≤−ϵ1 2 ξ(t)Ep(t) + C4ξ(t) Z Ω Z t 0 gp(t −s)\|∇u(t) −∇u(s)\|2 ds dx. (3.14) Since ξ(t), E(t) are non-increasing functions, from (3.10) it follows that for t ≥0, d ξ(t)Ep−1(t)J(t) = ξ(t)Ep−1(t) d J(t) + J(t) d ξ(t)Ep−1(t) Since ξ(t), E(t) are non-increasing functions, from (3.10) it follows that for t ≥0, d d d Since ξ(t), E(t) are non-increasing functions, from (3.10) it follows that for t ≥0, d dt ξ(t)Ep−1(t)J(t) = ξ(t)Ep−1(t) d dtJ(t) + J(t) d dt ξ(t)Ep−1(t) ≤ξ(t)Ep−1(t) d dtJ(t), Since ξ(t), E(t) are non-increasing functions, from (3.10) it follows that for t ≥0, d dt ξ(t)Ep−1(t)J(t) = ξ(t)Ep−1(t) d dtJ(t) + J(t) d dt ξ(t)Ep−1(t) ≤ξ(t)Ep−1(t) d dtJ(t), ≤ξ(t)Ep−1(t) d dtJ(t), STABILITY OF VISCOELASTIC EQUATIONS EJDE-2020/85 STABILITY OF VISCOELASTIC EQUATIONS 11 EJDE-2020/85 and and ξ(t) Z Ω Z t 0 gp(t −s)\|∇u(t) −∇u(s)\|2 ds dx ≤ Z Ω Z t 0 ξ(t −s)gp(t −s)\|∇u(t) −∇u(s)\|2 ds dx ≤− Z Ω Z t 0 g′(t −s)\|∇u(t) −∇u(s)\|2 ds dx ≤−2 d dtE(t). Hence, by (3.14), for t ≥t0, we have d dt ξ(t)Ep−1(t)J(t) + 2C4E(t) ≤−ϵ1 2 ξ(t)Ep(t). (3.15) Hence, by (3.14), for t ≥t0, we have d dt ξ(t)Ep−1(t)J(t) + 2C4E(t) ≤−ϵ1 2 ξ(t)Ep(t). (2) The decay rates given in Theorem 3.4 are optimal in according to [13, Ex- ample 3.1, Remark 3.2] and [8, Remark 3.3(ii)]. 3. Main results and their proofs (3.15) (3.15) Now, we deﬁne ow, we de e R(t) := ξ(t)Ep−1(t)J(t) + 2C4E(t). Then, R(t) ∼E(t). By (3.15), for t ≥t0, we obtain , R(t) := ξ(t)Ep−1(t)J(t) + 2C4E(t). Then, R(t) ∼E(t). By (3.15), for t ≥t0, we obtain d dtR(t) ≤−ϵ2ξ(t)Rp(t), □ where ϵ2 > 0 is a constant. This completes the proof. □ where ϵ2 > 0 is a constant. This completes the proof. where ϵ2 > 0 is a constant. This completes the proof. □ Remark 3.5. (1) Theorem 3.4 extends the results in [13, 14, 16], where g′(t) was assumed to satisfy (3.10) with p ∈[1, 3/2), since Theorem 3.4 holds for all p ∈[1, 2). Moreover, the decay rates obtained in [13] are Remark 3.5. (1) Theorem 3.4 extends the results in [13, 14, 16], where g′(t) was assumed to satisfy (3.10) with p ∈[1, 3/2), since Theorem 3.4 holds for all p ∈[1, 2). Moreover, the decay rates obtained in [13] are E(t) ≤Ke−λ R t t0 ξ(s)ds, p = 1, E(t) ≤K 1 1 + R t t0 ξ2p−1(s)ds 1 2p−2 , 1 < p < 3 2. In addition, if Z +∞ 0 1 tξ2p−1(t) + 1 dt < +∞, 1 < p < 3 2, (3.16) (3.16) reference [13] shows the improved estimate E(t) ≤K 1 1 + R t t0 ξp(s)ds 1 p−1 , 1 < p < 3 2. Since ξ(t) is nonnegative and non-increasing, it is clear that ξp(s) ≲ξ(s), and then 1 1 + R t 0 ξ(s)ds 1 p−1 ≲ 1 1 + R t t0 ξp(s)ds 1 p−1 . Therefore, the decay rates given in Theorem 3.4 is stronger than the previous conclusion in the [13, Theorem 3.1] for all p ∈[1, 2). On the other hand, we obtain the stronger estimate without the other restrictions on ξ(t) (as (3.16) in [13, Theorem 3.1]). As can be seen, Theorem 3.4 here give stronger conclusions essentially under weaker conditions on g(t). (2) The decay rates given in Theorem 3.4 are optimal in according to [13, Ex- ample 3.1, Remark 3.2] and [8, Remark 3.3(ii)]. K. P. JIN, J. LIANG, T.-J. XIAO 12 Theorem 3.6. Let the assumptions of Theorem 3.2 hold, and g′(t) ≤−H(g(t)), t ≥0, (3.17) Theorem 3.6. 3. Main results and their proofs Let the assumptions of Theorem 3.2 hold, and g′(t) ≤−H(g(t)), t ≥0, (3.17) (3.17) where H ∈C1 (R+) is a positive function with H(0) = 0, and it is also a linear or strictly increasing and strictly convex C2 function on (0, r], for some r < 1. Then there are constants k1, k2, k3, ε0 > 0 such that where H ∈C1 (R+) is a positive function with H(0) = 0, and it is also a linear or strictly increasing and strictly convex C2 function on (0, r], for some r < 1. Then there are constants k1, k2, k3, ε0 > 0 such that E(t) ≤k3G−1(k1t + k2), t ≥0, (3.18) E(t) ≤k3G−1(k1t + k2), t ≥0, (3.18) where G(t) = Z 1 t 1 sH′(ε0s)ds. E(t) ≤k3G−1(k1t + k2), t ≥0, (3.18) h (3.18) where Proof. By Theorem 3.2, we obtain Proof. By Theorem 3.2, we obtain Z +∞ 0 E(t)dt ≤CE(0) and E(t) ≤CE(0)(t + 1)−1. So, Z Ω Z t 0 \|∇u(t) −∇u(s)\|2 ds dx ≤CE(0) < +∞. (3.19) (3.19) According to (3.17) and (3.19), we can and do take t1 > t0 large enough such that for any t ≥t1, According to (3.17) and (3.19), we can and do take t1 > t0 large enough such that for any t ≥t1, Z Ω Z t t1 \|∇u(t) −∇u(s)\|2 ds dx < min{r, H(r)}, (3.20) − Z Ω Z t−t1 0 g′(t −s)\|∇u(t) −∇u(s)\|2 ds dx < min{r, H(r)}, (3.21) Z Ω Z t−t1 0 g(t −s)\|∇u(t) −∇u(s)\|2 ds dx < min{r, H(r)}, (3.22) max{g(t), −g′(t)} < min{r, H(r)}. (3.23) Z Ω Z t t1 \|∇u(t) −∇u(s)\|2 ds dx < min{r, H(r)}, (3.20) (3.20) (3.23) Using (3.17), (3.20)-(3.23) and Jensen’s inequality, for t ≥t1, we obtain Using (3.17), (3.20)-(3.23) and Jensen’s inequality, for t ≥t1, we obtain − Z Ω Z t−t1 0 g′(t −s)\|∇u(t) −∇u(s)\|2 ds dx ≥ Z Ω Z t−t1 0 H(g(t −s))\|∇u(t) −∇u(s)\|2 ds dx ≥H Z Ω Z t−t1 0 g(t −s)\|∇u(t) −∇u(s)\|2 ds dx . (3.24) (3.24) Then for t ≥t1, Z Ω Z t−t1 0 g(t −s)\|∇u(t) −∇u(s)\|2 ds dx ≤H−1 − Z Ω Z t−t1 0 g′(t −s)\|∇u(t) −∇u(s)\|2 ds dx . (3.25) (3.25) Moreover, by [14, P. 3. Main results and their proofs 1860, equation (3.24)], for t ≥t1, we obtain d dtW1(t) ≤−ϵ3E(t) + C5 Z Ω Z t−t1 0 g(t −s)\|∇u(t) −∇u(s)\|2 ds dx, (3.26) (3.26) where W1(t) ∼E(t) and ϵ3 > 0 is a constant. where W1(t) ∼E(t) and ϵ3 > 0 is a constant. where W1(t) ∼E(t) and ϵ3 > 0 is a constant. STABILITY OF VISCOELASTIC EQUATIONS EJDE-2020/85 13 By (3.25) and (3.26), for t ≥t1, we have d dtW1(t) ≤−ϵ3E(t) + C5H−1 − Z Ω Z t−t1 0 g′(t −s)\|∇u(t) −∇u(s)\|2 ds dx . (3.27) By (3.25) and (3.26), for t ≥t1, we have By (3.25) and (3.26), for t ≥t1, we have d dtW1(t) (3.27) dt ≤−ϵ3E(t) + C5H−1 − Z Ω Z t−t1 0 g′(t −s)\|∇u(t) −∇u(s)\|2 ds dx . (3.27) Now, we deﬁne W2(t) := H′ ε0 E(t) E(0) W1(t) + ME(t), ( ) where 0 < ε0 < r, M > 0 are constants, which will be speciﬁc later. where 0 < ε0 < r, M > 0 are constants, which will be speciﬁc later. Clearly W2(t) ∼E(t) because of the assumption on H Therefore where 0 < ε0 < r, M > 0 are constants, which will be speciﬁc later. Clearly, W2(t) ∼E(t) because of the assumption on H. Therefore, for t ≥t1, 0 , , p Clearly, W2(t) ∼E(t) because of the assumption on H. Therefore, for t ≥t1, d dtW2(t) d dtW2(t) = H′ ε0 E(t) E(0) d dtW1(t) + ε0 E′(t) E(0) H′′ ε0 E(t) E(0) W1(t) + ME′(t) ≤C5H′ ε0 E(t) E(0) H−1 − Z Ω Z t−t1 0 g′(t −s)\|∇u(t) −∇u(s)\|2 ds dx −ϵ3E(t)H′ ε0 E(t) E(0) + ME′(t), (3.28) (3.28) where we have used E′(t) ≤0, H′′ ≥0, and (3.27). where we have used E′(t) ≤0, H′′ ≥0, and (3.27). where we have used E′(t) ≤0, H′′ ≥0, and (3.27). ( ) , , ( ) Next, we estimate the ﬁrst term on the right of (3.28). Let H⋆be the convex conjugate of H in the sense of Young (see [2, P. 61-64] and [14, P. 1863]). Then H⋆(s) = s(H′)−1(s) −H[(H′)−1(s)], s ∈(0, H′(r)), (3.29) and it satisﬁes ab ≤H⋆(a) + H(b), for a ∈(0, H′(r)], b ∈(0, r]. 3. Main results and their proofs (3.30) (3.30) Setting a = H′ ε0 E(t) E(0) , b = H−1 − Z Ω Z t−t1 0 g′(t −s)\|∇u(t) −∇u(s)\|2 ds dx , and using (3.29), (3.30) and (3.21), we obtain and using (3.29), (3.30) and (3.21), we obtain H′ ε0 E(t) E(0) H−1 − Z Ω Z t−t1 0 g′(t −s)\|∇u(t) −∇u(s)\|2 ds dx ≤H⋆ H′ ε0 E(t) E(0) − Z Ω Z t−t1 0 g′(t −s)\|∇u(t) −∇u(s)\|2 ds dx ≤ε0 E(t) E(0)H′ ε0 E(t) E(0) −2E′(t). (3.31) (3.31) From (3.28) and (3.31), it follows that for t ≥t1, d dtW2(t) ≤−(ϵ3E(0) −C5ε0) E(t) E(0)H′ ε0 E(t) E(0) + (M −2C5)E′(t). (3.32) (3.32) Therefore, if we take M > 0 large enough and ε0 > 0 small suﬃciently, then we obtain, for t ≥t1, Therefore, if we take M > 0 large enough and ε0 > 0 small suﬃciently, then we obtain, for t ≥t1, d dtW2(t) ≤−ϵ4 eH E(t) E(0) , (3.33) (3.33) K. P. JIN, J. LIANG, T.-J. XIAO EJDE-2020/85 14 where ϵ4 > 0 is a constant and eH(t) = tH′(ε0t). We deﬁne W(t) := γ W2(t) E(0) , where γ > 0 small enough such that where γ > 0 small enough such that where γ > 0 small enough such that where γ > 0 small enough such that W(t) < E(t) E(0). W(t) < E(t) E(0). W(t) < E(t) E(0). Clearly, W(t) ∼E(t) ∼W2(t), and eH(t), eH′(t) ≥0. So, by (3.33), we know that there exists ϵ5 > 0 such that for t ≥t1 Clearly, W(t) ∼E(t) ∼W2(t), and eH(t), eH′(t) ≥0. So, by (3.33), we know that there exists ϵ5 > 0 such that for t ≥t1 d dtW(t) ≤−ϵ5 eH (W(t)) . (3.34) (3.34) □ This gives the estimate (3.18). Thus the proof is complete. □ This gives the estimate (3.18). Thus the proof is complete. □ Remark 3.7. In [14, Theorem 3.1], if the relaxation function g(t) satisﬁes (3.17), then the decay rate is E(t) ≤k3H−1 1 (k1t + k2), t ≥0. E(t) ≤k3H−1 1 (k1t + k2), t ≥0. Detailed information about H1 can be found in [14, Theorem 3.1]. In addition, if Z 1 0 H1(t)dt < +∞, (3.35) (3.35) hen the improved estimate (3.18) iss obtained then the improved estimate (3.18) iss obtained. then the improved estimate (3.18) iss obtained. 3. Main results and their proofs As showed in Theorem 3.6, the improved estimate (3.18) is directly obtained without the extra assumption condition (3.35) (except (3.17)). Therefore, Theorem 3.6 improves [14, Theorem 3.1] essentially, with weaker conditions on the relaxation function. Moreover, Theorem 3.6 gives stronger conclusions. Acknowledgments. The work was supported by the NSF of China (11771091, 11971306, 11831011), by the China Postdoctoral Science Foundation (2018M632094), and by the Shanghai Key Laboratory for Contemporary Applied Mathematics (08DZ2271900). Acknowledgments. The work was supported by the NSF of China (11771091, 11971306, 11831011), by the China Postdoctoral Science Foundation (2018M632094), and by the Shanghai Key Laboratory for Contemporary Applied Mathematics (08DZ2271900). [8] K. P. Jin, J. Liang, T. J. Xiao; Coupled second order evolution equations with fading memory: Optimal energy decay rate, J. Diﬀerential Equations, 257(4) (2014), 1501–1528. References [1] R. O. Araujo, T. F. Ma, Y. Qin; Long-time behavior of a quasilinear viscoelastic equation with past history, J. Diﬀerential Equations, 254(10) (2013), 4066–4087. [ ] with past history, J. Diﬀerential Equations, 254(10) (2013), 4066–4087. [2] V I Arnold; Mathematical Methods of Classical Mechanics Springer-Verlag New York [2] V. I. Arnold; Mathematical Methods of Classical Mechanics, Springer-Verlag, New York, 1989. [3] A. N. Carvalho, J. W. Cholewa, T. Dlotko; Strongly damped wave problems: bootstrapping and regularity of solutions, J. Diﬀerential Equations, 244(9) (2008), 2310–2333. [4] M. M. Cavalcanti, V. N. D. Cavalcanti, J. Ferreira; Existence and uniform decay for nonlinear viscoelastic equation with strong damping, Math. Methods Appl. Sci., 24 (2001), 1043–1053. [5] M. M. Cavalcanti, V. N. Domingos Cavalcanti, F. A. F. Nascimento, I. Lasiecka; Intrinsic de- cay rate estimates for the wave equation with competing viscoelastic and frictional dissipative eﬀects, Discrete Contin. Dyn. Syst. Ser. B, 19(7) (2014), 1987–2012. [6] B. W. Feng; General decay rates for a viscoelastic wave equation with dynamic boundary conditions and past history, Mediterr. J. Math., 15 (2018), Article 103. [7] Y. Gao, J. Liang, T. J. Xiao; A new method to obtain uniform decay rates for multidimen- sional wave equations with nonlinear acoustic boundary conditions, SIAM J. Control Optim., 56(2) (2018), 1303–1320. [8] K. P. Jin, J. Liang, T. J. Xiao; Coupled second order evolution equations with fading memory: Optimal energy decay rate, J. Diﬀerential Equations, 257(4) (2014), 1501–1528. STABILITY OF VISCOELASTIC EQUATIONS 15 EJDE-2020/85 [9] K. P. Jin, J. Liang, T. J. Xiao; Uniform stability of semilinear wave equations with arbitrary local memory eﬀects versus frictional dampings. J. Diﬀerential Equations, 266 (2019), 7230– 7263. [10] K. P. Jin, J. Liang, T. J. Xiao; Asymptotic behavior for coupled systems of second order abstract evolution equations with one inﬁnite memory, J. Math. Anal. Appl., 475(1) (2019), 554–575. [11] M. Kaﬁni; Rate of decay for solutions of viscoelastic evolution equations, Electron. J. Diﬀer- ential Equations, 2013 (134) (2013), 1–17. [12] Y. X. Li; Existence and asymptotic stability of periodic solution for evolution equations with delays, J. Funct. Anal., 261(5) (2011), 1309–1324. [13] S. A. Messaoudi, W. Al-Khulaiﬁ; General and optimal decay for a quasilinear viscoelastic equation, Appl. Math. Lett., 66 (2017), 16–22. [14] S. A. Messaoudi, M. I. Mustafa; A general stability result for a quasilinear wave equation with memory, Nonlinear Anal. Real World Appl., 14(4) (2013), 1854–1864. [15] S. A. Messaoudi, N.-E. References Tatar; Global existence and uniform stability of solutions for a quasi- linear viscoelastic problem, Math. Methods Appl. Sci. 30 (2007), 665–680. [16] S. A. Messaoudi, N.-E. Tatar; Exponential and polynomial decay for a quasilinear viscoelastic equation, Nonlinear Anal. 68 (2008), 785–793. [17] M. I. Mustafa, G. A. Abusharkh; Plate equations with frictional and viscoelastic dampings, Appl. Anal. 96(7) (2017), 1170–1187. [18] J. E. Mu˜noz Rivera, M. G. Naso; Asymptotic stability of semigroups associated with linear weak dissipative systems with memory, J. Math. Anal. Appl. 326(1) (2007), 691–707. [19] A. Peyravi; General decay and blow up of solutions for a system of viscoelastic wave equations with nonlinear boundary source terms, J. Math. Anal. Appl. 451(2) (2017), 1056–1076. [20] T. J. Xiao, J. Liang; The Cauchy Problem for Higher-Order Abstract Diﬀerential Equations, Lecture Notes in Mathematics, 1701, Springer, Berlin, Germany, 1998. [21] T. J. Xiao, J. Liang; A solution to an open problem for wave equations with generalized Wentzell boundary conditions, Math. Ann., 327 (2003), 351–363. ( ) [22] T. J. Xiao, J. Liang; Second order diﬀerential operators with Feller-Wentzell type boundary conditions, J. Funct. Anal., 254 (2008), 1467–1486. ( ) [23] T. J. Xiao, J. Liang; Coupled second order semilinear evolution equations indirectly damped via memory eﬀects, J. Diﬀerential Equations, 254(5) (2013), 2128–2157. [24] Q. L. Zhang; Stability of Riemann solutions to pressureless Euler equations with Coulomb- type friction by ﬂux approximation, Electron. J. Diﬀerential Equations, 2019 (65) (2019), 1–22. Kun-Peng Jin School of Science, Chongqing University of Posts and Telecommunications, Chongqing 400065, China Kun-Peng Jin School of Science, Chongqing University of Posts and Telecommunications, Chongqing 400065, China Email address: kjin11@fudan.edu.cn Email address: kjin11@fudan.edu.cn Jin Liang (corresponding author) School of Mathematical Sciences, Shanghai Jiao Tong University, Shanghai 200240, China Jin Liang (corresponding author) School of Mathematical Sciences, Shanghai Jiao Tong University, Shanghai 200240, China J g (corr s o g u or) School of Mathematical Sciences, Shanghai Jiao Tong University, Shanghai 200240, China Email address: jinliang@sjtu.edu.cn Ti-Jun Xiao Shanghai Key Laboratory for Contemporary Applied Mathematics, School of Mathe- matical Sciences, Fudan University, Shanghai 200433, China Ti-Jun Xiao Shanghai Key Laboratory for Contemporary Applied Mathematics, School of Mathe- matical Sciences, Fudan University, Shanghai 200433, China Ti-Jun Xiao Shanghai Key Laboratory for Contemporary Applied Mathematics, School of Mathe- matical Sciences, Fudan University, Shanghai 200433, China Email address: tjxiao@fudan.edu.cn
https://openalex.org/W3195045201	https://www.frontiersin.org/articles/10.3389/fpsyg.2021.702106/pdf	English	null	A Web-Based Auditory and Visual Emotion Perception Task Experiment With Children and a Comparison of Lab Data and Web Data	Frontiers in psychology	2,021	cc-by	10,300	ORIGINAL RESEARCH published: 18 August 2021 doi: 10.3389/fpsyg.2021.702106 Edited by: Sho Tsuji, Edited by: Sho Tsuji, The University of Tokyo, Japan The University of Tokyo, Japan Reviewed by: Yang Yang, Nanyang Technological University, Singapore Paddy Ross, Durham University, United Kingdom Correspondence: Akihiro Tanaka akih.tanaka@gmail.com Reviewed by: Yang Yang, Nanyang Technological University, Singapore Paddy Ross, Durham University, United Kingdom Yang Yang, Nanyang Technological University, Singapore Paddy Ross, Durham University, United Kingdom Paddy Ross, Durham University, United Kingdom Correspondence: Akihiro Tanaka akih.tanaka@gmail.com Correspondence: Akihiro Tanaka akih.tanaka@gmail.com Keywords: online experiments, emotion perception, cognitive development, auditory perception, visual perception, vocal expression, facial expression INTRODUCTION Specialty section: This article was submitted to Developmental Psychology, a section of the journal Frontiers in Psychology The COVID-19 pandemic that began in 2020 has forced people to move much of their daily, face-to-face communication online. Psychological experiments are no exception. Many behavioral scientists had to stop their research and decide whether to postpone it or to move it online. Although many researchers have been trying to conduct studies remotely, suﬃcient examination of the validity of online developmental research is absent to date. In the present study, we introduce an online trial of perception tasks for children. We conducted a simple experiment featuring an auditory and a visual emotion perception task using video chat and an online experiment platform with children (4–12 years old) and adult participants. We then examined the validity of these data (online data) with the data from our previous, similar laboratory trial (Kawahara et al., 2021). Received: 29 April 2021 Accepted: 19 July 2021 Published: 18 August 2021 A Web-Based Auditory and Visual Emotion Perception Task Experiment With Children and a Comparison of Lab Data and Web Data Hisako W. Yamamoto1,2, Misako Kawahara1,2 and Akihiro Tanaka1 1 Tokyo Woman’s Christian University, Tokyo, Japan, 2 Japan Society for the Promotion of Science, Tokyo, Japan Due to the COVID-19 pandemic, the signiﬁcance of online research has been rising in the ﬁeld of psychology. However, online experiments with child participants are rare compared to those with adults. In this study, we investigated the validity of web- based experiments with child participants 4–12 years old and adult participants. They performed simple emotional perception tasks in an experiment designed and conducted on the Gorilla Experiment Builder platform. After short communication with each participant via Zoom videoconferencing software, participants performed the auditory task (judging emotion from vocal expression) and the visual task (judging emotion from facial expression). The data collected were compared with data collected in our previous similar laboratory experiment, and similar tendencies were found. For the auditory task in particular, we replicated differences in accuracy perceiving vocal expressions between age groups and also found the same native language advantage. Furthermore, we discuss the possibility of using online cognitive studies for future developmental studies. Citation: Moreover, even when participants remained until the end of the experiment, some of them, known as “satisﬁers,” might not devote the cognitive eﬀort in the tasks (Miura and Kobayashi, 2016). In addition to considering issues with online participants, we should consider the variety of their environments. In most of online studies, participants use their own devices. For this reason, while the validity of online data has been ensured for within-subject designs (e.g., Semmelmann and Weigelt, 2017), the case for between-subject design has not been clearly made. These factors may lead to greater variance in web experiments compared to lab experiments (Germine et al., 2012). Thus, while Internet-based experiments are easy to participate in, there may be some problems due to this ease (see Paolacci and Chandler, 2014). In this study, we report on our attempt at moving an experiment involving children’s perception tasks online. Our experiment consisted of video chat communication, and main tasks were controlled through the online experiment platforms. First, the experimenter communicated with the child participants and their parents via Zoom1 to check the child’s participation and to build rapport with them. Next, the experimenter guided participants to the browser experiment webpage built with Gorilla Experiment Builder2,3 (Anwyl-Irvine et al., 2020) and instructed them to engage in two simple emotion perception tasks. To investigate the validity of the obtained data, we compared web data for each task lab data for very similar tasks (Kawahara et al., 2021). We hypothesized that this online experiment method would reduce the issues usually associated with an online experiment. Speciﬁcally, we predicted that participants would perform as well in the online experiment as in the lab experiment and that the accuracy of each task would not diﬀer between web and lab data. Now then, what is the situation with online research for child participants? We were able to ﬁnd some trials and projects that shifted developmental research online. For example, Tran et al. (2017) tried to move an infant study online by recruiting participants through Amazon Mechanical Turk. They measured the length of time that 5–8-month-olds remained looking at various stimuli and reported success in capturing changes in their attention depending on the stimulus presented, even in an online data collection environment. Concerning behavioral measures, Klindt et al. Citation: Yamamoto HW, Kawahara M and Tanaka A (2021) A Web-Based Auditory and Visual Emotion Perception Task Experiment With Children and a Comparison of Lab Data and Web Data. Front. Psychol. 12:702106. doi: 10.3389/fpsyg.2021.702106 Even before the pandemic, online experiment research targeting adults was becoming popular due to its advantages. Unlike laboratory experiments, in which participants tend to be limited to residents around universities (e.g., Henrich et al., 2010), in online studies researchers can recruit participants without geographical constraints. Moreover, online experiments pair well with crowdsourcing services. Such services enable researchers to collect large amounts of data at low costs within a short time (Stewart et al., 2017). Such advantages have led many cognitive psychology August 2021 \| Volume 12 \| Article 702106 1 Frontiers in Psychology \| www.frontiersin.org Online Perceptual Experiment With Children Yamamoto et al. researchers to adopt data collected through online experiments (e.g., Mills and D’Mello, 2014; Shin and Ma, 2016; Laeng et al., 2018; Lavan et al., 2018; McPherson and McDermott, 2018; Carbon, 2020). to choose online research to pursue their research questions? The lack of online developmental research may be caused by the following diﬃculties. First, it is diﬃcult for participants to form a rapport with the experimenter during online experiments. A rapport is important for ensuring that child participants are as relaxed as possible while engaging in tasks. Second, we cannot always check whether a participant is really a child (and not an adult), and participant age is a critical factor in developmental research. Third, diﬀerences in performance between diﬀerent aged participants may be diﬃcult to observe because experiments with a between-subject design are not considered suitable for online research. However, as the pandemic continues, the beneﬁts of online developmental research may surpass such disadvantages if we can ensure the data is valid. Thus, it is imperative to accumulate data from online developmental studies featuring various online tasks to determine its suitability for use in future research. However, can online experiments ensure the validity and quality of the data they generate? To answer this question, some studies have compared online cognitive experiment data with laboratory experiment data and reported their success in replicating results (e.g., Crump et al., 2013; Simcox and Fiez, 2014; de Leeuw and Motz, 2016). Previous studies have also demonstrated some disadvantages of online studies. One such problem is high dropout rates (Reips, 2002; Zhou and Fishbach, 2016). Frontiers in Psychology \| www.frontiersin.org 1https://zoom.us/ 2https://gorilla.sc/ 3Anwyl-Irvine et al. (2020) showed that child participants had completed a ﬂanker task created with Gorilla Experiment Builder (the youngest participant in the ﬁnal sample was 4.38 years old) and revealed the development of the children’s performance. This was not a remote online study because participation in the experiment took place in a laboratory setting with an experimenter, not in participants’ homes. Nonetheless, the results do suggest that even child participants can engage in cognitive tasks controlled by this platform. 5According to EMFACS (Friesen and Ekman, 1984), the activation of AU 17 is related to negative expressions such as distress and rage. Considering this fact, it is possible that the activation of AU 17 leads to the judgment of facial expressions as negative. Nevertheless, for stimuli in the present study (Kawahara et al., 2021), we conﬁrmed that both Japanese and Dutch adult participants’ accuracy of perceiving angry faces did not diﬀer between stimulus cultures. Therefore, we consider that the frequency of activation in AU 17 did not signiﬁcantly impact participants’ judgment in the present study. MATERIALS AND METHODS voice expression. The speakers were four women (two native Japanese speakers and two native Dutch speakers). In each video clip, each speaker speaks one of four utterances containing only emotionally neutral linguistic information, including Hello (Japanese, “Hai, moshimoshi”; Dutch, “Hallo, dat ben ja”) and Good-by (Japanese, “Sayonara”; Dutch “Een goede dag”); What is this? (Japanese, “Korenani”; Dutch “Hey, wat is dit?”); and Is that so? (Japanese, “Sounandesuka”; Dutch, “Oh, is dat zo?”). A total of 32 video clips [in two languages (Japanese and Dutch) × two emotions (angry and happy) × two speakers × four utterances] were used. Participants Web Data The 36 children aged 4–12 years old (30 girls and 6 boys) and the 16 undergraduate or graduate students (age range: 18–29, M age = 21.63, 13 women and 3 men) participated in the experiment. Since one 5-years-old girl’s parent reported that she used built- in laptop speakers because her earphones did not ﬁt her, her data for both tasks were excluded from the analysis. In the analysis, 4–8-year-old children were classiﬁed as the younger child group (N = 21, M age = 6.48 years old) and the 9–12-year- old children were classiﬁed as the older child group (N = 14, M age = 10.29 years old). Data were collected from undergraduate or graduate students to compare the data collected from children with data collected from adults. Auditory stimuli were created by turning oﬀthe images and adding a gray rectangle image of the same size. Visual stimuli were created by muting sounds. Auditory stimuli comprised 32 video clips with vocal expression information only. Visual stimuli comprised 32 video clips with facial expression information only. The resolution of each video clip was 640 × 480 pixels. In the web experiment, auditory and visual stimuli were encoded in MP4 ﬁles for web page presentation. Child participant data were collected during the online science event of the National Museum of Emerging Science and Innovation (Miraikan) in Tokyo, Japan. We recruited participants through the Miraikan web page and SNS services (Twitter, Facebook). This event was held from August to December 2020. Adult participants were recruited through a snowball-sampling method and the Crowdworks crowdsourcing service website.4 Citation: (2017) reported that the large amount of data they collected from online participants revealed changes in cognitive skills (e.g., working memory, false belief, etc.) over the human lifespan. They collected the data through the BRAiN’US online platform for smartphones, and participants also included children (participants ranged from 5 to 85 years old). However, their study did not focus on the validity of online experiments with children, nor did it compare their data with lab data; rather their aim was to obtain a large dataset from a wide range of participants. More recently, Nussenbaum et al. (2020) investigated the decision-making strategies of participants aged 8–25 during an online task. They compared their results with data from previous lab experiments and were able to replicate age-related changes in strategy even in the online experiment. Moreover, some new online platforms for child research, such as Lookit (Scott et al., 2017), Discoveries Online (Rhodes et al., 2020), and Childlab (Sheskin and Keil, 2018) have been developed with the aim of enabling participation in remote studies for children who are not able to easily travel to a laboratory. In the emotion perception tasks, participants were asked to judge emotions by watching dynamic facial expressions or listening to vocal expressions. We chose these tasks for our online developmental research for two reasons. First, the development of emotional perception has not been investigated in online research. Second, the emotional judgment task enables us to examine the eﬀect of stimulus presented through a web browser on auditory (vocal expression) perception and visual (facial expression) perception independently. To compare web data with lab data for each modality, participants engaged in an auditory task judging emotions by listening to sounds only, and a visual task judging emotions by watching facial dynamics only (with no sound). These studies notwithstanding, less remote developmental research is being conducted than remote cognitive studies targeting adults. Why have developmental researchers hesitated August 2021 \| Volume 12 \| Article 702106 Frontiers in Psychology \| www.frontiersin.org 2 Online Perceptual Experiment With Children Yamamoto et al. 4https://crowdworks.co.jp/ Lab Data We compared lab data from the unimodal session of our previous experiment (Kawahara et al., 2021) with our web data. Data collected from 179 children aged 5–12 years old (75 girls and 104 boys) and from 33 undergraduate or graduate students (age range: 18–32, M age = 22.39, 17 women, 16 men) were included in the analysis. Child participants’ lab data were collected during the science event held at the Miraikan in 2015. We recruited participants through the Miraikan web page. For data analysis, the 5–8 year-old children comprised the younger child group (N = 100, M age = 6.36), and the 9–12 year-old children comprised the older child group (N = 79, M age = 10.66). Adult participants were recruited using a snowball-sampling method. As with participants in the web experiment, all participants in the lab experiment spoke Japanese as their native language. Adult participants and parents of child participants gave written informed consent in accordance with the Declaration of Helsinki. Lab Data The web experiment stimuli and the lab experiment stimuli were almost same but diﬀered in ﬁle format. In the lab experiment, the auditory stimuli ﬁles were in WAV format and the visual stimuli ﬁles in AVI format. Moreover, in the lab experiment auditory stimuli were presented with a blank, white display, and in web experiment a gray rectangle was displayed while the auditory stimuli were presented. The latter was to prevent web participants from becoming anxious due to watching a mere blank display in an experiment in which the experimenter is not present, unlike in a lab experiment. All participants spoke Japanese as their native language. All adult participants and parents of child participants were informed of the purpose of the study and gave informed consent in accordance with the Declaration of Helsinki by checking a box on the consent page during the browser experiment session. The Validation of Stimuli The validation of our stimulus set was veriﬁed in Kawahara et al.’s (2021) study, which investigated cross-cultural audiovisual emotion perception. Overall, there was no remarkable diﬀerence between Japanese and Dutch stimuli. For auditory stimuli, the average fundamental frequency (f0) was higher in Japanese than in Dutch for the happy voice stimuli (z = –3.36, p < 0.001), but not for the angry voice stimuli (Table 1). Considering that both Japanese and Dutch adult participants in Kawahara et al. (2021) responded to their ingroup voice stimuli more correctly than to their outgroup stimuli, this diﬀerence reﬂected their natural expressions in each culture. For visual stimuli, a certiﬁed FACS (Facial Action Coding System; Ekman and Friesen, 1978) coder coded all activated AUs during each stimulus. There was no diﬀerence in activated AUs except for AU175 in angry faces (z = –3.00, p = 0.01) between Japanese and Dutch visual stimuli. Thus, the stimulus set was validated. 6We used response alternatives written in hiragana characters (angry: おこっている[okotteiru]; happy: よろこんでいる[yorokondeiru]) because both young children and adults can read them easily. According to previous studies on Japanese children’s literacy, more than half of 3–4-year-olds could read most of hiragana characters (Kakihana et al., 2009), and about 90% of children in 5-year-old classes could read them (Ota et al., 2018) without formal education. Since Japanese hiragana characters have high transparency (each hiragana character corresponds to a syllable in Japanese phonology), Japanese children who know hiragana characters can be assumed to also read words. Considering Japanese children’s literacy, we inferred that the participants in this study could read response alternatives. Procedure Web Data After these tasks, in response to the questionnaire, participants’ parents reported problems during the experiment, whether participants had worn earphones, headphones, or had used other devices, whether parents had instructed their children to press a speciﬁc key during the main trials (“Did you ask your child to press any speciﬁc key during a task, for example, by saying ‘Press this key’?”); if they had any concerns, they were asked to ﬁll out a form. According to the questionnaire, we conﬁrmed that all participants included in the analysis had worn earphones or headphones, and that no parent instructed their child to press any speciﬁc key. None of parents reported any problems and concerns related with the tasks. The ﬂow of the procedure is shown in Figure 1. Before the experiment, child participants’ parents and adult participants received an instructions and documents ﬁle that included how to participate in the event and research brief. At the starting time, each participant and their parent joined the Zoom meeting room. The experimenter and the staﬀcommunicated with each participant using their web cameras and microphones to help participants relax. After a short communication, the experimenter provided attendees with instructions (e.g., not to click the web browser back button during the experiment, not to inﬂuence their children’s responses), checked that participants understood the positions of keys for response (D and K) on their keyboards, and guided them to the experiment web page by providing the URL link in the meeting room chatbox. After checking that each participant succeeded in accessing the experiment page, the experimenter instructed each participant to quit the meeting room to avoid low internet connection speeds during the experiment. They were also instructed to return to the same meeting room if they had any problems or reached the browser experiment’s ﬁnal display. Adult participants similarly joined a Zoom meeting room before the browser experiment and received instructions. They were to proceed with the browser experiment by themselves and return to the same meeting room if they had any problems or had reached the last display in the browser experiment. The procedure was similar to that of the child participants except that for adult participants the instructions were rewritten In the browser experiment session, participants’ parents proceeded with the experiment by themselves following instructions on the display. We used Gorilla Experiment Builder to control the experimental program and collect data. Stimuli W b D t The auditory and visual stimuli used were based on the audiovisual stimuli originally used by Tanaka et al. (2010). These audiovisual stimuli (stimuli used as the “congruent condition” in their study) were short video clips featuring a speaker expressing anger or happiness through face and August 2021 \| Volume 12 \| Article 702106 Frontiers in Psychology \| www.frontiersin.org 3 Online Perceptual Experiment With Children Yamamoto et al. TABLE 1 \| The average fundamental frequency (f0) of auditory stimuli (Hz). Angry Happy Japanese 242.8 336.9 Dutch 233.1 261.4 consent and indicated that environment requirements were met (suﬃcient device battery, headphones or earphones connection, environmental silence, web browser maximization) using checkboxes. Next, parents checked the sound volume with child participants, MP4 ﬁle playback, and keyboard operation following displayed instructions. After preparation, participants engaged in the auditory task and the visual task in each task section. The order of tasks was counterbalanced. At the beginning of each task section, participants watched a task instruction movie that included a simple speaking animation describing the task. The ﬂow of each task is shown in Figure 1 (see panels 2 and 3, Auditory Task and Visual Task). Frontiers in Psychology \| www.frontiersin.org Lab Data Researchers provided headphones (SONY MDR-ZX660) (used at a comfortable listening level) to present auditory stimuli and computers (Latitude 3540, Dell) to present visual stimuli and control the experiment program using Hot Soup Processor (Onion Software). Apparatus Web Data Participants used their own earphones or headphones to listen to auditory stimuli and their own computers to watch visual stimuli and control the browser experiment program. We asked participants to use a computer monitor and earphones (or headphones) and recommended that they use the latest version of Google Chrome. We did not specify the models of the devices. In the auditory task, participants were instructed to listen to a voice and judge whether the speaker was angry or happy. A ﬁxation point was displayed at the center of the monitor for 500 ms, after which an auditory stimulus was presented. When the response alternatives written in hiragana characters6 were displayed, participants responded by pressing D or K keys (i.e., the allocation of response alternatives was counterbalanced). Five hundred ms after participant’s response, the next test trial began, for a total of 32 trials. In the visual task, participants were instructed to observe the face of a (muted) speaker and judge whether they were angry or happy. A ﬁxation point was displayed at the center of the monitor for 500 ms, and each visual stimulus was presented successively. As with the auditory task, responses were indicated by pressing keys. 500 ms blank displays were inserted between trials, for 32 trials. For each task, the main trials were conducted following two practice trials. The resolution of participant displays ranged from 915 × 515 to 1920 × 1080. The participants’ used Windows (45 participants), Mac OS (3), Android (2), and iOS (1) operating systems and Google Chrome (27), Microsoft Edge (19), Microsoft Internet Explorer (4), and Safari (1) web browsers. Procedure Web Data The browser experiment session consisted of a preparation section, the auditory task, the visual task, and a questionnaire. In the preparation section, participants’ parents gave informed August 2021 \| Volume 12 \| Article 702106 4 Online Perceptual Experiment With Children Yamamoto et al. t ﬂow (The instructions and alternatives were written in Japanese in the actual experiment). FIGURE 1 \| Experiment ﬂow (The instructions and alternatives were written in Japanese in the actual experiment). Frontiers in Psychology \| www.frontiersin.org August 2021 \| Volume 12 \| Article 702106 5 Online Perceptual Experiment With Children Yamamoto et al. using online tools were not signiﬁcantly diﬀerent from those of the lab experiment. (e.g., converting some hiragana characters to kanji characters for readability7), and they were not asked a question about parents’ instruction (“Did you ask your child to press any speciﬁc key during a task, for example, by saying ‘Press this key’?”) in the questionnaire after the task. p As for other factors, results showed signiﬁcant main eﬀects for age group (F(2, 257) = 31.56, p < 0.001, ηp2 = 0.20). The post hoc analysis (Shaﬀer’s Modiﬁed Sequentially Rejective Bonferroni Procedure) revealed that the older child group of participants responded correctly to more stimulus than the younger child group and that adult participants responded correctly more often than younger and older child participants (ps < 0.001). The main eﬀect of stimulus culture was also signiﬁcant (F(1, 257) = 281.61, p < 0.001, ηp2 = 0.52), showing that participants responded correctly to the Japanese voice more often than to the Dutch voice. Interaction between age group and stimulus culture was marginally signiﬁcant (F(2, 257) = 2.89, p = 0.057, ηp2 = 0.02). To check whether the impact of stimulus culture was diﬀerent among age group, we conducted a simple main eﬀect analysis. The simple main eﬀect analysis also showed that all groups selected more correct answers in responses to the Japanese voice than in response to the Dutch voice (Younger child group: F(1, 119) = 157.56, p < 0.001; Older child group: F(1, 91) = 97.22, p < 0.001; Adult group: F(1, 47) = 59.11, p < 0.001). Moreover, a simple main eﬀect of age was signiﬁcant for both the Japanese voice (F(2, 257) = 9.01, p < 0.001) and the Dutch voice (F(2, 257) = 42.04, p < 0.001). 7We did it because it is easier for native Japanese speaking adults to read sentences such as instructions that use both kanji and hiragana than sentences written in hiragana characters only. RESULTS We calculated the rate of correct responses for each participant. Then, this rate was arcsine transformed to increase the normality of its distribution (accuracy). To investigate whether the experiment method aﬀected diﬀerent aged participants’ performance diﬀerently, we conducted a 2 (method: web, lab) × 3 (age group: younger child, older child, adult) × 2 (stimulus culture: Japanese, Dutch) mixed-factorial ANOVA on accuracy for each task. To further examine the marginal interaction between age group and stimulus culture, we conducted a two-way ANOVA (method × age group) on the ingroup advantage. This was calculated by subtracting the accuracy of Dutch voices from that of Japanese voices. The main eﬀect of age groups was marginally signiﬁcant (F(2, 257) = 2.89, p = 0.057, ηp2 = 0.02). Post hoc analysis (Shaﬀer’s Modiﬁed Sequentially Rejective Bonferroni Procedure) showed that the diﬀerence between younger children and adults was marginally signiﬁcant (p = 0.051), suggesting that the ingroup advantage in younger children was more salient than that in adults. The diﬀerences between other pairs were not signiﬁcant (younger child group – older child group: p = 0.149, older child group – adult group: p = 0.393). The main eﬀects of method (F(1, 257) = 0.07, p = 0.789, ηp2 < 0.001) and the interaction of method and age group (F(2, 257) = 0.22, p = 0.800, ηp2 = 0.002) were not signiﬁcant. Lab Data The experiment was conducted in an experimental room at the Miraikan for the child participants and in an experimental room at the Tokyo Woman’s Christian University for the adult participants. The procedure was almost the same as with the web experiment but with three slight diﬀerences. First, in the (Kawahara et al., 2021) lab experiment, auditory and visual tasks were conducted after audiovisual emotional perception tasks in which participants judged speakers’ emotions after being presented with face and voice simultaneously. Participants in the web experiment did not engage in audiovisual emotion perception tasks like those in the lab experiment. We cannot rule out the priming eﬀect in the lab data induced by the audiovisual stimuli that had been presented before. However, considering that the number of presentations of “angry” and “happy” stimuli was the same in the audiovisual emotion perception task, a response bias is not possible. Second, cards showing the alternatives (“angry” and “happy” written in hiragana characters) were put on a keyboard in the lab experiment; these alternatives were shown on the display in the web experiment. Third, the experimenter was physically present next to each participant and controlled the experiment program throughout the experiment session in the lab experiment. The presentation of stimuli was controlled using the Hot Soup Processor (Onion Software). These diﬀerences were summarized in Figure 2. Procedure Web Data Post hoc analysis (Shaﬀer’s Modiﬁed Sequentially Rejective Bonferroni Procedure) revealed that older child participants responded correctly to the Japanese voice more than the younger child group (p = 0.049), and that adult participants responded correctly more often than younger (p < 0.001) and older child participants (p = 0.034). A similar accuracy diﬀerence between age groups was observed with the Dutch voice. Older child participants responded correctly to the Dutch voice more often than the younger child group, and adult participants responded correctly more often than younger and older child participants (ps < 0.001). All age groups responded correctly more often to the Japanese voice than the Dutch voice, and accuracy with both voices increased with age. Auditory Task The Bayesian ANOVA revealed that the BFinclusion values of the eﬀect of method (BFinclusion = 0.165), the interaction eﬀect between method and stimulus culture August 2021 \| Volume 12 \| Article 702106 Frontiers in Psychology \| www.frontiersin.org 7 Online Perceptual Experiment With Children Yamamoto et al. FIGURE 4 \| Visual task Accuracy (Error bars indicate standard deviations and a dashed line indicates a perfect score). FIGURE 4 \| Visual task Accuracy (Error bars indicate standard deviations and a dashed line indicates a perfect score). FIGURE 3 \| Auditory task Accuracy (Error bars indicate standard deviations and a dashed line indicates a perfect score). FIGURE 3 \| Auditory task Accuracy (Error bars indicate standard deviations and a dashed line indicates a perfect score). FIGURE 3 \| Auditory task Accuracy (Error bars indicate standard deviations and a dashed line indicates a perfect score). FIGURE 4 \| Visual task Accuracy (Error bars indicate standard deviations and a dashed line indicates a perfect score). FIGURE 3 \| Auditory task Accuracy (Error bars indicate standard deviations and a dashed line indicates a perfect score). TABLE 2 \| Evidence for the presence of particular effects in the accuracy of the auditory task (Data averaged over all the models including/excluding a particular predictor). participants responded correctly to the Dutch face more often than to the Japanese face. Interaction between age group and stimulus culture (F(2, 257) = 3.05, p = 0.049, ηp2 = 0.023) was also signiﬁcant, but the main eﬀect for age group was not (F(2, 257) = 0.19, p = 0.829, ηp2 = 0.001). A simple main eﬀect analysis revealed that older children (p = 0.007) and adults (p = 0.003) responded correctly to the Dutch face more often than to the Japanese face, while younger children’s accuracy did not diﬀer between stimulus cultures (p = 0.744). The accuracy for faces did not diﬀer among age groups both for Japanese (p = 0.624) and Dutch stimuli (p = 0.165). Auditory Task The results for the auditory task are shown in Figure 3. In the auditory task, the main eﬀects of method (F(1, 257) = 2.07, p = 0.151, ηp2 = 0.008), the interaction of method and age group (F(2, 257) = 0.09, p = 0.917, ηp2 = 0.001), of method and stimulus culture (F(1, 257) = 0.07, p = 0.789, ηp2 < 0.001), and of the second-order interaction of method, age group, and stimulus culture (F(2, 257) = 0.22, p = 0.800, ηp2 = 0.002) were not signiﬁcant. Thus, the results of the auditory task To further examine the eﬀect of method on accuracy, we conducted a Bayesian repeated measures ANOVA on accuracy using JASP (2018) with default prior scales. Table 2 shows the inclusion probabilities and the inclusion Bayes factor (Clyde et al., 2011; van den Bergh et al., 2019). The inclusion Bayes 7We did it because it is easier for native Japanese speaking adults to read sentences such as instructions that use both kanji and hiragana than sentences written in hiragana characters only. August 2021 \| Volume 12 \| Article 702106 Frontiers in Psychology \| www.frontiersin.org 6 Online Perceptual Experiment With Children Yamamoto et al. FIGURE 2 \| The differences in procedures between web and lab data. FIGURE 2 \| The differences in procedures between web and lab data. actors reﬂect the average across possible models and reveal (BFinclusion = 0 111) the interaction eﬀect between method FIGURE 2 \| The differences in procedures between web and lab data. (BFinclusion = 0.111), the interaction eﬀect between method and age group (BFinclusion = 0.069), and the second-order interaction of method, age group, stimulus culture, and age (BFinclusion = 0.004) were all small, supporting no eﬀect of the diﬀerence between the web and lab experiments. Additionally, the data provide strong evidence for the eﬀects of age group and stimulus culture (BFinclusions > 100), although they are not factors reﬂect the average across possible models and reveal whether models with a particular predictor are more likely to have produced the observed data than those without. This approach is especially useful when the number of potential variables under consideration is large. Auditory Task P(incl) P(excl) P(incl\| data) P(excl\| data) BFincl Stimulus Culture 0.737 0.263 1.000 0.000 ∞ Age Group 0.737 0.263 1.000 0.000 ∞ Method 0.737 0.263 0.316 0.684 0.165 Stimuli × Age Group 0.316 0.684 0.336 0.664 1.095 Stimuli × Method 0.316 0.684 0.049 0.951 0.111 Method × Age Group 0.316 0.684 0.031 0.969 0.069 Stimuli × Method × Age Group 0.053 0.947 > 0.001 1.000 0.004 To further examine the eﬀect of method on accuracy, similar to the auditory task, we conducted a Bayesian repeated measures ANOVA using JASP with default prior scales. Table 3 shows the inclusion probabilities and inclusion Bayes factor. The Bayesian ANOVA revealed that the eﬀect of method type (BFinclusion = 0.084), the interaction eﬀect between method and stimulus culture (BFinclusion = 0.083), the interaction eﬀect between method and age group (BFinclusion = 0.033), and the second-order interaction of method, age group, stimulus culture, and age (BFinclusion = 0.006) were all small, supporting no eﬀect of the diﬀerence between the web and lab experiments. Consistent with the results of classical ANOVA, the data provided moderate evidence for the eﬀect of stimulus culture (BFinclusion = 5.893). However, the eﬀect of the interaction between age group and stimulus culture was not informative (BFinclusion = 0.128). suﬃciently informative to allow a strong conclusion about the eﬀect of the interaction between age group and stimulus culture (BFinclusion = 1.095). Reaction Time the Dutch voice (Younger child group: F(1, 20) = 6.01, p = 0.024; Older child group: F(1, 13) = 17.37, p = 0.011; Adult group: F(1, 15) = 8.51, p = 0.011). Moreover, a simple main eﬀect of age was signiﬁcant for both the Japanese voice (F(2, 48) = 4.35, p = 0.018) and the Dutch voice (F(2, 48) = 5.08, p < 0.001). Post hoc analysis (Shaﬀer’s Modiﬁed Sequentially Rejective Bonferroni Procedure) revealed that adult participants responded to Japanese voice faster than older children (p = 0.029) and younger children (p = 0.029), and adult participants responded to Dutch voice faster than older children (p = 0.011) and younger children (p = 0.022). There was no signiﬁcant diﬀerence between the younger and older child groups for both Japanese voice (p = 0.962) and Dutch voice (p = 0.351). We conducted a one-way ANOVA on the ingroup advantage to further examine the interaction between age group and stimulus culture. This was calculated by subtracting the reaction time to Japanese voices from that of Dutch voices. The main eﬀect of age groups was signiﬁcant (F(2, 48) = 3.91, p = 0.027, ηp2 = 0.01). Post hoc analysis (Shaﬀer’s Modiﬁed Sequentially Rejective Bonferroni Procedure) showed that the ingroup advantage in older children was larger than that in both adults (p = 0.025) and younger children (marginally signiﬁcant; p = 0.053). The diﬀerence between younger children and adults was not signiﬁcant (p = 0.338). Thus, the ingroup advantage based on reaction times was most salient in older children, unlike the analysis of accuracy. We showed average reaction times of each task in Figure 5. We excluded outlier reaction time data (each participant’s average reaction time ±2.5 SD), while including trials in which participants pressed the wrong key, considering the following reasons. First, children’s responses classiﬁed as “incorrect responses” may include the results of their careful consideration. Second, given the age diﬀerences in accuracy, the number of correct responses, that is, the number of trials included in the analysis diﬀered among age groups in the auditory task. We conducted a 3 (age group) × 2(stimulus culture) mixed-factorial ANOVA on reaction time for each task. In the auditory task, the main eﬀects for age group (F(2, 48) = 4.91, p = 0.011, ηp2 = 0.17) was signiﬁcant. Visual Task The results of the visual task are shown in Figure 4. In the visual task, the main eﬀects of method (F(1, 257) = 0.36, p = 0.551, ηp2 = 0.001), the interaction of method and age group (F(2, 257) = 1.10, p = 0.335, ηp2 = 0.008), method and stimulus culture (F(1, 257) = 1.00, p = 0.320, ηp2 = 0.004), and the second-order interaction of method, age group and stimuli (F(2, 257) = 1.99, p = 0.138, ηp2 = 0.015) were not signiﬁcant. Thus, the results of the visual task using online tools were not signiﬁcantly diﬀerent from those of the lab experiment. Thus, results showed that the experiment method (web or lab) did not aﬀect participants’ performance in either the auditory task Results indicated a signiﬁcant main eﬀect for stimulus culture (F(1, 257) = 13.92, p < 0.001, ηp2 = 0.051), showing that August 2021 \| Volume 12 \| Article 702106 Frontiers in Psychology \| www.frontiersin.org 8 Online Perceptual Experiment With Children Yamamoto et al. TABLE 3 \| Evidence for the presence of particular effects in the accuracy of the visual task (Data averaged over all the models including/excluding a particular predictor). FIGURE 5 \| The average of participants’ reaction time in web data (Error bars indicate standard deviations). P(incl) P(excl) P(incl\| data) P(excl\| data) BFincl Stimulus Culture 0.737 0.263 0.943 0.057 5.893 Age Group 0.737 0.263 0.300 0.700 0.153 Method 0.737 0.263 0.191 0.809 0.084 Stimuli × Age Group 0.316 0.684 0.056 0.944 0.128 Stimuli × Method 0.316 0.684 0.037 0.963 0.083 Method × Age Group 0.316 0.684 0.015 0.985 0.033 Stimuli × Method × Age Group 0.053 0.947 >0.001 1.000 0.006 or the visual task; that is, the web data obtained in the present study did not diﬀer from our previously obtained lab data. That is, our method can enable researchers to obtain data that would be comparable to those of a laboratory experiment in the emotion perception tasks. Reaction Time Here we reported reaction times in the online experiment. Since we did not measure reaction times in the lab experiment, we cannot compare them between methods. Moreover, we did not instruct participants to respond to each stimulus as quickly as they could. Thus, the reaction times data here are for reference only. Nevertheless, this is useful as data of online developmental experiment, and it also enable us to investigate whether we could ﬁnd age diﬀerence in performance for the visual task, in which no age diﬀerence in accuracy was found due to the ceiling eﬀect. FIGURE 5 \| The average of participants’ reaction time in web data (Error bars indicate standard deviations). the Dutch voice (Younger child group: F(1, 20) = 6.01, p = 0.024; Older child group: F(1, 13) = 17.37, p = 0.011; Adult group: F(1, 15) = 8.51, p = 0.011). Moreover, a simple main eﬀect of age was signiﬁcant for both the Japanese voice (F(2, 48) = 4.35, p = 0.018) and the Dutch voice (F(2, 48) = 5.08, p < 0.001). Post hoc analysis (Shaﬀer’s Modiﬁed Sequentially Rejective Bonferroni Procedure) revealed that adult participants responded to Japanese voice faster than older children (p = 0.029) and younger children (p = 0.029), and adult participants responded to Dutch voice faster than older children (p = 0.011) and younger children (p = 0.022). There was no signiﬁcant diﬀerence between the younger and older child groups for both Japanese voice (p = 0.962) and Dutch voice (p = 0.351). We conducted a one-way ANOVA on the ingroup advantage to further examine the interaction between age group and stimulus culture. This was calculated by subtracting the reaction time to Japanese voices from that of Dutch voices. The main eﬀect of age groups was signiﬁcant (F(2, 48) = 3.91, p = 0.027, ηp2 = 0.01). Post hoc analysis (Shaﬀer’s Modiﬁed Sequentially Rejective Bonferroni Procedure) showed that the ingroup advantage in older children was larger than that in both adults (p = 0.025) and younger children (marginally signiﬁcant; p = 0.053). The diﬀerence between younger children and adults was not signiﬁcant (p = 0.338). Thus, the ingroup advantage based on reaction times was most salient in older children, unlike the analysis of accuracy. Frontiers in Psychology \| www.frontiersin.org Reaction Time The post hoc analysis (Shaﬀer’s Modiﬁed Sequentially Rejective Bonferroni Procedure) revealed that adult group of participants responded more quickly than the younger (p = 0.019) and older child groups (p = 0.019). There was no signiﬁcant diﬀerence between the younger and older child groups (p = 0.587). The main eﬀect of stimulus culture was also signiﬁcant (F(1, 48) = 27.67, p < 0.001, ηp2 = 0.37), showing that participants responded more quickly to the Japanese voice than to the Dutch voice. Interaction between age group and stimulus culture was also signiﬁcant (F(2, 48) = 3.91, p = 0.027, ηp2 = 0.14). To check whether the impact of stimulus culture was diﬀerent among age groups, we conducted a simple main eﬀect analysis. The simple main eﬀect analysis also showed that all groups yielded faster responses to the Japanese voice than in response to In the visual task, the main eﬀects of age group (F(2, 48) = 2.18, p = 0.124, ηp2 = 0.08) and of stimulus culture (F(1, 48) < 0.01, p = 0.991, ηp2 < 0.001), and the interaction between them (F(2, 48) = 0.84, p = 0.438, ηp2 = 0.03) were not signiﬁcant. Thus, all groups responded visual stimulus quickly and there was August 2021 \| Volume 12 \| Article 702106 9 Online Perceptual Experiment With Children Yamamoto et al. studies using low intensity facial expressions, with more variety of emotions, or with smaller sized pictures in the future. no diﬀerence among age groups and between stimulus culture. This is consistent with that high accuracy was observed in all age groups. Taken together, there was no diﬀerences among age groups both in accuracy and reaction times in the visual task. Although our main purpose was to investigate the validity of data obtained through online experiments, our data provide interesting ﬁndings on perceptual development. First, in the auditory task, the diﬀerence in the ingroup advantage was marginally signiﬁcantly diﬀerent between younger children and adults. That is, the ingroup advantage in younger children may be more salient than that in adults. This tendency may be related to the ﬁndings that young children prefer people who spoke their native language (e.g., Kinzler et al., 2007). Second, in the visual task, participants gave more correct responses to Dutch faces than to Japanese faces. DISCUSSION The purpose of this study was to investigate the validity of online developmental studies through an emotion perception experiment. To that end, we conducted an experiment controlled by Gorilla Experiment Builder with child and adult participants who engaged in simple auditory and visual emotion perception tasks following communication and instruction through Zoom. As predicted, results showed no signiﬁcant diﬀerences in participants’ performance between our web data results and out lab data results (Kawahara et al., 2021). In the auditory task, we found performance diﬀerences between age groups (between-subject factor) and better performance with stimuli spoken in their native language (within-subject factor). These ﬁndings were consistent with previous laboratory studies reporting performance improvements with age (Sauter et al., 2013; Chronaki et al., 2014) and superior perception of vocal emotional expression with native language stimulus (Sauter et al., 2010). In the visual task, accuracy was high and near- perfect among all age groups both in our web and lab data. To date, although online experiments have replicated laboratory experiment results with adult participants (e.g., Crump et al., 2013), developmental studies with child participants have been limited. By including child participants in our online study of emotional perception tasks, the present study adds new evidence regarding the validity of data collected in online developmental studies. Notably, accuracy of perception of vocal expression in the lab experiment was replicated in the web experiment even though participants were not required to use a speciﬁc device and were allowed to use any earphones or headphones. The results of the present study may relieve researchers’ hesitation to conduct developmental experiments online, at least in the ﬁeld of auditory emotional perception. Of course, we need additional examination to determine the suitability of online platforms for other types of auditory perception research. In tasks such as phoneme perception, judgment of speaker identities, or perceiving vocal expression from among multiple choices, participants’ responses may be aﬀected by the devices they use (see Woods et al., 2017). We showed new possibilities for using simple, general (not specialized for children) online tools that may enable researchers to move their laboratory studies online. However, we should clarify the limitations of the methods presented here. First, this study targeted children who could be instructed verbally and could respond by pressing keys on a keyboard. Reaction Time These results are unexpected considering that previous studies have insisted on the ingroup eﬀect in facial recognition tasks (e.g., Elfenbein and Ambady, 2002). However, more recent studies have demonstrated that Japanese raters did not show the ingroup advantage in the perception of facial expressions (Matsumoto et al., 2009; Hutchison et al., 2018). Overall, Japanese people’s facial expressions may not necessarily be perceived correctly by ingroup members. Our results of the visual task may also reﬂect this. Moreover, the interaction between age group and stimulus culture on accuracy was also signiﬁcant in the visual task, suggesting that older children and adults responded more correctly to Dutch facial expressions than to Japanese facial expressions. These may be interesting if Japanese people judge their outgroup facial expressions more accurately compared to Japanese stimuli with age. Japanese children may come to know that Japanese people tend to conceal their true emotions (e.g., Matsumoto, 1990) and that they inhibit their facial expressions, and this may cause “outgroup advantage.” This may lead to Japanese people’s tendency to prioritize voice in audiovisual emotion perception as for Japanese stimuli shown in previous studies (Yamamoto et al., 2020; Kawahara et al., 2021). We cannot clarify this speculation based on the present study because the Bayesian ANOVA did not provide strong evidence for the interaction between age group and stimulus culture in both tasks. However, we need to investigate these perceptual developmental suggestions in the further study. Frontiers in Psychology \| www.frontiersin.org August 2021 \| Volume 12 \| Article 702106 DISCUSSION Considering that Japanese preschool children read written words from relatively early on, we did not check the level of literacy for each participant. However, checking this would be important for researchers to apply this method to children living in various environments. For studies targeting preliterate children as participants, researchers should select a video-recorder type experiment model and record participants’ oral or pointing responses. Second, we had to rely on parents’ self-reports and could not independently check children’s actual states during the browser experiment because we instructed parents to turn oﬀtheir web cameras. Although we ensured that parents did not ask their children to respond in line with parents’ answers during the main trials by questionnaires, y y y We cannot strongly conclude that online developmental research is valid for the task of perceiving facial expressions because we observed a ceiling eﬀect; that is, performance was near perfect among all age groups in the present task. Our stimuli for the visual task were quite clear—Tanaka et al. (2010) originally created them by adding random dynamic noises to be degraded—and only two response alternatives (angry or happy) were available. The reasoning behind Kawahara et al.’s (2021) use of visual stimuli without noises was to avoid unpleasantness for the children, and so the present study followed that procedure. However, to investigate the impact of browser experiments on the presentation of visual stimuli in detail, we should conduct online August 2021 \| Volume 12 \| Article 702106 August 2021 \| Volume 12 \| Article 702106 10 Online Perceptual Experiment With Children Yamamoto et al. we did not have a way to conﬁrm this was the case. Moreover, it is possible that parents would have given their children instructions without being aware of it. This could be avoided by keeping the web cameras on during the experiment. However, this could aﬀect the quality of the presentation of the stimuli due to internet connection speed issues. These are the trade-oﬀs, and in the present study we gave prioritized the quality of the stimuli. Such choices should be made in accordance with the aim of each study. Moreover, we should not forget the burden on parents during online experiments, and minimizing this burden should be considered when designing online experiments. In addition, as described in the section “Materials and Methods,” our web experiment procedure was not exactly the same as that of our laboratory experiment (Figure 2). DISCUSSION Nevertheless, there were no signiﬁcant diﬀerences between our web data and lab data in the present study, which suggests that the diﬀerence in procedure does not have a critical impact on the results in the experiments investigating the development of emotion perception. not ﬁnd age diﬀerences between child groups for reaction times. Moreover, while we observed a salient ingroup advantage in younger children compared with adults (although the tendency was marginally signiﬁcant) for accuracy, this was not reﬂected in reaction times. Rather, for reaction times, we found that older children’s ingroup advantage was more salient than the other two age groups. Since we do not have reaction time data of our lab experiment, it remains unclear whether such tendency is observed also in a lab experiment or is unique to an online experiment. As a limitation of online research, one previous study pointed out the diﬃculty in controlling a short presentation of stimuli such as a masked priming procedure (Crump et al., 2013). Another study investigating the contrast threshold reported a high rate of data exclusions due to each participant’s experimental environment (Sasaki and Yamada, 2019). Further studies are needed to examine which indices and tasks are adequate for online experiments. Despite the limitations, we demonstrated that online experiments are useful for child research using auditory and visual (movie) stimuli. Combinations of online tools will lead researchers to new developmental research styles. Moreover, due to the validity of this online research using unimodal auditory and visual stimuli, application to future audiovisual perception research is expected. As an online experiment research, the procedure of the present study has two particularities. First, child participants’ parents take on the “experimenter” role. Second, researchers and participants communicate with each other through video chat before the tasks. Previous online psychological experiment studies with adults have pointed out high dropout rates (e.g., Reips, 2002), large variances due to various environments among participants, and diﬃculties in a between-subject design study. On the contrary, it is worth noting that the methods we adopted resulted in very few cases of participant data being excluded. Moreover, the variance of performance seemed to be similar to that of the lab experiment even though participants used their laptops and earphones (headphones). Our only requests before the experiment were to enter the video chat at the appointed time and to prepare earphones or headphones. DISCUSSION Considering the eﬀort involved in making an appointment with each participant, and in instructing both parents and child participants to proceed with the experiments, our method does not have beneﬁts such as large data collecting in a short period, unlike usual crowdsourced online experiments. Nevertheless, the results of the present study suggest that this eﬀort can reduce issues associated with online research, such as a dropout rate and variance of data. Given that even adult participants engaged in tasks seriously without an experimenter, video chat communication before the main experiment may be speciﬁcally eﬀective. Even though our method does not have the aforementioned beneﬁts associated with crowdsourced online research, we regard its biggest advantage to be the fact that both experimenters and participants are not aﬀected by geographical constraints. In fact, as long as they have an internet connection, researchers can conduct studies with people living in various countries and continue to collect data even under a pandemic. ETHICS STATEMENT The studies involving human participants were reviewed and approved by Tokyo Woman’s Christian University Research Ethics Committee. Written informed consent or digital informed consent to participate in this study was provided by the participants’ legal guardian/next of kin. Frontiers in Psychology \| www.frontiersin.org DATA AVAILABILITY STATEMENT The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. AUTHOR CONTRIBUTIONS HY was involved in designing the web experiment programs, collecting the data in the web experiment, analyzing the data, and drafting the manuscript. MK was involved in collecting the data and designing programs in the laboratory experiment. AT was involved in the creation of stimuli. All authors were involved in the experimental design, interpretation of the results, revised, and approved the ﬁnal version of the manuscript. We should note that the reproducibility of results in online experiments may depend on indices. We used the rate of correct responses, and we did not compare other indices such as reaction times or ﬁxations. Given that our web data showed that all age groups responded more quickly to the Japanese stimuli in the auditory task, reaction time may be used even in online experiments. However, unlike the results for accuracy, we did REFERENCES Lavan, N., Burston, L. F. K., and Garrido, L. (2018). How many voices did you hear? Natural variability disrupts identity perception from unfamiliar voices. Br. J. Dev. Psychol. 110, 576–593. doi: 10.1111/bjop.12348 Lavan, N., Burston, L. F. K., and Garrido, L. (2018). How many voices did you hear? Natural variability disrupts identity perception from unfamiliar voices. Anwyl-Irvine, A. L., Massonnié, J., Flitton, A., Kirkham, N., and Evershed, J. K. (2020). Gorilla in our midst: an online behavioral experiment builder. Behav. Res. Methods 52, 388–407. doi: 10.3758/s13428-019-01237-x Br. J. Dev. Psychol. 110, 576–593. doi: 10.1111/bjop.12348 Matsumoto, D. (1990). Cultural similarities and diﬀerences in display rules. Motiv. Emot. 14, 195–214. doi: 10.1007/BF00995569 Carbon, C.-C. (2020). Wearing face masks strongly confuses counterparts in reading emotions. Front. Psychol. 11:566886. doi: 10.3389/fpsyg.2020.56 6886 Matsumoto, D., Olide, A., and Willingham, B. (2009). Is there an ingroup advantage in recognizing spontaneously expressed emotions? J. Nonverbal. Behav. 33:181. doi: 10.1007/s10919-009-0068-z McPherson, M. J., and McDermott, J. H. (2018). Diversity in pitch perception revealed by task dependence. Nat. Hum. Behav. 2, 52–66. doi: 10.1038/s41562- 017-0261-8 Chronaki, G., Hadwin, J. A., Garner, M., Maurage, P., and Sonuga-Barke, E. J. S. (2014). The development of emotion recognition from facial expressions and non-linguistic vocalizations during childhood. Br. J. Dev. Psychol. 33, 218–236. doi: 10.1111/bjdp.12075 Mills, C., and D’Mello, S. (2014). On the validity of the autobiographical emotional memory task for emotion induction. PLoS One 9:e95837. doi: 10.1371/journal. pone.0095837 Clyde, M. A., Ghosh, J., and Littman, M. L. (2011). Bayesian adaptive sampling for variable selection and model averaging. J. Comput. Graph. Stat. 20, 80–101. doi: 10.1198/jcgs.2010.09049 Miura, A., and Kobayashi, T. (2016). Survey satisﬁcing inﬂates stereotypical responses in online experiment: the case of immigration study. Front. Psychol. 7:1563. doi: 10.3389/fpsyg.2016.01563 Crump, M. J. C., McDonnell, J. V., and Gureckis, T. M. (2013). Evaluating amazon’s mechanical turk as a tool for experimental behavioral research. PLoS One 8:e57410. doi: 10.1371/journal.pone.0057410 Nussenbaum, K., Scheuplein, M., Phaneuf, C. V., Evans, M. D., and Hartley, C. A. (2020). Moving developmental research online: comparing in-lab and web-based studies of model-based reinforcement learning. Collabra. Psychol. 6:17213. doi: 10.1525/collabra.17213 de Leeuw, J. R., and Motz, B. A. (2016). Psychophysics in a web browser? Comparing response times collected with javascript and psychophysics toolbox in a visual search task. Behav. Res. Methods 48, 1–12. doi: 10.3758/s13428-015- 0567-2 Ota, S., Uno, A., and Inomata, T. (2018). REFERENCES Attainment level of hiragana reading / spelling in kindergarten children. Jpn. J. Logop. Phoniatr. 59, 9–15. doi: 10.5112/ jjlp.59.9 Ekman, P., and Friesen, W. V. (1978). Facial Action Coding System: Investigator’s Guide. Palo Alto, CA: Consulting Psychologists Press. Paolacci, G., and Chandler, J. (2014). Inside the turk: understanding mechanical turk as a participant pool. Psychol. Sci. 23, 184–188. doi: 10.1177/09637214145 31598 Elfenbein, H. A., and Ambady, N. (2002). On the universality and cultural speciﬁcity of emotion recognition: a meta-analysis. Psychol. Bull. 128, 203–235. doi: 10.1037/0033-2909.128.2.203 Friesen, W. V., and Ekman, P. (1984). EMFACS-7. Unpublished manuscript. Human Interaction Laboratory. San Francisco, CA: University of California. Reips, U. D. (2002). Standards for internet-based experimenting. Exp. Psychol. 49, 243–256. doi: 10.1027/1618-3169.49.4.243 Rhodes, M., Rizzo, M. T., Foster-Hanson, E., Moty, K., Leshin, R. A., Wang, M., et al. (2020). Advancing developmental science via unmoderated remote research with children. J. Cogn. Dev. 21, 477–493. doi: 10.1080/15248372.2020. 1797751 Germine, L., Nakayama, K., Duchaine, B. C., Chabris, C. F., Chatterjee, G., and Wilmer, J. B. (2012). Is the web as good as the lab? comparable performance from web and lab in cognitive/perceptual experiments. Psychon. Bull. Rev. 19, 847–857. doi: 10.3758/s13423-012-0296-9 Sasaki, K., and Yamada, Y. (2019). Crowdsourcing visual perception experiments: a case of contrast threshold. PeerJ 7:e8339. doi: 10.7717/peerj.8339 Henrich, J., Heine, S. J., and Norenzayan, A. (2010). The Weirdest People in the World? (RatSWD Working Paper Series, 139). Berlin: Rat für Sozial- und Wirtschaftsdaten (RatSWD). Sauter, D. A., Eisner, F., Ekman, P., and Scott, S. K. (2010). Cross-cultural recognition of basic emotions through nonverbal emotional vocalizations. Proc. Natl. Acad. Sci. U.S.A. 107, 2408–2412. doi: 10.1073/pnas.0908239106 Hutchison, A., Gerstein, L., and Kasai, M. (2018). A cross-cultural comparison of u.s. and japanese trainees’ emotion-recognition ability. Jpn. Psychol. Res. 60, 63–76. doi: 10.1111/jpr.12182 Sauter, D. A., Panattoni, C., and Happé, F. (2013). Children’s recognition of emotions from vocal cues. Br. J. Dev. Psychol. 31, 97–113. JASP (2018). JASP (Version 0.9) [Computer software]. Available online at: https: //jasp-stats.org (accessed June 21, 2021). Scott, K., Chu, J., and Schulz, L. (2017). Lookit (Part 2): assessing the viability of online development research, results from three case studies. Open Mind 1, 15–29. doi: 10.1162/opmi_a_00001 Kakihana, S., Ando, J., Koyama, M., Iitaka, S., and Sugawara, I. (2009). Cognitive factors relating to the development of early literacy in the Kana syllabary. Jpn. J. Educ. Psychol. 57, 295–308. doi: 10.5926/jjep.57.295 Semmelmann, K., and Weigelt, S. (2017). ACKNOWLEDGMENTS We thank all the participants and staﬀmembers of the National Museum of Emerging Science and Innovation (Miraikan). The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpsyg. 2021.702106/full#supplementary-material FUNDING This work was supported by JSPS KAKENHI (No. 20J01281) and Grant-in-Aid for Scientiﬁc Research on Innovative Areas No. 17H06345 “Construction of the Face-Body Studies in Transcultural Conditions”. August 2021 \| Volume 12 \| Article 702106 11 Online Perceptual Experiment With Children Yamamoto et al. REFERENCES Online psychophysics: reaction time eﬀects in cognitive experiments. Behav. Res. Methods 49, 1241–1260. doi: 10. 3758/s13428-016-0783-4 Kawahara, M., Sauter, D., and Tanaka, A. (2021). Culture shapes emotion perception from faces and voices: changes over development. Cogn. Emot. doi: 10.1080/02699931.2021.1922361 [Epub ahead of print]. Sheskin, M., and Keil, F. (2018). TheChildLab.com: a video chat platform for developmental research. PsyArXiv [Preprint]. doi: 10.31234/osf.io/rn7w5 Kinzler, K. D., Dupoux, E., and Spelke, E. S. (2007). The native language of social cognition. Proc. Natl. Acad. Sci. U.S.A. 104, 12577–12580. doi: 10.1073/pnas. 0705345104 Shin, H., and Ma, W. J. (2016). Crowdsourced single-trial probes of visual working memory for irrelevant features. J. Vis. 16, 1–8. doi: 10.1167/16.5.10 Simcox, T., and Fiez, J. A. (2014). Collecting response times using amazon mechanical turk and adobe ﬂash. Behav. Res. Methods 46, 95–111. doi: 10.3758/ s13428-013-0345-y Klindt, D., Devaine, M., and Daunizeau, J. (2017). Does the way we read others’ mind change over the lifespan? Insights from a massive web poll of cognitive skills from childhood to late adulthood. Cortex 86, 205–215. doi: 10.1016/j. cortex.2016.09.009 Stewart, N., Chandler, J., and Paolacci, G. (2017). Crowdsourcing samples in cognitive science. Trends Cogn. Sci. 21, 736–748. doi: 10.1016/j.tics.2017.0 6.007 Laeng, B., Kiambarua, K. G., Hagen, T., Bochynska, A., Lubell, J., Suzuki, H., et al. (2018). The “face race lightness illusion”: an eﬀect of the eyes and pupils? PLoS One 13:e0201603. doi: 10.1371/journal.pone.02 01603 Tanaka, A., Koizumi, A., Imai, H., Hiramatsu, S., Hiramoto, E., and de Gelder, B. (2010). I feel your voice. Cultural diﬀerences in the multisensory perception of emotion. Psychol. Sci. 21, 1259–1262. doi: 10.1177/0956797610380698 August 2021 \| Volume 12 \| Article 702106 Frontiers in Psychology \| www.frontiersin.org 12 Yamamoto et al. Online Perceptual Experiment With Children Tran, M., Cabral, L., Patel, R., and Cusack, R. (2017). Online recruitment and testing of infants with Mechanical Turk. J. Exp. Child Psychol. 156, 168–178. doi: 10.1016/j.jecp.2016.12.003 Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. j j van den Bergh, D., van Doorn, J., Marsman, M., Draws, T., van Kesteren, E., Derks, K., et al. (2019). A tutorial on conducting and interpreting a bayesian anova in JASP. PsyArXiv [Preprint]. doi: 10.31234/osf.io/spreb. Zhou, H., and Fishbach, A. (2016). The pitfall of experimenting on the web: how unattended selective attrition leads to surprising (yet false) research conclusions. J. Pers. Soc. Psychol. 111, 493–504. doi: 10.1037/pspa0000056 Frontiers in Psychology \| www.frontiersin.org REFERENCES Publisher’s Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their aﬃliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Woods, K. J. P., Siegel, M. H., Traer, J., and McDermott, J. H. (2017). Headphone screening to facilitate web-based auditory experiments. Atten. Percept. Psychophys. 79, 2064–2072. doi: 10.3758/s13414-017-1361-2 Yamamoto, H. W., Kawahara, M., and Tanaka, A. (2020). Audiovisual emotion perception develops diﬀerently from audiovisual phoneme perception during childhood. PLoS One 15:e0234553. doi: 10.1371/journal.pone.023 4553 Copyright © 2021 Yamamoto, Kawahara and Tanaka. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Zhou, H., and Fishbach, A. (2016). The pitfall of experimenting on the web: how unattended selective attrition leads to surprising (yet false) research conclusions. J. Pers. Soc. Psychol. 111, 493–504. doi: 10.1037/pspa0000056 August 2021 \| Volume 12 \| Article 702106 Frontiers in Psychology \| www.frontiersin.org 13
https://openalex.org/W4240541518	https://www.matec-conferences.org/articles/matecconf/pdf/2021/11/matecconf_simpro21_01008.pdf	English	null	Measurement of the occupational risk level specific to workplaces	MATEC web of conferences	2,021	cc-by	3,222	Measurement of the occupational risk level specific to workplaces Angelica-Nicoleta Călămar1, Sorin Simion1, Marius Kovacs1, Alexandru Simion1 1National Institute for Research and Development in Mine Safety and Protection to Explosion – INSEMEX Petroşani, 32-34 G-ral Vasile Milea Street, Postcode: 332047, Petroşani, Hunedoara County, Romania Abstract. Any workplace implies a greater or lesser number of occupational hazards, respectively workers are exposed to occupational risks, putting their health in danger. If we talk about industrial environments (construction, energy, metallurgy, textiles, wood, etc.), these hazards are found to a greater extent, often exceeding the allowable limit, but occupational pollutants such as dust in suspension and microclimate parameters can also be found in offices, whether they are open space or not. In this sense, the current paper reviews some workplaces and quantifies the level of risk to which workers are exposed to show and demonstrate the importance of occupational health prevention in order not to reach further measures, in time, when these occupational diseases occur. The research conducted led to the conclusion that the determined pollutants (dust, gas, microclimate) exceeded the maximum allowable concentration required by national legislation, both at workplaces in various technological flows and in offices. The high concentrations found in offices are caused by staff traffic, large number of employees, as well as the lack of natural ventilation. Corresponding author: angela.calamar@insemex.ro © 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 342, 01008 (2021) UNIVERSITARIA SIMPRO 2021 MATEC Web of Conferences 342, 01008 (2021) UNIVERSITARIA SIMPRO 2021 https://doi.org/10.1051/matecconf/202134201008 1 Introduction Occupational health requires multidisciplinary research to prevent health damage that may result from excessive, acute, or chronic exposure to chemicals. Early detection of harmful exposures can significantly reduce the occurrence of adverse effects by reducing exposure level and by taking precautionary measures. Exposure monitoring is a procedure that consists of routine assessment and interpretation of biological and/or environmental parameters in order to detect possible health risks [1, 2]. Work capacity and workers' health are influenced by nature of work, its regulation, relationships between work teams and structure of jobs. Ensuring the health of workers exposed to occupational hazards is an important goal in increasing the quality of life. The occupational environment in which the worker's activity takes place is characterized by complex interaction of specific physical-chemical factors that can negatively influence the health of employees. These factors (microclimate parameters, toxic substances, MATEC Web of Conferences 342, 01008 (2021) UNIVERSITARIA SIMPRO 2021 https://doi.org/10.1051/matecconf/202134201008 suspended dust, noise, inadequate lighting, etc.) are also called harmful or dangerous factors [3]. suspended dust, noise, inadequate lighting, etc.) are also called harmful or dangerous factors [3]. [ ] Along with the dangerous and harmful factors, working conditions are determined by technological processes, the core business of workers or the type of work. To quantify the level of occupational risk, a number of economic sectors were selected, namely, physical-chemical analysis laboratory, steel plant, automotive and IT activities. Investigation of selected objectives involved gathering information related to work environment, choosing the dominant toxins, choosing the appropriate equipment and working methods, taking samples, analysing and interpreting these samples. Occupational risk assessment is a tool that demonstrates the application of prevention principles at company/enterprise level [4]. This means that any company must anticipate the dangers that can lead to accidents at work or occupational diseases, instead of reacting after such events have taken place. An essential step in implementing a responsible approach to occupational health and safety is occupational risk assessment. 2 Investigating working conditions To investigate a workplace, it is necessary to: have knowledge of the technological process, choose the pollutants, sample assays, analyse and interpret data. To quantify the level of pollution of workplace atmosphere, the measured concentrations are related to the legislation in force, respectively GD 359/2015 and GD 157/2020 [3]. In case of simultaneous presence of several noxious substances, their action and interaction will be taken into account both from an analytical point of view and especially in terms of their action upon the human body (synergism by addition or potentiation, antagonism, etc.) [5, 6]. In choosing the noxious substances, both the raw material used in technological flows and possible impurities that it may contain, were taken into account. Exact knowledge of the noxious substances action upon humans, as well as their virulence are very important things, because the noxious substances collecting system and the method of determination chosen can largely depend on them. In the industrial environment, the main way toxic substances penetrate the body is the respiratory tract (80 - 90% of occupational poisonings occur in this way), followed by transcutaneous and gastrointestinal paths [5, 7, 8]. In work places, the microclimate is influenced by regional climate and its variations, but it depends, first of all, on the heat sources, the heating and the ventilation found inside. Microclimate conditions in workplaces must ensure the maintenance of human body’s thermal balance, in accordance with the activity carried out. Unfavourable microclimate is defined as the set of microclimate factors whose combined action exceeds the adaptive capacity of the body, overloading the thermoregulation system in order to maintain thermal balance. Strains upon the thermoregulation system generate thermal stress [9, 10]. 3 Methodology for determining occupational hazards Determination of occupational hazards and microclimate parameters in the work environment was performed in accordance with test procedures of the Toxicology Laboratory within INCD INSEMEX, authorized by the Ministry of Health, Public Health and Public Health Control. Determination of respirable dusts in the work environment is performed gravimetrically, according to SR EN 689/2018 and STAS 10813/76. The principle consists in retaining dust 2 https://doi.org/10.1051/matecconf/202134201008 MATEC Web of Conferences 342, 01008 (2021) UNIVERSITARIA SIMPRO 2021 UNIVERSITARIA SIMPRO 2021 on an FM membrane filter by passing a determined volume of air. The sampling assembly retains coarse dust from total dust, allowing the breathable fraction to pass through the filter. Fig. 1. Sampling probes (air flow meters) type TUFF Fig. 1. Sampling probes (air flow meters) type TUFF Fig. 2. AMI 300 device for determining microclimate parameters Fig. 2. AMI 300 device for determining microclimate parameters Fig. 2. AMI 300 device for determining microclimate parameters Fig. 2. AMI 300 device for determining microclimate parameters Fig. 2. AMI 300 device for Fig. 1. Sampling probes (air flow meters) type TUFF Fig. 2. AMI 300 device for determining microclimate parameters g determining microclimate parameters For performing noise tests, the integrating sound level meter type 2250 was used, with 4189 type microphone and weighting networks A, C and Z. To determine the weighted sound exposure A and/or the equivalent continuous A-weighted sound pressure level, the measurements were performed with the microphone placed in the position normally occupied by the worker's head. To determine the microclimate parameters, the AMI 300 device (Fig. 2.) from INCD INSEMEX Petrosani was used, which is a multifunctional instrument for measuring temperature (in the range of -20 ÷ +800C), humidity (in the range of 3 ÷ 98%) and air velocity (in the range of 0.2 ÷ 30 m/s), and measuring gaseous pollutants, both portable gas analysers with electrochemical sensors (MSA ORION plus) and the Optima 2100 DV inductive coupled plasma atomic emission spectrometer were used. 4 Results and discussion During 2019, the team of the Toxicology Laboratory within INCD INSEMEX carried out several measurement campaigns (table 1.) at different economic agents, having different objects of activity. The current paper summarizes results found in several economic sectors (physical-chemical analysis laboratory, steel plant, automotive and IT activities) regarding the determination of pollutants in workplaces, to emphasize the importance of annually checks performed for these pollutants, so that employers are be able to take measures prevent or reduce noxae, where appropriate. 3 https://doi.org/10.1051/matecconf/202134201008 MATEC Web of Conferences 342, 01008 (2021) UNIVERSITARIA SIMPRO 2021 Table 1. Determination of occupational hazards in the work environment N o. Economi c sector Work area / technologi cal operation Determi ned noxae Measuri ng unit Average determined concentrati on Max. allowable concentrat ion according to GD 157/2020, GD 493/2006 Observati ons 15 mi n. 8 ore 1. Steel plant Electrical furnace Breathabl e dust in suspensio n [mg / m3] - 13,8 1 5 The activity takes place in closed halls without the possibility of optimal retention / exhaust of dust generated by technologi cal processes. Pot preparatio n cast [mg / m3] - 6,41 5 Crane cabin rolling bridge [mg / m3] - 8,16 5 Flue Gas Capture and Treatment Plant [mg / m3] - 8,59 5 Electrical furnace Noise dB(A) - 103, 4 87 Pot preparatio n cast dB(A) - 89,8 87 Crane cabin rolling bridge dB(A) - 83,3 75 Flue Gas Capture and Treatment Plant dB(A) - 87,7 87 Electrical furnace CO [ppm] 29 - 26 The activity of the evaluated Sections is carried out in closed halls without the possibility of an optimal retention / exhaust of CO2 [ppm] 642 0 - 5000 Ferric oxide [mg / m3] 7 - 10 Pot preparatio n cast CO [ppm] 10 - 26 CO2 [ppm] 540 - 5000 Ferric oxide [mg / m3] 1,2 - 10 Flame cutting machine CO [ppm] 14 - 26 CO2 [ppm] 542 - 5000 Ferric oxide [mg / m3] 7,6 - 10 Distributio n room CO [ppm] 2 - 26 CO2 [ppm] 479 - 5000 IVERSITARIA SIMPRO 2021 4 https://doi.org/10.1051/matecconf/202134201008 MATEC Web of Conferences 342, 01008 (2021) UNIVERSITARIA SIMPRO 2021 Ferric oxide [mg / m3] 4,2 - 10 the gases generated by the technologi cal processes. 2. MATEC Web of Conferences 342, 01008 (2021) UNIVERSITARIA SIMPRO 2021 MATEC Web of Conferences 342, 01008 (2021) UNIVERSITARIA SIMPRO 2021 Conferenc e room Air temperatu re [0C] - 23,3 - Central air conditioni ng system Radiant average temperatu re [0C] - 23,9 8 Relative humidity [%] - 38,2 Air flow speed [m/s] - 0,01 4 Kindergart en Air temperatu re [0C] - 19,1 5 - Individual air conditioni ng system (AC) Radiant average temperatu re [0C] - 20,4 8 Relative humidity [%] - 46,9 5 Air flow speed [m/s] - 0,00 6 nalysis of results (table 1.) showed that in the steel plant activity sector, the maximum allowed limits for dust, noise and gas (CO and CO2) are exceeded only in the electric furnace. In the activity carried out in the automotive section, there is a single exceeding of the noise level for the 80t press, and in the physical-chemical analysis laboratory there are exceedances for Pb and some volatile compounds. Regarding the IT activity carried out in open space offices, values of dust in suspension concentration exceed the limit set by in force legislation, by 1.4 mg / m3. According to the website of the European Agency for Safety and Health at Work (EU- OSHA) regarding the limits for microclimate parameters, it is recommended that during warm periods of the year (when air cooling is required) temperature reaches 23-260C, and in cold periods of the year (when the air needs to be heated) the temperature should be in the range of 20-240C. Also, relative humidity of the air is recommended to be in the range of 30- 70% (avoiding, as much as possible, the limits of the range), and speed of the air currents in the room to be between 0.01 - 0.2 THX. PMV (Predicted mean vote) and PPD (Predicted percentage dissatisfied) calculation indices were used to interpret data obtained from IT activities, and for this it was necessary to estimate the metabolic rate and clothing isolation indices: - the metabolic rate index was estimated at 1.2, which represents the specific value office activity (offices, schools, laboratories, housing); - the insulation index of daily clothing was estimated at 1, which represents the specific value for clothing: shoes, trousers, blouse or shirt, socks and underwear to which 0.1 is added, represented by the index of the office chair. 4 Results and discussion Automot ive Sandblasti ng section Breathabl e dust in suspensio n [mg / m3] - 3,25 5 Activity carried out in halls, with permanentl y controlled temperatur e and humidity Injection moulding machine [mg / m3] - 1,33 5 Eccentric press 125 t Noise dB(A) - 79,9 87 Eccentric press 80 t dB(A) - 88,0 87 Hydraulic press 60 t dB(A) - 72,8 87 3. Physical- chemical analysis laborator y Physical- chemical analysis laboratory HCl [mg / m3] 4,96 - 8 Industrial ventilation system NO2 [mg / m3] 7,04 - 5 Furnace room Pb [mg / m3] - 0,32 9 0,15 Weighing room - Document ary tests Pb [mg / m3] - 0,46 8 0,15 4. Carpentr y worksho p - wood parts treatment Oiling wood parts Toluene [mg / m3] 458 - 384 Inefficient ventilation system Xylene [mg / m3] 371 - 442 Acetone [mg / m3] 121 0 - 1210 Varnishing room Toluene [mg / m3] 96 - 384 Xylene [mg / m3] 266 - 442 Acetone [mg / m3] 285 4 - 1210 Ethyl alcohol [mg / m3] 652 - 9500 5. IT activities Offices (open space) Breathabl e dust in suspensio n [mg / m3] - 6,40 5 Central air conditioni ng system Air temperatu re [0C] - 23,9 5 - Radiant average temperatu re [0C] - 26,6 8 Relative humidity [%] - 39,2 5 Air flow speed [m/s] - 0,18 9 VERSITARIA SIMPRO 2021 5 https://doi.org/10.1051/matecconf/202134201008 MATEC Web of Conferences 342, 01008 (2021) UNIVERSITARIA SIMPRO 2021 - the insulation index of daily clothing was estimated at 1, which represents the specific value for clothing: shoes, trousers, blouse or shirt, socks and underwear to which 0.1 is added, represented by the index of the office chair. In kindergarten, the calculated value of PPD is 6.28% (fig. 3.), which represents the percentage of people dissatisfied with microclimate parameters of the work environment, and the PMV value indicates a "cool" work environment. In kindergarten, the calculated value of PPD is 6.28% (fig. 3.), which represents the percentage of people dissatisfied with microclimate parameters of the work environment, and the PMV value indicates a "cool" work environment. 6 6 MATEC Web of Conferences 342, 01008 (2021) UNIVERSITARIA SIMPRO 2021 https://doi.org/10.1051/matecconf/202134201008 Fig.3. Graphic representation of the PMV and PPD index in kindergarten Fig.3. Graphic representation of the PMV and PPD index in kindergarten Therefore, the analysed work environments are in line with the activities carried out (low energy consumption activities - office work and children's playgrounds), keeping a thermal balance between the internal heat production of the human body and heat loss in the work environment. In conclusion, in the work environment where microclimate parameters were determined, 6.28% of the employed staff could be dissatisfied with the microclimate conditions, which proves the microclimate is favourable for development of office activities and education of children, but to increase the PMV index to a warmer environment it is recommended to increase the temperature in the work environment. For a full understanding of the specific terminology in accordance with SR EN ISO 7730: 2006, the terms PPD and PMV are defined below. PMV (Predicted mean vote) - is an index that predicts the average value of the votes of a large group of people on the thermal sensation scale. PPD (Predicted percentage dissatisfied) - is an index that establishes a quantitative prediction of the percentage of thermally dissatisfied people who feel the work environment is too cold or too hot. Conclusions Concentrations of noxious substances (dust, gas, noise) from the analysed workplaces exceed the limit allowed by the legislation in force, especially in the steel sector. Also, values of respirable dust concentration in the IT sector - offices, exceed the maximum limit allowed by the applicable legislation. In this case, a number of technical and organizational measures are recommended, including replacement of existing carpeting in the workspace with parquet or other flooring that does not retain dust, installation of ventilation systems so as to ensure efficient ventilation, periodic replacement of filters in the air conditioning system. Environmental conditions (temperature, humidity, pressure, thermal environment and thermal stress) have a significant influence on work performance. Analysis of results according to the thermal sensation felt by workers (according to SR EN ISO 7730: 2006), shows that in most workplaces it is “warm / warmish”, except for the Kindergarten, where a “cool” thermal sensation is felt. In order to reduce the percentage of people who are dissatisfied, from a thermal point of view, it is recommended to reduce temperature and increase humidity in work spaces where the thermal environment is described as “warm”. Also, at the Kindergarten location where a “cool” thermal sensation is felt, it is recommended to increase temperature to an approximate value of 23 ÷ 260C. MATEC Web of Conferences 342, 01008 (2021) UNIVERSITARIA SIMPRO 2021 https://doi.org/10.1051/matecconf/202134201008 The case study showed that even in office work (multinational companies), there may be concentrations of noxious substances, especially dust, which exceed the allowable limit, creating discomfort for workers and affecting their health over time. The risk assessment may include a justification by the employer that nature and extent of risks due to chemical agents do not require another detailed risk assessment, if occupational exposure limit values or national biological limit values are not exceeded. p g The risk assessment and implicitly the determination of noxious substances are updated, in particular if there have been significant changes in the working conditions or when results of workers’ health status surveillance make this necessary. 7 7 References 1. L. Cioca, R. Moraru, Occupational psychosocial risk management, “Lucian Blaga” University of Sibiu Publishing House, ISBN 978-973-739-924-3, (2010) 2. A. N. Calamar, L. Toth, D. Pupazan, Environmental protection and risk factor analysis, Focus Publishing House, Petrosani, ISBN 978-973-677-313-6, (2016) 3. DECISION no. 157 of February 27th, 2020 for the amendment of the Government Decision no. 1,218 / 2006 regarding the establishment of minimum occupational safety and health requirements for ensuring protection of workers against risks related to presence of chemical agents, as well as for modification and completion of the Government Decision no. 1,093 / 2006 on the establishment of minimum safety and health requirements for protection of workers from risks related to exposure to carcinogens or mutagens at work. 4. A. N. Calamar, G.A. Gaman, L. Toth; D. Pupazan, S. Simion, Assessment of workers` occupational exposure in the context of industrial toxicology, World multidisciplinary earth sciences symposium WMESS 2016, Prague, Czech Republic, September, Vol.44, Article number: 032004, ISSN 1755-1307, (2016) 5. Guide General aspects of industrial toxicology. Methods of analysis used in industrial toxicology, Ministry of Health - National Institute of Public Health, (2013) 6. R.I. Moraru, Safety and health at work: university treaty, Focus Publishing House, Petrosani, ISBN 978-973-677-272-6, (2013) 7. M. Kovacs, L. Toth, G. Ghetie, A. N. Draghici, V. Traian, L., Gheorghe Best management practices applied to prevent and reduce concentrations of dust and gases released from power plants, Environmental Engineering and Management Journal, July, vol.13, No.6, pg.1421-1458, ISSN 1582-9596, (2014) R. Stanescu Dumitru, Harmful agents in the professional environment, Romanian Medical Life Publishing House - Bucharest, ISBN 978-973-160-013-0, (2008) 9. C. Lupu, A.N. Draghici, G. Ghetie, M. Kovacs, L. Toth, C. Ilie, The influence of microclimate parameters in workplace, Bulletin of the Polytechnic Institute of Iasi "Gheorghe Asachi" Technical University of Iasi, Fasc. 4, Ed. Polithnium, ISSN 1453-1690, (2013) 10. M. Nakielska, K. Pawlowski, Conditions of the Internal Microclimate in the Museum, Journal of Ecological Engineering, Vol. 21, Issue: 1, pg.205-209, (2020) 8 8
https://openalex.org/W3122389018	https://www.preprints.org/manuscript/201805.0142/v1/download	English	null	A Review Paper on “Graphene Field Emission for Electron Microscopy”	Applied sciences	2,018	cc-by	10,855	Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 9 May 2018 doi:10.20944/preprints201805.0142 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 9 May 2018 doi:10.20944/preprints201805.0142 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 9 May 2018 doi:10.20944/preprints201805.0142.v1 Review 1 A Review Paper on “Graphene Field Emission for 2 Electron Microscopy” 3 Xiuyuan Shao 1 and Anjam Khursheed 1,2,* 4 1 Department of Electrical and Computer Engineering, National University of Singapore, 4 Engineering 5 Drive 3, Singapore 117583, Singapore 6 2 Engineering Science Programme, National University of Singapore, 9 Engineering Drive 1, Singapore 7 117575, Singapore 8 * Correspondence: eleka@nus.edu.sg (A.K.); Tel.: +65-6516-2295 (A.K.) 9 10 Abstract: Although good field emission from graphene has been demonstrated from a wide variety 11 of different micro-fabricated structures, very few of them can be used to improve the design of cold 12 field emitters for electron microscopy applications. Most of them consist of densely packed nano- 13 emitters, which produce a large array of defocused overlapping electron beams, and therefore 14 cannot be subsequently defocused down to a single nano-meter electron probe. This paper reviews 15 the kind of single-tip cathode structures suitable in cold field emission guns for instruments such as 16 the Scanning Electron Microscopy, Transmission Electron Microscope or the Scanning Transmission 17 Electron Microscopy, and reviews progress in fabricating them from graphene based materials. 18 Keywords: graphene; cold field emission; single-tip cathode; electron microscopy 19 20 1. Introduction 21 The Dyke group proposed a variety of applications of a cold field emission (CFE) source and 22 formed the first company to produce CFE based commercial products in the early 1960s [1]. The 23 successful application of CFE cathodes to electron microscopy, including both Scanning Electron 24 Microscopy (SEM) and Scanning Transmission Electron Microscopy (STEM), was achieved by a 25 group led by Crewe et al. in the late 1960s [2,3]. For over 40 years, state-of-the-art metal tip CFE 26 sources used in electron microscopy suffer from many well-known practical challenges, such as an 27 inherent Ultrahigh Vacuum (UHV) condition requirement (<10-9 Torr) and relatively large current 28 instabilities (~40% fall-off within the first hour), which have impeded their wide-ranging use [4]. As 29 a result, most of the latest generation electron microscopes use the more stable and reliable Schottky 30 electron source, which has a lower brightness (~108 A m-2 sr-1 V-1) and higher energy spread (~0.5 eV) 31 [5-7]. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 9 May 2018 doi:10.20944/preprints201805.0142 Although CFE sources have the desirable characteristics of a low energy spread (~0.3 eV) and 32 high brightness (~109 A m-2 sr-1 V-1), their lifetimes need to be improved (> 1 year) and their current 33 stabilities need to be reduced (< 1%) [4,5,8]. 34 Nanostructures in the form of nanotubes [9-12], nanotips [13], and nanowires [14,15] have the 35 potential to be used as cathodes in CFE sources. Their large field enhancement factors stem from 36 nanometer-scale emission sites which can, in some cases, produce electron beams with brightness 37 values, one order of magnitude higher than the state-of-the-art single crystal tungsten cathode tips. 38 Despite their promising electron optics performance, they have not been successfully integrated into 39 any commercial electron microscopes, and this is largely due to the same technological difficulties 40 faced by single crystal tungsten cathode-tips (stringent vacuum, low lifetime and high beam current 41 fluctuations). However, in addition, they require the delicate transfer/mounting process of an 42 individual emitter being attached to the tip of a sharpened supporting metal wire, and this is very 43 difficult do without incurring significant misalignment or damage. 44 Review 1 A Review Paper on “Graphene Field Emission for 2 Electron Microscopy” 3 Xiuyuan Shao 1 and Anjam Khursheed 1,2,* 4 1 Department of Electrical and Computer Engineering, National University of Singapore, 4 Engineering 5 Drive 3, Singapore 117583, Singapore 6 2 Engineering Science Programme, National University of Singapore, 9 Engineering Drive 1, Singapore 7 117575, Singapore 8 * Correspondence: eleka@nus.edu.sg (A.K.); Tel.: +65-6516-2295 (A.K.) 9 10 Abstract: Although good field emission from graphene has been demonstrated from a wide variety 11 of different micro-fabricated structures, very few of them can be used to improve the design of cold 12 field emitters for electron microscopy applications. Most of them consist of densely packed nano- 13 emitters, which produce a large array of defocused overlapping electron beams, and therefore 14 cannot be subsequently defocused down to a single nano-meter electron probe. This paper reviews 15 the kind of single-tip cathode structures suitable in cold field emission guns for instruments such as 16 the Scanning Electron Microscopy, Transmission Electron Microscope or the Scanning Transmission 17 Electron Microscopy, and reviews progress in fabricating them from graphene based materials. 18 Keywords: graphene; cold field emission; single-tip cathode; electron microscopy 19 20 1. Introduction 21 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 9 May 2018 doi:10.20944/preprints201805.0142.v1 Since the discovery of two-dimensional (2D) graphene in 2004, graphene has attracted a lot of attention as a potential candidate for CFE source emitters, due to its high aspect ratio (the lateral size to the thickness) [16-18] and excellent thermal, mechanical, and electrical properties [19-22]. Excellent field emission from graphene has been demonstrated from a wide variety of different micro- fabricated structures, and most of them consist of densely packed nano-emitters that produce a large array of defocused overlapping electron beams, and therefore cannot be subsequently defocused down to a single nano-meter electron probe [23-25], making them unsuitable for electron microscopy/lithography applications. py g p y pp This paper primarily focuses on reviewing developments in the field emission of graphene based 53 emitters for electron microscopy/lithography applications. In the first part of the paper, it will 54 summarize basic field emission theory in relation to single crystal W field emitters (CFE) oriented in 55 the <310> or <111> directions and other well-developed one-dimensional (1D) nanostructure CFE 56 electron emitters. In the second part, different types of graphene field emitters will be introduced, 57 including graphene film emitters, graphene point emitters and graphene ring emitters. The main 58 theme is primarily on how graphene can be used to make single-tip cathodes in electron sources 59 suitable for electron microscopy/lithography applications, whose electron beams can be defocused 60 down to a single nano-meter electron probe. Finally, a summary and outlook are given towards the 61 future development of high brightness-high resolution CFE electron gun on graphene-based 62 nanomaterials. 63 1. Introduction 21 The Dyke group proposed a variety of applications of a cold field emission (CFE) source and 22 formed the first company to produce CFE based commercial products in the early 1960s [1]. The 23 successful application of CFE cathodes to electron microscopy, including both Scanning Electron 24 Microscopy (SEM) and Scanning Transmission Electron Microscopy (STEM), was achieved by a 25 group led by Crewe et al. in the late 1960s [2,3]. For over 40 years, state-of-the-art metal tip CFE 26 sources used in electron microscopy suffer from many well-known practical challenges, such as an 27 inherent Ultrahigh Vacuum (UHV) condition requirement (<10-9 Torr) and relatively large current 28 instabilities (~40% fall-off within the first hour), which have impeded their wide-ranging use [4]. As 29 a result, most of the latest generation electron microscopes use the more stable and reliable Schottky 30 electron source, which has a lower brightness (~108 A m-2 sr-1 V-1) and higher energy spread (~0.5 eV) 31 [5-7]. Although CFE sources have the desirable characteristics of a low energy spread (~0.3 eV) and 32 high brightness (~109 A m-2 sr-1 V-1), their lifetimes need to be improved (> 1 year) and their current 33 stabilities need to be reduced (< 1%) [4,5,8]. 34 Nanostructures in the form of nanotubes [9-12], nanotips [13], and nanowires [14,15] have the 35 potential to be used as cathodes in CFE sources. Their large field enhancement factors stem from 36 nanometer-scale emission sites which can, in some cases, produce electron beams with brightness 37 values, one order of magnitude higher than the state-of-the-art single crystal tungsten cathode tips. 38 Despite their promising electron optics performance, they have not been successfully integrated into 39 any commercial electron microscopes, and this is largely due to the same technological difficulties 40 faced by single crystal tungsten cathode-tips (stringent vacuum, low lifetime and high beam current 41 fluctuations). However, in addition, they require the delicate transfer/mounting process of an 42 individual emitter being attached to the tip of a sharpened supporting metal wire, and this is very 43 difficult do without incurring significant misalignment or damage. 44 2.1. Theory of cold field electron emission 65 derived an analytical formula to calculate the 94 virtual source size [34], given by the following expression: 95 ݀୴= 1.67 ௥౪౟౦ ௠ഀට ழா౪வ ௘௏౛౮౪ , (3) (3) where ݎ୲୧୮ is the tip radius and < ܧ୲> is an initial average tangential energy defined as < ܧ୲> 97 = ݁ħܨ/√(8݉߶), with ܨ the local electric field strength, ϕ the work function, ݉ఈ the angular 98 magnification and ħ the reduced Planck constant. In these formulas, ݉ఈ and F can be estimated from 99 direct ray tracing simulations and when used together with an experimentally measured angular 100 intensity profile, the source brightness Br can be estimated. For high brightness electron beam 101 applications, statistical Coulomb interactions lead to radial broadening of the virtual source size due 102 to lateral electron-electron interactions, which in turn, lower the brightness [35]. The calculation of 103 statistical Coulomb interactions within an electron source is not straightforward, since the electron 104 potential and beam size vary in the gun region. A semi-analytical technique known as the “slice 105 method” reported in the recent papers by Kruit, was introduced to do this [36]. First, the electric 106 potential distribution in the gun region is solved numerically, and then the beam size in the region is 107 determined by the simulated direct ray tracing of electron trajectories that leave the cathode-tip. The 108 gun region is subsequently divided into small segments over which the voltage and beam size is 109 assumed to remain constant, and an analytical expression is used to estimate the trajectory 110 displacement effect due to the lateral electron-electron interactions. 111 It should be noted that the method just outlined avoids the need to measure the virtual source 112 diameter dv experimentally, this is because direct measurement of the virtual source diameter not 113 trivial. One way to achieve it is to put the source in a transmission electron microscope as the emitter 114 and obtain a magnified source image of it [7]. A different method to measure dv is to use a 115 demagnifying lens to focus the electron beam from the gun unit into a spot which is source-size 116 dominated (relatively low contributions from lens aberrations), and then measure the spot size of the 117 beam by scanning it across a knife edge [37,38]. 2.1. Theory of cold field electron emission 65 derived an analytical formula to calculate the 94 virtual source size [34], given by the following expression: 95 ݀୴= 1.67 ௥౪౟౦ ௠ഀට ழா౪வ ௘௏౛౮౪ , (3) 96 where ݎ୲୧୮ is the tip radius and < ܧ୲> is an initial average tangential energy defined as < ܧ୲> 97 = ݁ħܨ/√(8݉߶), with ܨ the local electric field strength, ϕ the work function, ݉ఈ the angular 98 magnification and ħ the reduced Planck constant. In these formulas, ݉ఈ and F can be estimated from 99 direct ray tracing simulations and when used together with an experimentally measured angular 100 intensity profile, the source brightness Br can be estimated. For high brightness electron beam 101 applications, statistical Coulomb interactions lead to radial broadening of the virtual source size due 102 to lateral electron-electron interactions, which in turn, lower the brightness [35]. The calculation of 103 statistical Coulomb interactions within an electron source is not straightforward, since the electron 104 potential and beam size vary in the gun region. A semi-analytical technique known as the “slice 105 method” reported in the recent papers by Kruit, was introduced to do this [36]. First, the electric 106 potential distribution in the gun region is solved numerically, and then the beam size in the region is 107 determined by the simulated direct ray tracing of electron trajectories that leave the cathode-tip. The 108 gun region is subsequently divided into small segments over which the voltage and beam size is 109 assumed to remain constant, and an analytical expression is used to estimate the trajectory 110 displacement effect due to the lateral electron-electron interactions. 111 ܤ୰= ସூᇲ ஠ௗ౬మ௏౛౮౪ , (2) 92 (2) where ܫᇱ is the angular intensity, obtained by dividing the emission current over the acceptance 93 solid angle subtending the source. Bronsgeest et al. derived an analytical formula to calculate the 94 virtual source size [34], given by the following expression: 95 where ܫᇱ is the angular intensity, obtained by dividing the emission current over the acceptance 93 solid angle subtending the source. Bronsgeest et al. 2.1. Theory of cold field electron emission 65 Another method is using a point projection 118 microscope, and this technique for measuring dv is to place a knife edge at a small distance (a few 119 micrometers) from an emitter and to measure the angular width of the Fresnel fringes patterns 120 [12,39]. In this technique, the intensity distribution shape of the source is assumed to be a Gaussian 121 function, and the values of magnification and electron wavelength are needed to extract dv. 122 Another important electron gun parameter, apart from brightness, is its energy spread. The 3 energy spread, through the chromatic aberration of the objective lens, degrades the final spatial 4 resolution of an electron optical system, particularly at high relative beam energy spreads (say higher 5 than 10-4). Ideally, the energy distribution (∆ܧ) of the emitted electron beam should be as narrow as 6 possible while maintaining sufficient brightness. From the theoretical point of view, the total energy 7 distribution (TED) of electron emission in the thermal field regime was first derived by Young based 8 on the free-electron model as [40]: 9 ܲ(ܧ) = ௃ಷಿ ௗ൥ ௘௫௣൬ ಶషಶ೑ ೏ ൰ ଵା௘௫௣൬ ಶషಶ೑ ೖ೅൰ ൩ , (4) 2.1. Theory of cold field electron emission 65 Electron sources are an essential tool for investigation into a broad range of field emission 66 devices [26-30]. For electron microscopy applications, electron sources can be divided into two main 67 categories, known as thermionic and field emission [31]. A thermionic electron gun uses Joule-heating 68 induced thermal excitation to allow electrons to overcome the energy barrier between the metal tip 69 and the vacuum. The electron beam it produces is inherently incoherent, has a source size in the tens 70 of microns range, a relatively large energy spread (2 eV) and poor brightness (~106 A m-2 sr-1 V-1). A 71 CFE electron gun overcomes these limitations by decreasing the virtual source size to about 3 nm (by 72 significantly decreasing the emission tip to about 100 nm radius) and a low energy spread to about 73 0.3 eV (operating the source at room temperature), in which electrons pass from a solid surface to the 74 vacuum by creating a high local electric field [1]. The mechanism of the electron emission process is 75 based on the Fermi-Dirac distribution for a free electron gas in the potential energy barrier between 76 the metal tip and the vacuum [32]. 77 Fowler and Nordheim first found a classical theory describing the behavior of field emission 78 currents from a metallic surface in 1928 [33]. In the theory, the relationship between the emission 79 current (I) and the tip field strength (F) is expressed as, 80 ܫ= ܣ ଵ.ହ×ଵ଴షల థ ܨଶexp (− ଺.ସସ×ଵ଴వథభ.ఱ ி ) , (1) 81 (1) For successful electron microscopy applications, a high source brightness (Br) and a low energy 89 spread (∆ܧ) are always needed to provide a high-resolution focused electron beam. Calculation of Br 90 requires knowledge of the virtual source size (dv), which is given by the following expression [34]: 91 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 9 May 2018 doi:10.20944/preprints201805.0142.v1 doi:10.20944/preprints201805.0142.v1 ܤ୰= ସூᇲ ஠ௗ౬మ௏౛౮౪ , (2) 92 where ܫᇱ is the angular intensity, obtained by dividing the emission current over the acceptance 93 solid angle subtending the source. Bronsgeest et al. 130 In particular, 159 their use as potential electron field emitters exhibit excellent geometric, electrical and mechanical 160 properties, as compared to conventional metallic cold field emitters [43-46]. However, there are also 161 significant technical challenges that have yet to be resolved. 162 Generally, preparation of an individual nanoscopic CFE electron emitter is an extremely labor- 163 intensive delicate process. To mount a single nanowire or nanotube onto a supporting tip, the most 164 common method is to use microscopic manipulators under the observation of an electron microscope. 165 Fig. 2a and 2c show a single LaB6 nanowire [15] and a single CNT nanotube [9] mounted on a 166 sharpened W tip, respectively. The ends of the emitter were deposited with platinum (or carbon) by 167 electron beam induced deposition (EBID) in order to make good thermal contact and the final emitter 168 structure is fitted into an electron gun unit is shown in Fig. 2b, and its inset. Figs. 2d and 2e show the 169 mounting procedure for a carbon cone nanotip (CCnT) [13], where a CCnT was welded onto the apex 170 of the supporting tip by using a gas injecting system. The whole process has a very high probability 171 of incurring misalignment and damage 172 These nanoscale point-cathode emitters also have the limitation that they need to be operated in 173 an UHV level, in the 10-11 to 10-10 Torr range. For a LaB6 point-cathode emitter with a dimeter of nearly 174 60 nm, a maximum reduced brightness (Br) of 2.77×1011 A m-2 sr-1 V-1 and energy spread value of 0.367 175 eV have been reported in a vacuum with a base pressure of 10-10 Torr. The short-term stability profile 176 of the LaB6 point-cathode is 0.32% over 60 seconds, and the emission current deviates from the mean 177 within 5% throughout a week-long test. For the CNT and CCnT point-cathode emitters with 178 diameters of around 10 nm, the source electron optics parameters were reported to be 8.2×109 A m-2 179 148 Figure 1. SEM photos of the W<310> (a) and W<111> (b) CFE sources. (c) The source reduced 149 brightness Br versus the corresponding FWHM(E) values for the two CFE sources. Reproduced with 150 permission from [4], Copyright Elsevier, 2009. 151 Figure 1. SEM photos of the W<310> (a) and W<111> (b) CFE sources. 130 (c) The source reduced brightness Br versus the corresponding FWHM(E) values for the two CFE sources. Reproduced with permission from [4], Copyright Elsevier, 2009. The source reduced brightness (Br) versus the FWHM(E) values for the W<310> and W<111> 152 sources are plotted in Fig 1c. For the state-of-the-art W<310> field emitter, a reduced brightness value 153 of 4×109 A m-2 sr-1 V-1 has been reported, and the measured energy spread ranges from 0.33 eV to 0.48 154 eV [4]. The relatively large values of Br mainly come from the small values of virtual source size dv 155 (~3 nm) and the large values of angular current density (~100 µA/sr). 156 Nanostructures in the form of 1D Nanowires, Nanotubes, and Nanocones have attracted much attention in past decades and have been demonstrated for many potential applications. In particular, their use as potential electron field emitters exhibit excellent geometric, electrical and mechanical properties, as compared to conventional metallic cold field emitters [43-46]. However, there are also significant technical challenges that have yet to be resolved. Generally, preparation of an individual nanoscopic CFE electron emitter is an extremely labor- 163 intensive delicate process. To mount a single nanowire or nanotube onto a supporting tip, the most 164 common method is to use microscopic manipulators under the observation of an electron microscope. 165 Fig. 2a and 2c show a single LaB6 nanowire [15] and a single CNT nanotube [9] mounted on a 166 sharpened W tip, respectively. The ends of the emitter were deposited with platinum (or carbon) by 167 electron beam induced deposition (EBID) in order to make good thermal contact and the final emitter 168 structure is fitted into an electron gun unit is shown in Fig. 2b, and its inset. Figs. 2d and 2e show the 169 mounting procedure for a carbon cone nanotip (CCnT) [13], where a CCnT was welded onto the apex 170 of the supporting tip by using a gas injecting system. The whole process has a very high probability 171 of incurring misalignment and damage 172 These nanoscale point-cathode emitters also have the limitation that they need to be operated in an UHV level, in the 10-11 to 10-10 Torr range. 130 According to Knauer’s model of a 136 spherical electric field around an emitter of tip radius rtip, the energy broadening (in eV) due to 137 Coulomb interactions is given by [41]: 138 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 9 May 2018 doi:10.20944/preprints201805.0142 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 9 May 2018 doi:10.20944/preprints201805.0142.v1 ∆ܧ୆୭ୣ୰ୱୡ୦= 15.9 ൫ூᇲ൯మ/య ௥౪౟౦ భ/య௏౛౮౪ భ/య (5) 139 (5) 2.3. The state-of-the-art electron source used in electron microscopes 2.3. The state-of-the-art electron source used in electron microscopes 140 2.3. The state-of-the-art electron source used in electron microscopes 140 The state-of-the-art electron sources used in high-resolution electron microscopy are made from 141 a single crystal W field emitter (CFE) oriented in the <310> direction with a work function of 4.5 eV 142 [4]. Figs. 1a and 1b show typical SEM images of the W<310> and W<111> CFE sources, respectively. 143 As observed in the SEM images, the emitters have a spherical apex with a radius of around 130 nm 144 at the end of the cathode-tip. For a single crystal W field emitter, the emitter axis orientation is 145 required to be <310> or <111> at the emitter apex, due to their lower work function values, and this 146 can be achieved by using a zone melting technique [42]. 147 y g g q 148 Figure 1. SEM photos of the W<310> (a) and W<111> (b) CFE sources. (c) The source reduced 149 brightness Br versus the corresponding FWHM(E) values for the two CFE sources. Reproduced with 150 permission from [4], Copyright Elsevier, 2009. 151 The source reduced brightness (Br) versus the FWHM(E) values for the W<310> and W<111> 152 sources are plotted in Fig 1c. For the state-of-the-art W<310> field emitter, a reduced brightness value 153 of 4×109 A m-2 sr-1 V-1 has been reported, and the measured energy spread ranges from 0.33 eV to 0.48 154 eV [4]. The relatively large values of Br mainly come from the small values of virtual source size dv 155 (~3 nm) and the large values of angular current density (~100 µA/sr). 156 2.4. Nanoscale point-cathode emitters 157 Nanostructures in the form of 1D Nanowires, Nanotubes, and Nanocones have attracted much 158 attention in past decades and have been demonstrated for many potential applications. 130 130 (4) where kT = 0.155 eV at room temperature, JFN is the well-known Fowler-Nordheim emission 131 current density and d is the tunneling parameter given by: d = 9.76×10-11 F/ϕ1/2 t(y). The variable t(y) is 132 a slowly-varying function of y = 3.79×10-5 F1/2/ϕ and can be approximated by the formula t(y) = 1 + 133 0.1107 y1.33. The analytical formula is valid provided kT/d < 0.7 and y < 1. The longitudinal electron- 134 electron interactions (known as Boersch effect) will add to the intrinsic source energy spread which 135 causes an additional enlargement of the energy spread [35]. According to Knauer’s model of a 136 spherical electric field around an emitter of tip radius rtip, the energy broadening (in eV) due to 137 Coulomb interactions is given by [41]: 138 where kT = 0.155 eV at room temperature, JFN is the well-known Fowler-Nordheim emission 131 current density and d is the tunneling parameter given by: d = 9.76×10-11 F/ϕ1/2 t(y). The variable t(y) is 132 a slowly-varying function of y = 3.79×10-5 F1/2/ϕ and can be approximated by the formula t(y) = 1 + 133 0.1107 y1.33. The analytical formula is valid provided kT/d < 0.7 and y < 1. The longitudinal electron- 134 electron interactions (known as Boersch effect) will add to the intrinsic source energy spread which 135 causes an additional enlargement of the energy spread [35]. According to Knauer’s model of a 136 spherical electric field around an emitter of tip radius rtip, the energy broadening (in eV) due to 137 Coulomb interactions is given by [41]: 138 where kT = 0.155 eV at room temperature, JFN is the well-known Fowler-Nordheim emission 131 current density and d is the tunneling parameter given by: d = 9.76×10-11 F/ϕ1/2 t(y). The variable t(y) is 132 a slowly-varying function of y = 3.79×10-5 F1/2/ϕ and can be approximated by the formula t(y) = 1 + 133 0.1107 y1.33. The analytical formula is valid provided kT/d < 0.7 and y < 1. The longitudinal electron- 134 electron interactions (known as Boersch effect) will add to the intrinsic source energy spread which 135 causes an additional enlargement of the energy spread [35]. 130 For a LaB6 point-cathode emitter with a dimeter of nearly 60 nm, a maximum reduced brightness (Br) of 2.77×1011 A m-2 sr-1 V-1 and energy spread value of 0.367 eV have been reported in a vacuum with a base pressure of 10-10 Torr. The short-term stability profile of the LaB6 point-cathode is 0.32% over 60 seconds, and the emission current deviates from the mean within 5% throughout a week-long test. For the CNT and CCnT point-cathode emitters with diameters of around 10 nm, the source electron optics parameters were reported to be 8.2×109 A m-2 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 9 May 2018 doi:10.20944/preprints201805.0142.v1 sr-1 V-1, 0.48 eV, and 1.6×109 A m-2 sr-1 V-1, 0.32 eV respectively. The vacuum pressure requirements 180 are even more stringent for CNTs and CCnT point-cathode emitters, typically around 10-11 Torr. It 181 was found there exists some unrecoverable structure damage to the CNT during electron emission 182 under the high vacuum conditions of 9×10-8 Torr, and this leads to emission reduction or failure [47]. 183 In addition to its inherent UHV requirements, the CNT emitter is reported to have relatively large 184 fluctuations in emission current, e.g. up to 24% for an emission current of about 2 µA over 6 hours 185 with a sampling rate of 3×10-3 Hz. 186 187 Figure 2. Well-developed nanowire, nanotube, and nanocone point-cathode cold field emitters. (a) 188 Illustration showing the electron beam deposition process to fix a LaB6 NW onto a W needle. (b) SEM 189 image showing the finished LaB6 NW emitter and the complete emitter assembly used for field- 190 emission SEM, scale bar, 10 µm. Reproduced with permission from [15], Copyright Springer Nature, 191 2015; (c) TEM image of a very thin and short nanotube sample. Reproduced with permission from [9], 192 Copyright Elsevier, 2003; (d) View of part of the starting CCnT object. (e) Resulting new tip after 193 welding the carbon microfiber to the tungsten tip. The inset displays the whole set-up inside the dual 194 beam. Reproduced with permission from [13], Copyright Elsevier, 2012. 195 Figure 2. Well-developed nanowire, nanotube, and nanocone point-cathode cold field emitters. (a) Illustration showing the electron beam deposition process to fix a LaB6 NW onto a W needle. (b) SEM image showing the finished LaB6 NW emitter and the complete emitter assembly used for field- emission SEM, scale bar, 10 µm. 130 Reproduced with permission from [15], Copyright Springer Nature, 2015; (c) TEM image of a very thin and short nanotube sample. Reproduced with permission from [9], Copyright Elsevier, 2003; (d) View of part of the starting CCnT object. (e) Resulting new tip after welding the carbon microfiber to the tungsten tip. The inset displays the whole set-up inside the dual beam. Reproduced with permission from [13], Copyright Elsevier, 2012. These nanoscale CFE electron sources have not as yet been successfully integrated in any 196 commercial electron microscopes due to the aforementioned significant technical challenges, and 197 studies of nanowire, nanotube, and nanocone based cold field emitters have remained at the research 198 level. Meanwhile, other developments in CFE electron emitter research have come from the use of 199 materials such as graphene. 200 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 9 May 2018 doi:10.20944/preprints201805 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 9 May 2018 doi:10.20944/preprints201805.0142.v1 216 3.1. Graphene film emitters To achieve successful electron emission from graphene, it is crucial to orientate thin graphene 217 edges or control the dimension of graphene protrusions on some supporting substrate, in order to 218 sufficiently enhance the applied electric field to produce quantum tunneling. Until now, many 219 emerging technologies have become available for fabricating vertically aligned graphene nanosheets 220 (VG) or graphene protrusions. The plasma-enhanced chemical vapor deposition (PECVD) is the most 221 widely used way to synthesize large-scale VG graphene films [50], which offers the advantages of a 222 lower substrate temperature and higher growth selectivity (Fig. 3a). Koh et al. presented a pulsed 223 laser deposition (PLD) method [51] to directly fabricate graphene thin film onto metal nano-sized 224 Spindt tips by using the transformation of solid carbon deposited from a pulsed laser system at low 225 temperature (Fig. 3b). The main drawback of these two deposition methods remains the difficulty of 226 controlling the graphene film ordering and density with problems arising due to the screening effect 227 [52,53]. 228 Figure 3. SEM images of microfabricated graphene film emitters. (a) Top view SEM micrograph of as grown FLG by microwave PECVD. Reproduced with permission from [50], Copyright AIP, 2008; (b) Graphene onto metal nano-Spindt tip by pulsed laser deposition. Reproduced with permission from [51], Copyright Wiley, 2014; (c) Graphene films prepared by electrophoretic deposition. Reproduced with permission from [54], Copyright Wiley, 2009; (d) Screen-printed graphene films. Reproduced with permission from [55], Copyright IOP, 2009; (e) Hybrid structures of graphene stretched on patterned and size-controllable Si nanowires. Reproduced with permission from [24], Copyright Springer Nature, 2015. Figure 3. SEM images of microfabricated graphene film emitters. (a) Top view SEM micrograph of as grown FLG by microwave PECVD. Reproduced with permission from [50], Copyright AIP, 2008; (b) Graphene onto metal nano-Spindt tip by pulsed laser deposition. Reproduced with permission from [51], Copyright Wiley, 2014; (c) Graphene films prepared by electrophoretic deposition. Reproduced with permission from [54], Copyright Wiley, 2009; (d) Screen-printed graphene films. Reproduced with permission from [55], Copyright IOP, 2009; (e) Hybrid structures of graphene stretched on patterned and size-controllable Si nanowires. Reproduced with permission from [24], Copyright Springer Nature, 2015. Wu et al. 3. Graphene based cold field emitters 201 Fast development of 2D graphene in recent years has led to a variety of different applications 202 for field emission based devices [19]. Compared with other metallic or 1D nanoscale CFE electron 203 sources, graphene is well known for possessing several desirable properties: (1) it has excellent 204 thermal, mechanical, and electrical characteristics [16,20], (2) its work function can be significantly 205 lowered both by direct contact with metals (effectively doping it), and through the application of 206 intense electric fields [48,49], (3) it is extremely flexible. Graphene CFE electron sources fall into two 207 main categories: the single-tip cathode field nano-emitter (which includes point cathodes and ring 208 cathodes) and graphene film cathodes, which consist of densely packed graphene nano-emitters. 209 Only the former can be used for electron microscopy applications, as the latter produces a large array 210 of defocused overlapping electron virtual sources that cannot be subsequently defocused down to a 211 single nano-sized probe. 212 213 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 9 May 2018 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 9 May 2018 doi:10.20944/preprints201805.0142.v1 not suitable for electron microscopy applications, graphene film emitters are very useful for many 251 non-focused electron beam applications, like flat panel display [27,60], X-ray sources [61-63], etc. 252 3.2. Graphene point-cathode field emitters 253 Until now, graphene point-cathode field emitters for electron microscopy applications have been 254 rarely studied due to the difficulties inherent in controlling graphene morphology. Tsai et al. [64] 255 reported an idea of overlaying an ultra-thin (~1 nm) graphene flake on to a blunted tungsten probe 256 by van der Waals forces, as shown in Fig. 4a, which would make it applicable to single-column 257 electron beam microscopy applications. However, this loosely hanging ultra-thin freestanding 258 graphene flake is not suitable for practical applications, since the graphene can easily be damaged or 259 detached from its supporting probe. More importantly, the emitting surface is not circular, it spans a 260 few micrometers along one direction, and this means it can’t be subsequently defocused down to a 261 single nano-meter electron spot. 262 not suitable for electron microscopy applications, graphene film emitters are very useful for many 251 non-focused electron beam applications, like flat panel display [27,60], X-ray sources [61-63], etc. 252 Until now, graphene point-cathode field emitters for electron microscopy applications have been 254 rarely studied due to the difficulties inherent in controlling graphene morphology. Tsai et al. [64] 255 reported an idea of overlaying an ultra-thin (~1 nm) graphene flake on to a blunted tungsten probe 256 by van der Waals forces, as shown in Fig. 4a, which would make it applicable to single-column 257 electron beam microscopy applications. However, this loosely hanging ultra-thin freestanding 258 graphene flake is not suitable for practical applications, since the graphene can easily be damaged or 259 detached from its supporting probe. More importantly, the emitting surface is not circular, it spans a 260 few micrometers along one direction, and this means it can’t be subsequently defocused down to a 261 single nano-meter electron spot. 262 263 Figure 4. Images of graphene edge field emitters and field emission characteristics. (a) Field emission 264 from individual freestanding graphene edge. Reproduced with permission from [64], Copyright 265 Wiley, 2013; (b) Field emission from atomically thin edges of reduced graphene oxide. Reproduced 266 with permission from [69], Copyright ACS publications, 2011. 267 Figure 4. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 9 May 2018 doi:10.20944/preprints201805 applied the electrophoretic deposition (EPD) technique [54] to fabricate large area 238 homogeneous single-layer graphene films, and this processing technique is more economic and 239 versatile compared to PECVD (Fig. 3b). Screen printing technology is a sophisticated technology 240 widely used in large-scale applications coating on various substrates. A screen-printed graphene 241 cathode was fabricated by Qian et al. [55] using high-yield graphene prepared by a modified 242 Hummers method and a hydrazine hydrate reduction process (Fig. 3d). Another efficient way to 243 produce uniform nanometer-scale graphene protrusions is through transferring graphene onto some 244 sharp supporting substrates (Fig. 3e) [23,24,56]. However, the exfoliated graphene by using either 245 screen printing technologies, or electrophoretic deposition, or simple transfer have been found to 246 lead to many defects to the as-grown graphene, that degrades its physical properties [57-59]. For 247 graphene film emitters, high current densities, around 23 mA cm-2, have been reported with good 248 stability over a period of several hours [54]. Current density here, is calculated by dividing the total 249 measured emission current by the average area on the substrate occupied by emitter sites. Although 250 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 9 May 2018 In doing so, they have overcome many Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 9 May 2018 doi:10.20944/preprints201805.0142.v1 of the practical difficulties associated with conventional metallic tungsten CFE sources, such as 289 misalignment, damage, stringent vacuum requirements, and current instabilities. They reported 290 stable field emission results from a graphene coated nickel wire tip in HV conditions (4×10-8 Torr), 291 where the tip diameter is in the submicron to micron range, over a factor of three times larger than 292 conventional single crystal tungsten tip sizes, sizes that are comparable to the Schottky field emitter 293 tip. 294 Figure 5. Graphene coated point cathode field emitter. (a) SEM images of graphene coated point cathode emitter. Reproduced with permission from [71], Copyright Springer Nature, 2018; (b) Source brightness and energy spread. Reproduced with permission from [71], Copyright Springer Nature, 2018. Figure 5. Graphene coated point cathode field emitter. (a) SEM images of graphene coated point cathode emitter. Reproduced with permission from [71], Copyright Springer Nature, 2018; (b) Source brightness and energy spread. Reproduced with permission from [71], Copyright Springer Nature, 2018. The Graphene-Ni point cathode was fabricated by a two-step process, which consists firstly of 300 an electrochemical etching process to obtain a sharp Ni tip, and secondly, a graphene coating step of 301 chemical vapor deposition (CVD). The Graphene-Ni point cathode was shown to have a maximum 302 emission current of about 5 µA, and the experimental results do not indicate any obvious need for 303 regular flashing (heating) of the cathode. More importantly, experimental results demonstrate that 304 the large diameter Graphene-Ni point cathodes are capable of emitting over a few micro-amps of 305 current stably for over 72 hours, and no emitter failure was observed. In their study, direct ray tracing 306 simulations of electron trajectory paths leaving the emitter surface were carried out to estimate ݉ఈ 307 and F, from which the virtual source size dv and gun brightness Br were then calculated by using Eqs. 308 2 and 3. The source brightness values range from 7×107 A m-2 sr-1 V-1 to 2.51×109 A m-2 sr-1 V-1 for a 309 cathode tip radii range of 130 nm to 800 nm. There is a trade-off between the source brightness value 310 and the tip size. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 9 May 2018 Images of graphene edge field emitters and field emission characteristics. (a) Field emission from individual freestanding graphene edge. Reproduced with permission from [64], Copyright Wiley, 2013; (b) Field emission from atomically thin edges of reduced graphene oxide. Reproduced with permission from [69], Copyright ACS publications, 2011. For most single point field emitters, the emission current is surface sensitive and the adsorption 268 of any gas molecules will increase the surface work function [14,65]. A high emission current under 269 excessive applied voltage ionizes many gas molecules around the emission surface and eventually 270 triggers a vacuum arc. Therefore, the practical use of cold field emitters in electron microscopes 271 typically requires cathode flashing at regular intervals to remove the residual contaminant molecules. 272 Endo et al. [66] reported that CFE electron sources can be cleaned by in situ Joule heating during the 273 emission process in high vacuum (HV) conditions, which is well known as a “conditioning” process 274 in many cold field emission experiments [67-69]. For example, during the first and second cycle of 275 voltage ramping up from the graphene flake overlaid on the tungsten probe (Fig. 4a), the current- 276 applied voltage characteristic presents a typical conditioning process, which is observed to deviate 277 from the conventional FN plot. The third cycle of electron emission shows a well fitted FN plot with 278 an emission current up to 6 µA. Similar work has been done by other researchers studying the field 279 emission characteristics from reduced graphene oxide (Fig. 4b) and carbon nanowalls, and they have 280 obtained high emission current, around several tens of micro-amps [69,70]. However, none of these 281 kinds of emission conditioning experiments on CFE graphene flakes have direct application for 282 electron microscopy, where a single point graphene source is required. 283 Recently, the research group at the National University of Singapore, led by A. Khursheed, have presented the development of a completely new type of cold field emission electron source (Fig. 5a), a Graphene-Ni point cathode for electron microscopy systems [71]. They present a way of overcoming the morphological problems associated with micro-fabricating single-tip graphene emitters, by coating it on to a sharpened wire metal wire tip. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 9 May 2018 doi:10.209 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 9 May 2018 doi:10.20944/preprints201805.0142.v1 3.3. Graphene ring-cathode field emitters 323 Khursheed first proposed the idea of using a ring-cathode emitter for combining high spatial 324 resolution with high beam current for focused electron beam applications in 2013 [73,74]. Khursheed 325 presented electron focused beam designs based upon using ring-cathodes together with off-axis 326 aberration correction techniques. The first step towards a practical implementation of this idea is to 327 develop a ring-cathode emitter. The proposal of a field emission ring-cathode gun unit has the 328 advantage of intrinsically being a large area field emitter, and it is therefore expected to overcome 329 the well-known disadvantage of conventional point-cathode field emitters having relatively low total 330 current (compared to earlier larger cathode electron gun designs). The much larger emission area of 331 a ring-cathode field emitter, compared to a point-cathode field emitter is also expected to provide it 332 with greater current stability. 333 Nguyen et al. [75] reported a versatile and robust method based on graphene growth on Ni 334 templates and thermo-assisted removal of poly(methyl methacrylate) (PMMA) for fabrication of 335 macroscopic and freestanding tubular graphene (TG) architectures. The TG architectures have a 336 diameter of 50 µm with a wall thickness in the range of 2.1-2.9 nm. The individual TG emitter can 337 produce an extremely high emission current, up to 0.46 mA at a very low external applied electric 338 field of 0.68 V µm-1. This most likely comes from ultrathin edges on graphitic walls, which consist of 339 a great number of field emission sites. Another important observation is that the TG CFE electron 340 source has good current stability and the stability is maintainable for days under HV conditions 341 (2.2×10-8 Torr). However, one limitation of TG architectures fabricated by Nguyen et al. is that the 342 edges are very jagged rather than being smooth, making it hard to be directly used as a CFE electron 343 source for electron microscopy applications. Moreover, transfer and alignment of such a large 344 diameter graphene tube is an extremely delicate process. 345 Khursheed first proposed the idea of using a ring-cathode emitter for combining high spatial 324 resolution with high beam current for focused electron beam applications in 2013 [73,74]. Khursheed 325 presented electron focused beam designs based upon using ring-cathodes together with off-axis 326 aberration correction techniques. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 9 May 2018 doi:10.209 The first step towards a practical implementation of this idea is to 327 develop a ring-cathode emitter. The proposal of a field emission ring-cathode gun unit has the 328 advantage of intrinsically being a large area field emitter, and it is therefore expected to overcome 329 the well-known disadvantage of conventional point-cathode field emitters having relatively low total 330 current (compared to earlier larger cathode electron gun designs). The much larger emission area of 331 a ring-cathode field emitter, compared to a point-cathode field emitter is also expected to provide it 332 with greater current stability. 333 Nguyen et al. [75] reported a versatile and robust method based on graphene growth on Ni 334 templates and thermo-assisted removal of poly(methyl methacrylate) (PMMA) for fabrication of 335 macroscopic and freestanding tubular graphene (TG) architectures. The TG architectures have a 336 diameter of 50 µm with a wall thickness in the range of 2.1-2.9 nm. The individual TG emitter can 337 produce an extremely high emission current, up to 0.46 mA at a very low external applied electric 338 field of 0.68 V µm-1. This most likely comes from ultrathin edges on graphitic walls, which consist of 339 a great number of field emission sites. Another important observation is that the TG CFE electron 340 source has good current stability and the stability is maintainable for days under HV conditions 341 (2.2×10-8 Torr). However, one limitation of TG architectures fabricated by Nguyen et al. is that the 342 edges are very jagged rather than being smooth, making it hard to be directly used as a CFE electron 343 source for electron microscopy applications. Moreover, transfer and alignment of such a large 344 diameter graphene tube is an extremely delicate process. 345 Figure 6. Graphene ring-cathode field emitter. (a) Macroscopic, freestanding, and tubular graphene architectures. Reproduced with permission from [75], Copyright ACS publications, 2015; (b) A few- layer graphene ring-cathode field emitter. Reproduced with permission from [76], Copyright Elsevier, 2016; Figure 6. Graphene ring-cathode field emitter. (a) Macroscopic, freestanding, and tubular graphene architectures. Reproduced with permission from [75], Copyright ACS publications, 2015; (b) A few- layer graphene ring-cathode field emitter. Reproduced with permission from [76], Copyright Elsevier, 2016; Shao et al. [76] reported an in-situ fabrication technique, eliminating any transfer and alignment 351 process. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 9 May 2018 On the one hand, a larger tip size has a greater emission area, thereby having a better 311 current stability and can operate in much less stringent vacuum conditions, predicted by the root 312 mean square (RMS) noise ratio varying inversely with the emission area [72]. One the other hand, a 313 larger tip size has a larger virtual source size and lower source brightness value. This trade-off is 314 applicable to all CFE sources, and most of the previous studies on CNT, LaB6, and CCnT tips sought 315 to maximized gun brightness at the expense of gun current stability and stringent vacuum 316 requirements. Another stand out feature of the Graphene-Ni point cathodes is their low energy 317 spread (Fig. 5b). The lowest reported energy spread is 0.246 eV obtained from a tip with a radius of 318 500 nm, and these properties make cold field emission sources the best type of electron source for 319 forming nano-size electron probes combined with high current intensity. Therefore, the promising 320 prospect of using Graphene-based CFE cathodes for high brightness high resolution electron 321 microscopy applications has recently been established. 322 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 9 May 2018 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 9 May 2018 doi:10.20944/preprints201805.0142.v1 achieved, it is expected to a wide variety of applications in subjects such as lithography, mass spectrometer, X-ray tube and accelerator physics. achieved, it is expected to a wide variety of applications in subjects such as lithography, mass spectrometer, X-ray tube and accelerator physics. 357 358 4. Conclusions High brightness, low energy spread and good current stability are critical for CFE electron 360 sources for electron microscopy applications. Although considerable research has been carried out 361 into developing single crystal tungsten and other 1D nanostructure-based field emitters, they have 362 as yet, faced many practical technological difficulties, such as stringent UHV requirements, poor 363 current stability and fast emission decay. Therefore, other ways to make CFE electron sources need 364 to be investigated. With the fast development of 2D graphene in recent years, graphene has been 365 proven to have ultra-high aspect ratios, good electric conductivity, chemical inertness and mechanical 366 hardness, and these properties make graphene based field emitters an attractive possible alternative. 367 Three types of graphene-based field emitters have been highlighted in this review: graphene film 368 emitters, graphene point/edge-cathode emitters, and graphene ring-cathode emitters. Most graphene 369 field emitters proposed and studied so however, involve field emission from multiple emission sites, 370 and are therefore not suitable for electron microscopy applications. 371 This paper has highlighted some recent promising developments in the category of single-point 372 and ring-shaped graphene field emitters for electron microscopy applications, however, the 373 investigation of graphene field emitters is still in an early stage of development, and there is still 374 much room for further improvement. More emission characterization tests need to be performed, 375 ones that can measure the source virtual source size, transverse coherent length, emission under a 376 variety of different vacuum conditions, and emitter lifetime need to be made. An electron gun unit 377 that can accommodate promising graphene emitters, which has its optical axis well aligned to the 378 central axis of the cathode still needs to be developed. Beyond that, electron guns based upon the 379 new class of graphene based field emitters need to be fitted on to electron microscopes, and their 380 performance critically compared to convention systems, in terms of parameters such as image 381 resolution and signal-to-noise. 382 Acknowledgments: This work is funded by the National Research Foundation (NRF) Competitive Research 383 Acknowledgments: This work is funded by the National Research Foundation (NRF) Competitive Research 383 Programme (CRP) award # NRFCRP13-2014-04 titled “Micro-fabricated Ring Carbon Nanotube electron/ion 384 sources”. 385 Conflicts of Interest: The authors declare no conflict of interest. 386 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 9 May 2018 doi:10.209 They obtained a graphene ring emitter, which consists of a graphene tube grown in-situ on 352 a sharp nickel wire tip. The diameter of the tubular graphene is about 5 µm while the edge of the tube 353 is approximately 2.7 nm. They have obtained high emission current of ~30 µA at the relatively low 354 applied field of ~1.75 V/mm. The ring-cathode field emitter is a new type of field emitter source, and 355 suitable electron ring-focused beam columns will first need to be designed for it. When that is 356 References 387 Dyke, W.; Dolan, W. Field emission. Adv. Electron. Electron Phys. 1956, 8, 89-185. 1. Dyke, W.; Dolan, W. Field emission. Adv. Electron. Electron Phys. 1956, 8, 89-185. 388 Crewe, A.; Wall, J.; Welter, L. A high-resolution scanning transmission electron microscope. J. Appl. Phys 1968, 39, 5861-5868. 3. Crewe, A.V.; Isaacson, M.; Johnson, D. A simple scanning electron microscope. Rev. Sci. Instrum. 1969, 40, 391 241-246. 392 4. Swanson, L.; Schwind, G. A review of the cold-field electron cathode. Adv. Imaging Electron Phys. 2009, 393 159, 63-100. 394 5. Schwind, G.; Magera, G.; Swanson, L. Comparison of parameters for schottky and cold field emission 395 sources. J. Vac. Sci. Technol. B 2006, 24, 2897-2901. 396 6. Tuggle, D.; Swanson, L. Emission characteristics of the zro/w thermal field electron source. J. Vac. Sci. 397 Technol. B 1985, 3, 220-223. 398 7. Fransen, M.; Overwijk, M.; Kruit, P. Brightness measurements of a zro/w schottky electron emitter in a 399 transmission electron microscope. Appl. Surf. Sci. 1999, 146, 357-362. 400 8. Crewe, A.; Eggenberger, D.; Wall, J.; Welter, L. Electron gun using a field emission source. Rev. Sci. Instrum. 401 1968, 39, 576-583. 402 9. De Jonge, N.; Van Druten, N. Field emission from individual multiwalled carbon nanotubes prepared in 403 an electron microscope. Ultramicroscopy 2003, 95, 85-91. 404 10. Bonard, J.-M.; Dean, K.A.; Coll, B.F.; Klinke, C. Field emission of individual carbon nanotubes in the 405 scanning electron microscope. Phys. Rev. Lett. 2002, 89, 197602. 406 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 9 May 2018 doi:10.20944/preprints201805.0142.v1 11. Fransen, M.; Van Rooy, T.L.; Kruit, P. Field emission energy distributions from individual multiwalled 407 carbon nanotubes. Appl. Surf. Sci. 1999, 146, 312-327. 408 2. De Jonge, N.; Lamy, Y.; Schoots, K.; Oosterkamp, T.H. High brightness electron beam from a multi-wa carbon nanotube. Nature 2002, 420, 393. 13. Houdellier, F.; De Knoop, L.; Gatel, C.; Masseboeuf, A.; Mamishin, S.; Taniguchi, Y.; Delmas, M.; 411 Monthioux, M.; Hÿtch, M.; Snoeck, E. Development of tem and sem high brightness electron guns using 412 cold-field emission from a carbon nanotip. Ultramicroscopy 2015, 151, 107-115. 413 14. Yeong, K.; Thong, J. Field-emission properties of ultrathin 5 nm tungsten nanowire. J. Appl. Phys. 2006, 414 100, 114325. 415 15. Zhang, H.; Tang, J.; Yuan, J.; Yamauchi, Y.; Suzuki, T.T.; Shinya, N.; Nakajima, K.; Qin, L.-C. An ultrabright 416 and monochromatic electron point source made of a lab 6 nanowire. Nat. References 387 Nanotechnol. 2016, 11, 273. 417 16. Novoselov, K.S.; Geim, A.K.; Morozov, S.; Jiang, D.; Katsnelson, M.; Grigorieva, I.; Dubonos, S.; Firsov; AA. 418 Two-dimensional gas of massless dirac fermions in graphene. Nature 2005, 438, 197. 419 17. Xiao, Z.; She, J.; Deng, S.; Tang, Z.; Li, Z.; Lu, J.; Xu, N. Field electron emission characteristics and physical 420 mechanism of individual single-layer graphene. Acs Nano 2010, 4, 6332-6336. 421 18. Lei, W.; Li, C.; Cole, M.T.; Qu, K.; Ding, S.; Zhang, Y.; Warner, J.H.; Zhang, X.; Wang, B.; Milne, W.I. A 422 graphene-based large area surface-conduction electron emission display. Carbon 2013, 56, 255-263. 423 19. Geim, A.K.; Novoselov, K.S. The rise of graphene. Nat. Mater. 2007, 6, 183. 424 20. Balandin, A.A.; Ghosh, S.; Bao, W.; Calizo, I.; Teweldebrhan, D.; Miao, F.; Lau, C.N. Superior thermal 425 conductivity of single-layer graphene. Nano Lett. 2008, 8, 902-907. 426 21. Neto, A.C.; Guinea, F.; Peres, N.M.; Novoselov, K.S.; Geim, A.K. The electronic properties of graphene. 427 Rev. Mod. Phys. 2009, 81, 109. 428 22. Lee, C.; Wei, X.; Kysar, J.W.; Hone, J. Measurement of the elastic properties and intrinsic strength of 429 monolayer graphene. science 2008, 321, 385-388. 430 23. Ye, D.; Moussa, S.; Ferguson, J.D.; Baski, A.A.; El-Shall, M.S. Highly efficient electron field emission from 431 graphene oxide sheets supported by nickel nanotip arrays. Nano Lett. 2012, 12, 1265-1268. 432 24. Lv, S.; Li, Z.; Liao, J.; Wang, G.; Li, M.; Miao, W. Optimizing field emission properties of the hybrid 433 structures of graphene stretched on patterned and size-controllable sinws. Sci. Rep. 2015, 5, 15035. 434 25. Zhang, Y.; Du, J.; Tang, S.; Liu, P.; Deng, S.; Chen, J.; Xu, N. Optimize the field emission character of a 435 vertical few-layer graphene sheet by manipulating the morphology. Nanotechnology 2011, 23, 015202. 436 26. Hernandez-Garcia, C.; O Shea, P.G.; Stutzman, M.L. Electron sources for accelerators. Phys. today 2 44. 27. Choi, W.; Chung, D.; Kang, J.; Kim, H.; Jin, Y.; Han, I.; Lee, Y.; Jung, J.; Lee, N.; Park, G. Fully sealed, high- 439 brightness carbon-nanotube field-emission display. Appl. Phys. Lett. 1999, 75, 3129-3131. 440 28. Saito, Y.; Uemura, S. Field emission from carbon nanotubes and its application to electron sources. Carbon 441 2000, 38, 169-182. 442 29. Barber, M.; Bordoli, R.; Sedgwick, R.; Tyler, A. Fast atom bombardment of solids as an ion source in mass 443 spectrometry. References 387 Zhang, H.; Tang, J.; Zhang, Q.; Zhao, G.; Yang, G.; Zhang, J.; Zhou, O.; Qin, L.C. Field emission of electrons 472 from single lab6 nanowires. Adv. Mater. 2006, 18, 87-91. 473 473 474 47. Jin, C.; Wang, J.; Wang, M.; Su, J.; Peng, L.-M. In-situ studies of electron field emission of single carbon 474 nanotubes inside the tem. Carbon 2005, 43, 1026-1031. 475 48. Giovannetti, G.; Khomyakov, P.; Brocks, G.; Karpan, V.v.; Van den Brink, J.; Kelly, P.J. Doping graphene 476 with metal contacts. Phys. Rev. Lett. 2008, 101, 026803. 477 49. Kwon, K.C.; Choi, K.S.; Kim, B.J.; Lee, J.-L.; Kim, S.Y. Work-function decrease of graphene sheet usin metal carbonates. J. Phys. Chem. C 2012, 116, 26586-26591. 50. Malesevic, A.; Kemps, R.; Vanhulsel, A.; Chowdhury, M.P.; Volodin, A.; Van Haesendonck, C. Field 480 emission from vertically aligned few-layer graphene. J. Appl. Phys. 2008, 104, 084301. 481 51. Koh, A.T.; Foong, Y.M.; Yusop, Z.; Tanemura, M.; Chua, D.H. Low temperature direct of graphene onto 482 metal nano-spindt tip with applications in electron emission. Adv. Mater. Interfaces 2014, 1. 483 52. Suh, J.S.; Jeong, K.S.; Lee, J.S.; Han, I. Study of the field-screening effect of highly ordered carbon nanotube 484 arrays. Appl. Phys. Lett. 2002, 80, 2392-2394. 485 y pp y 53. Bonard, J.M.; Weiss, N.; Kind, H.; Stöckli, T.; Forró, L.s.; Kern, K.; Chatelain, A. Tuning the field emission 486 properties of patterned carbon nanotube films. Adv. Mater. 2001, 13, 184-188. 487 54. Wu, Z.S.; Pei, S.; Ren, W.; Tang, D.; Gao, L.; Liu, B.; Li, F.; Liu, C.; Cheng, H.M. Field emission of single- 488 layer graphene films prepared by electrophoretic deposition. Adv. Mater. 2009, 21, 1756-1760. 489 55. Qian, M.; Feng, T.; Ding, H.; Lin, L.; Li, H.; Chen, Y.; Sun, Z. Electron field emission from screen-printed 490 graphene films. Nanotechnology 2009, 20, 425702. 491 56. Hwang, J.O.; Lee, D.H.; Kim, J.Y.; Han, T.H.; Kim, B.H.; Park, M.; No, K.; Kim, S.O. Vertical zno 492 nanowires/graphene hybrids for transparent and flexible field emission. J. Mater. Chem. 2011, 21, 3432- 493 3437. 494 57. Hallam, T.; Berner, N.C.; Yim, C.; Duesberg, G.S. Strain, bubbles, dirt, and folds: A study of graphene 495 polymer-assisted transfer. Adv. Mater. Interfaces 2014, 1. 496 58. Li, X.; Zhu, Y.; Cai, W.; Borysiak, M.; Han, B.; Chen, D.; Piner, R.D.; Colombo, L.; Ruoff, R.S. Transfer of 497 large-area graphene films for high-performance transparent conductive electrodes. References 387 Nature 1981, 293, 270. 444 30. Khursheed, A. Scanning electron microscope. Google Patents: 2007. 445 30. Khursheed, A. Scanning electron microscope. Google Patents: 2007. 445 32. Murphy, E.L.; Good Jr, R. Thermionic emission, field emission, and the transition region. Phys. Rev. 1956, 447 102, 1464. 448 33. Fowler, R.H.; Nordheim, L. Electron emission in intense electric fields. Proc. R. Soc. Lond. A 1928, 119, 173- 449 181. 450 34. Bronsgeest, M.; Barth, J.; Swanson, L.; Kruit, P. Probe current, probe size, and the practical brightness for 451 probe forming systems. J. Vac. Sci. Technol. B 2008, 26, 949-955. 452 35. Kruit, P.; Jansen, G.H. Space charge and statistical coulomb effects. Handbook of Charged Particle Optics 453 1997, 2. 454 36. Cook, B.; Verduin, T.; Hagen, C.; Kruit, P. Brightness limitations of cold field emitters caused by coulomb 455 interactions. J. Vac. Sci. Technol. B 2010, 28, C6C74-C76C79. 456 37. Kratschmer, E.; Rishton, S.; Kern, D.; Chang, T. Quantitative analysis of resolution and stability in 457 nanometer electron beam lithography. J. Vac. Sci. Technol. B 1988, 6, 2074-2079. 458 38. Rishton, S.; Beaumont, S.; Wilkinson, C. Measurement of the profile of finely focused electron beams in a 459 scanning electron microscope. J. Phys. E: Sci. Instrum. 1984, 17, 296. 460 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 9 May 2018 doi:10.20944/preprints201805.0142.v1 39. Fransen, M.; Damen, E.; Schiller, C.; van Rooy, T.; Groen, H.; Kruit, P. Characterization of ultrasharp field 461 emitters by projection microscopy. Appl. Surf. Sci. 1996, 94, 107-112. 462 40. Young, R.D. Theoretical total-energy distribution of field-emitted electrons. Phys. Rev. 1959, 113, 1 41. Knauer, W. Energy broadening in field emitted electron and ion-beams. Optik 1981, 59, 335-354. 41. Knauer, W. Energy broadening in field emitted electron and ion-beams. Optik 1981, 59, 335-354. 464 42. Dyke, W.; Charbonnier, F.; Strayer, R.; Floyd, R.; Barbour, J.; Trolan, J. Electrical stability and lif heated field emission cathode. J. Appl. Phys. 1960, 31, 790-805. 43. Ajayan, P. Nanotubes from carbon. Chem. Rev. 1999, 99, 1787-1800. 43. Ajayan, P. Nanotubes from carbon. Chem. Rev. 1999, 99, 1787-1800. 467 44. Berber, S.; Kwon, Y.-K.; Tománek, D. Unusually high thermal conductivity of carbon nanotubes. Phys. Rev. 468 Lett. 2000, 84, 4613. 469 45. Ebbesen, T.; Lezec, H.; Hiura, H.; Bennett, J.; Ghaemi, H.; Thio, T. Electrical conductivity of individual 470 carbon nanotubes. Nature 1996, 382, 54. 471 472 46. 65. Kleint, C. Electron emission noise. Surf. Sci. 1988, 200, 472-489. 513 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 9 May 2018 doi:10.20944/preprints201805.0142 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 9 May 2018 doi:10.20944/preprints201805.0142.v1 66. Endo, Y.; Honjo, I.; Goto, S. Microelectron gun with silicon field emitter. J. Vac. Sci. Technol. 514 3082-3085. 515 67. Purcell, S.; Vincent, P.; Journet, C.; Binh, V.T. Hot nanotubes: Stable heating of individual multiw 516 nanotubes to 2000 k induced by the field-emission current. Phys. Rev. Lett. 2002, 88, 105502. 517 68. Semet, V.; Binh, V.T.; Vincent, P.; Guillot, D.; Teo, K.; Chhowalla, M.; Amaratunga, G.; M 518 Legagneux, P.; Pribat, D. Field electron emission from individual carbon nanotubes of a vertica 519 array. Appl. Phys. Lett. 2002, 81, 343-345. 520 69. Yamaguchi, H.; Murakami, K.; Eda, G.; Fujita, T.; Guan, P.; Wang, W.; Gong, C.; Boisse, J.; Mille 521 M. Field emission from atomically thin edges of reduced graphene oxide. Acs Nano 2011, 5, 494 522 70. Wang, Y.; Yang, Y.; Zhao, Z.; Zhang, C.; Wu, Y. Local electron field emission study of two-di 523 carbon. Appl. Phys. Lett. 2013, 103, 033115. 524 71. Shao, X.; Srinivasan, A.; Ang, W.K.; Khursheed, A. A high-brightness large-diameter graphene co 525 cathode field emission electron source. Nat. Commun. 2018, 9, 1288. 526 72. Swanson, L.; Martin, N. Field electron cathode stability studies: Zirconium/tungsten thermal-fiel 527 J. Appl. Phys. 1975, 46, 2029-2050. 528 73. Khursheed, A. Design of a focused electron beam column for ring-cathode sources. Ultramicros 529 128, 10-23. 530 74. Khursheed, A.; Ang, W.K. Annular focused electron/ion beams for combining high spatial resol 531 high probe current. Microsc. Microanal. 2016, 22, 948-954. 532 75. Nguyen, D.D.; Suzuki, S.; Kato, S.; To, B.D.; Hsu, C.C.; Murata, H.; Rokuta, E.; Tai, N.-H.; Yosh 533 Macroscopic, freestanding, and tubular graphene architectures fabricated via thermal annealing. 534 2015, 9, 3206-3214. 535 76. Shao, X.; Srinivasan, A.; Zhao, Y.; Khursheed, A. A few-layer graphene ring-cathode field e 536 focused electron/ion beam applications. Carbon 2016, 110, 378-383. 537 538 539 66. Endo, Y.; Honjo, I.; Goto, S. Microelectron gun with silicon field emitter. J. Vac. Sci. Technol. B 1998, 16, 3082-3085. 67. Purcell, S.; Vincent, P.; Journet, C.; Binh, V.T. Hot nanotubes: Stable heating of individual multiwall carbon nanotubes to 2000 k induced by the field-emission current. Phys. Rev. Lett. 2002, 88, 105502. 68. Semet, V.; Binh, V.T.; Vincent, P.; Guillot, D.; Teo, K.; Chhowalla, M.; Amaratunga, G.; Milne, W.; 8 Legagneux, P.; Pribat, D. References 387 Nano Lett. 2009, 9, 498 4359-4363. 499 59. Suk, J.W.; Kitt, A.; Magnuson, C.W.; Hao, Y.; Ahmed, S.; An, J.; Swan, A.K.; Goldberg, B.B.; Ruoff, R.S. 500 Transfer of cvd-grown monolayer graphene onto arbitrary substrates. ACS nano 2011, 5, 6916-6924. 501 J g J g Transfer of cvd-grown monolayer graphene onto arbitrary substrates. ACS nano 2011, 5, 6916-6924. 501 60. Yang, C.; Liu, N.; Zeng, W.; Long, F.; Song, Z.; Su, J.; Li, L.; Zou, Z.; Fang, G.; Xiong, L. Superelastic and 502 ultralight electron source from modifying 3d reduced graphene aerogel microstructure. Nano Energy 2017, 503 33, 280-287. 504 61. Lee, H.R.; Lee, S.W.; Shikili, C.; Kang, J.S.; Lee, J.; Park, K.C. Enhanced electron emission of paste cnt 505 emitters with nickel buffer layer and its x-ray application. J. Nanosci. Nanotechnol. 2016, 16, 12053-12058. 506 61. Lee, H.R.; Lee, S.W.; Shikili, C.; Kang, J.S.; Lee, J.; Park, K.C. Enhanced electron emission of paste cnt 505 emitters with nickel buffer layer and its x-ray application. J. Nanosci. Nanotechnol. 2016, 16, 12053-12058. 506 62. Sugie, H.; Tanemura, M.; Filip, V.; Iwata, K.; Takahashi, K.; Okuyama, F. Carbon nanotubes as electron 507 508 emitters with nickel buffer layer and its x-ray application. J. Nanosci. Nanotechnol. 2016, 16, 12053-12058. 506 62. Sugie, H.; Tanemura, M.; Filip, V.; Iwata, K.; Takahashi, K.; Okuyama, F. Carbon nanotubes as electron 507 source in an x-ray tube. Appl. Phys. Lett. 2001, 78, 2578-2580. 508 62. Sugie, H.; Tanemura, M.; Filip, V.; Iwata, K.; Takahashi, K.; Okuyama, F. Carbon nanotubes as electron 507 source in an x-ray tube. Appl. Phys. Lett. 2001, 78, 2578-2580. 508 63. Lee, Y.Z.; Burk, L.; Wang, K.-h.; Cao, G.; Lu, J.; Zhou, O. Carbon nanotube based x-ray sources: Applications 509 in pre-clinical and medical imaging. Nucl. Instrum. Meth. A 2011, 648, S281-S283. 510 64. Tsai, J.T.; Chu, T.Y.; Shiu, J.Y.; Yang, C.S. Field emission from an individual freestanding graphene edge. 511 Small 2012, 8, 3739-3745. 512 65. Kleint, C. Electron emission noise. Surf. Sci. 1988, 200, 472-489. 513 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 9 May 2018 doi:10.20944/preprints201805.0142 Field electron emission from individual carbon nanotubes of a vertically aligned 9 array. Appl. Phys. Lett. 2002, 81, 343-345. 0 69. Yamaguchi, H.; Murakami, K.; Eda, G.; Fujita, T.; Guan, P.; Wang, W.; Gong, C.; Boisse, J.; Miller, S.; Acik, 21 M. Field emission from atomically thin edges of reduced graphene oxide. Acs Nano 2011, 5, 4945-4952. 22 70. Wang, Y.; Yang, Y.; Zhao, Z.; Zhang, C.; Wu, Y. Local electron field emission study of two-dimensional 523 carbon. Appl. Phys. Lett. 2013, 103, 033115. 524 71. Shao, X.; Srinivasan, A.; Ang, W.K.; Khursheed, A. A high-brightness large-diameter graphene coated point 525 cathode field emission electron source. Nat. Commun. 2018, 9, 1288. 526 72. Swanson, L.; Martin, N. Field electron cathode stability studies: Zirconium/tungsten thermal-field cathode. 527 J. Appl. Phys. 1975, 46, 2029-2050. 528 73. Khursheed, A. Design of a focused electron beam column for ring-cathode sources. Ultramicroscopy 2013, 529 128, 10-23. 530 73. Khursheed, A. Design of a focused electron beam column for ring-cathode sources. Ultramicroscopy 2013, 529 128, 10-23. 530 74 Khursheed A ; Ang W K Annular focused electron/ion beams for combining high spatial resolution with 531 74. Khursheed, A.; Ang, W.K. Annular focused electron/ion beams for combining high spatial resolution w high probe current. Microsc. Microanal. 2016, 22, 948-954. 75. Nguyen, D.D.; Suzuki, S.; Kato, S.; To, B.D.; Hsu, C.C.; Murata, H.; Rokuta, E.; Tai, N.-H.; Yoshimura, M. 533 Macroscopic, freestanding, and tubular graphene architectures fabricated via thermal annealing. ACS nano 534 2015, 9, 3206-3214. 535 76. Shao, X.; Srinivasan, A.; Zhao, Y.; Khursheed, A. A few-layer graphene ring-cathode field emitter for 536 focused electron/ion beam applications. Carbon 2016, 110, 378-383. 537 538 539
https://openalex.org/W2900314329	https://europepmc.org/articles/pmc6254677?pdf=render	English	null	Multiple mechanisms can disrupt oncogenic pathways in multiple myeloma	Oncotarget	2,018	cc-by	1,337	Oncotarget, 2018, Vol. 9, (No. 88), pp: 35801-35802 www.oncotarget.com Phuc H. Hoang and Richard S. Houlston Multiple myeloma (MM) is a clinically and biologically heterogeneous malignancy resulting from the infiltration of clonal plasma cells in the bone marrow [1]. Thus far the molecular mechanisms responsible for the initiation and heterogeneous evolution of MM are poorly understood. Recent large-scale analyses of MM have focussed mainly on the protein-coding components of the genome, identifying recurrently mutated genes including KRAS, NRAS, PRDM1, CCND1, and TP53 as drivers of tumourigenesis [1]. Many of these mutations are, however typically found at low frequency (<10% of tumours), and hence do not fully explain the clinical and biological diversity of MM. With the increasing availability of MM whole-genome sequencing (WGS) initiatives such as The Relating Clinical Outcomes in Multiple Myeloma to Personal Assessment of Genetic Profile Study (CoMMpass), we have sought to systematically search for driver mutations in the MM non-coding as well as coding regions of the genome. In our recent study, we reported an integrated analysis of the WGS of 765 and whole-exome sequencing (WES) of 804 MM patients from CoMMpass, identifying novel non-coding drivers altering expression of target genes as well as coding drivers [2]. We also demonstrated that pathways central to MM tumourigenesis could be targeted by both coding and non-coding drivers, further enhancing our understanding of alternative oncogenic pathways driving MM.ii To search for non-coding drivers, we first defined the cis-regulatory elements (CREs) and promoters using information from promoter capture Hi-C in naïve B-cells [3] and transcription start site (TSS) proximity respectively. The approach enabled us to narrow down the genomic searches and mitigate against the high statistical burden in establishing significantly mutated regions. We identified promoters associated with 34 genes and CREs associated with 271 genes and as recurrently mutated by single nucleotide variants (SNVs). Many of the target genes are enriched for established oncogenic pathways in MM such as PAX5 and BCL6 in B-cell differentiation. Recurrent mutation of the NBPF1 promoter corresponded to 1.7-fold increase in gene expression of NBPF1. Mutations in CREs of six target genes (PAX5, ST6GAL1, Figure 1: Key oncological pathways in multiple myeloma can be targeted by both coding and non-coding mechanisms. Figure adapted from Hoang et al [2]. Figure 1: Key oncological pathways in multiple myeloma can be targeted by both coding and non-coding mechanisms. Figure adapted from Hoang et al [2] Figure 1: Key oncological pathways in multiple myeloma can be targeted by both coding and non-coding mechanisms. Phuc H. Hoang and Richard S. Houlston Hoang, email phuc.hoang@icr.ac.uk Keywords: multiple myeloma; cancer genetics; non-coding drivers; coding drivers; integrated analysis Received: October 08, 2018 Published: November 09, 2018 drivers; coding drivers; integrated analysis g pi To better understand the interplay of alternative somatic mechanisms underlying MM, we extended our analysis to categorise chromosomal copy number alterations, structural variations (SVs), and protein-coding drivers in CoMMpass dataset. We observed aberrant copy number alterations characteristic of MM [1]: gain of odd number chromosomes (in 59% tumours); deletions at 13q (63%), 14q (43%), 16q (38%), and 8p (38%); and amplification overlapping MYC and PVT1 at 8q24.21. We detected novel SVs affecting genes including inversions disrupting CYLD, MYC translocations disrupting CD96, translocations intergenic to PRDM1 and FBXW7, and translocations associated with upregulation of MAP3K14 (7-fold) and CCND2 (12-fold). We also identified 40 significantly mutated coding genes, 11 of which are novel (BAX, C8orf86, FAM154B, FTL, HIST1H4H, LEMD2, PABPC1, RPN1, RPS3A, SGPP1, TBC1D29). Phuc H. Hoang and Richard S. Houlston Figure adapted from Hoang et al [2]. Figure 1: Key oncological pathways in multiple myeloma can be targeted by both coding and non-coding mechanisms. Figure adapted from Hoang et al [2]. www.oncotarget.com Oncotarget 35801 COBLL1, HOXB3, and ATP13A2) were also associated with differential gene expression. Notably, mutations in the PAX5 CRE identified resulted in a 4.6-fold reduced expression, consistent with PAX5 being a tumour suppressor as in other B-cell malignancies [4-6]. In contrast, disruption of ST6GAL1 CRE by SNVs led to a 1.4-fold upregulation, consistent with ST6GAL1 is overexpressed in various cancers [7] and aberrant glycosylation in MM [8]. COBLL1, HOXB3, and ATP13A2) were also associated with differential gene expression. Notably, mutations in the PAX5 CRE identified resulted in a 4.6-fold reduced expression, consistent with PAX5 being a tumour suppressor as in other B-cell malignancies [4-6]. In contrast, disruption of ST6GAL1 CRE by SNVs led to a 1.4-fold upregulation, consistent with ST6GAL1 is overexpressed in various cancers [7] and aberrant glycosylation in MM [8]. prevalent in MM than previously described (present in 96% of samples) and is associated with mutation affecting PAX5 CREs. Finally, we also identified novel mutational signatures reflective of homologous recombination deficiency (signature 3 and 8) and of unknown aetiologies (signature 16 and 30) in >30% of tumours. In summary, through identifying novel mutations disrupting coding and non-coding genomes, we have demonstrated that key biological pathways in MM could be targeted by alternative mechanisms. Our study thus further delineates the genomic complexity and heterogeneity underlying the disease, providing important information for potential development of novel therapeutic agents and offering perspectives for personalised therapy. We also found copy number variations at CREs regulate expression of seven candidate genes (MYC, PACS2, TEX22, KDM3B, RAB36, PLD4, and SP110). Notably, MYC oncogene was overexpressed in tumours having either deletion of upstream putative silencers or amplification of downstream putative enhancers. The amplified enhancers coincides with the Myc enhancer cluster essential for MLL–AF9-driven leukaemia in mice [9]. Our results demonstrate that MYC can be amplified via dysregulation of non-coding regulatory regions, in addition to the well-established mechanisms of MYC upregulation through chromosomal translocations and gene amplification. Phuc H. Hoang: Division of Genetics and Epidemiology, The Institute of Cancer Research, London, UK; Division of Molecular Pathology, The Institute of Cancer Research, London, UK Correspondence to: Phuc H. Hoang, email phuc.hoang@icr.ac.uk Correspondence to: Phuc H. REFERENCES 1. Manier S, et al. Nat Rev Clin Oncol. 2017; 14:100-113. 1. Manier S, et al. Nat Rev Clin Oncol. 2017; 14:100-113. 1. Manier S, et al. Nat Rev Clin Oncol. 2017; 14:100 113. 2. Hoang PH, et al. Leukemia. 2018; 32:2459-2470. https://doi.org/10.1038/s41375-018-0103-3. 3. Javierre BM, et al. Cell. 2016; 167:1369-1384.e19. 4. Puente XS, et al. Nature. 2015; 526:519-524. 5. Shah S, et al. Nat Genet. 2013; 45:1226-1231. 6. Dang J, et al. Blood. 2015; 125:3609-3617. 7. Lu J, et al. J Biol Chem. 2014; 289:34627-34641. 8. Aurer I, et al. Coll Antropol. 2007; 31:247-251. 9. Bahr C, et al. Nature. 2018; 553:515-520. , ; 2. Hoang PH, et al. Leukemia. 2018; 32:2459-2470. https://doi.org/10.1038/s41375-018-0103-3. 3. Javierre BM, et al. Cell. 2016; 167:1369-1384.e19. 4. Puente XS, et al. Nature. 2015; 526:519-524. 5. Shah S, et al. Nat Genet. 2013; 45:1226-1231. 6. Dang J, et al. Blood. 2015; 125:3609-3617. 7. Lu J, et al. J Biol Chem. 2014; 289:34627-34641. 8. Aurer I, et al. Coll Antropol. 2007; 31:247-251. 9. Bahr C, et al. Nature. 2018; 553:515-520. 2. Hoang PH, et al. Leukemia. 2018; 32:2459-2470. https://doi.org/10.1038/s41375-018-0103-3. 3. Javierre BM, et al. Cell. 2016; 167:1369-1384.e19. . Aurer I, et al. Coll Antropol. 2007; 31:247-251. 9. Bahr C, et al. Nature. 2018; 553:515-520. Performing an integrated analysis, allowed us to identify several key pathways somatically targeted by both coding and non-coding mutations (Figure 1). This is exemplified by plasma cell differentiation pathway in which we identified significant non-coding mutations associated with BCL6 and PAX5, complementary to established coding drivers affecting IRF4 and PRDM1. Copyright: Hoang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and re production in any medium, provided the original author and source are credited. Following on from these analyses, to gain insight into aetiological basis of somatic mutations in MM, we examined mutational signatures. Our findings are consistent with an association between the APOBEC family of cytidine deaminases signature 2 with coding drivers in DNAH5, SAMHD1, TP53, and BRAF along with and myeloma subtypes – t(14;16), t(14;20), and t(4;14). Additionally, we noted, AID-related signature 9 was more www.oncotarget.com www.oncotarget.com Oncotarget 35802
https://openalex.org/W2145921127	https://escholarship.org/content/qt8kj2n564/qt8kj2n564.pdf?t=mm2t2b	English	null	RegTransBase – a database of regulatory sequences and interactions based on literature: a resource for investigating transcriptional regulation in prokaryotes	BMC genomics	2,013	cc-by	5,480	UC Davis UC Davis Previously Published Works Title RegTransBase -- a database of regulatory sequences and interactions based on literature: a resource for investigating transcriptional regulation in prokaryotes Permalink https://escholarship.org/uc/item/8kj2n564 Journal BMC Genomics, 14(1) * Correspondence: ildubchak@lbl.gov 2Lawrence Berkeley National Laboratory, Berkeley, CA 94710, USA 7Department of Energy Joint Genome Institute, 2800 Mitchell Drive, Walnut Creek, CA 94598, USA Full list of author information is available at the end of the article © 2013 Cipriano et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Due to the constantly growing number of sequenced microbial genomes, comparative genomics has been playing a major role in the investigation of regulatory interactions in bacteria. Regulon inference mostly remains a field of semi-manual examination since absence of a knowledgebase and informatics platform for automated and systematic investigation restricts opportunities for computational prediction. Additionally, confirming computationally inferred regulons by experimental data is critically important. Description: RegTransBase is an open-access platform with a user-friendly web interface publicly available at http://regtransbase.lbl.gov. It consists of two databases – a manually collected hierarchical regulatory interactions database based on more than 7000 scientific papers which can serve as a knowledgebase for verification of predictions, and a large set of curated by experts transcription factor binding sites used in regulon inference by a variety of tools. RegTransBase captures the knowledge from published scientific literature using controlled vocabularies and contains various types of experimental data, such as: the activation or repression of transcription by an identified direct regulator; determination of the transcriptional regulatory function of a protein (or RNA) directly binding to DNA or RNA; mapping of binding sites for a regulatory protein; characterization of regulatory mutations. Analysis of the data collected from literature resulted in the creation of Putative Regulons from Experimental Data that are also available in RegTransBase. Conclusions: RegTransBase is a powerful user-friendly platform for the investigation of regulation in prokaryotes. It uses a collection of validated regulatory sequences that can be easily extracted and used to infer regulatory interactions by comparative genomics techniques thus assisting researchers in the interpretation of transcriptional regulation data. Keywords: Transcriptional regulation, Prokaryotes, Comparative genomics RegTransBase – a database of regulatory sequences and interactions based on literature: a resource for investigating transcriptional regulation in prokaryotes RegTransBase – a database of regulatory sequences and interactions based on literature: a resource for investigating transcriptional regulation in prokaryotes Michael J Cipriano1, Pavel N Novichkov2, Alexey E Kazakov2, Dmitry A Rodionov3,4, Adam P Arkin2,5, Mikhail S Gelfand4,6 and Inna Dubchak2,7* Michael J Cipriano1, Pavel N Novichkov2, Alexey E Kazakov2, Dmitry A Rodionov3,4, Adam P Arkin2,5, Mikhail S Gelfand4,6 and Inna Dubchak2,7* Powered by the California Digital Library University of California eScholarship.org Cipriano et al. BMC Genomics 2013, 14:213 http://www.biomedcentral.com/1471-2164/14/213 Cipriano et al. BMC Genomics 2013, 14:213 DATABASE Open Access * Correspondence: ildubchak@lbl.gov 2 © 2013 Cipriano 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. Background considered to belong to a regulon. Predicting the regulon of a transcription factor that binds DNA by detecting TFBSs in most cases requires the alignment of known binding sites to create a positional weight matrix (PWM). It is very important to filter out irrelevant sites and find TFBSs that are of higher confidence, and com- parative genomics is the method of choice for this. Activation and repression of gene expression in bacteria is usually mediated by DNA-binding transcription fac- tors (TFs) that specifically recognize TF-binding sites (TFBSs) in upstream regions of target genes. Genes and operons directly co-regulated by the same TF are With the advent of new and cheaper sequencing tech- nologies and ongoing sequencing projects such as GEBA [1], which aims to close the gaps in the bacterial tree of Page 2 of 8 Cipriano et al. BMC Genomics 2013, 14:213 http://www.biomedcentral.com/1471-2164/14/213 Table 1 Content of RegTransBase 2012 2006 Articles Curated: 7213 2958 Experiments: 18877 7129 Organisms: Phylum 11 9 Genus 224 Not reported Species 666 128 Genes: 59754 7467 Sites: 13321 6470 Regulators: 1488 678 Effectors: 875 558 PWM 151 151 Putative Regulons Total 6390 N/A Avg. Regulon/Genome 13 N/A Median Regulon/Genome 5 N/A Table 1 Content of RegTransBase life, a lot of bacterial organisms are now being se- quenced [2]. Of note is that not only are organisms with no close sequenced relatives being sequenced, but spe- cifically groups of closely related organisms and multiple strains of the same species. This trend of sequencing can be successfully exploited when using comparative ana- lyses, and already has been used in studying and pre- dicting transcriptional regulation [3-6]. life, a lot of bacterial organisms are now being se- quenced [2]. Of note is that not only are organisms with no close sequenced relatives being sequenced, but spe- cifically groups of closely related organisms and multiple strains of the same species. This trend of sequencing can be successfully exploited when using comparative ana- lyses, and already has been used in studying and pre- dicting transcriptional regulation [3-6]. While many transcriptional regulation experiments are performed on model organisms, the existing experimental evidence can be transferred to other organisms by com- parative methods. However, even closely related organisms can have different transcriptional regulation [7], thus pre- diction of binding sites and regulon inference in bacteria until recently has been mostly done by careful manual ana- lysis [8-10]. Background Availability of experimental data on regulation for a wider range of organisms would be very helpful in automatic verification of computationally derived predic- tions of regulation. These verifications require well- designed databases accessible to prediction and analysis programs. of transcriptional regulation and experimentally charac- terized TF binding sites. The main steps of the data col- lection. Described in detail in our previous article [22], are the following: search for relevant articles in PubMed [25], entry of data through a specialized annotator inter- face, quality control, mapping sites and genes to ge- nomes, additional manual corrections (if necessary) and presentation of the data in the final format. The entry quality is controlled by a number of consistency and completeness checks. The genomic location of a specific feature (site or gene) is then recorded by the annotator as a signature (a DNA sequence fragment of sufficient length) that is then used to map all the features in the database to a wide range of the NCBI RefSeq genomes [26,27]. Eukaryotic transcriptional regulation data has been sum- marized in both commercial and open-source databases, such as TransFac [11], Pazar [12], and ORegAnno [13], widely used by the community. There are several gene regulation databases that focus on distinct microbial organ- isms such as E. coli [14,15], B. subtilis [16], Mycobacterium tuberculosis [17], and corynebacteria [18]. On the other hand, PRODORIC [19], PePPER [20] and SwissRegulon [21] cover a wide range of bacterial genomes. RegTransBase, first introduced in 2007 [22], was built with the goal to cover a wide microbial diversity and pro- vide a collection of curated experimental data to use in ex- ternal computational tools. The current advanced version of RegTransBase: (i) contains a much larger set of manually collected experimental results (Table 1); (ii) has a brand new interface with novel capabilities for multi-level data navigation such as the new Classification Browser and new data aggregation tools such as the Putative Regulons Browser; (iii) is linked to associated analytical systems. Each database entry describes a single experiment that is an experimentally determined relationship between several database elements. A single entry may describe an experiment and control, identical results obtained by different methods or the results of the application of one technique to several similar objects. Only original results are recorded, normally from the ‘Results’ or ‘Discussion’ sections of an article. Background It is important to mention that we have recently deve- loped two new resources – the RegPredict Web tool to support genomic reconstruction of transcriptional regu- lons in groups of closely related prokaryotic genomes [23], and the RegPrecise database to capture, visualize and analyze transcription factor regulons that were recon- structed [24]. We are working on the integration of RegTransBase, RegPredict and RegPrecise into a powerful platform for regulon reconstruction and analysis. The types of experimental techniques form a con- trolled vocabulary. The following categories of experi- ments were accepted: (i) regulation of gene expression by a known regulator; (ii) demonstration that a gene en- codes a regulatory protein (excluding proteins that do not directly bind DNA, e.g. protein kinases); (iii) experi- mental mapping of DNA binding sites for known regula- tors; (iv) identification of mutations in regulatory genes influencing expression of regulated genes; (v) computa- tional prediction of binding sites. Construction and content Experimental data annotation The main objective during the article annotation phase for RegTransBase was to collect experimental evidences Cipriano et al. BMC Genomics 2013, 14:213 http://www.biomedcentral.com/1471-2164/14/213 Page 3 of 8 Cipriano et al. BMC Genomics 2013, 14:213 http://www.biomedcentral.com/1471-2164/14/213 Cipriano et al. BMC Genomics 2013, 14:213 http://www.biomedcentral.com/1471-2164/14/213 The classes of elements in the database are: regulators (regulatory proteins and RNAs directly binding to DNA, with a well-defined binding site); effectors (molecules not binding DNA or physical effects such as stress, etc.); and positional elements. The latter are described as re- gions in DNA sequences. Positional elements form a hierarchy: locus > operon > transcript > gene and site; an elements may be a sub-elements of other elements of the same or higher levels (e.g., a site and a gene may be a sub-element of a operon). scanning published articles, and recorded its genomic lo- cation. The curator then annotated binding sites and their sequence, downstream gene, location in a published genome, and any published experimental evidence. In addition, curators supplied groups of organisms that they believe could be used when searching for homologous binding sites based on phylogenetic distance of organism and presence of a conserved transcription factor. Lastly, the curator recorded default scores and the expected dis- tance a binding site would be from the start of a gene based on examination of the existing binding sites. All elements are linked to the corresponding expe- riments and together they are linked to the original article. As mentioned above, positional elements are mapped to genomes, thus if two independent articles describe regulation of the same gene, the data contained in these articles will be interlinked via this gene, but sites and other experimental data will be reported as independent entries. A PWM is automatically created in the RegTransBase database based on the TFBSs alignment. We then searched all recommended bacterial genomes using MAST [28]. We recorded all hits that passed the following criteria into the RegTransBase database: e-value of 1e-5 or better, it did not overlap coding regions and it was upstream of a predic- ted gene. With each record, we provide the binding site location with a reference to a published sequence (usually NCBI RefSeq [26]), the sequence, the gene which is affected by the binding site, the evidence for the binding if any, any relevant articles pertaining to that site, and the tran- scription factor which binds the site. Putative regulons from experimental data The Putative Regulons section of RegTransBase provides a list of experimental sites along with a non-redundant list of target genes for each regulator. The process we undertook in developing this list of putative regulons from the manually curated data includes three steps. First, we selected a subset of experiments using the following criteria: (i) the experiment describes a single regulator, (ii) a regulator and its regulated genes belong to the same genome, (iii) no computational predictions are included. Utility and discussion Our goal is to provide a comprehensive resource to the greater genomic community to allow for easy transfer of known binding site information as well as tools for dis- covering interesting regulatory interactions in groups of organisms. We believe that by using a comparative ap- proach, new genomes could be more easily annotated, and this approach can help facilitate the discovery and expansion of regulons in a wide range of organisms. Third, we compiled a list of putative regulons by uni- fying all ‘regulator-gene’ pairs with the same regulator. Database statistics As of November 2012, RegTransBase contains information on 666 bacterial species from 224 genera. This resource al- lows for access to the information on 19000 different expe- riments from about 7200 articles from as far back as 1977 until the present day (more details in Table 1). Second, from this subset we extracted the pairs ‘regulator-regulated gene’ for each genome, taking into account operon structure, that extend the list of regu- lated genes by adding other members of a particular op- eron. In some cases we see a particular pair of a regulator and an associated regulated gene in multiple entries in RegTransBase. We removed such redundant pairs from the list of regulator-regulated genes based on positional mapping. Construction and content Experimental data annotation We also provide for download the sequence logo for the alignment, pro- files and alignments in many different formats, and recommended options in using the profiles for search- ing other genomes (cut-off scores, distance from gene, taxonomy). Our original publication on RegTransBase [22] and the Help pages at http://regtransbase.lbl.gov provide more details on the procedure of experimental data annotation. Database access and features Each record in the Manually Curated PWM section of the database comprises a TFBS training set (alignment) cre- ated by an expert curator using published experimen- tal data and manual in silico analyses. The curator first gathered information about a known transcription factor where a set of binding sites was known, created a sum- mary of a description of this transcription factor by RegTransBase is freely accessible via a user-friendly web interface at http://regtransbase.lbl.gov. Besides browsing, searching for various data of interest, and carrying out ana- lytical tasks (see below), users can download the Annota- tors Database, which includes all of the annotated data elements and experiments as a sql dump file to perform Cipriano et al. BMC Genomics 2013, 14:213 http://www.biomedcentral.com/1471-2164/14/213 Cipriano et al. BMC Genomics 2013, 14:213 http://www.biomedcentral.com/1471-2164/14/213 Page 4 of 8 their own analysis, as well as the Annotators Database Schema Description, and Alignments of Binding Site through the ‘Download’ page. their own analysis, as well as the Annotators Database Schema Description, and Alignments of Binding Site through the ‘Download’ page. Two categories refer to experimental methodology and the goals of experiments. The ‘Experiment techniques’ classification uses a controlled vocabulary of methods used in experiments. This classification has a two-level struc- ture with the upper level containing method categories (i.e. protein analysis, RNA analysis) and lower level containing individual techniques such as Western blotting, DNAase footprinting etc. The ‘Experiment result’ classifi- cation describes what the experiment resulted in (i.e. pro- moter mapping, regulatory site mapping, gene/operon repression). Data navigation BMC Genomics 2013, 14:213 http://www.biomedcentral.com/1471-2164/14/213 Page 6 of 8 Use Case 1: use of Putative Regulon for the search of a TF binding motif Putative regulons from experimental data d f f f b d Identification of transcription factor binding motifs is an important step in the computational reconstruction of regulatory elements. The ‘Putative Regulons’ section of RegTransBase provides sets of upstream sequences of target genes for each regulon. These sets can be used for the identification of conserved DNA motifs that may bind transcriptional regulators. This scheme was successfully tested for the TnrA regulon from B. subtilis. Search methods RegTransBase provides a user with a broad range of search options such as search by Gene name, effector name, or a full text search of an abstract. Search for genes involved in regulatory experiments can be done using the gene name, function, product, accession num- ber, or any other GenBank annotation. Searching for ef- fectors by their name extracts the information on regulator, experiment, and genome with all associated links. Full text search allows for running complex que- ries against the abstracts and experiment descriptions such as ‘+mga +promoter’. 1. Find genome of interest on the Putative Regulons page. 2. Find regulon of interest based on the regulator name. 3. Get a set of upstream sequences by clicking the ‘Download’ link in the ‘Upstream sequences column of regulons table. 4. Start RegPredict [23], select genomes of interest. 5. Open ‘Discover Profiles’, paste upstream sequences (at least three sequences). 6. Select profile parameters (palindrome 6. Select profile parameters (palindrome recommended), start search. 7. Select profile with highest informational content and run search for sites in selected genomes. Data navigation We developed a new navigation interface to easily select a set of experimental records based on six categories (classifi- cations) covering different aspects of the database. Three categories (classifications) describe genomes that were studied in relevant experiments (Figure 1). The ‘Effector’ classification uses a tree-like hierarchy of effectors where classes of the hierarchy are mainly based on the Chemicals and Drugs Category of MESH [31]. The ‘Taxonomy’ category is based on the NCBI Taxonomy [29] and describes phylogenetic relationships. A user can choose a taxon of interest starting from the super kingdom level (Bacteria or Archaea) and move down to the species level. The ‘Relevance’ category refers to the attributes of genome projects that provide infor- mation about the wide area of research a particular genome is a part of, such as Antibiotic production, Agri- cultural, etc. [30]. The ‘Phenotypes’ category includes at- tributes that describe phenotypic properties of the organisms [30]. User can browse all categories in the database by choosing a term in one classification and then narrowing a result by choosing terms in other classifications as additional filters. At any time, the user can click on the number beside the classification to get articles fitting all criteria currently selected. For example, we want to know if there is any data on experiments with cis-elements that are involved in Figure 1 Home page of RegTransBase. Data navigation panel with its major classifications in the middle of the page. Figure 1 Home page of RegTransBase. Data navigation panel with its major classifications in the middle of the page. Cipriano et al. BMC Genomics 2013, 14:213 http://www.biomedcentral.com/1471-2164/14/213 Page 5 of 8 Figure 2 Step-by-step data navigation in search for the experiments where cis-elements are involved in the fructose-dependent l ti Figure 2 Step-by-step data navigation in search for the experiments where cis-elements are involved in the fructose-dependent regulation. Figure 2 Step-by-step data navigation in search for the experiments where cis-elements are involved in the fructose-dependent regulation. A subsequent choosing of the ‘Regulatory site map- ping’ term in the ‘Result’ classification produces a list of 3 experiments where cis-elements involved in fructose- dependent regulation were studied. fructose-dependent regulation. By using the ‘Effectors’ classification in three steps: ‘Carbohydrates’ -> ‘Mono- saccharides’ -> ‘Fructose’ we find a list of 20 experiments (Figure 2). Cipriano et al. BMC Genomics 2013, 14:213 http://www.biomedcentral.com/1471-2164/14/213 Cipriano et al. BMC Genomics 2013, 14:213 http://www.biomedcentral.com/1471-2164/14/213 Cipriano et al. Acknowledgements The authors are grateful to Igor Lukashin for obtaining the genome alignment data, MicrobesOnline team for useful discussions and encouragement, and to Tatiana Smirnova for the artistic and highly functional RegTransBase Web site. 6. Start RegPredict (click ‘Start Application’) 7. Click ‘Select genomes’. 8. Find recommended taxonomical group (Bacillales - see the ‘Recommended options’ section on ABC0302 page in RegTransBase) and add all genomes from that group (or as many genomes as possible). ‘This work conducted by ENIGMA- Ecosystems and Networks Integrated with Genes and Molecular Assemblies (http://enigma.lbl.gov), a Scientific Focus Area Program at Lawrence Berkeley National Laboratory, was supported by the Office of Science, Office of Biological and Environmental Research, of the U. S. Department of Energy under Contract No. DE-AC02-05CH11231.’ The work was also supported by the Director, Office of Science, Office of Biological and Environmental Research, Life Sciences Division, U.S. 9. Click ‘Run Profile’. 9. Click ‘Run Profile’. 10. Select the ‘Sequences’ tab and paste your alignment of binding sites in the FASTA format. Department of Energy under Contracts No. DE-AC02-05CH11231 and No. DE-SC0004999. 11. Click ‘Generate profile’. Conclusions RegTransBase, a user-friendly open-access database, pro- vides biologists involved in the investigation of microbial regulation and systems biology with convenient access to experimental data collected in thousands of original studies. It allows a user to interact with a valuable collec- tion of manually curated data on a range of experiments related to the transcriptional regulation of bacteria. These data, with associated analytical tools, provide a valuable resource to assist in investigation of gene functions in the constantly growing number of available genome assem- blies. RegTransBase collection of PWMs is currently used by various tools for TF binding prediction and motif comparison (for example, MEME-ChIP [32] and TOMTOM [33] from MEME Suite, FITBAR [34], ISGA Received: 15 November 2012 Accepted: 22 March 2013 Published: 2 April 2013 Authors’ contributions 2. Find a regulator of interest (for example, ABC0302). MJC worked on the database and interface design, general data organization and access; PSN participated in the database and interface design and construction, and lead the putative regulon collection and RegPredict access projects; AEK was responsible for data collection and manual curation; DAR proposed several critical directions of the project and actively participated in discussions; APA was involved with the MicrobesOnline integration and general discussions; MSG conceived and performed general coordination of the project. ID supervised the project and was involved with all aspects of database design, construction and implementation. MJC, AEK and ID wrote the manuscript. All authors read and approved the final manuscript. 3. Open the page with the ABC0302 binding sites alignment (http://regtransbase.lbl.gov/cgi-bin/ regtransbase?page=show_alignment& matrix_id=95). 4. Download an alignment in FASTA format (First option in Download section at the bottom of the page). 5. Go to the RegPredict website (http://regpredict.lbl. gov/). Author details 1 12. Set search parameters ‘Position from’ and ‘Position to’ (see ‘Recommended options’ section on ABC0302 page in RegTransBase). 1Department of Microbiology, University of California Davis, Davis, CA 95616, USA. 2Lawrence Berkeley National Laboratory, Berkeley, CA 94710, USA. 3Sanford-Burnham Medical Research Institute, La Jolla, CA 92037, USA. 4A.A. Kharkevich Institute for Information Transmission Problems, Russian Academy of Sciences, Moscow 127994, Russia. 5Department of Bioengineering, University of California, Berkeley, CA 94720, USA. 6M.V. Lomonosov Moscow State University, Vorobyevy Gory 1073, Moscow 119991, Russia. 7Department of Energy Joint Genome Institute, 2800 Mitchell Drive, Walnut Creek, CA 94598, USA. 13. Click ‘Run’. Manually curated position weight matrices (PWM) Positional weight matrices from RegTransBase collec- tions can be used for computational prediction of TFBSs Figure 3 Access to the RegTransBase PWMs and browsing capabilities. Figure 3 Access to the RegTransBase PWMs and browsing capabilities. Cipriano et al. BMC Genomics 2013, 14:213 http://www.biomedcentral.com/1471-2164/14/213 Page 7 of 8 [35], STAMP [13]. MicrobesOnline, an integrated portal for comparative and functional genomics [36], is cross- linked with RegTransBase. using RegPredict [23] or other software for PWM-based TFBS search. Figure 3 shows an access page to the RegTransBase PWMs and the associated data. A user se- lects a PWM of interest from the list and opens a webpage with PWM description. PWMs are available for download in different formats including a binding site alignment in FASTA format, matrices in MAST and Transfac formats and as a frequency matrix. As regulon inference is of significant importance for deciphering the regulation of biological processes, we be- lieve that a current improved and expanded version of RegTransBase is a useful tool for the research community. Use Case 2: use of manually curated PWM for compu- tational reconstruction of a regulon Competing interests The authors declare that they have no competing interests. Competing interests The authors declare that they have no competing interests. Availability and requirements RegTransBase is available at http://regtransbase.lbl.gov. 1. Open a list of the binding site alignments (http:// regtransbase.lbl.gov/cgi-bin/regtransbase? page=alignment_browse). 1. Wu D, Hugenholtz P, Mavromatis K, Pukall R, Dalin E, Ivanova NN, Kunin V, Goodwin L, Wu M, Tindall BJ, et al: A phylogeny-driven genomic encyclopaedia of Bacteria and Archaea. Nature 2009, 462(7276):1056–1060. 2. Pagani I, Liolios K, Jansson J, Chen IM, Smirnova T, Nosrat B, Markowitz VM, Kyrpides NC: The Genomes OnLine Database (GOLD) v.4: status of genomic and metagenomic projects and their associated metadata. Nucleic Acids Res 2012, 40(Database issue):D571–D579. 3. Liu J, Xu X, Stormo GD: The cis-regulatory map of Shewanella genomes. Nucleic Acids Res 2008, 36(16):5376–5390. 4. Rodionov DA: Comparative genomic reconstruction of transcriptional regulatory networks in bacteria. Chem Rev 2007, 107(8):3467–3497. References 1. Wu D, Hugenholtz P, Mavromatis K, Pukall R, Dalin E, Ivanova NN, Kunin V, Goodwin L, Wu M, Tindall BJ, et al: A phylogeny-driven genomic encyclopaedia of Bacteria and Archaea. Nature 2009, 462(7276):1056–1060. 1. Wu D, Hugenholtz P, Mavromatis K, Pukall R, Dalin E, Ivanova NN, Kunin V, Goodwin L, Wu M, Tindall BJ, et al: A phylogeny-driven genomic encyclopaedia of Bacteria and Archaea. Nature 2009, 462(7276):1056–1060. 2. Pagani I, Liolios K, Jansson J, Chen IM, Smirnova T, Nosrat B, Markowitz VM, Kyrpides NC: The Genomes OnLine Database (GOLD) v.4: status of genomic and metagenomic projects and their associated metadata. Nucleic Acids Res 2012, 40(Database issue):D571–D579. 3. Liu J, Xu X, Stormo GD: The cis-regulatory map of Shewanella genomes. Nucleic Acids Res 2008, 36(16):5376–5390. 4. Rodionov DA: Comparative genomic reconstruction of transcriptional regulatory networks in bacteria. Chem Rev 2007, 107(8):3467–3497. Page 8 of 8 Cipriano et al. BMC Genomics 2013, 14:213 http://www.biomedcentral.com/1471-2164/14/213 5. Rodionov DA, Novichkov PS, Stavrovskaya ED, Rodionova IA, Li X, Kazanov MD, Ravcheev DA, Gerasimova AV, Kazakov AE, Kovaleva GY, et al: Comparative genomic reconstruction of transcriptional networks controlling central metabolism in the Shewanella genus. BMC Genomics 2011, 12(Suppl 1):S3. 25. Coordinators NR: Database resources of the national center for biotechnology information. Nucleic Acids Res 2013, 41(Database issue):D8– D20. 26. Pruitt KD, Tatusova T, Maglott DR: NCBI Reference Sequence (RefSeq): a curated non-redundant sequence database of genomes, transcripts and proteins. Nucleic Acids Res 2005, 33(Database issue):D501–D504. 6. Xu X, Ji Y, Stormo GD: Discovering cis-regulatory RNAs in shewanella genomes by support vector machines. PLoS Comput Biol 2009, 5(4):e1000338. 27. Wheeler DL, Barrett T, Benson DA, Bryant SH, Canese K, Church DM, DiCuccio M, Edgar R, Federhen S, Helmberg W, et al: Database resources of the national center for biotechnology information. Nucleic Acids Res 2005, 33(Database issue):D39–D45. 7. Gelfand MS: Evolution of transcriptional regulatory networks in microbial genomes. Curr Opin Struct Biol 2006, 16(3):420–429. 28. Bailey TL, Gribskov M: Combining evidence using p-values: application to sequence homology searches. Bioinformatics 1998, 14(1):48–54. 8. Gerasimova A, Kazakov AE, Arkin AP, Dubchak I, Gelfand MS: Comparative genomics of the dormancy regulons in mycobacteria. J Bacteriol 2011, 193(14):3446–3452. 29. Federhen S: The NCBI Taxonomy database. Nucleic Acids Res 2012, 40 (Database issue):D136–D143. 9. Suvorova IA, Tutukina MN, Ravcheev DA, Rodionov DA, Ozoline ON, Gelfand MS: Comparative genomic analysis of the hexuronate metabolism genes and their regulation in gammaproteobacteria. doi:10.1186/1471-2164-14-213 Cite this article as: Cipriano et al.: RegTransBase – a database of regulatory sequences and interactions based on literature: a resource for investigating transcriptional regulation in prokaryotes. BMC Genomics 2013 14:213. Cite this article as: Cipriano et al.: RegTransBase – a database of regulatory sequences and interactions based on literature: a resource for investigating transcriptional regulation in prokaryotes. BMC Genomics 2013 14:213. 15. Robison K, McGuire AM, Church GM: A comprehensive library of DNA-binding site matrices for 55 proteins applied to the complete Escherichia coli K-12 genome. J Mol Biol 1998, 284(2):241–254. 16. Sierro N, Makita Y, de Hoon M, Nakai K: DBTBS: a database of transcriptional regulation in Bacillus subtilis containing upstream intergenic conservation information. Nucleic Acids Res 2008, 36(Database issue):D93–D96. 17. Sharma D, Mohanty D, Surolia A: RegAnalyst: a web interface for the analysis of regulatory motifs, networks and pathways. Nucleic Acids Res 2009, 37(Web Server issue):W193–W201. 18. Baumbach J: CoryneRegNet 4.0 - A reference database for corynebacterial gene regulatory networks. BMC Bioinforma 2007, 8:429. 18. Baumbach J: CoryneRegNet 4.0 - A reference database for corynebacterial gene regulatory networks. BMC Bioinforma 2007, 8:429. 19. Grote A, Klein J, Retter I, Haddad I, Behling S, Bunk B, Biegler I, Yarmolinetz S, Jahn D, Munch R: PRODORIC (release 2009): a database and tool platform for the analysis of gene regulation in prokaryotes. Nucleic Acids Res 2009, 37(Database issue):D61–D65. 20. de Jong A, Pietersma H, Cordes M, Kuipers OP, Kok J: PePPER: a webserver for prediction of prokaryote promoter elements and regulons. BMC Genomics 2012, 13:299. 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 21. Pachkov M, Erb I, Molina N, van Nimwegen E: SwissRegulon: a database of genome-wide annotations of regulatory sites. Nucleic Acids Res 2007, 35(Database issue):D127–D131. 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 21. References J Bacteriol 2011, 193(15):3956–3963. 30. Liolios K, Tavernarakis N, Hugenholtz P, Kyrpides NC: The Genomes On Line Database (GOLD) v.2: a monitor of genome projects worldwide. Nucleic Acids Res 2006, 34(Database issue):D332–D334. 31. Rogers FB: Medical subject headings. Bull Med Libr Assoc 1963, 51:114–116. 10. Vitreschak AG, Mironov AA, Lyubetsky VA, Gelfand MS: Comparative genomic analysis of T-box regulatory systems in bacteria. RNA 2008, 14(4):717–735. 32. Machanick P, Bailey TL: MEME-ChIP: motif analysis of large DNA datasets. Bioinformatics 2011, 27(12):1696–1697. 33. Gupta S, Stamatoyannopoulos JA, Bailey TL, Noble WS: Quantifying similarity between motifs. Genome Biol 2007, 8(2):R24. 11. Wingender E: The TRANSFAC project as an example of framework technology that supports the analysis of genomic regulation. Brief Bioinform 2008, 9(4):326–332. y 34. Oberto J: FITBAR: a web tool for the robust prediction of prokaryotic regulons. BMC Bioinforma 2010, 11:554. y 34. Oberto J: FITBAR: a web tool for the robust prediction of prokaryotic regulons. BMC Bioinforma 2010, 11:554. 12. Portales-Casamar E, Arenillas D, Lim J, Swanson MI, Jiang S, McCallum A, Kirov S, Wasserman WW: The PAZAR database of gene regulatory information coupled to the ORCA toolkit for the study of regulatory sequences. Nucleic Acids Res 2009, 37(Database issue):D54–D60. 35. Hemmerich C, Buechlein A, Podicheti R, Revanna KV, Dong Q: An Ergatis- based prokaryotic genome annotation web server. Bioinformatics 2010, 26(8):1122–1124. 35. Hemmerich C, Buechlein A, Podicheti R, Revanna KV, Dong Q: An Ergatis- based prokaryotic genome annotation web server. Bioinformatics 2010, 26(8):1122–1124. 36. Dehal PS, Joachimiak MP, Price MN, Bates JT, Baumohl JK, Chivian D, Friedland GD, Huang KH, Keller K, Novichkov PS, et al: MicrobesOnline: an integrated portal for comparative and functional genomics. Nucleic Acids Res 2010, 38(Database issue):D396–D400. 13. Griffith OL, Montgomery SB, Bernier B, Chu B, Kasaian K, Aerts S, Mahony S, Sleumer MC, Bilenky M, Haeussler M, et al: ORegAnno: an open-access community-driven resource for regulatory annotation. Nucleic Acids Res 2008, 36(Database issue):D107–D113. Friedland GD, Huang KH, Keller K, Novichkov PS, et al: MicrobesOnline: an integrated portal for comparative and functional genomics. Nucleic Acids Res 2010, 38(Database issue):D396–D400. 14. Gama-Castro S, Jimenez-Jacinto V, Peralta-Gil M, Santos-Zavaleta A, Penaloza-Spinola MI, Contreras-Moreira B, Segura-Salazar J, Muniz-Rascado L Martinez-Flores I, Salgado H, et al: RegulonDB (version 6.0): gene regulation model of Escherichia coli K-12 beyond transcription, active (experimental) annotated promoters and Textpresso navigation. Nucleic Acids Res 2008, 36(Database issue):D120–D124. doi:10.1186/1471-2164-14-213 Pachkov M, Erb I, Molina N, van Nimwegen E: SwissRegulon: a database of genome-wide annotations of regulatory sites. Nucleic Acids Res 2007, 35(Database issue):D127–D131. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: 22. Kazakov AE, Cipriano MJ, Novichkov PS, Minovitsky S, Vinogradov DV, Arkin A, Mironov AA, Gelfand MS, Dubchak I: RegTransBase–a database of regulatory sequences and interactions in a wide range of prokaryotic genomes. Nucleic Acids Res 2007, 35(Database issue):D407–D412. • Convenient online submission 23. Novichkov PS, Rodionov DA, Stavrovskaya ED, Novichkova ES, Kazakov AE, Gelfand MS, Arkin AP, Mironov AA, Dubchak I: RegPredict: an integrated system for regulon inference in prokaryotes by comparative genomics approach. Nucleic Acids Res 2010, 38(Web Server issue):W299–W307. 24. Novichkov PS, Laikova ON, Novichkova ES, Gelfand MS, Arkin AP, Dubchak I, Rodionov DA: RegPrecise: a database of curated genomic inferences of transcriptional regulatory interactions in prokaryotes. Nucleic Acids Res 2010, 38(Database issue):D111–D118.
https://openalex.org/W2886451843	https://europepmc.org/articles/pmc6534009?pdf=render	English	null	Perioperative factors associated with delayed graft function in renal transplant patients	Brazilian Journal of Nephrology	2,018	cc-by	4,301	Resumo Authors Milton Halyson Benevides de Freitas1 Luciana Cavalcanti Lima1 Tania Cursino de Menezes Couceiro1 Wilton Bernadino da Silva2 João Marcelo de Andrade1 Marcio Handerson Benevides de Freitas2 Introdução: O sucesso do transplante renal e a boa função do enxerto dependem de uma boa técnica cirúrgica, anestesia que assegure a estabilidade hemodinâmica do receptor e condições adequadas de enxerto e receptor. Diversos fatores podem interferir na perfusão do enxerto e comprometer sua viabilidade. O objetivo deste estudo foi avaliar os fatores perioperatórios associados à função retardada do enxerto (FRE) em pacientes transplantados renais. Métodos: Estudo de coorte histórica em 310 pacientes submetidos a transplante entre 2011 e 2013. A FRE foi definida como a necessidade de diálise durante a primeira semana pós-transplante. Utilizou-se regressão logística e técnica Stepwise para construir modelos estatísticos. Resultados: A análise multivariada revelou fatores associados à FRE: técnica de anestesia combinada (OR = 3,81,95% CI, 1,71 a 9,19), regime de fluidos < 50 mL.kg-1 (OR = 3,71,95% CI, 1,68 a 8,61), diálise por mais de 60 meses (OR = 4,77,95% IC, 1,93 a 12,80), basiliximab (OR = 3,34,95% IC, 1,14 a 10,48), tempo de isquemia fria > 12 horas (OR = 5,26,95 % IC, 2,62 a 11,31), doador vivo (OR = 0,19,95% CI, 0,02 a 0,65) e diurese precoce (OR = 0,02,95% IC, 0,008 a 0,059). A precisão desse modelo é de 92,6%, calculada usando a área sob a curva ROC. A incidência de FRE na população estudada foi de 76,1%. Conclusões: Técnica combinada de anestesia, diálise por mais de 60 meses, basiliximab e tempo de isquemia fria> 12 horas são fatores de risco para FRE; regimes de fluidos liberais e rins de doadores vivos são protetores. Introduction: Successful renal transplant and consequent good graft function depend on a good surgical technique, an anesthetic that ensures the hemodynamic stability of the receiver, and appropriate conditions of graft and recipient. Several factors can in- terfere with the perfusion of the graft and compromise its viability. The objective of this study was to evaluate perioperative fac- tors associated with delayed graft function (DGF) in renal transplantation patients. Methods: This is a historical cohort study of patients who underwent renal trans- plantation between 2011 and 2013. Three hundred and ten transplants were ana- lyzed. DGF was defined as the need for di- alysis during the first week post-transplant. Perioperative factors associated with delayed graft function in renal transplant patients Fatores perioperatórios associados à função retardada do enxerto em pacientes transplantados renais Perioperative factors associated with delayed graft function in renal transplant patients Fatores perioperatórios associados à função retardada do enxerto em pacientes transplantados renais 1 Instituto de Medicina Integral Professor Fernando Figueira, Recife, PE, Brasil. 2 Universidade Federal de Pernambuco, Recife, PE, Brasil. Submitted on: 01/08/2018. Approved on: 06/12/2018. Introduction Introduction Renal transplantation is the treatment of choice for patients with end-stage kidney disease and is asso- ciated with better quality of life, a greater cost/ben- efit ratio, and longer survival.1 Proper functioning of the graft is essential for patients to re-establish body homeostasis and thus benefit from receiving the transplant.2 Standard monitoring included continuous elec- trocardiography, heart rate (HR), peripheral oxygen saturation, and noninvasive blood pressure measure- ment. Induction and maintenance of general anesthe- sia were performed using sevoflurane in concentra- tions of 1 to 2% in a mixture of 50% oxygen and 50% air. The most commonly used intravenous an- esthetics were fentanyl, propofol and atracurium. Patients underwent balanced general anesthesia (GA) or combined anesthesia (CA), defined by the assistant anesthesiologist depending on hemodynamic stability, comorbidities, and coagulation profile. Regional an- esthesia was performed by lumbar epidural injection of bupivacaine 0.125%, follow by general anesthe- sia. The protocol of immunosuppressive drugs used in our institution is Thymoglobulin and Basiliximab. Thymoglobulin is generally used when the patient has a panel reactive antibody (PRA) > 25% and Basiliximab when the PRA < 25%. Early diuresis is a good marker of successful re- nal transplantation as it reflects the re-establishment of graft function.3 Kidney failure immediately after transplantation, on the other hand, is known as de- layed graft function (DGF), which is associated with acute kidney rejection, renal failure and an increased risk of graft loss.4 The fluid regimen, living or de- ceased donor, the anesthetic technique, comorbidities, cold ischemia time (CIT) and the anesthetic drugs used during surgery are some of the factors that may interfere with the perfusion of the graft and compro- mise its viability.5 The aim of this study was to evaluate the periop- erative factors associated with delayed graft function in patients who underwent renal transplantation. Methods Data were collected from patients’ pre-anesthesia evaluation form, intraoperative anesthesia records, and nephrology medical records. The independent variables included anesthesia technique, intraopera- tive hydration regimen, CIT, duration and type of di- alysis, PRA, living or deceased donor, hemodynamic instability, traumatic or non-traumatic death of the donor, receptor comorbidities, type of immunosup- pressant used, and weight and age of the donor and receptor. The dependent variable was DGF. Following approval by the hospital ethics committee, this historical cohort study was conducted on patients aged 18-60 years who were scheduled for renal trans- plantation at our institution in Recife, Brazil between 2011 and 2013. Patients with cardiac disease (ejec- tion fraction < 30), kidney-pancreas transplantation, and kidney retransplantation were excluded from this study. Hemodynamic instability was considered to be a drop in systolic blood pressure (SBP) of at least 30% compared to the initial blood pressure in the operat- ing room and/or requiring vasoactive drugs for a pe- riod equal to or greater than 15 minutes.6 Restrictive fluid regimen was defined as the intraoperative hy- dration < 50 mL·kg-1. There are many definitions of DGF in the literature; one of the most well established and adopted in our research was the need for dialysis during the first week post-transplant. Immediate graft function (IGF) is defined as serum creatinine decrease less than 70% of preoperative value in the first week. We used the Fisher’s exact and Chi-square (χ2) tests to compare categorical variables and the Student’s t and Mann-Whitney tests for mean comparisons (table 1). The independent variables were subjected to logistic regression modeling using a backward step- wise method, according to the Akaike information criterion (AIC).8 To evaluate the quality of the regres- sion adjustment, the Hosmer and Lemeshow test was used.9 The accuracy and performance of each model were evaluated using the area under the ROC curve. Expanded criteria deceased organ donors (ECD) are a source of kidneys that permit more patients to benefit from transplantation. ECD is defined as all Resumo Logistic regression with a stepwise tech- nique was used to build statistical models. Results: Multivariate analysis revealed the following risk factor for DGF: combined anesthesia technique (OR = 3.81, 95%CI, 1.71 to 9.19), a fluid regimen < 50 mL·kg-1 (OR = 3.71, 95%CI, 1.68 to 8.61), dialy- sis for more than 60 months (OR = 4.77, 95%CI, 1.93 to 12.80), basiliximab (OR = 3.34, 95%CI, 1.14 to 10.48), cold ischemia time > 12 hour (OR = 5.26, 95%CI, 2.62 to 11.31), living donor (OR = 0.19, 95%CI, 0.02 to 0.65), and early diuresis (OR = 0.02, 95%CI, 0.008 to 0.059). The accuracy of this model was 92.6%, calculated using the area under the ROC curve. The incidence of DGF in the study population was 76.1%. Conclusions: Combined anesthesia tech- nique, dialysis for more than 60 months, basiliximab, and cold ischemia time > 12 hours are risk factor for DGF, while liberal fluid regimens and kidneys from living do- nors are protective factors. 1 Instituto de Medicina Integral Professor Fernando Figueira, Recife, PE, Brasil. 2 Universidade Federal de Pernambuco, Recife, PE, Brasil. Keywords: Kidney Transplantation; Re- nal Dialysis; Immunosuppressive Agents; Delayed Graft Function. Palavras-chave: Transplante de Rim; Di- álise Renal; Imunossupressores; Função Retardada do Enxerto. Delayed graft function in renal transplant patients deceased donors older than 60 years or donors older than 50 years with two of the following conditions: hypertension, stroke as the cause of death, or prer- etrieval serum creatinine greater than 1.5 mg/dL-1.7 Results Three hundred forty-four patients underwent re- nal transplantation but 34 were excluded from the Braz. J. Nephrol. (J. Bras. Nefrol.) 2018;40(4):360-365 361 Delayed graft function in renal transplant patients Table 1 Demographic and clinical features of the study population IGF (74) DGF (236) p-value Recipient Gender 0.1767° Female 27 (36.4%) 73 (30.9%) Male 47 (63.5%) 163 (69%) Age (years) 42.6 (± 15.1) 45.9 (± 12.6) 0.2962°° Weight (kg) 63.5 (± 13.4) 65.2 (± 14.2) 0.3041°° BMI (kg.m-2) * 22.9 (± 4.2) 24 (± 4.3) 0.4239°° Hypertension 0.3424° - No 19 (25.6%) 53 (22.4%) - Yes 55 (74.4%) 183 (77.6%) Diabetes 0.1964° - No 63 (82.8%) 194 (82.2%) - Yes 11 (17.1%) 42 (17.7%) Dialysis type 0.0826° - Peritoneal dialysis 6 (0.8%) 8 (3.3%) - Hemodialysis 68 (91.2%) 228 (96.7%) PRA > 25 0.1556° - No 18 (24.3%) 42 (17.7%) - Yes 56 (75.6%) 194 (82.2%) Donor Deceased < 0.0001° - No 28 (37.8%) 4 (12.5%) - Yes 46 (62.1%) 232 (83.4%) Age (years) 39.3 (± 15) 42.1 (± 14.5) 0.5368°° Cr 1.5 (± 0.8) 2.2 (± 1.9) 0.4495°° Transplantation Cold Ischemia Time (hours) 11.7 (± 11.2) 21.2 (± 7.8) < 0.0001° Restrictive fluid regimen < 0.0001° No 43 (58.1%) 64 (27.1%) Yes 31 (41.8%) 172 (72.8%) Surgical time (min) 153.9 (± 31.6) 146.1 (± 33.5) 0.3730°° SD = Standard deviation (±),°Chi-square (χ2) tests; °° Student's t; IGF = Immediate graft function; PRA = panel reactive antibody. analysis (Figure 1). The demographic and clinical de- tails of the included patients are shown in Table 1. Both groups were comparable concerning recipient sex, age, and positive versus negative PRA. ECD organs were present in 109 transplanted patients, 56 in the DGF group (p-value = 0.44). Traumatic death occurred in 42.7% (120) of the do- nors. Stroke was the cause of death of 46.3% (130) and death from others causes was 11%. Of 310 patients, 67.7% were male, 89% received kidneys from deceased donors, and 112 (36%) received general anesthesia. Upon univariate analysis (Table 1), only the origin of the kidney, restrictive fluid regimen, and the CIT were more prevalent among DGF com- pared with IGF patients. Hemodynamic instability oc- curred in 110 patients during the perioperative period, without correlation with any group (p-value = 0.48). Results Flowchart of patient inclusion Table 2 Factors associated with Delayed Graft Function (DGF) * p < 0.05 Estimate of coefficients p-value OR (95% CI) Risk factors - Combined Anesthesia 1.3381 < 0.0001* 3.81 (1.71-9.19) - Restrictive fluid regimen (< 50 mL·kg-1) 1.3118 0.00153 * 3.71 (1.68-8.61) - Immunosuppressant (Basiliximab) 1.2082 0.03201 * 3.34 (1.14-10.48) - Dialysis > 60 months 1.5641 0.00110 * 4.77 (1.93-12.80) - Cold ischemia time (> 12h) 1.6618 < 0.0001* 5.26 (2.62-11.31) Protective factors - Living donor -1.2047 0.03434 * 0.19 (0.02-0.65) - Early diuresis -3.7312 < 0.0001* 0.02 (0.008-0.059) Figure 1. Flowchart of patient inclusion Table 2 Factors associated with Delayed Graft Function (DGF) * p < 0.05 Estimate of coefficients p-value OR (95% CI) Risk factors - Combined Anesthesia 1.3381 < 0.0001* 3.81 (1.71-9.19) - Restrictive fluid regimen (< 50 mL·kg-1) 1.3118 0.00153 * 3.71 (1.68-8.61) - Immunosuppressant (Basiliximab) 1.2082 0.03201 * 3.34 (1.14-10.48) - Dialysis > 60 months 1.5641 0.00110 * 4.77 (1.93-12.80) - Cold ischemia time (> 12h) 1.6618 < 0.0001* 5.26 (2.62-11.31) Protective factors - Living donor -1.2047 0.03434 * 0.19 (0.02-0.65) - Early diuresis -3.7312 < 0.0001* 0.02 (0.008-0.059) hours (43.1%). Analysis of recipient factors revealed hours (43.1%). Analysis of recipient factors revealed associations of DGF with dialysis time > 60 months (OR = 4.77, 95%CI, 1.93 to 12.80). During the intraoperative time, a restrictive fluid regime (OR = 3.71, 95%CI, 1.68 to 8.61), and the use of combined anesthesia (OR = 3.81, 95%CI, 1.71 to 9.19) were related to DGF. Cold ischemia time > 12 hours (OR = 5.26, 95%CI, 2.62 to 11.31) and basil- iximab (OR = 3.34, 95%CI, 1.14 to 10.48) were risk factors to DGF. Among donor factors, living donor was a protective factor against DGF (OR = 0.19, 95%CI, 0.02 to 0.65) and patients with early diuresis had a lower risk of DGF (OR = 0.02, 95%CI, 0.008 to 0.059). To assess the performance of the multiple logistic regression analysis, we calculated the area un- der ROC curve, which yielded accuracies of 92.6% (Figure 2). Results The base logistic regression model obtained had the following independent variables: dialysis time, type of anesthesia, immunosuppressive drugs, flu- id regimen, early diuresis, and origin of the kidney (Table 2). The incidence of DGF in our study was 76.1%; most of the patients needed dialysis in the first 24 Braz. J. Nephrol. (J. Bras. Nefrol.) 2018;40(4):360-365 362 Delayed graft function in renal transplant patients Figure 1. Flowchart of patient inclusion hours (43.1%). Analysis of recipient factors revealed associations of DGF with dialysis time > 60 months (OR = 4.77, 95%CI, 1.93 to 12.80). During the intraoperative time, a restrictive fluid regime (OR = 3.71, 95%CI, 1.68 to 8.61), and the use of combined anesthesia (OR = 3.81, 95%CI, 1.71 to 9.19) were related to DGF. Cold ischemia time > 12 hours (OR = 5 26 95%CI 2 62 to 11 31) and basil logistic regression analysis, we calculated the area un- der ROC curve, which yielded accuracies of 92.6% (Figure 2). Discussion In this retrospective study conducted in 310 kidney transplant patients, we identified several factors re- Figure 1. Flowchart of patient inclusion Table 2 Factors associated with Delayed Graft Function (DGF) * p < 0.05 Estimate of coefficients p-value OR (95% CI) Risk factors - Combined Anesthesia 1.3381 < 0.0001* 3.81 (1.71-9.19) - Restrictive fluid regimen (< 50 mL·kg-1) 1.3118 0.00153 * 3.71 (1.68-8.61) - Immunosuppressant (Basiliximab) 1.2082 0.03201 * 3.34 (1.14-10.48) - Dialysis > 60 months 1.5641 0.00110 * 4.77 (1.93-12.80) - Cold ischemia time (> 12h) 1.6618 < 0.0001* 5.26 (2.62-11.31) Protective factors - Living donor -1.2047 0.03434 * 0.19 (0.02-0.65) - Early diuresis -3.7312 < 0.0001* 0.02 (0.008-0.059) hours (43.1%). Analysis of recipient factors revealed associations of DGF with dialysis time > 60 months (OR = 4.77, 95%CI, 1.93 to 12.80). During the intraoperative time, a restrictive fluid regime (OR = 3.71, 95%CI, 1.68 to 8.61), and the use of combined anesthesia (OR = 3.81, 95%CI, 1.71 to logistic regression analysis, we calculated the area un- der ROC curve, which yielded accuracies of 92.6% (Figure 2). Discussion In this retrospective study conducted in 310 kidney Figure 1. Discussion In this retrospective study conducted in 310 kidney transplant patients, we identified several factors re- lated with delayed graft function. Although causality cannot be established, our data suggested associations in agreement with other reports. DGF is one of the most common complications following renal transplantation and is often associat- ed with acute rejection and increased risk of graft loss. The incidence of DGF in our study was 76.1%, with Braz. J. Nephrol. (J. Bras. Nefrol.) 2018;40(4):360-365 363 Delayed graft function in renal transplant patients rates of 0.01% for living donor kidneys and 83.4% for deceased donor kidneys. In a Brazilian multicenter study of 6 transplantation centers, totaling 612 kid- ney transplants with grafts of deceased donors carried out between 2000 and 2002, revealed an incidence of DGF of 53.9, 62.3, and 51.6% in 2000, 2001, and 2002, respectively.10 One center had an incidence of 81.6%, similar to our results. These high rates of DGF of grafts from deceased donors are explained mainly by the difficulty of maintaining hemodynamic stability of the donor, prolonged cold ischemia time, use of ECD kidneys, and the definition for DGF (need for dialysis during the first week post-transplant). Ojo et al.11 have demonstrated that recipients of ECD kidneys benefit from extra life-years when compared to wait-listed dialysis patients, although they pres- ent higher rates of delayed graft function, more acute rejection episodes, and decreased long-term graft function. Living donor kidneys and early diuresis were iden- tified as protective factors for DGF. The short cold ischemia time and the consequent less pronounced ischemia-reperfusion injury (IRI) produce fewer cyto- kines and free radicals, which limit the deleterious ef- fects of IRI. A similar association between graft origin and DGF was reported by Ojo et al.12, who conducted a retrospective cohort study of 37,216 patients. In that study, there was a 23% increased risk of DGF for every 6 hours of cold ischemia time in deceased donor kidneys. DGF rates were also affected by the type of an- esthesia used. Combined anesthesia showed a three- fold increased risk of graft dysfunction in our study. This is the first study to correlate anesthetic technique with DGF. The association may be explained by the reduction in catecholamine levels and vasodilatation following neuraxial blockade, which reduces graft perfusion.13 A restrictive fluid regimen increased the risk of DGF approximately 4-fold. Discussion Kidney perfusion depends linearly on mean arterial pressure, which is influenced by intravascular volume, sympathetic tone, and renal autoregulation.14 Due to the denervation during trans- plantation, self-regulation and sympathetic tone are Figure 2. ROC curve showing the sensitivity and false positive rate (1- specificity) of factors associated with Delayed Graft Function (area under the curve: 92.6%). Figure 2. ROC curve showing the sensitivity and false positive rate (1- specificity) of factors associated with Delayed Graft Function (area under the curve: 92.6%). Figure 2. ROC curve showing the sensitivity and false positive rate (1- specificity) of factors associated with Delayed Graft Function (area under the curve: 92 6%) Figure 2. ROC curve showing the sensitivity and false positive rate (1 specificity) of factors associated with Delayed Graft Function (area under the curve: 92.6%). rates of 0.01% for living donor kidneys and 83.4% for deceased donor kidneys. In a Brazilian multicenter study of 6 transplantation centers, totaling 612 kid- ney transplants with grafts of deceased donors carried out between 2000 and 2002, revealed an incidence of DGF of 53.9, 62.3, and 51.6% in 2000, 2001, and 2002, respectively.10 One center had an incidence of 81.6%, similar to our results. These high rates of DGF of grafts from deceased donors are explained mainly by the difficulty of maintaining hemodynamic stability of the donor, prolonged cold ischemia time, use of ECD kidneys, and the definition for DGF (need for dialysis during the first week post-transplant). Ojo et al.11 have demonstrated that recipients of ECD kidneys benefit from extra life-years when compared to wait-listed dialysis patients, although they pres- ent higher rates of delayed graft function, more acute rejection episodes, and decreased long-term graft function. rates of 0.01% for living donor kidneys and 83.4% for deceased donor kidneys. In a Brazilian multicenter study of 6 transplantation centers, totaling 612 kid- ney transplants with grafts of deceased donors carried out between 2000 and 2002, revealed an incidence of DGF of 53.9, 62.3, and 51.6% in 2000, 2001, and 2002, respectively.10 One center had an incidence of 81.6%, similar to our results. These high rates of DGF of grafts from deceased donors are explained mainly by the difficulty of maintaining hemodynamic stability of the donor, prolonged cold ischemia time, use of ECD kidneys, and the definition for DGF (need for dialysis during the first week post-transplant). References 1. Coupe N, O’Brien M, Gibson P, de Lima J. Anesthesia for pediatric renal transplantation with and without epidural analgesia-- a review of 7 years experience. Paediatr Anaesth 2005;15:220-8. 2. Rabbat CG, Thorpe KE, Russel JD, Churchill DN. Comparison of mortality risk for dialysis patients and cadaveric first renal transplant recipients in Ontario, Canada. J Am Soc Nephrol 2000;11:917-22. 3. Dawidson IJ, Ar’Rajab A. Perioperative fluid and drug therapy during cadaver kidney transplantation. Clin Transpl 1992:267- 84. Duration of dialysis pre-transplantation was also identified as a risk factor for DGF in our study. In a retrospective study of 30,294 kidney transplant pa- tients, Keith et al.16 found that the incidence of DGF was 26.4% higher in patients who received dialysis for more than 72 months. 4. Perico N, Cattaneo D, Sayegh MH, Remuzzi G. Delayed graft function in kidney transplantation. Lancet 2004;364:1814-27. 5. Gil JS, Pereira BJ. Death in the first year after kidney trans- plantation: implication for patient waiting list. Transplantation 2003;75:113-7. 6. Smith TW, Kelly RA, Stevenson LW, Braunwald E. Manage- ment of heart failure. In: Braunwald E. Heart Disease: A Text- book of Cardiovascular Medicine. Philadelphia: W.B. Saun- ders; 1997. p. 442-514. Basiliximab was identified as risk factor for DGF (OR 3.34, p-value = 0.03201). Similarly, in a retro- spective cohort study of 327 patients, Chen et al.17 found a higher risk of DGF in the basiliximab group (37.1% versus 26.1%, p-value = 0.035). According to Lebranchu et al.,18 both anti-lymphocyte drugs (Thymoglobulin and Basiliximab) are effective for in- ducing immunosuppression and are associated with DGF because they release cytokines and induce neph- rotoxicity. Thymoglobulin is associated with a lower incidence of graft dysfunction because of its anti-ad- hesin molecules, which prevent leukocytes from ad- hering to cell surfaces.19 7. Metzger RA, Delmonico FL, Feng S, Port FK, Wynn JJ, Merion RM. Expanded criteria donors for kidney transplantation. Am J Transplant 2003;3:114-25. 8. Bozdogan H. Model selection and Akaike’s Information Cri- terion (AIC): The general theory and its analytical extensions. Psychometrika 1987;52:345-70. y 9. Hosmer DW, Lemeshow S. Applied Logistic Regression. New York: Wiley; 1989. 10. Azevedo LS, Castro MC, Monteiro de Carvalho DB, d’Avila DO, Contieri F, Gonçalves RT, et al. Incidence of delayed graft function in cadaveric kidney transplants in Brazil: a multicenter analysis. Transplant Proc 2005;37:2746-7. 11. Ojo AO, Hanson JA, Meier-Kriesche, Okechukwu CN, Wolfe RA, Leichtman AB, et al. Discussion Ojo et al.11 have demonstrated that recipients of ECD kidneys benefit from extra life-years when compared to wait-listed dialysis patients, although they pres- ent higher rates of delayed graft function, more acute rejection episodes, and decreased long-term graft function. ischemia-reperfusion injury (IRI) produce fewer cyto- kines and free radicals, which limit the deleterious ef- fects of IRI. A similar association between graft origin and DGF was reported by Ojo et al.12, who conducted a retrospective cohort study of 37,216 patients. In that study, there was a 23% increased risk of DGF for every 6 hours of cold ischemia time in deceased donor kidneys. DGF rates were also affected by the type of an- esthesia used. Combined anesthesia showed a three- fold increased risk of graft dysfunction in our study. This is the first study to correlate anesthetic technique with DGF. The association may be explained by the reduction in catecholamine levels and vasodilatation following neuraxial blockade, which reduces graft perfusion.13 A restrictive fluid regimen increased the risk of DGF approximately 4-fold. Kidney perfusion depends linearly on mean arterial pressure, which is influenced by intravascular volume, sympathetic tone, and renal autoregulation.14 Due to the denervation during trans- plantation, self-regulation and sympathetic tone are Living donor kidneys and early diuresis were iden- tified as protective factors for DGF. The short cold ischemia time and the consequent less pronounced Braz. J. Nephrol. (J. Bras. Nefrol.) 2018;40(4):360-365 364 Delayed graft function in renal transplant patients lost and the renal flow undergoes great influence of volemia.15 In a clinical study of 40 patients, Othman et al.14 evaluated the influence of the hydration regime on hemodynamic stability and early function of the graft and found that patients who received the largest infusion of fluids moments before the renal artery was unclamped presented lower hemodynamic instability and higher levels of systolic blood pressure, medium blood pressure, and central venous pressure. References Survival in recipients of marginal ca- daveric donor kidneys compared with other recipients and wait- listed transplant candidates. J Am Soc Nephrol 2001;12:589- 97. 12. Ojo AO, Wolfe RA, Held PJ, Port FK, Schmouder RL. Delayed graft function: risk factors and implications for renal allograft survival. Transplantation 1997;63:968-74. The current study had a few limitations. First, some unmeasured variables may have affected the accuracy of our models. Second, perioperative care practices may be different in other institutions, po- tentially accounting for the differences in outcomes. Third, the lack of water balance, which would be the ideal variable to characterize peri-operative volume management. Our study has several strengths: this is the first study to examine the effect of anesthesia on DGF, and our end-points are statistically relevant for clinical practice. Future controlled prospective studies should be conducted to further evaluate these results. 13. Yost CS, Niemann CU. Anesthesia for Abdominal Organ Transplantation. In: Miller RD, ed. Miller’s Anesthesia. Phila- delphia: Churchill Livingstone; 2010. p. 2155-84. 14. Othman MM, Ismael AZ, Hammouda GE. The impact of tim- ing of maximal crystalloid hydration on early graft function during kidney transplantation. Anesth Analg 2010;110:1440- 6. 15. Morita K, Seki T, Nonomura K, Koyanagi T, Yoshioka M, Saito H. Changes in renal blood flow in response to sympatho- mimetics in the rat transplanted and denervated kidney. Int J Urol 1999;6:24-32. 16. Keith D, Cantarovich M, Paraskevas S, Tchervenkov J. Duration of dialysis pretransplantation is an important risk factor for de- layed recovery of renal function following deceased donor kidney transplantation. Transplant Int 2008;21:126-32. 17. Chen G, Gu J, Qiu J, Wang C, Fei J, Deng S, et al. Efficacy and safety of thymoglobulin and basiliximab in kidney transplant pa- tients at high risk for acute rejection and delayed graft function. Exp Clin Transplant 2013;11:310-4. Conclusion In summary, this study showed that liberal fluid regi- mens, kidneys from living donors, and cold ischemia of less than 12 hours are protective factors for DGF. Dialysis pre-transplantation > 60 months and com- bined anesthesia are risk factors. Basiliximab is a risk factors for DGF. 18. Lebranchu Y, Bridoux F, Büchler M, Le Meur Y, Etienne I, Tou- pance O, et al. Immunoprophylaxis with basiliximab compared with antithymocyte globulin in renal transplant patients receiving MMF-containing triple therapy. Am J Transplant 2002;2:48-56. 19. Batiuk TD, Bennet WM, Norman DJ. Cytokine nephropathy during antilymphocyte therapy. Transplant Proc 1993;25:27- 30. Braz. J. Nephrol. (J. Bras. Nefrol.) 2018;40(4):360-365 365
https://openalex.org/W4281645226	https://zenodo.org/records/6629104/files/ARIMS0714.pdf	Russian	null	ЎЗБЕКИСТОНДА БОЛА ҲУҚУҚЛАРИ КАФОЛАТЛАРИ	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	1,135	ACADEMIC RESEARCH IN MODERN SCIENCE International scientific-online conference ЎЗБЕКИСТОНДА БОЛА ҲУҚУҚЛАРИ КАФОЛАТЛАРИ Д.М.Эргашев АндДУ Ижтимоий-иқтисодиёт факультети талабаси https://doi.org/10.5281/zenodo.6629104 Abstract Президентимизнинг 2017 йил 7 февралдаги Фармони билан тасдиқланган 2017 — 2021 йилларда Ўзбекистон Республикасини ривожлантиришнинг бешта устувор йўналиши бўйича Ҳаракатлар стратегиясининг 4-устивор йўналиши “Ижтимоий соҳани ривожлантиришнинг устувор йўналишлари” деб номланиб, унинг 4.2.– банди Аҳолини ижтимоий ҳимоя қилиш ва соғлиқни сақлаш тизимини такомиллаштириш, хотин-қизларнинг ижтимоий-сиёсий фаоллигини ошириш сифатида белгиланган. Key words: ҳуқуқий тарбия, болалар, фуқаролик, жамият, ички ва ташқи сиёсати. Мазкур бандда оила саломатлигини мустаҳкамлаш, оналик ва болаликни муҳофаза қилиш, оналар ва болаларнинг сифатли тиббий хизматдан фойдаланишни кенгайтириш, уларга ихтисослаштирилган ва юқори технологияларга асосланган тиббий ёрдам кўрсатиш, чақалоқлар ва болалар ўлимини камайтириш бўйича комплекс чора-тадбирларни янада кенгроқ амалга ошириш каби муҳим масалалар белгилаб берилган бўлса, “Таълим ва фан соҳасини ривожлантириш” деб номланган 4.4.- бандида эса мактабгача таълим муассасалари тармоғини кенгайтириш ва ушбу муассасаларда болаларнинг ҳар томонлама интеллектуал, эстетик ва жисмоний ривожланиши учун шарт-шароитларни тубдан яхшилаш, болаларнинг мактабгача таълим билан қамраб олинишини жиддий ошириш ва фойдаланиш имкониятларини таъминлаш, педагог ва мутахассисларнинг малака даражасини юксалтириш каби мухум масалалар устивор вазифа қилиб белгиланди. Ўзбекистон Республикаси БМТнинг тўла ҳуқуқли аъзосига айланганидан кейин, инсон ҳуқуқ ва эркинликларини ҳимоялаш бўйича халқаро тизимни шакллантирувчи 60 дан ортиқ халқаро ҳуқуқий ҳужжатларга қўшилди, Бола ҳуқуқлари Конвенцияси шулар жумласидан. Республиканинг ярмидан кўп аҳолиси (67 %) 30 ёшгача бўлган ёшлар, шулардан 11 млн. - 18 ёшгача бўлгани ҳолда, бу аҳолининг умумий хисобида 48,2 % ни ташкил этади. Бундай вазиятда Конвенция қоидаларининг қўлланилиш муаммоси ўта долзарбдир. Болаларнинг 56 ACADEMIC RESEARCH IN MODERN SCIENCE International scientific-online conference ҳуқуқ ва эркинликларини таъминлаш муаммосини самарали ечмасдан туриб мамлакатнинг демократик ривожланишини тасаввур қилиб бўлмайди, шу сабабдан мамлакат ҳукумати томонидан мазкур ижтимоий муносабатлар доирасида катта ишлар амалга оширилди. Ўзбекистонда бола ҳуқуқларини ҳимоялашнинг қонуний тизими асосини Ўзбекистон Республикаси Конституцияси, қонун ва қонуности ҳужжатлари ташкил этади, уларда инсоннинг асосий ҳуқуқ ва эркинликлари акс этган. Мазкур соҳадаги бевосита қонун бўлиб 1991 йил 20 ноябрдаги «Ўзбекистон Республикасида ёшларга оид давлат сиёсатининг асослари тўғрисида»гиЎзбекистон Республикасининг қонуни хисобланади. Унга мувофиқ, «Ўзбекистон Республикасида ёшларга оид давлат сиёсати қуйидаги қоидаларга асосланади: миллати, ирқи, тили, дини, ижтимоий мавқеи, жинси, маълумоти ва сиёсий эътиқодидан қатъи назар ёшлар тўғрисида ғамхўрлик қилиш; ёшларни ҳуқуқий ва ижтимоий жиҳатдан ҳимоя қилиш; миллий, маданий анъаналарнинг авлоддан авлодга ўтиши, авлодларнинг маънавий алоқаси; ёшларнинг ташаббусларини қўллаб- қувватлаш, ёшлар Ўзбекистон Республикаси Конституцияси ва қонунлари доирасида ўз манфаатларини амалга ошириш йўлларини эркин танлаб олишларига кафолат бериш; жамиятни ривожлантиришга, айниқса республика ёшлари ҳаётига оид сиёсат ва дастурларни ишлаб чиқиш ҳамда амалга оширишда ёшларнинг бевосита иштирок этиши; ҳуқуқ ва бурчларнинг эркинлик ва фуқаролик масъулиятининг бирлиги». Abstract Қуйидаги ҳолатда биз Ўзбекистон қонунчилигида бола ҳуқуқлари масалалари таъминланганлигини кўришимиз мумкин. Республикада меҳнат муносабатларини тартибга солувчи асосий қонун бўлиб Ўзбекистон Республикасининг Меҳнат кодекси хисобланади (Ўзбекистон Республикаси МК). Ишлаб чиқаришда ёки ишлаб чиқариш давомидаги таълимда меҳнат ва дам олишни тўғри ташкил этилган ҳолатдаги вояга етмаганларнинг жисмоний меҳнати нафақат уларнинг соғлигига зарар етказмайди, балки кучи, чидамлилига ва иродасининг мустаҳкамланишига ҳам хизмат қилади. Аммо, ҳар бир бола жисмоний ва ақлий заифлиги сабабли маҳсус ҳуқуқий ҳимояга муҳтож. Давлат болага зўравонлик, ёмон даволаш, мажбурий меҳнат ёки уларнинг ҳаёти ва соғлигига зарар етказиши мумкин бўлган ACADEMIC RESEARCH IN MODERN SCIENCE International scientific-online conference ҳар қандай ишдан ҳимояланганлик ҳуқуқини таъминлаб бериши лозим, буни болаларга ўқиб-ўрганиш ва енгил меҳнат билан шуғулланишга имкон яратиб бериш билан таъминлаш мумкин. Бир томондан 18 ёшга тўлмаган ишчилар ҳар қандай чекловларсиз, катталар каби тўла ҳуқуқларга эга бўлашлари лозим. Иккинчи томондан эса, вояга етмаганларни махсус ҳимояга олиш зарурати, табиийки уларга бир қанча қўшимча ҳуқуқ ва кафолатларни яратиб берилишини талаб этади ҳамда баъзи ҳолларда катталарга қўлланиладиганидан фарқ қилувчи қоидаларнинг тадбиқ этилишини кўзда тутади . Ҳозирда юқорида қайд етилган аждодларимизнинг илмий меросини, ижтимоий-сиёсий фаолиятини ўрганиш ва ёшлар билан таништириш хайриҳоҳлиги ҳозирги замон зиёлиларининг асосий долзарб вазифаларидан саналади. Ушбу мақола бугунги бозор иқтисодиётида кичик бизнес ва бизнеснинг асосий ўрнини қамраб олади. Шу жумладан, кичик бизнес ва бизнеснинг ривожланиши ва ҳуқуқий асослари илмий таҳлил қилиниб, айни пайтда кичик бизнес ва бизнесни молиявий қўллаб-қувватлаш, иккинчисига ўзгартиришлар киритилади ва миллий қонунчиликнинг ушбу соҳасига оид қоидалар қўшилади. Мақолада замонавий Ўзбекистонда қонун устуворлиги ва фуқаролик жамиятини шакллантиришнинг енг муҳим масалалари кўриб чиқилган. Ўзбекистон сиёсий ҳаётнинг бой тажрибасига, сиёсий онг хусусиятларига ега бўлиб, ўзига хос ва мураккаб шароитларда ривожланади. Шундай қилиб, Ғарб дунёси сиёсий тажрибасининг оддий нусхасини кўчириб олиш нотўғри бўлиб чиқди. Жамиятда ижтимоий барқарорликни таъминлаш мақсадида оила институти функцияларининг бажарилиши масаласига алоҳида эътибор қаратиб, оила аъзоларига оила ва унинг функцияларини бажарилиши ҳақида сабоқ бериш лозим. Ам Мақолада маънавий салоҳиятнинг моҳияти ва унинг замонавий жамиятдаги ўрни ўрганилган. Шукур Бугунги кунда "Миллий model" га асосланган таълим соҳасидаги ислоҳотларнинг моҳияти миллий қадриятларнинг устуворлигини таъминлаш емас, балки янги давр тараққиётини жаҳон илм-фани ва ACADEMIC RESEARCH IN MODERN SCIENCE International scientific-online conference таълим тизимидаги улкан ютуқлар ва миллий ўзлик асосига қурадиган таълим тизимини - рағбатлантириш. ACADEMIC RESEARCH IN MODERN SCIENCE International scientific-online conference таълим тизимидаги улкан ютуқлар ва миллий ўзлик асосига қурадиган таълим тизимини - рағбатлантириш. Ўзбекистон Республикаси МКнинг 6-моддаси ҳамма фуқароларга чекловларнинг барча шаклларидан ҳимояни кафолатлайди. Abstract Унда белгиланганки, барча фуқаролар жинси, ёши, ирқи ва бошқа белгиларидан қатъий назар меҳнат ҳуқуқларига эга бўлиш ва улардан фойдаланишда тенг имкониятларга эга. Корхона маъмурияти ишга қабул қилишда хизматчининг ишчанлик қобилияти, ва унинг меҳнати натижаларидан ташқари, бошқа сабаблар туфайли рад жавобини беришга ҳақлари йўқ. Бу Бола ҳуқуқлари тўғрисидаги Конвенцияга, яъни Конвенциянинг 138- моддаси ишга қабул қилишнинг минимал ёши ва 146-модданинг талабрига тўла мос келади. р ур 1. Ўзбекистон Республикасини 2017-2021 йилларда ривожлантиришнинг Ҳаракатлар стратегияси. Ўзбекистон Республикаси қонун ҳужжатлари тўплами, 2017 й., 6-сон, 70-модда 2. Болалар меҳнатининг энг ёмон шаклларига барҳам бериш. Халқаро Меҳнат Ташкилотининг 182-Конвенциясини қўллаш бўйича амалий қўлланма Парламентлар аъзолари учун қўппанма №3/2002 Тошкент, Инсон ҳуқуқлари бўйича Ўзбекистон Республикаси Миллий маркази, 2008 3. Tolibjonovich, M. T. (2021). EASTERN RENAISSANCE AND ITS CULTURAL HERITAGE: THE VIEW OF FOREIGN RESEARCHERS. ResearchJet Journal of Analysis and Inventions, 2(05), 211-215. 4. Tolibjonovich, Madumarov T., and Gulomjonov O. R. Ogli. "Lombard Microcredit Organization Its Concept and Its Importance Today." JournalNX, vol. 6, no. 10, 2020, pp. 109-111. 4. Tolibjonovich, Madumarov T., and Gulomjonov O. R. Ogli. "Lombard Microcredit Organization Its Concept and Its Importance Today." JournalNX, vol. 6, no. 10, 2020, pp. 109-111. 5. Nasriddinovich, A. A. (2021). Structure, Models and Characteristics of Civil Society. STRUCTURE, 7(4). 6. Амирхўжаев Шукуржон Қурбонович, & Ғуломжонов Одилжон Рахимжон Ўғли (2022). Ижтимоий-фалсафий жиҳатдан оиланинг этномаданий хусусиятлари. Ta’lim fidoyilari, 14 (1), 133-143. 7. Madumarov Talantbek, & Ergashev Omonillo. (2022). SPIRITUAL POTENTIAL AND ITS ROLE IN MODERN. Yosh Tadqiqotchi Jurnali, 1(2), 58– 62. Retrieved from http://2ndsun.uz/index.php/yt/article/view/66 7. Madumarov Talantbek, & Ergashev Omonillo. (2022). SPIRITUAL POTENTIAL AND ITS ROLE IN MODERN. Yosh Tadqiqotchi Jurnali, 1(2), 58– 62. Retrieved from http://2ndsun.uz/index.php/yt/article/view/66 8. Mukhtarov, U. M. (2019). STUDYING OF SCIENTIFIC-PHILOSOPHICAL HERITAGE OF THE EAST RENAISSANCE INTELLECTUALS IN DEVELOPMENT 8. Mukhtarov, U. M. (2019). STUDYING OF SCIENTIFIC-PHILOSOPHICAL HERITAGE OF THE EAST RENAISSANCE INTELLECTUALS IN DEVELOPMENT 59 ACADEMIC RESEARCH IN MODERN SCIENCE International scientific-online conference OF PHILOSOPHICAL EDUCATION. Theoretical & Applied Science, (7), 120- 124. ACADEMIC RESEARCH IN MODERN SCIENCE International scientific-online conference 124.
https://openalex.org/W2949777931	https://ieeexplore.ieee.org/ielx7/6287639/8600701/08737948.pdf	English	null	Data Augmentation and Second-Order Pooling for Facial Expression Recognition	IEEE access	2,019	cc-by	6,255	Received June 7, 2019, accepted June 13, 2019, date of publication June 17, 2019, date of current version July 17, 2019. Received June 7, 2019, accepted June 13, 2019, date of publication June 17, 2019, date of current version July 17, 2019. Digital Object Identifier 10.1109/ACCESS.2019.2923530 The associate editor coordinating the review of this manuscript and approving it for publication was Abdullah Iliyasu. Data Augmentation and Second-Order Pooling for Facial Expression Recognition INDEX TERMS Facial expression recognition, data augmentation, second-order pooling, deep convolutional neural networks. Data Augmentation and Second-Order Pooling for Facial Expression Recognition XIAOYUN TONG , SONGLIN SUN , (Senior Member, IEEE), AND MEIXIA FU National Engineering Laboratory for Mobile Network Security, Beijing University of Posts and Telecommunications, Beijing 100876, Chi Key Laboratory of Trustworthy Distributed Computing and Service (BUPT), Ministry of Education, Beijing University of Posts and Telec Beijing 100876, China XIAOYUN TONG , SONGLIN SUN , (Senior Member, IEEE), AND MEIXIA FU National Engineering Laboratory for Mobile Network Security, Beijing University of Posts and Telecommunications, Beijing 100876, China Key Laboratory of Trustworthy Distributed Computing and Service (BUPT), Ministry of Education, Beijing University of Posts and Telecommunications, Beijing 100876, China School of Information and Communication Engineering, Beijing University of Posts and Telecommunications, Beijing 100876, China j g School of Information and Communication Engineering, Beijing University of Posts and Telecommunications, Beijing 100876, China Corresponding author: Xiaoyun Tong (email: xiaoyun_t@bupt.edu.cn) This work was supported in part by the National Natural Science Foundation of China under Project 61471066, and in part by the Open Project Fund of the National Key Laboratory of Electromagnetic Environment, China, under Grant 201600017. ABSTRACT Facial expression is the main medium of information transmission in human communication, playing an important role in human’s daily life. Facial expression recognition is still challenging due to the various obstacle, illumination, and posture. However, most of the existing works focus on deeper or wider network structures and rarely explores the high-level feature statistics. In this paper, we propose a second-order pooling convolution neural network to explore the correlation information between the facial features after deep network learning. At the ﬁnal stage of the network, we add a new covariance pooling layer to replace the ﬁrst-order pooling of standard convolution networks. In the pooling layer of covariance, the Newton iteration method is used to approximate the square root instead of EIG or SVD, which makes it more suitable for GPU. Due to the small amount of facial expression data, this paper uses different data augmentation methods to increase the amount of training data and improve the generalization ability of the model. The proposed method, data augmentation and second-order pooling (DASOP), was evaluated on the real-world affective faces database (RAFDB) and the static facial expressions in the wild (SFEW), yielding correct rates of 88.625% and 59.518%, respectively. We achieve state-of-the-art performance superior to existing methods. INDEX TERMS Facial expression recognition, data augmentation, second-order pooling, deep convolutional neural networks. II. RELATED WORK The proposed method DASOP consists of two parts: data aug- mentation and second-order pooling. Therefore, this paper introduces the related work of data augmentation and the second-order pooling separately, and summarizes as follows. So, the performance of single model is limited to the loss of discriminant information. At present, deep facial expression recognition faces two key problems: (1) over-ﬁtting due to lack of sufﬁcient training data; (2) facial expression changes are subtle and changeable, the ﬁrst-order information is insufﬁcient to provide more discriminant information. To overcome these issues, this paper proposes data augmentation and second-order pooling based deep convolutional neural networks for facial expres- sion recognition. An overview of the proposed methods is shown in Figure 1. The input image is transformed into a Densenet121 neural network, then the second-order informa- tion of the output feature is calculated, and the ﬁnal output category is obtained. In summary, the main contribution of our work consists of: I. INTRODUCTION Newton iteration method is used to solve the covariance matrix to make it more suitable for GPU. (3) The proposed DASOP achieves the state-of-art perfor- mance on two public datasets: RAFDB and SFEW. The remainder of this paper is introduced as follows. Section II summarize the work most relevant to our work. Section III descripts the proposed model, including data aug- mentation, and covariance descriptors. The improved perfor- mance of the proposed DASOP on two public datasets, and comparison with the state-of-art solutions is demonstrated in Section IV. Finally, Section V concludes this paper and gives the directions of future work. I. INTRODUCTION amount of public database of facial expression recognition is limited, so data augmentation can be used to increase the diversity of training samples, improve the robustness and generalization ability of the model, and avoid over-ﬁtting [3]. For example, we can crop the image in different ways so that the objects appear at different positions in the image at different scales, which can reduce the sensitivity of the model to the target position. Facial expression is the most natural and direct way to reﬂect people’s inner emotions and thoughts. Therefore, the research of facial expression recognition technology based on visual information is another research topic after face detection and recognition [1]. Facial expression recognition has potential application value in many ﬁelds, such as intelligent med- ical care, intelligent monitoring in security ﬁeld, human- computer interaction, digital entertainment, etc. In facial expression recognition, it is a very challenging task because the visual differences between different subclasses are small and easily affected by posture, perspective, illumination, and so on [2]. Convolutional neural network (CNNs) have enough capac- ity to represent the complex variations of samples by learning features hierarchically [4]. Therefore, CNNs have made out- standing achievements in facial expression recognition. The common models mainly adopt the average or max pooling layer in designing network architectures. However, these lay- ers only capture the ﬁrst-order statistics of input features, which limit the learning capacity of models [5]. In addition, the structure of face not only contains the global discrimi- nant information, but also the local discriminant information. Deep convolutional neural networks generally require a large amount of training data to get ideal results. However, the VOLUME 7, 2019 86821 86821 X. Tong et al.: DASOP for Facial Expression Recognition FIGURE 1. Data augmentation and second-order pooling based Densenet121 networks for facial expression recognition. RE 1. Data augmentation and second-order pooling based Densenet121 networks for facial expression recognition. FIGURE 1. Data augmentation and second-order pooling based Densenet121 networks for facial expression recognit TABLE 1. Comparison of accuracies with various standard network architectures on RAFDB and SFEW. TABLE 1. Comparison of accuracies with various standard network architectures on RAFDB and SFEW. (2) Facial expression recognition adopts end-to-end pool- ing of second-order statistics. Newton iteration method is used to solve the covariance matrix to make it more suitable for GPU. (2) Facial expression recognition adopts end-to-end pool- ing of second-order statistics. B. SECOND-ORDER POOLING Deep convolutional neural networks have achieved great success in large-scale object classiﬁcation and other areas of computer vision. In fact, this network can be seen as a process of learning low level features through hierarchical convolution and pooling, and getting high level representation through a global average pooling that is sent it to the classiﬁer for classiﬁcation. Global Gaussian Distribution Embedding Network (G2DeNet) [15]: take advantage of matrix normalization but suffer from large dimensionality since they use the square- root of a large pooled matrix. At the same time, the authors use the ﬁrst and second-order information and normalize it by 0.5 matrix power. Global Gaussian Distribution Embedding Network (G2DeNet) [15]: take advantage of matrix normalization but suffer from large dimensionality since they use the square- root of a large pooled matrix. At the same time, the authors use the ﬁrst and second-order information and normalize it by 0.5 matrix power. The focus here is global average pooling. This layer of work was ﬁrst proposed on [8], and is now widely used in mainstream deep networks, such as Inception, Resnet, Densenet and so on. However, the problem of global average pooling is that the previous network acquires a strong expres- sive feature through continuous learning, but at last it makes a global mean when it expresses the image. Statistically speaking, the mean is only a ﬁrst-order information that is often simple, fast and effective. However, it is easy to ignore the relevant information between channels by calculating the mean of each channel. Based on this point, many authors propose a second-order statistical method to replace the ﬁrst- order global average pooling, and thus make a series of work. One of the earliest works employing second-order pool- ing for semantic segmentation used it as regional descriptor, combined with LBP, SIFT and MSIFT [9]. Previous well- known work included DeepO2P [5] and Bilinear Model [10] and various improvements. In [11], authors propose a new class of CNN architectures to employ covariance pooling. The method of global covariance pooling is ﬁrst applied to large-scale visual recognition tasks in [12]. It not only given The focus here is global average pooling. This layer of work was ﬁrst proposed on [8], and is now widely used in mainstream deep networks, such as Inception, Resnet, Densenet and so on. A. DATA AUGMENTATION Convolutional neural networks can classify objects placed in different directions, or different scales, or different illu- minations robustly, that is, convolutional neural networks have invariant properties [3]. This is the premise of data augmentation. Data augmentation is mainly to improve the robustness and generalization ability of the model, and reduce the over-ﬁtting phenomenon of the network. By transforming the training data, the network with stronger generalization ability can be obtained, which can better adapt to the application scenarios. Over-ﬁtting is mainly caused by two reasons: too little data and too complex model. Hence, data augmentation is the most direct and effective method to avoid over-ﬁtting. (1) We add multiple data augmentation technologies to datasets, and set a group of experiments on different networks to illustrate the effectiveness of data augmentation. The result of experiments is shown in Table 1. 86822 86822 VOLUME 7, 2019 X. Tong et al.: DASOP for Facial Expression Recognition Commonly data augmentation methods listed as follows: some theoretical analysis, but also prove to be effective in large-scale vision problems, such as the classiﬁcation of Ima- geNet. In [13], authors propose symmetric positive deﬁnite manifold network, that is, covariance pool is used in facial expression recognition, combining manifold networks with traditional convolutional networks, and using spatial pools in a single image feature map in an end-to-end depth learning approach. The following is a brief description of the covari- ance processing for different second-order pooling. some theoretical analysis, but also prove to be effective in large-scale vision problems, such as the classiﬁcation of Ima- geNet. In [13], authors propose symmetric positive deﬁnite manifold network, that is, covariance pool is used in facial expression recognition, combining manifold networks with traditional convolutional networks, and using spatial pools in a single image feature map in an end-to-end depth learning approach. The following is a brief description of the covari- ance processing for different second-order pooling. Rotation: Random rotates the image at a certain angle. Reﬂection: change the orientation of the image content. Flip: Flip the image horizontally or vertically. Scale: The image can be outward or inward according to the speciﬁed scale factor; or the scale space is constructed by ﬁltering the image with using the speciﬁed scale factor, according to the idea of SIFT feature extraction. Crop: Random select the region of the image for clipping and scaling to a certain scale. A. DATA AUGMENTATION Deep Second Order Pooling (DeepO2P) [5]: adopts Log-Euclidean (Log-E) metric for exploiting geometry of covariance spaces, which only brings side effect on covari- ance representations. It is not suitable for large-scale datasets, such as ImageNet. Shift: shifts the image in a certain way on the image plane. Noise: Random perturbation of each pixel of the image. The common noise modes are salt and pepper noise and Gauss noise. Color jitter: Randomly change the brightness, contrast and saturation of the image. Bilinear Pooling (BP) [10]: performs element-wise square root normalization, without considering the manifold of covariance matrices. Bilinear convergence calculates the outer product of different spatial locations and calculates the average convergence for different spatial locations to obtain bilinear features. The outer product captures the pairwise correlation between the feature channels, and this is trans- lationally invariant. Bilinear convergence provides a stronger representation of the feature than the linear model, and can be optimized end-to-end to achieve performance. Random Erasing [6]: Random selection of an area on a picture and random erasure of image information. Generative Adversarial Networks (GAN) [7]: An image generation model consists of two parts, a generator and a dis- criminator, which generate images through two-part mutual game learning. In order to get more data, we just need to make minor changes to our existing dataset, such as ﬂips, translations or rotations. Data augmented images are distinct and indepen- dent images for untrained neural networks. Compact Bilinear Pooling (CBP) [14]: use the outer product to combine the feature maps of the two stream CNNs, then transform the bilinear features through the square root of the symbol, and add L2 normalization, input classiﬁer. Compared with the BP, the loss of the CBP is basically unchanged, but the size of the parameters is reduced by two orders of magnitude, and CBP supports an end-to-end training structure. B. SECOND-ORDER POOLING However, the problem of global average pooling is that the previous network acquires a strong expres- sive feature through continuous learning, but at last it makes a global mean when it expresses the image. Statistically speaking, the mean is only a ﬁrst-order information that is often simple, fast and effective. However, it is easy to ignore the relevant information between channels by calculating the mean of each channel. Based on this point, many authors propose a second-order statistical method to replace the ﬁrst- order global average pooling, and thus make a series of work. Moments Embedding Network (MoNet) [16]: using a sub-matrix square root layer, matrix normalization is performed before bilinear pooling, combined with com- pact pooling, reduces dimensionality signiﬁcantly with- out compromising performance. The description matrix is augmented, which contains both ﬁrst and second-order information. In addition, tensor sketch can be used to design a compact bilinear merge operation. Its performance is better than G2DeNet. Symmetric Positive Deﬁnite Manifold Network (SPDNET) [13]: covariance features can be ﬂattened through Bilinear Mapping Layer, and then introduced non- linearity by Eigenvalue Rectiﬁcation, Log Eigenvalue Layer endows elements in Riemannian manifold with a Lie Group structure. Symmetric Positive Deﬁnite Manifold Network (SPDNET) [13]: covariance features can be ﬂattened through Bilinear Mapping Layer, and then introduced non- linearity by Eigenvalue Rectiﬁcation, Log Eigenvalue Layer endows elements in Riemannian manifold with a Lie Group structure. Symmetric Positive Deﬁnite Manifold Network (SPDNET) [13]: covariance features can be ﬂattened through Bilinear Mapping Layer, and then introduced non- linearity by Eigenvalue Rectiﬁcation, Log Eigenvalue Layer endows elements in Riemannian manifold with a Lie Group structure. One of the earliest works employing second-order pool- ing for semantic segmentation used it as regional descriptor, combined with LBP, SIFT and MSIFT [9]. Previous well- known work included DeepO2P [5] and Bilinear Model [10] and various improvements. In [11], authors propose a new class of CNN architectures to employ covariance pooling. The method of global covariance pooling is ﬁrst applied to large-scale visual recognition tasks in [12]. It not only given Matrix Power Normalization [12]: the covariance matrix of depth description vector is normalized by 0.5 matrix power to obtain bilinear convergence feature. The covariance is by Matrix Power Normalization [12]: the covariance matrix of depth description vector is normalized by 0.5 matrix power to obtain bilinear convergence feature. The covariance is by 86823 VOLUME 7, 2019 X. III. PROPOSED METHOD eigen-decomposed to obtain the eigenvalue, and then the square root of eigenvalue is obtained. It solves the problem of high dimension statistics of small samples, and effectively uses the geometric structure of covariance matrix [17]. How- ever, almost all platforms based on GPU have poor support for eigen decomposition, so its efﬁciency is very poor. Facial expression recognition mainly includes three parts: face detection, feature extraction, expression recognition. That is, given a picture, face detection is performed ﬁrst, and then align based on facial markers. The extracted face is augmented and standardized, and then fed into the deep neural network. To consider the interaction between fea- tures, we use second-order pooling, and then employ the Newton-Schulz iteration to explore second-order statistical information. Because the difference between classes is not signiﬁcant, the ﬁrst-order information is not applicable, but the second-order information can bring more discriminatory and valuable information to the classiﬁer. Iterative Matrix Square Root Normalization of Covari- ance Pooling [18]: because there is no efﬁcient implementa- tion of eigenvalue decomposition and SVD on GPU, Schur decomposition or eigenvalue decomposition are still needed when Lyapunov equation is solved in reverse. It is pro- posed that both forward and backward processes are based on Newton iteration method. The matrix is normalized by 0.5 matrix power. A common framework of above references is to use CNN to extract feature map, and then use each location in feature map as a sample to calculate the covariance matrix of the entire feature map. After the covariance matrix is obtained, the eigenvalues are reduced, parameterized or normalized and then output. B. SECOND-ORDER POOLING Tong et al.: DASOP for Facial Expression Recognition FIGURE 2. Data augmentation in train and test datasets. FIGURE 2. Data augmentation in train and test datasets. III. PROPOSED METHOD IV. EXPERIMENTS In this section, we conduct many experiments of the proposed method on two public datasets and present the state-of-the-art performance though comparing with the previous work. The proposed system is implemented on Pytorch using NVIDIA GeForce GTX 1080Ti with 11GB memory. In our experi- ments, the input image is 224∗224, the batch size is 32. The training epoch is set to 100. The initial learning rate is 0.1 and decays by a factor of 0.1 every 30 epochs. DNNs adopted in Table 1 is trained from scratch. B. COVARIANCE POOLING Equation (4) only involves matrix product operations, not matrix inverse, hence is suitable for implementations on GPUs. Compared to the exact square root of the EIG calculation, the approximate solution can only be obtained by 5 iterations [13]. After deep convolutional neural network, feature will be obtained. Firstly, covariance matrix is calculated. Unlike ﬁrst- order pooling, covariance matrix can describe the correlation between each channel. The reason for exponentiation is to solve the problem of small sample high dimension in covariance estimation. Reference [8] has been veriﬁed that when the power is 0.5, that is, the square root operation, the effect is optimal. Deﬁning the output of the convolutional network is a tensor of h×w×d dimensions, where h is the height, w is the width, and d is the number of channels. This tensor is reshaped into a n × d dimensional feature matrix, where n = h × w. Then calculate the covariance of this matrix: A. DATASETS A = 1 tr (6)6 (2) In this paper, facial expression datasets in the wild are chosen, which can better approximation to the real-world scenarios, and also more challenging. We present the evaluation results of RAFDB and SFEW datasets, and reveal the relevance and importance of facial features. In this paper, facial expression datasets in the wild are chosen, which can better approximation to the real-world scenarios, and also more challenging. We present the evaluation results of RAFDB and SFEW datasets, and reveal the relevance and importance of facial features. (2) Let λi be eigenvalues of 6, arranged in nondecreasing order. As tr (6) = P i λi, then: Let λi be eigenvalues of 6, arranged in nondecreasing order. As tr (6) = P i λi, then: ∥6 −I∥2 = max (6 −I) = 1 − λ1 P i λi < 1 (3) Real-world Affective Faces Database (RAFDB) [19] is a large-scale facial expression database with around 30K great- diverse facial images downloaded from the Internet. This dataset contains 15331 images and that have been labeled for seven basic expressions. There are 12271 samples in training set and 3068 samples in test set. The RAF-DB is 100∗100 RGB image, is shown in ﬁgure 3(a). There are 7 rows, each row representing a category with 7 images per category. Figure 3(b) is the data distribution. It shows the number of images per emotion in the training set and test set. As can be seen from Figure 3(b), the data distribution is very uneven. (3) Therefore, the proposed algorithm satisﬁes the conver- gence condition. A. DATA AUGMENTATION Because of too little data and too many network param- eters, it is easy to lead to over-ﬁtting. The facial expres- sions publicity dataset is small, and the categories are not balanced, so this paper adopts data augmentation methods to expand the dataset to avoid over-ﬁtting. Another advan- tage of data augmentation is to expand the amount of data in the database, which makes the training network more robust. The data augmentation of training used in this paper are as follows: resize, random horizontal ﬂip, random crop and random Erasing. Random erasing is complementary to random clipping and random horizontal ﬂipping. The test- ing data is cropping the given image four corners and the central crop plus the ﬂipped version of these, then average the predictions of 10 images, the largest output classiﬁca- tion is corresponding expression. This method effectively reduces classiﬁcation errors. Data augmentation is shown in Figure 2. Facial expression recognition is more directly related to the deformation of facial feature points than to the existence or absence of speciﬁc feature points. Therefore, we believe that if geometric moments are applied to facial expression recognition, the second-order statistics are more suitable than other statistics for capturing the regional distortion of key points in facial features. In the generated convolution network, global covariance pooling is a better choice than global average pooling. There are three reasons: (1) better performance and stronger gener- alization ability; (2) good theoretical explanation in statistics and geometry; (3) fast convergence and high computational efﬁciency. 86824 VOLUME 7, 2019 VOLUME 7, 2019 X. Tong et al.: DASOP for Facial Expression Recognition 1) PRE-NORMALIZATION In order to ensure the global convergence of the proposed algorithm, we pre-normalize covariance matrix by dividing its trace before the Newton iteration, i.e., 3) POST-COMPENSATION 6 = 1 n −1 n X i=1 (xi −µ) (xi −µ)T = 1 n −1XT X (1) Since the pre-normalization changes the covariance size, in order to eliminate this effect, post-compensation is added after the Newton iteration method to compensate the trace value back, i.e., C = √tr (6)YN where x1, x2, . . . , xn ∈Rd be the set of features, X denotes the mean-subtracted data matrix. Finally, the output of the second-order pooling layer is fed into the classiﬁer for classiﬁcation. At present, almost all GPU-based platforms have very poor support for eigenvalue decomposition, so the second-order pooling layer in this paper uses iterative method to solve the square root of covariance. Since the iteration itself is not global convergence, it can be guaranteed by dividing the trace of the matrix by the covariance matrix; however, the covariance is changed after dividing by the trace, so the value of the trace is compensated by post-compensation after the iteration. Detailed description is as follows. 2) NEWTON-SCHULZ ITERATION The idea of Newton’s method is simple. Given an initial point, the tangent at that point is used to approximate the function, and then the root of the tangent is found as an iteration. Newton’s iteration method is to make the equation converge gradually through continuous iteration. SFEW [20] extracted from a temporal facial expressions database Acted Facial Expressions in the Wild which we have extracted from movies. The database covers unconstrained facial expressions, varied head poses, large age range, occlu- sions, varied focus, different resolution of face and close to real world illumination. In total, SFEW contains 1766 images and that have been labeled for seven basic expressions. There are 958 samples in training set and 436 samples in validation set. Since the test set are not labeled, here the results on the validation set is reported in this paper. Let A be an SPD matrix, the square root of A is Y = Udiag λ1/2 i UT , but both EIG and SVD are not well sup- ported on GPU [12]. Hence, we computing Y by Newton Schulz iteration, a case of coupled iteration as follows: Yk = 1 2Yk−1 (3I −Zk−1Yk−1) Zk = 1 2 (3I −Zk−1Yk−1) Zk−1 (4) (4) where Z = I for k = 1, . . . , N. where Z = I for k = 1, . . . , N. 86825 VOLUME 7, 2019 X. Tong et al.: DASOP for Facial Expression Recognition FIGURE 4. Confusion matrix for Densenet121 and DASOP on the RAFDB and SFEW. FIGURE 3. RAFDB samples and distribution. FIGURE 3. RAFDB samples and distribution. FIGURE 3. RAFDB samples and distribution. FIGURE 3. RAFDB samples and distribution. FIGURE 3. RAFDB samples and distribution. B. RESULTS AND DISCUSSION Since the images provided by the dataset contain many back- grounds, it needs to be preprocessed, that is, face extraction. For SFEW, we use the MTCNN proposed in [21] to detect faces. For RAFDB, the dataset provides the aligned faces. FIGURE 4. Confusion matrix for Densenet121 and DASOP on the RAFDB and SFEW. on RAFDB and 16-32% on SFEW, respectively. The SFEW datasets are fewer, so the improvement of the accuracy rate is more obvious. on RAFDB and 16-32% on SFEW, respectively. The SFEW datasets are fewer, so the improvement of the accuracy rate is more obvious. We evaluate ﬁve current classical CNN architecture as backbone network for DASOP in Table 1. we present the comparison of accuracies with various standard network architectures on RAFDB and SFEW. Original means the original data input to different networks for training. Aug is for data augmentation using standard techniques including random crop, random ﬂip and random erasing. DASOP is data augmentation and covariance pooling. (3) Compared with the ﬁrst-order pooling, the accuracy of the second-order pooling is improved by 0.7-6% on RAFDB and 1-6% on SFEW, respectively, which shows the effective- ness of the second-order pooling and provides more useful information. Figure 4 shows that the accuracy of happiness and angry is signiﬁcantly higher than others, but the accuracy of fear and disgust is very low. For this problem, we think that the ﬁrst reason is that the number of different expressions in the data set is not balanced, this kind of imbalance is enough to make the classiﬁcation error. The second reason is that surprise, disgust, fear and sad have certain similarities. In real life, people also ﬁnd it difﬁcult to distinguish these four kinds of expressions, especially when they are not acquainted with It can be seen from Table 1 that: (1) the densenet121 + DASOP of method proposed in this paper obtains best classiﬁcation results with accuracy 88.625% on RAFDB and 59.518% on SFEW. This shows that the deep convolution network has a very obvious effect on the classiﬁcation. The corresponding confusion matrixes are shown in Figure 3. (2) Data augmentation is very effective to improve the classiﬁcation effect. The accuracy rate is increased by 1-7% (2) Data augmentation is very effective to improve the classiﬁcation effect. The accuracy rate is increased by 1-7% VOLUME 7, 2019 VOLUME 7, 2019 86826 X. B. RESULTS AND DISCUSSION Tong et al.: DASOP for Facial Expression Recognition FIGURE 5. The loss function curve for original data and data augmentation with Densenet121 on the RAFDB. FIGURE 6. comparison of densenet121 + DASOP with other pooling. FIGURE 6. comparison of densenet121 + DASOP with other pooling. C. COMPARE TO OTHER POOLING In this section, we evaluate different second-order pooling methods for facial expression recognition. Figure 6 shows the comparison of the proposed method with the ﬁrst-order global average pooling (GAvP) method and the second-order pooling methods, BP with CNN (BCNN) and CBP. Accord- ing to the Table 1 experimental results, Densenet121 was chosen as the basic network. As can be seen from Figure 6, the result of second-order pooling is higher than ﬁrst-order pooling with accuracy 2-4% on RAFDB and 5-10% on SFEW, respectively. And our method achieves the highest accuracy in second-order pooling methods. FIGURE 5. The loss function curve for original data and data augmentation with Densenet121 on the RAFDB. each other, it is even more difﬁcult to correctly recognize expressions. Moreover, we ﬁnd that misjudgments always occur in some classes, which may be really difﬁcult to dis- tinguish and confuse. In RAFDB, the loss function curve of original data and data augmentation based on densnet121 is shown in Figure 5. From Figure 5 (a), it can be seen that there is a serious over-ﬁtting problem in the original data, so data augmenta- tion technology is used to avoid over-ﬁtting in this paper. Figure 5 (b) shows that data augmentation can solve this problem very well. FUTURE WORKS In this work, we use traditional supervised methods for data enhancement. However, in the real world, data in the natural state exists in a variety of situations, and cannot be handled by the above simple methods. Therefore, data enhancement can be achieved by the following unsupervised methods: [17] Q. Wang, P. Li, W. Zuo, and L. Zhang, ‘‘RAID-G: Robust estimation of approximate inﬁnite dimensional Gaussian with application to material recognition,’’ in Proc. IEEE Conf. Comput. Vis. Pattern Recognit. (CVPR), Jun. 2016, pp. 4433–4441. [18] P. Li, J. Xie, Q. Wang, and Z. Gao, ‘‘Towards faster training of global covariance pooling networks by iterative matrix square root normal- ization,’’ in Proc. IEEE Conf. Comput. Vis. Pattern Recognit. (CVPR), Jun. 2018, pp. 947–955. (1) Through the distribution of model learning data, the images which are consistent with the distribution of training data set are randomly generated. The representative method, GAN. [19] S. Li, W. Deng, and J. Du, ‘‘Reliable crowdsourcing and deep locality- preserving learning for expression recognition in the wild,’’ in Proc. IEEE Conf. Comput. Vis. Pattern Recognit. (CVPR), Jul. 2017, pp. 2852–2861. [20] A. Dhall, R. Goecke, S. Lucey, and T. Gedeon, ‘‘Static facial expression analysis in tough conditions: Data, evaluation protocol and benchmark,’’ in Proc. IEEE Int. Conf. Comput. Vis. Workshops (ICCV Workshops), Nov. 2011, pp. 2106–2112. (2) Through the model, learn the data enhancement method suitable for the current task, the representative method, AutoAugment [27]. (2) Through the model, learn the data enhancement method suitable for the current task, the representative method, AutoAugment [27]. [21] K. Zhang, Z. Zhang, Z. Li, and Y. Qiao, ‘‘Joint face detection and alignment using multitask cascaded convolutional networks,’’ IEEE Signal Process. Lett., vol. 23, no. 10, pp. 1499–1503, Oct. 2016. Also, we can add Global Second-order Pooling [28] across from lower to higher layers of deep convolutional net- works aiming to learn more discriminative representations by exploiting the second-order statistics of holistic image throughout a deep convolutional network. This will be an interesting direction, and the results should be improved in theory. [22] Y. Fan, J. C. K. Lam, and V. O. K. Li, ‘‘Multi-region ensemble convolu- tional neural network for facial expression recognition,’’ in Proc. Int. Conf. Artif. Neural Netw. Cham, Switzerland: Springer, Sep. 2018, pp. 84–94. [23] Y. Li, J. Zeng, S. Shan, and X. Chen, ‘‘Occlusion aware facial expression recognition using CNN with attention mechanism,’’ IEEE Trans. D. COMPARE TO OTHER REFERENCE Table 2 shows the comparison of the accuracy of each class between this paper and other references on RAFDB and SFEW. It can be seen that our algorithm is 1.625% higher than the current highest result, achieves the state-of-art results. TABLE 2. The comparison of the accuracy of each class between this paper and other references on RAFDB and SFEW. VOLUME 7, 2019 86827 he accuracy of each class between this paper and other references on RAFDB and SFEW. TABLE 2. The comparison of the accuracy of each class between this paper and other references on RAFDB and SFEW. X. Tong et al.: DASOP for Facial Expression Recognition This veriﬁes the effective and signiﬁcant of the proposed method. From all results based-deep learning, we can obtain that this stems from the effectiveness of deep convolu- tional networks and second-order information for feature extraction. [6] Z. Zhong, L. Zheng, G. Kang, S. Li, and Y. Yang, ‘‘Random eras- ing data augmentation,’’ 2017, arXiv:1708.04896. [Online]. Available: https://arxiv.org/abs/1708.04896 [7] I. Goodfellow, J. Pouget-Abadie, and M. Mirza, ‘‘Generative adversarial nets,’’ in Proc. Adv. Neural Inf. Process. Syst., 2014, pp. 2672–2680. [8] M. Lin, Q. Chen, and S. Yan, ‘‘Network in network,’’ in Proc. ICLR, Mar. 2014, pp. 1–10. [9] J. Carreira, R. Caseiro, J. Batista, and C. Sminchisescu, ‘‘Semantic seg- mentation with second-order pooling,’’ in Proc. Eur. Conf. Comput. Vis., 2012, pp. 430–443. V. CONCLUSION The novelty of the proposed model is that second-order sta- tistical information is introduced into the network as image representation. Compared with the classical method, only the ﬁrst-order statistical information is mined in the learning process, and the visual features with stronger resolving power can be learned. By introducing global covariance aggregation and matrix power normalization techniques, the proposed model is signiﬁcantly better than the classical convolutional network in performance, and the convergence speed is faster. Data augmentation reduces the risk of over-ﬁtting and makes the model robust to occlusion. Experiment conducted on RAFDB and fer2013 with various architectures validate the effectiveness of our method. pp [10] T. Y. Lin, A. RoyChowdhury, and S. Maji, ‘‘Bilinear CNN models for ﬁne- grained visual recognition,’’ in Proc. IEEE Int. Conf. Comput. Vis. (ICCV), Dec. 2015, pp. 1449–1457. [11] K. Yu and M. Salzmann, ‘‘Second-order convolutional neural networks,’’ Clin. Immunol. Immunopathol., vol. 66, no. 3, pp. 230–238, 2017. [12] P. Li, J. Xie, Q. Wang, and W. Zuo, ‘‘Is second-order information helpful for large-scale visual recognition?’’ in Proc. IEEE Int. Conf. Comput. Vis., Oct. 2017, pp. 2070–2078. [13] D. Acharya, Z. Huang, and D. Pani-Paudel, ‘‘Covariance pooling for facial expression recognition,’’ in Proc. IEEE Conf. Comput. Vis. Pattern Recognit. Workshops, Jun. 2018, pp. 367–374. [14] Y. Gao, O. Beijbom, and N. Zhang, ‘‘Compact bilinear pooling,’’ in Proc. IEEE Conf. Comput. Vis. Pattern Recognit. (CVPR), Jun. 2016, pp. 317–326. [15] Q. Wang, P. Li, and L. Zhang, ‘‘G2DeNet: Global Gaussian distribution embedding network and its application to visual recognition,’’ in Proc. CVPR, Jul. 2017, pp. 6507–6516. [16] M. Gou, F. Xiong, and O. Camps, ‘‘Monet: Moments embedding net- work,’’ in Proc. IEEE Conf. Comput. Vis. Pattern Recognit., Jun. 2018, pp. 3175–3183. FUTURE WORKS Image Process., vol. 28, no. 5, pp. 2439–2450, May 2019. [24] J. Zeng, S. Shan, and X. Chen, ‘‘Facial expression recognition with incon- sistently annotated datasets,’’ in Proc. Eur. Conf. Comput. Vis. (ECCV), Sep. 2018, pp. 222–237. [25] Z. Yu and C. Zhang, ‘‘Image based static facial expression recognition with multiple deep network learning,’’ in Proc. ACM Int. Conf. Multimodal Interact., Nov. 2015, pp. 435–442. [2] Y. Zong, X. Huang, W. Zheng, Z. Cui, and G. Zhao,, ‘‘Learning from hierarchical spatiotemporal descriptors for micro-expression recognition,’’ IEEE Trans. Multimedia, vol. 20, no. 11, pp. 3160–3172, Nov. 2018. [1] S. Li and W. Deng, ‘‘Deep facial expression recognition: A survey,’’ 2018, arXiv:1804.08348. [Online]. Available: https://arxiv.org/abs/1804.08348 [1] S. Li and W. Deng, ‘‘Deep facial expression recognition: A survey,’’ 2018, arXiv:1804.08348. [Online]. Available: https://arxiv.org/abs/1804.08348 [2] Y. Zong, X. Huang, W. Zheng, Z. Cui, and G. Zhao,, ‘‘Learning from hierarchical spatiotemporal descriptors for micro-expression recognition,’’ IEEE Trans. Multimedia, vol. 20, no. 11, pp. 3160–3172, Nov. 2018. [3] K. He, X. Zhang, S. Ren, and J. Sun ‘‘Deep residual learning for image recognition,’’ in Proc. CVPR, Jun. 2016, pp. 770–778. [4] A. Krizhevsky, I. Sutskever, and G. E. Hinton, ‘‘Imagenet classiﬁcation with deep convolutional neural networks,’’ in Proc. NIPS, 2012, pp. 1–9. [5] C. Ionescu, O. Vantzos, and C. Sminchisescu, ‘‘Matrix backpropagation for deep networks with structured layers,’’ in Proc. IEEE Int. Conf. Comput. [3] K. He, X. Zhang, S. Ren, and J. Sun ‘‘Deep residual learning for image recognition,’’ in Proc. CVPR, Jun. 2016, pp. 770–778. [4] A. Krizhevsky, I. Sutskever, and G. E. Hinton, ‘‘Imagenet classiﬁcation with deep convolutional neural networks,’’ in Proc. NIPS, 2012, pp. 1–9. REFERENCES [1] S. Li and W. Deng, ‘‘Deep facial expression recognition: A survey,’’ 2018, arXiv:1804.08348. [Online]. Available: https://arxiv.org/abs/1804.08348 [26] X. Liu, B. V. K. V. Kumar, J. You, and P. Jia, ‘‘Adaptive deep metric learning for identity-aware facial expression recognition,’’ in Proc. IEEE Conf. Comput. Vis. Pattern Recognit. Workshops (CVPRW), Jul. 2017, pp. 522–531. [2] Y. Zong, X. Huang, W. Zheng, Z. Cui, and G. Zhao,, ‘‘Learning from hierarchical spatiotemporal descriptors for micro-expression recognition,’’ IEEE Trans. Multimedia, vol. 20, no. 11, pp. 3160–3172, Nov. 2018. [3] K. He, X. Zhang, S. Ren, and J. Sun ‘‘Deep residual learning for image recognition,’’ in Proc. CVPR, Jun. 2016, pp. 770–778. [27] E. D. Cubuk, B. Zoph, and D. Mane, ‘‘AutoAugment: Learning augmen- tation policies from data,’’ 2018, arXiv:1805.09501. [Online]. Available: https://arxiv.org/abs/1805.09501 [4] A. Krizhevsky, I. Sutskever, and G. E. Hinton, ‘‘Imagenet classiﬁcation with deep convolutional neural networks,’’ in Proc. NIPS, 2012, pp. 1–9. [28] Z. Gao, J. Xie, Q. Wang, and P. Li, ‘‘Global second-order pooling convolutional networks,’’ 2018, arXiv:1811.12006. [Online]. Available: https://arxiv.org/abs/1811.12006 [5] C. Ionescu, O. Vantzos, and C. Sminchisescu, ‘‘Matrix backpropagation for deep networks with structured layers,’’ in Proc. IEEE Int. Conf. Comput. Vis. (ICCV), Dec. 2015, pp. 2965–2973. 86828 VOLUME 7, 2019
https://openalex.org/W2028046413	https://jcheminf.biomedcentral.com/counter/pdf/10.1186/1758-2946-4-17	English	null	Avogadro: an advanced semantic chemical editor, visualization, and analysis platform	Journal of cheminformatics	2,012	cc-by	11,477	Correspondence: marcus.hanwell@kitware.com 1Department of Chemistry, University of Pittsburgh, 219 Parkman Avenue, Pittsburgh, PA, 15260, USA 2Department of Scientiﬁc Computing, Kitware, Inc., 28 Corporate Drive, Clifton Park, NY, 12065, USA Full list of author information is available at the end of the article © 2012 Hanwell et al.; licensee Chemistry 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. Hanwell et al. Journal of Cheminformatics 2012, 4:17 http://www.jcheminf.com/content/4/1/17 Hanwell et al. Journal of Cheminformatics 2012, 4:17 http://www.jcheminf.com/content/4/1/17 SOFTWARE Open Access Avogadro: an advanced semantic chemical editor, visualization, and analysis platform Marcus D Hanwell1,2, Donald E Curtis3, David C Lonie4, Tim Vandermeersch5, Eva Zurek4 and Geoﬀrey R Hutchison1 Abstract Background: The Avogadro project has developed an advanced molecule editor and visualizer designed for cross-platform use in computational chemistry, molecular modeling, bioinformatics, materials science, and related areas. It oﬀers ﬂexible, high quality rendering, and a powerful plugin architecture. Typical uses include building molecular structures, formatting input ﬁles, and analyzing output of a wide variety of computational chemistry packages. By using the CML ﬁle format as its native document type, Avogadro seeks to enhance the semantic accessibility of chemical data types. Results: The work presented here details the Avogadro library, which is a framework providing a code library and application programming interface (API) with three-dimensional visualization capabilities; and has direct applications to research and education in the ﬁelds of chemistry, physics, materials science, and biology. The Avogadro application provides a rich graphical interface using dynamically loaded plugins through the library itself. The application and library can each be extended by implementing a plugin module in C++ or Python to explore diﬀerent visualization techniques, build/manipulate molecular structures, and interact with other programs. We describe some example extensions, one which uses a genetic algorithm to ﬁnd stable crystal structures, and one which interfaces with the PackMol program to create packed, solvated structures for molecular dynamics simulations. The 1.0 release series of Avogadro is the main focus of the results discussed here. Conclusions: Avogadro oﬀers a semantic chemical builder and platform for visualization and analysis. For users, it oﬀers an easy-to-use builder, integrated support for downloading from common databases such as PubChem and the Protein Data Bank, extracting chemical data from a wide variety of formats, including computational chemistry output, and native, semantic support for the CML ﬁle format. For developers, it can be easily extended via a powerful plugin mechanism to support new features in organic chemistry, inorganic complexes, drug design, materials, biomolecules, and simulations. Avogadro is freely available under an open-source license from http://avogadro.openmolecules.net. Software architecture O h The core of Avogadro is written in portable C++ code with platform-speciﬁc diﬀerences abstracted away by Qt, OpenGL, and Open Babel. The CMake build system makes the build process relatively simple on all supported platforms. Avogadro has been successfully built and tested on Linux, Apple Mac OS X, and Microsoft Windows in common 32 and 64 bit hardware architectures. At the time of writing, it is apparent that other researchers have perceived similar needs. Several new applications are available today that focus on both build- ing and visualizing molecular structure. These include CCP1GUI [21], Gabedit [22] and some highly speciﬁc edi- tors such as MacMolPlt [23] which focus on particular computational packages (i.e., GAMESS-US for MacMol- Plt). Whilst oﬀering many interesting and useful fea- tures, these projects suﬀer from the same issues centering around eﬀective reuse of existing code, well commented and documented code, and easy extension to add new features and adapt for specialized areas. The core of Avogadro is written in portable C++ code with platform-speciﬁc diﬀerences abstracted away by Qt, OpenGL, and Open Babel. The CMake build system makes the build process relatively simple on all supported platforms. Avogadro has been successfully built and tested on Linux, Apple Mac OS X, and Microsoft Windows in common 32 and 64 bit hardware architectures. Hanwell et al. Journal of Cheminformatics 2012, 4:17 http://www.jcheminf.com/content/4/1/17 Hanwell et al. Journal of Cheminformatics 2012, 4:17 http://www.jcheminf.com/content/4/1/17 Hanwell et al. Journal of Cheminformatics 2012, 4:17 http://www.jcheminf.com/content/4/1/17 Licensing costs can be prohibitive. If the company were to change its direction or focus, this can lead to a loss of a signiﬁcant research investment in a commercial product. Furthermore, in most cases, these programs use custom, proprietary ﬁle formats, and semantic and chemical data can be lost in conversion to other data formats. chemistry, biochemistry, and molecular dynamics, among many others [28-47]. From the beginning, the project has strived to make a robust, ﬂexible framework for both building and visual- izing molecular structures. Much of the initial focus has been placed on preparing input and analyzing output from quantum calculations. Other applications such as prepar- ing input for MD simulations and visualizing periodic structures will also be presented, demonstrating the ﬂex- ibility of the Avogadro platform. The development team has also been members of the Blue Obelisk movement, fol- lowing the three pillars outlined by the group: Open Data, Open Standards, and Open Source [48,49]. The selection of free, open-source, cross-platform, three-dimensional, molecular builders was quite limited when the Avogadro project was founded in late 2006. Ghemical [17] was one of the only projects satisfying these needs at the time. Two of the authors (Hutchison and Curtis) contributed to Ghemical previously, but had found that it was not easily extensible. This led them to found a new project to address the issues they had observed in Ghemical and other packages. The Molden [18] applica- tion was also available, able to build up small molecules and analyze output from several quantum codes. How- ever, it suﬀers from a restrictive license and it uses an antiquated graphical toolkit, which is not native on most modern operating systems. Background aforementioned viewers are all freely available, and most of them are available under open-source licenses and work on the most common operating systems (Linux, Apple Mac OS X, Microsoft Windows, and BSD). Many ﬁelds such as chemistry, materials science, physics, and biology, need eﬃcient computer programs to both build and visualize molecular structures. The ﬁeld of molecular graphics is dominated by viewers with lit- tle or no editing capabilities, such as RasMol [1], Jmol [2], PyMOL [3], VMD [4], QuteMol [5], BALLView [6], VESTA [7], and XCrySDen [8,9], among many others. The The choice of software capable of building chemical structures in three dimensions is far smaller. There are existing commercial packages, such as CAChe/Scigress [10], ChemBio3D [11], GaussView [12], HyperChem [13], CrystalMaker [14], Materials Studio [15], and Spartan [16], which are polished and capable of constructing many diﬀerent types of molecular structures. They are, however, not available for all operating systems (most of them only run on Microsoft Windows), and are not easily extensi- ble, customized, or integrated into automated workﬂows. Page 2 of 17 Software architecture O h One area that seems to suﬀer in many code bases in chemistry is software architecture. This can lead to less maintainable code, poor code reuse, and a much higher barrier to entry. Problems were identiﬁed in other projects with a view to minimize their impact when developing Avogadro. Modern software design processes were used in the initial planning stages of Avogadro, along with the choice of modern programming languages and libraries. Broad goals for the design of a molecular editor were identiﬁed following a case study of the available appli- cations. One of the main issues with both commercial and open-source applications is a lack of extensibility; many of the applications also only work on one or two operating systems. The creation of an open and exten- sible framework that implements many of the necessary foundations for a molecular builder and visualizer would facilitate more eﬀective research in this area. Further, the open, standardized Chemical Markup Language (CML) ﬁle format [19,20] would be used, to secure semantic and chemical data and allow easy interoperability with other chemistry software. Avogadro has close ties to several other free, cross- platform, open-source projects to reuse as much code as is practical. These projects include Qt [50] to pro- vide a free, cross-platform graphical toolkit; Open Babel [51] for chemical ﬁle input/output, geometry optimiza- tion, and other chemical perception; Eigen [52] for matrix and vector mathematics; OpenGL/GLSL for real-time, three-dimensional rendering; and POV-Ray for ray-traced rendering. Based on the previous experience of the authors and a review of available programs at the time, several fun- damental choices were made. The C++ programming language; the Qt graphical toolkit; OpenGL for 3D visu- alization; CMake as the build system; and Open Babel as the chemical library. Using this combination of languages and libraries requires the project to be licensed under the GNU GPLv2 [53] license and made openly available to all. Based on the previous experience of the authors and a review of available programs at the time, several fun- damental choices were made. The C++ programming language; the Qt graphical toolkit; OpenGL for 3D visu- alization; CMake as the build system; and Open Babel as the chemical library. Using this combination of languages and libraries requires the project to be licensed under the GNU GPLv2 [53] license and made openly available to all. Implementation The Avogadro framework uses the model, view, con- troller paradigm. The model is comprised of the core data classes such as Molecule, Atom, and Bond, views are made up of the engine/display plugins, and controllers are the tools (interactive mouse) and extensions (non- interactive, form based/menu based). Every plugin has full access to the core data model, but view and controller The Avogadro project was started in earnest in 2007, and over the ﬁrst 5 years of development has been downloaded over 270,000 times [24], been translated into over 20 lan- guages [25], and has over 20 contributors [26]. So far, it has been cited over 100 times [27], including applications in spectroscopy, catalysis, materials chemistry, theoretical Hanwell et al. Journal of Cheminformatics 2012, 4:17 http://www.jcheminf.com/content/4/1/17 Page 3 of 17 activities (Figure 1). The Avogadro::Color base class deﬁnes the virtual interface for applying colors to atoms, bonds, and other properties. Avogadro::Engine deﬁnes the common interface for all display types in Avogadro: simple ball and stick, Van der Waals visualizations, surfaces, and force visualizations. The Avogadro::Tool base class provides the interface for all interactive tools, focusing principally on mouse and keyboard interaction with Avogadro. Examples of tool plugins include the draw tool used to draw molecules atom by atom, and the navigation tool used to pan, rotate, and scale the view of the molecule. There are also several specialized tools such as the alignment tool. plugins are conceptually diﬀerent; views are responsible for displaying data and controllers are responsible for modifying/changing data. Plugins rely on Avogadro’s set of programming inter- faces and almost all functionality is implemented in self contained plugins that are loaded at runtime. The major- ity of plugins distributed with Avogadro are written in C++, but the API is also available in the Python script- ing language. This allows for a great deal of choice in how plugins are implemented. Each plugin is a singleton class that implements a particular set of functions–depending on the type of plugin–which allows for features to be implemented in a very modular way. Finally there is the Avogadro::Extension class, which deﬁnes the interface for dialog based plugins. These extensions can interact with the molecule, and are used for a variety of purposes from molecule prop- erties dialogs to input ﬁle generation dialogs for many quantum codes including NWChem, Gaussian, GAMESS, and others. Implementation This class of plugin is also applied to ﬁle import, and network aware extensions querying web databases for structures given their common name for example. Over the last few years Avogadro development has started to use nightly builds of the latest version of the code in order to automatically ﬂag issues introduced in new commits. Code review was also introduced in order to add a review step before new code is merged, along with softening the line between someone with commit rights and someone without (anyone can propose and upload a patch, but a small group can choose if/when the patch will be merged). Some automated testing has been added, but coverage at this point remains relatively low. API docu- mentation is automatically generated from comments in the code using Doxygen. At start up, several standard directories, which may be customized, are searched for plugins. The Qt plugin framework is used to check that the plugins have a recent enough version to be loaded, and the plugin type can be deduced once loaded. The user interface is then populated with appropriate entries; tools are added to the main tool- bar using their embedded icons, display types are added Display types Table 1 List of default display type (engine) plugins Name Description Axes Renders x, y, z Cartesian axes from the origin Ball and Stick Standard ball and stick representation Cartoon Secondary biological structure (α helix and β sheet) Dipole Render direction/magnitude of dipole moment if present Force Renders arrows showing forces on atoms from force ﬁeld Hydrogen Bond Renders hydrogen bonds as dotted lines Label Shows labels on atoms and bonds, conﬁgurable Overlay Overlay of color gradient used for electrostatic properties Polygon Renders closed polygons of metallic centers Ribbon Basic secondary structure ribbon rendering Ring Renders rings in structure, diﬀerent colors depending on ring size Simple Wireframe Very simple wireframe display Sticks Stick or liquorice rendering style for atoms and bonds Surface Renders triangular isosurface meshes Van der Waals Van der Waals sphere rendering (no bonds, Spheres space-ﬁlling) Wireframe Wireframe with more features such as bond order rendering Table 1 List of default display type (engine) plugins Name Description Axes Renders x, y, z Cartesian axes from the origin Ball and Stick Standard ball and stick representation Cartoon Secondary biological structure (α helix and β sheet) Dipole Render direction/magnitude of dipole moment if present Force Renders arrows showing forces on atoms from force ﬁeld Hydrogen Bond Renders hydrogen bonds as dotted lines Label Shows labels on atoms and bonds, conﬁgurable Overlay Overlay of color gradient used for electrostatic properties Polygon Renders closed polygons of metallic centers Ribbon Basic secondary structure ribbon rendering Ring Renders rings in structure, diﬀerent colors depending on ring size Simple Wireframe Very simple wireframe display Sticks Stick or liquorice rendering style for atoms and bonds Surface Renders triangular isosurface meshes Van der Waals Van der Waals sphere rendering (no bonds, Spheres space-ﬁlling) Wireframe Wireframe with more features such as bond order rendering There are also two tools for adjustment of structures (atom or bond centric), a selection tool supporting stan- dard selection interactions, and an auto-rotate tool that allows users to set the speed and angles about which to rotate the molecule. The interactive auto-optimization tool provides a sculpting interaction, where the user can begin a continuous geometry optimization and switch back to the draw or adjustment tools and change the shape and structure of the molecule while observing the new structure being optimized. Display types Display plugins are referred to as “engines” internally. Their primary focus is rendering graphics to the screen. As is the case with most molecular graphics, a large por- tion of the geometric primitives are spheres and cylinders, typically used to represent atoms and bonds. There are many other properties that can be rendered using the dis- play type plugins, for example, some of the engines also convey information about the underlying data the geomet- ric primitives represent to allow for the molecule to be edited. Table 1 shows a summary of the display plugins distributed with Avogadro. The navigation tool provides basic scene navigation, implementing rotation,panning, tilting, and zooming sup- port. The initial point of interaction (where the click occurs) changes the anchor point for navigation; navi- gation takes place about the center of molecule when clicking in empty space or about the center of any clicked atom. During interaction, the navigation tool provides visual cues to show what type of navigation is taking place. The navigation tool is also used as the default tool if the currently active tool does not handle the mouse event passed to it. Engines are performance critical as the render functions are called each time a frame is requested for display. Eﬃ- cient rendering is also critical since multiple display types One of the other central tools is the draw tool, which implements a free-hand molecule drawing input method supporting keyboard shortcuts, combo boxes, and a peri- odic table view to select elements. The user can use the left mouse button to add new atoms or bonds, or click on the bonds to change their order. The right mouse button can be used to delete atoms or bonds, and the directional keys can be used in combination with the mouse to quickly rotate/pan the molecule. Display types This can also be combined with the measurement tool to interactively observe bond lengths and angles evolve as the structure is updated and the geometry minimized. If the optimization tool is turned oﬀ, the measurement tool also allows the user to precisely adjust bond lengths and/or angles using the adjustment tools. Tools The tools are responsible for virtually all mouse and key- board interaction with the molecule. A list of all tools is given in Table 2. Plugin interface Avogadro plugins are divided into four diﬀerent types corresponding to four main classes that derive from this common base class, specializing their interface for speciﬁc Extensions Tools Painters Engines Open Babel Qt Eigen Force Fields Manipulate Tool Balls & Sticks POV-Ray OpenGL Scripting Python Tools Colors Elements Figure 1 General code architecture of Avogadro. General code architecture of Avogadro, indicating major plugin interfaces for colors, display engines, tools, and extensions. Red boxes indicate code dependencies of Avogadro, blue boxes indicate plugin API classes, and green boxes inidicate examples of each plugin type. Manipulate Tool Scripting Force Fields Figure 1 General code architecture of Avogadro. General code architecture of Avogadro, indicating major plugin interfaces for colors, display engines, tools, and extensions. Red boxes indicate code dependencies of Avogadro, blue boxes indicate plugin API classes, and green boxes inidicate examples of each plugin type. Hanwell et al. Journal of Cheminformatics 2012, 4:17 http://www.jcheminf.com/content/4/1/17 Page 4 of 17 Hanwell et al. Journal of Cheminformatics 2012, 4:17 http://www.jcheminf.com/content/4/1/17 can be combined to form a composite display. For exam- ple, ball and stick display overlaid with a transparent Van der Waals space-ﬁlling display and ring rendering to high- light all rings in the structure. Figure 2 (d) and (f) show two such combinations of multiple display types. to the display type list, and menu entries are added for all loaded extensions. The tool and display type plugins can both (option- ally) provide a dialog for conﬁguring the plugin. Dialogs are speciﬁc to each plugin and integrated into the user interface. Extensions Extensions represent quite a diverse range of plugins including input generation dialogs for various quantum chemistry codes such as GAMESS, Molpro, NWChem, etc., animation of the molecule, and visualization of molecular orbitals and electron density. Network aware extensions allow the user to click on a menu item to fetch Hanwell et al. Journal of Cheminformatics 2012, 4:17 Page 5 of 17 http://www.jcheminf.com/content/4/1/17 Hanwell et al. Journal of Cheminformatics 2012, 4:17 Page 5 of 17 http://www.jcheminf.com/content/4/1/17 Hanwell et al. Journal of Cheminformatics 2012, 4:17 http://www.jcheminf.com/content/4/1/17 Hanwell et al. Journal of Cheminformatics 2012, 4:17 http://www.jcheminf.com/content/4/1/17 Page 5 of 17 Figure 2 Standard molecular structure representations. Several molecular representations of thiophene, (a) wireframe, (b) stick/licorice, (c) ball and stick, (d) ball and stick with ring, (e) Van der Waals/CPK and (f) transparent Van der Waal’s with stick. Figure 2 Standard molecular structure representations. Several molecular representations of thiophene, (a) wireframe, (b) stick/licorice, (c) ball and stick, (d) ball and stick with ring, (e) Van der Waals/CPK and (f) transparent Van der Waal’s with stick. by chemical name and search for “tnt” or “propanol” and have structures returned by the NIH CACTUS Chem- ical Structure Resolver service [54]. A summary of the extensions distributed with Avogadro is shown in Table 3. are all implemented as extensions showing up in Avo- gadro menus. More recently a crystallography extension was added, giving access to a much wider range to func- tionality useful to practitioners in that area, including Miller Plane visualization, slab and surface generation. New builders for nanotubes, nanoparticles, and DNA are also planned for upcoming releases. Other extensions translate the entire scene to POV- Ray input, and call POV-Ray to render the molecule using ray tracing techniques to provide higher quality renderings for publication. Various molecular property dialogs are also implemented as plugins, drawing largely on Open Babel functionality to provide an overview of the molecule. Cartesian editors, addition and removal of hydrogens, fragment, SMILES, and peptide insertion Colors The color plugins primarily take either double precision numbers or integer values and return an RGB value. The plugins range from the standard color plugin that takes atomic number and returns the standard RGB value for that element through to mapping things like partial change and index to more easily view various aspects of the molecule’s structure. Table 2 List of default mouse tool plugins Name Description Draw Tool Build and edit atoms Navigate Tool Move the camera, rotate, pan, and zoom Bond Centric Alter bond lengths, angles, and torsions Manipulate Tool Move atoms and selected fragments Select Tool Select individual atoms, bonds, or fragments Auto Rotate Tool Continuously rotate a molecule for presentations Auto Optimize Tool Continuously optimize molecular geometry using molecular mechanics Measure Tool Determine bond lengths, angles, and dihedrals Align Tool Rotate and translate to a speciﬁed frame of reference Table 2 List of default mouse tool plugins Name Description Draw Tool Build and edit atoms Navigate Tool Move the camera, rotate, pan, and zoom Bond Centric Alter bond lengths, angles, and torsions Manipulate Tool Move atoms and selected fragments Select Tool Select individual atoms, bonds, or fragments Auto Rotate Tool Continuously rotate a molecule for presentations Auto Optimize Tool Continuously optimize molecular geometry using molecular mechanics Measure Tool Determine bond lengths, angles, and dihedrals Align Tool Rotate and translate to a speciﬁed frame of reference By deﬁning a plugin interface for coloring atoms, bonds, or residues, developers can easily oﬀer ﬂexible render- ing options to highlight important information without requiring a user to tediously set colors on speciﬁc atoms or functional groups. Default color plugins are listed in Table 4, illustrating the variety of options. Each plugin is usually only 40-50 lines of C++ code. Python scripting h b d Python bindings are provided for all of the core API. Python code can be used in two ways: the ﬁrst is the inter- active Python terminal, and the second is to write Python plugins; extensions, tools, or display types. Writing a Page 6 of 17 Hanwell et al. Journal of Cheminformatics 2012, 4:17 http://www.jcheminf.com/content/4/1/17 Hanwell et al. Journal of Cheminformatics 2012, 4:17 http://www.jcheminf.com/content/4/1/17 Table 3 List of default extension commands Name Description Create Surfaces Create surface meshes from molecular orbital/ electron density data GAMESS Prepare input ﬁles for GAMESS-US, featuring syntax highlighting, advanced properties Insert Fragment Insert molecular fragments from a library of common fragments Insert Peptide Build up and insert peptide fragments Molecular Mechanics Use Open Babel’s force ﬁelds for geometry optimization and conformer searches MOPAC Prepare input for and run MOPAC200x POV-Ray Ray-trace the displayed structure using POV-Ray Properties Angle Properties Table of all bond angles (editable) Atom Properties Table of all atoms with common properties Bond Properties Table of all bonds with common properties Molecule Properties Common properties of the molecule (including molecular weight, etc.) Torsion Properties Table of all dihedral angles (editable) Spectra Visualize spectra from output ﬁles Super Cell Builder Expand atoms with space group, replicate speciﬁed repeats and perform simple bonding Unit Cell Change crystallographic unit cell display and parameters Vibrations Show and animate molecular vibrations Table 3 List of default extension commands Name Description instructions on how to compile the latest source code are also provided on the main Avogadro web site [60,61] for the more adventurous, or those using an operating system that is not yet supported. The Qt toolkit gives Avogadro a native look and feel on the three major supported operating systems—Linux, Apple Mac OS X, and Microsoft Windows. The basic functionality expected in a molecular builder and viewer has been implemented, along with several less common features. It is very easy for new users to install Avogadro and build their ﬁrst molecules within minutes. Thanks to the Open Babel library [51], Avogadro supports a large portion of the chemical ﬁle formats that are in common use. The vast majority of this functionality has been writ- ten using the interface made available to plugin writers, and is loaded at runtime. We will discuss these plugin interfaces and descriptions of the plugin types later. Semantic chemistry d h d Avogadro has used CML [19,20] as its default ﬁle format from a very early stage; this was chosen over other ﬁle formats because of the extensible, semantic structure pro- vided by CML, and the support available in Open Babel [51]. The CML format oﬀers a number of advantages over others in common use, including the ability to extend the format. This allows Avogadro and other programs to be future-proof, adding new information and features neces- sary for an advanced semantically-aware editor at a later time, while still remaining readable in older versions of Avogadro. Through the use of Open Babel [51], a large array of ﬁle formats can be interpreted. When extending Avogadro to read in larger amounts of the output from quantum codes, it was necessary to devote signiﬁcant develop- ment resources to understanding and adding semantic meaning to the quantum code output. This work was developed in a plugin, which was later split out into Python plugin requires the same functionality to be imple- mented as a native C++ plugin [55]. The advantage of Python plugins is that it’s easier to make prototypes since no compilation is required. Python plugins can also easily be shared with other users. The Python bindings interface with the PyQt python bindings for the Qt toolkit, which enables Python code to use all of Qt’s features when writing a plugin. For example, a short Python script can present a window using Qt and render molecules using Avogadro [56-58]. Semantic chemistry d h d Table 4 List of default color plugins Name Description Atom Index Color Color based on atom ID (from atom 1, 2, etc.) Charge Color Color based on predicted electrostatic partial charge Custom Color Color all atoms a speciﬁc, custom color Distance Color Color based on distance from one end of the molecule Element Color Standard color scheme, giving each atom a color (Default) deﬁned by its element Residue Color Color based on amino acid or nucleic acid residue (i.e., glycine, histidine, etc.) SMARTS Color Color atoms matching a speciﬁc SMARTS pattern with a custom color Table 4 List of default color plugins Name Description Atom Index Color Color based on atom ID (from atom 1, 2, etc.) Charge Color Color based on predicted electrostatic partial charge Custom Color Color all atoms a speciﬁc, custom color Distance Color Color based on distance from one end of the molecule Element Color Standard color scheme, giving each atom a color (Default) deﬁned by its element Residue Color Color based on amino acid or nucleic acid residue (i.e., glycine, histidine, etc.) SMARTS Color Color atoms matching a speciﬁc SMARTS pattern with a custom color Table 4 List of default color plugins Name Description Table 4 List of default color plugins Avogadro also includes an interactive Python console (Figure 3, which allows users to directly script and manip- ulate the Avogadro environment [59]. The graphical user interface The ﬁrst thing most people will see is the main Avo- gadro application window, as shown in Figure 4. Binary installers are provided for Apple Mac OS X and Microsoft Windows, along with packages for all of the major Linux distributions. This means that Avogadro can be installed quite easily on most operating systems. Easy to follow Hanwell et al. Journal of Cheminformatics 2012, 4:17 http://www.jcheminf.com/content/4/1/17 Page 7 of 17 Figure 3 Python scripting terminal, printing atomic numbers. Simply left-clicking on the black part of the display allows the user to draw a carbon atom. If the user pushes the left mouse button down and drags, a bonded carbon atom is drawn between the start point and the ﬁnal position where the mouse is released. a small independent library called OpenQube [62,63]. More recently a large amount of work has been done by the Quixote project [64], JUMBO-Converters, and the Semantic Physical Science workshop to augment quantum codes to output more of this data directly from the code. Since CML can be extended, it is possible to reuse exist- ing conventions for molecular structure data, and add new conventions for the additional quantum data. A large amount of eﬀort has been expended to create an intuitive tool for drawing small molecules. Common chemical elements can be selected from a drop down list, or a periodic table can be displayed to select less com- mon elements. Clicking on an existing atom changes it to the currently selected element, dragging changes the atom back to its previous element and draws a new atom Building a molecule: atom by atom After opening Avogadro a window such as that shown in Figure 4 is presented. By default, the draw tool is selected. Figure 4 The Avogadro graphical user interface. Taken on Mac OS X, showing the editing interface for a molecule. Figure 4 The Avogadro graphical user interface. Taken on Mac OS X, showing the editing interface for a molecule. Hanwell et al. Journal of Cheminformatics 2012, 4:17 http://www.jcheminf.com/content/4/1/17 Hanwell et al. Journal of Cheminformatics 2012, 4:17 http://www.jcheminf.com/content/4/1/17 Page 8 of 17 bonded to the original. If the bonds are left-clicked then the bond order cycles between single, double, and triple. Shortcut keys are also available, e.g., typing the atomic symbol (e.g., “C-o” for cobalt) changes the selected ele- ment, or typing the numbers “1,” “2,” and “3” changes the bond order. adding new groups, or removing groups can be observed interactively. Several dialogs are implemented to provide information on molecule properties and to precisely change parame- ters, such as the cartesian coordinates of the atoms in the molecule. Right clicking on atoms or bonds deletes them. If the “Adjust Hydrogens” box is checked, the number of hydro- gens bonded to each atom is automatically adjusted to satisfy valency. Alternatively, this can also be done at the end of an editing session by using the “Add hydrogens” extension in the build menu. Building a molecule: from fragments In addition to building molecules atom-by-atom, users can insert pre-built fragments of common molecules, ligands, or amino-acid sequences, as shown in Figure 5. In all cases, after inserting the fragment, the atom-centered manipulate tool is selected, allowing the fragment to be moved or rotated into position easily. In addition to the draw tool, there are two tools for adjusting the position of atoms in existing molecules. The “atom centric manipulate” tool can be used to move an atom or a group of selected atoms. The “bond centric manipulate” tool can be used to select a bond, and then adjust all atoms positions relative to the selected bond in various ways (e.g., altering the bond length, bond angles, or dihedral angles). These three tools allow for a great deal of ﬂexibility in building small molecules interactively on screen. Users can also insert a SMILES [65,66] string for a molecule. In this case, a rough 3D geometry is generated using Open Babel and a quick force ﬁeld optimization. Preparing input for quantum codes l d l Several extensions were developed for Avogadro that assist the user in preparing input ﬁles for popular quantum codes such as GAMESS-US, [67] NWChem, [68] Gaussian, [69] Q-Chem, [70] Molpro, [71] and MOPAC200x [72]. The graphical dialogs present the fea- tures required to run basic quantum calculations; some examples are shown in Figure 6. Once the molecular structure is complete, the force ﬁeld extension can be used to perform a geometry opti- mization. By clicking on “Extensions” and “Optimize Geometry” a fast geometry optimization is performed on the molecule. The force ﬁeld and calculation parameters can be adjusted, but the defaults are adequate for most molecules. This workﬂow is typical when building up a small molecular structures for use as input to quantum calculations, or publication quality ﬁgures. The preview of the input ﬁle at the bottom of each dialog is updated as options are changed. This approach helps new users of quantum codes to learn the syntax of input ﬁles for diﬀerent codes, and to quickly generate use- ful input ﬁles as they learn. The input can also be edited by hand in the dialog before the ﬁle is saved and sub- mitted to the quantum code. The MOPAC extension can also run the MOPAC200x program directly if it is avail- able on the user’s computer, and then reload the output An alternative is to combine the “Auto Optimization” tool with the drawing tool. This presents a unique way of sculpting the molecule while the geometry is constantly minimized in the background. The geometry optimiza- tion is animated, and the eﬀect of changing bond orders, Figure 5 Dialogs for inserting pre-built fragments. The left shows molecules, and the right amino-acid sequences. Figure 5 Dialogs for inserting pre-built fragments. The left shows molecules, and the right amino-acid sequences. Hanwell et al. Journal of Cheminformatics 2012, 4:17 http://www.jcheminf.com/content/4/1/17 Page 9 of 17 Figure 6 Dialog for generating input for quantum codes. Dialogs for generating input for Q-Chem, NWChem, Molpro and MOPAC200x. Note that the dialogs are similar in interface, allowing users to use multiple computational chemistry packages. Figure 6 Dialog for generating input for quantum codes. Dialogs for generating input for Q-Chem, NWChem, Molpro and MOPAC200x. Note that the dialogs are similar in interface, allowing users to use multiple computational chemistry packages. ﬁle into Avogadro once the calculation is complete. Preparing input for quantum codes l d l This feature will be extended to other quantum codes in future versions of Avogadro. the coordinate origin if one atom is selected, and along the speciﬁed axis if two atoms are selected. The align- ment tool can be combined with the measure, select, and manipulate tools to create inputs for quantum codes where the position and orientation of the molecule is important. One example of this is calculations where an external electric ﬁeld is applied to the molecule. In these types of calculations, the alignment of the molecule can have a large eﬀect. Figure 8 shows the measurement tool in action with the alignment tool conﬁguration dialog visible in the lower-left corner. The GAMESS-US plugin is one of the most highly developed, featuring a basic dialog present in most of the other input deck generators, as well as an advanced dialog exposing many of the more unusual and complex calcula- tion types. In addition to the advanced dialog, the input deck can be edited inline and features syntax highlight- ing (Figure 7) as used in many popular editors aimed at software developers. This can indicate simple typing errors in keywords, as well as harder to spot whitespace errors that would otherwise cause the hand-edited input deck to fail when being read by GAMESS-US. More complex alignment tools for speciﬁc tasks could be created. The alignment tool was created in just a few hours for a speciﬁc research project. This is a prime exam- ple where extensibility was very important for performing research using a graphical computational chemistry tool. It would not be worth the investment to create a new application just to align molecular structures to an axis, but creating a plugin for an extensible project is not unreasonable. Visualization The Avogadro application uses OpenGL to render molec- ular representations to the screen interactively. OpenGL oﬀers a high-level, cross-platform API for rendering three-dimensional images using hardware accelerated graphics. OpenGL 1.1 and below is used in most of the rendering code, and so Avogadro can be used even on older computer systems, or those without more modern accelerated graphics. It is capable of taking advantage of some of the newer features available in OpenGL 2.0 as described below, but this has been kept as an optional extra feature when working on novel visualizations of molecular structure. Quantum codes were originally developed for line print- ers, and unfortunately little has changed since then in the standard log ﬁles. There are several formats developed for use in other codes and speciﬁcally for visualization and analysis, but there is little agreement on any standard ﬁle format in the computational quantum chemistry commu- nity. A plugin was developed in Avogadro to visualize the output of various quantum codes, and get the data into the right format for further visualization and analysis. Initially support was added and extended in Open Babel for Gaussian cube ﬁles. This format provides atomic coor- dinates and one or more regularly spaced grids of scalar values. This can be read in, and techniques such as the marching cubes algorithm can be used to compute trian- gular meshes of isosurfaces at values of electron density for example. Once the code has been developed to visu- alize these isosurfaces, it became clear that it would be useful to be able to calculate these cubes on the ﬂy, and at diﬀerent levels of detail depending upon the intended use. Alignment and measurements One of the specialized tools included in the standard Avo- gadro distribution is the alignment tool. This mouse tool facilitates the alignment of a molecular structure with Hanwell et al. Journal of Cheminformatics 2012, 4:17 http://www.jcheminf.com/content/4/1/17 Page 10 of 17 Hanwell et al. Journal of Cheminformatics 2012, 4:17 http://www.jcheminf.com/content/4/1/17 Figure 7 The GAMESS-US input deck generator. This input generator has an advanced panel and syntax highlighting. Standard representations In chemistry, there are several standard representations of molecular structure, originally based upon those possible with physical models. The Avogadro application imple- ments each of these representations shown in Figure 2 as a plugin. These range from the simple wireframe repre- sentation, stick/licorice, ball and stick, and Van der Waals spheres. The ﬁrst format, which was somewhat documented at the time it was developed, is the Gaussian formatted checkpoint format. This format is much easier to parse than the log ﬁles generated as the program runs, and provides all of the detail needed to calculate scalar val- ues of the molecular orbital or electron density at any point in space. Once a class structure had been developed It is also possible to combine several representations, such as ball and stick with ring rendering (Figure 2 (d)), and a semi-transparent Van der Waals space-ﬁlling repre- sentation with a stick representation to elucidate molecu- lar backbone (Figure 2 (f)). Hanwell et al. Journal of Cheminformatics 2012, 4:17 http://www.jcheminf.com/content/4/1/17 Page 11 of 17 Figure 8 The measurement tool. The measurement tool being used to measure bond angles and lengths (on Linux with KDE 4). Figure 8 The measurement tool. The measurement tool being used to measure bond angles and lengths (on Linux with KDE 4). independently of all other points, the results of which can be seen in Figure 9. An alternate approach to calculating the molecular orbitals was developed in a second plu- gin that has since been split oﬀinto a separate project named “OpenQube”. The “OpenQube” library has also been added as an optional backend in VTK during the 2011 Google Summer of Code, bringing support for sev- eral output ﬁle formats and calculation of cube ﬁles that can later be fed into more advanced data pipelines. for Gaussian type orbitals, the approach was extended to read in several other popular output ﬁle formats including Q-Chem, GAMESS-US, NWChem, and Mol- pro. MOPAC200x support was added later, along with support for the AUX format and Slater type orbitals used in that code. All of these codes output their ﬁnal conﬁg- urations using the standard linear combination of atomic orbitals, meaning that parallelization is extremely simple. for Gaussian type orbitals, the approach was extended to read in several other popular output ﬁle formats including Q-Chem, GAMESS-US, NWChem, and Mol- pro. GLSL, novel visualization GLSL, or OpenGL Shader Language, is a C-like syntax that can be used to develop code that will run on graphics cards and included in the OpenGL 2.0 speciﬁcation. It has been used to great eﬀect by the games industry, as well as in many areas of data visualization. Several recent papers highlight the potential in chemistry, such as QuteMol [5] in adding support for features such as ambient occlusion to add depth to images. Avogadro has support for vertex and fragment shader programs, and several examples are bundled with the package. If the user’s graphics card is capable, these pro- grams can be loaded at runtime and used to great eﬀect to visualize structure. Some of these include summariza- tion techniques such as isosurface rendering where only the edges orthogonal to the view plane are visible, giv- ing a much better rendering of both the molecular and electronic structure (Figure 10). Ray tracing A d Avogadro uses a painter abstraction that makes it much easier for developers to add new display types. It also abstracts away the renderer, making it possible to add support for alternative backends. Currently only OpenGL and POV-Ray are supported. Due to the abstraction, we are able to use the implicit surfaces available in ray trac- ers to render molecular structure at very high levels of clarity and with none of the triangle artifacts present in standard OpenGL rendered images. Much higher quality transparency and reﬂection also allow for the images to be used in poster and oral presentations as well as research articles (Figure 11). Hanwell et al. Journal of Cheminformatics 2012, 4:17 http://www.jcheminf.com/content/4/1/17 Slater set is populated with the correct ordering and the expected normalization scheme. The s, p, and d-type Gaussian orbitals are supported, with f and g support planned in order to support the increasing number of calculations using these higher-order orbitals. The Basis Set Exchange hosted by EMSL provides access to the basis sets in common use, although at present these basis sets are normally read in directly from the output ﬁles. There are several related projects for adding semantic meaning to this type of output, including the JUMBO- Converters project and Quixote. It is hoped that more codes will adopt semantic output in the future, using a common format so that data exchange, validation, and analysis become easier across several codes. This was the subject of a recent meeting with several computa- tional chemistry codes beginning to use FoX in order to output CML. Development has begun on code to read in CML output, either directly from the codes or from conversion of other formats using Open Babel or the JUMBO-Converters. If enough semantic structure can be added to CML, and the converters support a large enough range of the output, this could replace most of the parsing code present in OpenQube. Semantic mean- ing is one of the most diﬃcult to extract from log ﬁles, and coming together as a community will help projects like Avogadro to derive more meaning from the outputs of these codes. Standard representations MOPAC200x support was added later, along with support for the AUX format and Slater type orbitals used in that code. All of these codes output their ﬁnal conﬁg- urations using the standard linear combination of atomic orbitals, meaning that parallelization is extremely simple. The plugin was developed to take advantage of the map- reduce approach oﬀered by QtConcurrent in order to use all available processor cores. This oﬀers almost lin- ear scaling as each point in the grid can be calculated The plugin was developed to take advantage of the map- reduce approach oﬀered by QtConcurrent in order to use all available processor cores. This oﬀers almost lin- ear scaling as each point in the grid can be calculated A class hierarchy with a standard API is provided for quantum output. Adding support for new codes involved developing a new parser and ensuring the Gaussian or Figure 9 Molecular orbitals and surfaces. Rendering of a molecular orbital isosurface (left) and an electrostatic surface potential mapped onto the electron density (right). Figure 9 Molecular orbitals and surfaces. Rendering of a molecular orbital isosurface (left) and an electrostatic surface potential mapped onto the electron density (right). Page 12 of 17 Page 12 of 17 Page 12 of 17 Hanwell et al. Journal of Cheminformatics 2012, 4:17 http://www.jcheminf.com/content/4/1/17 Hanwell et al. Journal of Cheminformatics 2012, 4:17 http://www.jcheminf.com/content/4/1/17 Avogadro library in use The Avogadro library’s ﬁrst use was the Avogadro appli- cation, closely followed by the Kalzium periodic table program that is part of the KDE software collection. This initial work was funded in part by the Google Summer of Code program in 2007, and also resulted in the addi- tion of several other features in the Avogadro library to support Kalzium and general visualization and editing of molecular structure (Figure 12). Secondary biological structure Avogadro uses the PDB reader from Open Babel to read in the secondary biological structure. Two plugins exist to process and render this information. The ﬁrst is a plu- gin which renders a simple tube between the biomolecule backbone atoms. A second more advanced plugin calcu- lates meshes for the alpha helices and beta sheets. While the ﬁrst plugin is much faster, the advanced plugin more accurately produces output expected in the ﬁeld. This allows users ﬂexibility for rendering secondary biological structures. This feature is implemented in an extension, with an additional painter class deriving from the base class and a dialog allowing the user to edit the basic rendering controls. The POV-Ray input ﬁle can also be retained and edited to produce more complex images, or to allow for much ﬁner control of the rendering process if desired. Figure 10 Molecular orbitals rendering using GLSL shaders. Rendering of a molecular orbital isosurface using two GLSL shaders to highlight the edges of the surfaces. The X-ray eﬀect (left) and red and blue (right) showing the positive and negative molecular orbital shapes. Figure 10 Molecular orbitals rendering using GLSL shaders. Rendering of a molecular orbital isosurface using two GLSL shaders to highlight the edges of the surfaces. The X-ray eﬀect (left) and red and blue (right) showing the positive and negative molecular orbital shapes. Hanwell et al. Journal of Cheminformatics 2012, 4:17 http://www.jcheminf.com/content/4/1/17 Hanwell et al. Journal of Cheminformatics 2012, 4:17 http://www.jcheminf.com/content/4/1/17 Page 13 of 17 Figure 11 Ray-traced HOMO isosurfaces of varying cube density. Rendering of a molecular orbital isosurface using POV-Ray with cubes of low (left) and high (right) density. some results from their development of a Molpro interface using the Avogadro library [74]. Conclusions Avogadro has grown over its ﬁrst six years to become an important tool for building, editing, visualizing, and ana- lyzing chemical and molecular data. With over 270,000 downloads, language translations and localizations, and over 100 citations, it has become an integral part of the chemical software toolbox. Through use of the native CML ﬁle format and a wide variety of chemical data import, Avogadro can provide semantic chemical data editing and conversion. We seek to provide an inte- grated environment in the simulation and cheminfor- matics workﬂow. While more must be done, particularly in regards to documentation, tutorials, ease-of-use, and automation, we aim to improve the quality and feature set with each new release. Development Center (W912HZ-11-P-0019) for ﬁnancial support. EZ and DL acknowledge the NSF (DMR-1005413) for ﬁnancial support. Author details 1 1Department of Chemistry, University of Pittsburgh, 219 Parkman Avenue, Pittsburgh, PA, 15260, USA. 2Department of Scientiﬁc Computing, Kitware, Inc., 28 Corporate Drive, Clifton Park, NY, 12065, USA. 3Department of Computer Science, Coe College, 1220 First Avenue NE, Cedar Rapids, Iowa 52402, USA. 4Department of Chemistry, State University of New York at Buﬀalo, Buﬀalo, New York 14260-3000, USA. 5Avogadro development team. Received: 25 June 2012 Accepted: 31 July 2012 Published: 13 August 2012 Received: 25 June 2012 Accepted: 31 July 2012 Published: 13 August 2012 Currently, two upcoming versions of Avogadro are under development. The ﬁrst is Avogadro version 1.1, which adds additional features and reﬁnement, par- ticularly including crystallography support developed through the XtalOpt project. The second is a more sub- stantial development for Avogadro version 2.0, where many of the core data structures are being rewritten in order to oﬀer greater ﬂexibility and scalability. Our goal is to support an increasing scope of chemical sys- tems, including biomolecules (DNA, RNA, saccarides, etc.), materials (crystallography, polymers, surfaces), nanoscience (nanoparticles, nanotubes, graphene, etc.) with improved speed, intuitive ease-of-use and simpler non-reciprocal licensing terms. References 1. Sayle R, Milner-White EJ: RasMol: Biomolecular graphics for all. Trends Biochem Sci(TIBS) 1995, 20(9):374. 2. Hanson RM, Howard MT, Willighagen EL, et al.: Jmol: an open-source Java viewer for chemical structures in 3D. 2012. [http://www.jmol.org] 3. DeLano WL: The PyMOL Molecular Graphics System. 2002. [http:// www.pymol.org] 4. Humphrey W, Dalke A, Schulten K: VMD - Visual Molecular Dynamics. J Molec Graphics 1996, 14:33–38. 5. Tarini M, Cignoni P, Montani C: Ambient Occlusion and Edge Cueing for Enhancing Real Time Molecular Visualization. IEEE Trans Visualization and Comput Graphics 2006, 12(5):1237–1244. 6. Mehlhorn K, Lenhof HP, Kholbacher O, Hildebrandt A: BALLView. 2012. [http://www.ballview.org] 7. Momma K, Izuma F: VESTA 3 for three-dimensional visualization of crystal, volumetric and morphology data. J Appl Cryst 2011, 44:1272–1276. 8. Kokalj A: XCrySDen—a new program for displaying crystalline structures and electron densities. J Mol Graphics and Model 1999, 17(3–4):176–179. 9. Kokalj A: Computer graphics and graphical user interfaces as tools in simulations of matter at the atomic scale. Comput Mater Sci 2003, 28(2):155–168. 10. SCIGRESS 2012. [http://www.fujitsu.com/global/services/solutions/tc/ hpc/app/scigress] 11. ChemBio3D 2012. [http://www.cambridgesoft.com/software/ chembio3d] 12. GaussView 5 2012. [http://gaussian.com/g prod/gv5.htm] Authors’ contributions GRH and DEC are the founders of the Avogadro project. MDH is the current lead developer and maintainer of Avogadro. GRH, DL and TV are active developers. DL and EZ are founders of the XtalOpt project which is discussed in this work. TV developed the PackMol plugin. All authors read and approved the ﬁnal manuscript. Acknowledgements We wish to thank the many contributors to the Avogadro project, including developers, testers, translators, and users. We thank SourceForge for providing resources for issue tracking and managing releases, Launchpad for hosting language translations, and Kitware for additional dashboard resources. MDH and GRH thank the University of Pittsburgh for support. DEC would like to thank Jan Halborg Jensen for designing the GAMESS-US interface and supporting Avogadro in its infancy; believing Avogadro could be better than what was available. MDH acknowledges the Engineering Research Development Center (W912HZ-11-P-0019) for ﬁnancial support. EZ and DL acknowledge the NSF (DMR-1005413) for ﬁnancial support. 1. Sayle R, Milner-White EJ: RasMol: Biomolecular graphics for all. Trends Biochem Sci(TIBS) 1995, 20(9):374. 1. Sayle R, Milner-White EJ: RasMol: Biomolecular graphics for all. Trends Biochem Sci(TIBS) 1995, 20(9):374. 2. Hanson RM, Howard MT, Willighagen EL, et al.: Jmol: an open-source Java viewer for chemical structures in 3D. 2012. [http://www.jmol.org] 3. DeLano WL: The PyMOL Molecular Graphics System. 2002. [http:// www.pymol.org] 4. Humphrey W, Dalke A, Schulten K: VMD - Visual Molecular Dynamics. J Molec Graphics 1996, 14:33–38. 4. Humphrey W, Dalke A, Schulten K: VMD - Visual Molecular Dynamics. J Molec Graphics 1996, 14:33–38. 6. Mehlhorn K, Lenhof HP, Kholbacher O, Hildebrandt A: BALLView. 2012. [http://www.ballview.org] 7. Momma K, Izuma F: VESTA 3 for three-dimensional visualization of crystal, volumetric and morphology data. J Appl Cryst 2011, 44:1272–1276. 8. Kokalj A: XCrySDen—a new program for displaying crystalline structures and electron densities. J Mol Graphics and Model 1999, 17(3–4):176–179. Avogadro is freely available from http://avogadro. openmolecules.net/, and new contributors are welcome in all areas (users, developers, testers, translators, educators, students, researchers, dreamers). 9. Kokalj A: Computer graphics and graphical user interfaces as tools in simulations of matter at the atomic scale. Comput Mater Sci 2003, 28(2):155–168. 9. Kokalj A: Computer graphics and graphical user interfaces as tools in simulations of matter at the atomic scale. Comput Mater Sci 2003, 28(2):155–168. 10. SCIGRESS 2012. [http://www.fujitsu.com/global/services/solutions/tc/ hpc/app/scigress] Packmol Journal of Cheminformatics 2012, 4:17 http://www.jcheminf.com/content/4/1/17 Hanwell et al. Journal of Cheminformatics 2012, 4:17 http://www.jcheminf.com/content/4/1/17 Competing interests The authors declare that they have no competing interests. Competing interests The authors declare that they have no competing interests. Competing interests The authors declare that they have no competing interests. Packmol Packmol is a third-party package designed to create ini- tial “packed” conﬁgurations of molecules for molecular dynamics or other simulations [75,76]. Examples include surrounding a protein with solvent, solvent mixtures, lipid bilayers, spherical micelles, placing counterions, adding ligands to nanoparticles, etc. Typically, users may have equilibrated “solvent boxes” which have been run for long simulations to ensure proper density, and both short and long-range interactions between solvent molecules. Using such solvent boxes allows placing solute molecules, such g The Q-Chem package [70] has developed “QUI - The Q-Chem User Interface” [73] around Avogadro, originally as an Avogadro extension. This is a more advanced ver- sion of the input generator developed in Avogadro, with much tighter integration. Molpro [71] has also published Figure 12 The Kalzium application in KDE using Avogadro to render molecular structures. Figure 12 The Kalzium application in KDE using Avogadro to render molecular structures. Hanwell et al. Journal of Cheminformatics 2012, 4:17 http://www.jcheminf.com/content/4/1/17 Hanwell et al. Journal of Cheminformatics 2012, 4:17 http://www.jcheminf.com/content/4/1/17 Page 14 of 17 Figure 13 The PackMol lipid layer as produced by the PackMol extension. Figure 13 The PackMol lipid layer as produced by the PackMol extension. including estimating the number of molecules in a given volume. as proteins, in an approximately correct initial structure, such as that shown in Figure 13. The solute is added into the box, and solvent molecules with overlapping atoms are removed. While these utilities are often enough, creating complex input ﬁles is not always easy. For more compli- cated systems, Packmol can create an initial conﬁguration based on deﬁned densities, geometries (e.g., sphere, box, etc.), and the molecules to be placed. An Avogadro devel- oper wrote an external plugin to facilitate use of Packmol, The plugin is not currently distributed with Avogadro as a standard feature, although it is planned for some future version. It serves as an example of how Avogadro can facil- itate a workﬂow with a text-oriented package (Packmol), including saving ﬁles in the PDB format required by Pack- mol, generating an input ﬁle, and reading the output for visualization, analysis, and further simulations. Figure 14 The XtalOpt extension. XtalOpt extension showing a plot of stability vs. search progress for a TiO2 supercell. igure 14 The XtalOpt extension. XtalOpt extension showing a plot of stability vs. search progress for a TiO2 supercell. Page 15 of 17 Hanwell et al. XtalOpt p The XtalOpt [77,78] software package is implemented as a third-party C++ extension to Avogadro and makes heavy use of the libavogadro API. The extension implements an evolutionary algorithm tailored for crystal structure prediction. The XtalOpt development team chose Avo- gadro as a platform because of its open-source license, well-designed API, powerful visualization tools, and intu- itive user-interface. XtalOpt exists as a dialog window (Figure 14) and uses the main Avogadro window for visu- alizing candidate structures as they evolve. The API is well suited for XtalOpt’s needs, providing a simple mechanism to allow the user to view, edit, and export the struc- tures generated during the search. Taking advantage of the cross-platform capabilities of Avogadro and its dependen- cies, XtalOpt is available for Linux, Windows, and Mac. Hanwell et al. Journal of Cheminformatics 2012, 4:17 http://www.jcheminf.com/content/4/1/17 com/scholar?&q=22http://avogadro.openmolecules.net/22] Mera-Adasme R, Mendiz´abal F, Olea-Azar C, Miranda-Rojas S, Fuentealba 28. Mera-Adasme R, Mendiz´abal F, Olea-Azar C, Miranda-Rojas S, Fuentealba P: A Computationally Eﬃcient and Reliable Bond Order Measure. J Phys Chem A 2011, 115(17):4397–4405. 48. Guha R, Howard MT, Hutchison GR, Murray-Rust P, Rzepa H, Steinbeck C, Wegner J, Willighagen EL: The Blue Obelisk - Interoperability in Chemical Informatics. J Chem Inf and Model 2006, 46(3):991–998. P: A Computationally Eﬃcient and Reliable Bond Order Measure. J Phys Chem A 2011, 115(17):4397–4405. 29. Closser KD, Head-Gordon M: Ab Initio Calculations on the Electronically Excited States of Small Helium Clusters. J Phys Chem A 2010, 114(31):8023–8032. 49. O’Boyle N, Guha R, Willighagen E, Adams S, Alvarsson J, Bradley JC, Filippov I, Hanson R, Hanwell M, Hutchison G, James C, Jeliazkova N, Lang A, Langner K, Lonie D, Lowe D, Pansanel J, Pavlov D, Spjuth O, Steinbeck C, Tenderholt A, Theisen K, Murray-Rust P: Open Data, Open Source and Open Standards in chemistry: the Blue Obelisk ﬁve years on. J Cheminformatics 2011, 3:37. 30. Ide T, Takeuchi D, Osakada K, Sato T, Higuchi M: Aromatic Macrocycle Containing Amine and Imine Groups: Intramolecular Charge-Transfer and Multiple Redox Behavior. J Org Chem 2011, 76(22):9504–9506. 50. Qt Framework 2012. [http://qt-project.org/] 31. Menegazzo N, Zou Q, Booksh KS: Characterization of electrografted 4-aminophenylalanine layers for low non-speciﬁc binding of proteins. New J Chem 2012, 36(4):963–970. 51. O’Boyle NM, Banck M, James CA, Morley C, Vandermeersch T, Hutchison GR: Open Babel: an open chemical toolbox. J Cheminf 2011, 3:33. 52. Guennebaud G, Jacob B, et al.: Eigen v2. 2010. [http://eigen.tuxfamily.org] 32. Patel DGD, Ohnishi Yy, Yang Y, Eom SH, Farley RT, Graham KR, Xue J, Hirata S, Schanze KS, Reynolds JR: Conjugated polymers for pure UV light emission: Poly(meta-phenylenes). J Polym Sci Part B-Polym Phys 2011, 49(8):557–565. 53. GNU General Public License v2.0 2012. [http://www.gnu.org/licenses/ gpl-2.0.html] 54. Sitzmann M, Filippov IV, Nicklaus MC: Internet resources integrating many small-molecule databases. SAR QSAR Environ Res 2008, 19(1–2):1–9. 33. Popov AV, Gould EA, Salvitti MA, Hernandez R, Solntsev KM: Diﬀusional eﬀects on the reversible excited-state proton transfer. From experiments to Brownian dynamics simulations. Phys Chem Chem Phys 2011, 13(33):14914. 55. Python Extensions - Avogadro wiki 2012. [http://avogadro. openmolecules.net/wiki/Python Extensions] 56. Vandermeersch T: Python Example. 2009. [http://timvdm.blogspot. com/2009/05/using-avogadro-library-from-python.html] 34. Hu W, Webb LJ: Direct Measurement of the Membrane Dipole Field in Bicelles Using Vibrational Stark Eﬀect Spectroscopy. J PhysChem Lett 2011, 2(15):1925–1930. Hanwell et al. Journal of Cheminformatics 2012, 4:17 http://www.jcheminf.com/content/4/1/17 13. HyperChem 2012. [http://www.hyper.com/] 40. Kapla J, Stevensson B, Dahlberg M, Maliniak A: Molecular Dynamics Simulations of Membranes Composed of Glycolipids and Phospholipids. J Phys Chem B 2012, 116:244–252. 14. CrystalMaker 2012. [http://www.crystalmaker.com] 15. Materials Studio 2001–2007. [http://accelrys.com/products/materials- studio] 41. Mandal D, Mondal B, Das AK: Nucleophilic Degradation of Fenitrothion Insecticide and Performance of Nucleophiles: a Computational Study. J Phys Chem A 2012, 116(10):2536–2546. 16. Spartan 2012. [http://www.wavefun.com] 17. Hassinen T, et al.: Ghemical. 2012. [http://www.uku.ﬁ/thassine/projects/ ghemical] 42. Bernstein N, Kulp III JL, Cato Jr MA, Clark TD: Simulations of Nanocylinders Self-Assembled from Cyclic β-Tripeptides. J Phys Chem A 2010, 114(44):11948–11952. 18. Schaftenaar G, Noordik JH: Molden: a pre- and post-pro 18. Schaftenaar G, Noordik JH: Molden: a pre- and post-processing program for molecular and electronic structures. J Comput-Aided Mol Des 2000, 14:123–134. 43. Hlawacek G, Khokhar FS, van Gastel R, Poelsema B, Teichert C: Smooth Growth of Organic Semiconductor Films on Graphene for High-Eﬃciency Electronics. Nano Lett 2011, 11(2):333–337. 19. Murray-Rust P, Townsend J, Adams S, Phadungsukanan W, Thomas J: The semantics of Chemical Markup Language (CML): dictionaries and conventions. J Cheminformatics 2011, 3:43. 44. F¨orster S, Hahn T, Loose C, R¨oder C, Liebing S, Seichter W, Eißmann F, Kortus J, Weber E: Synthesis and characterization of new derivatives of azulene, including experimental and theoretical studies of electronic and spectroscopic behavior. J Phys Org Chem 2012: . [http:// onlinelibrary.wiley.com/doi/10.1002/poc.2934/abstract? deniedAccessCustomisedMessage=&userIsAuthenticated=false] 20. Murray-Rust P, Rzepa H: CML: Evolution and Design. J Cheminformatics 2011, 3:44. 21. Thomas J, Sherwood P: The CCP1 GUI Project. 2009. [http://www.cse. scitech.ac.uk/ccg/software/ccp1gui] 2. Allouche AR: Gabedit. 2012. [http://gabedit.sourceforge.net] 45. Burkhardt SE, Rodr´ıguez-Calero GG, Lowe MA, Kiya Y, Hennig RG, Abru˜na HD: Theoretical and Electrochemical Analysis of Poly(3,4- alkylenedioxythiophenes): Electron-Donating Eﬀects and Onset of p-Doped Conductivity. J Phys Chem C 2010, 114(39):16776–16784. 23. Bode BM, Gordon MS: Macmolplt: a graphical user interface for GAMESS. J Mol Graphics Model 1998, 16(3):133–138. 24. Avogadro Downloads 2012. [http://sourceforge.net/projects/avogadro/ ﬁles/stats/timeline?dates=2006-04-14+to+2012-03-29] 46. Burkhardt SE, Conte S, Rodr´ıguez-Calero GG, Lowe MA, Qian H, Zhou W, Gao J, Hennig RG, Abru˜na HD: Towards organic energy storage: characterization of 2,5-bis(methylthio)thieno[3,2-b]thiophene. J Mater Chem 2011, 21(26):9553. 25. Avogadro Translations 2012. [https://translations.launchpad.net/ avogadro/trunk/+translations] 26. Avogadro Contributors 2012. [http://www.ohloh.net/p/avogadro/ contributors] 47. Madison TA, Hutchison GR: Eﬀects of Charge Localization on the Orbital Energies of Bithiophene Clusters. J Phys Chem C 2011, 115(35):17558–17563. 27. Avogadro Citations from Google Scholar 2012. [http://scholar.google. Availability and requirements g 11. ChemBio3D 2012. [http://www.cambridgesoft.com/software/ chembio3d] Project Name: Avogadro Project Name: Avogadro Project home page: http://avogadro.openmolecules.net/ ct home page: http://avogadro.openmolecules.net/ 12. GaussView 5 2012. [http://gaussian.com/g prod/gv5.htm] Page 16 of 17 Hanwell et al. Journal of Cheminformatics 2012, 4:17 http://www.jcheminf.com/content/4/1/17 Hanwell et al. Journal of Cheminformatics 2012, 4:17 http://www.jcheminf.com/content/4/1/17 57. Python Scripting in Avogadro 2012. [http://avogadro.openmolecules. net/wiki/Category:Scripting] 35. Bing¨ol B, Durrell AC, Keller GE, Palmer JH, Grubbs RH, Gray HB: Electron Transfer Triggered by Optical Excitation of Phenothiazine-tris(meta- phenylene-ethynylene)-(tricarbonyl)(bpy)(py)rhenium(I). J Phys Chem B 2012:120503135353007 . g y g 58. PyQt Scripting Example 2012. [https://gist.github.com/3194164] 59. Vandermeersch T: Avogadro Python Terminal Tutorial 2012. [http:// avogadro.openmolecules.net/wiki/Tutorials:Console] Chem B 2012:120503135353007 . 36. Yao CJ, Sui LZ, Xie HY, Xiao WJ, Zhong YW, Yao J: Electronic Coupling between Two Cyclometalated Ruthenium Centers Bridged by 1,3,6,8-Tetra(2-pyridyl)pyrene (tppyr). Inorg Chem 2010, 49(18):8347–8350. 37. Fleisher A, Morgan P, Pratt D: High-Resolution Electronic Spectroscopy Studies of meta-Aminobenzoic Acid in the Gas Phase Reveal the Origins of its Solvatochromic Behavior. Chem Phys Chem 2011, 12(10):1808–1815. 38. Mayorkas N, Malka I, Bar I: Ionization-loss stimulated Raman spectroscopy for conformational probing of ﬂexible molecules. Phys Chem Chem Phys 2011, 13(15):6808–6815. 39. Tian H, Bora I, Jiang X, Gabrielsson E, Karlsson KM, Hagfeldt A, Sun L: Modifying organic phenoxazine dyes for eﬃcient dye-sensitized solar cells. J Mater Chem 2011, 21(33):12462. 60. Compiling Avogadro on Windows 2012. [http://avogadro. openmolecules.net/wiki/Compiling on Windows] 36. Yao CJ, Sui LZ, Xie HY, Xiao WJ, Zhong YW, Yao J: Electronic Coupling between Two Cyclometalated Ruthenium Centers Bridged by 1,3,6,8-Tetra(2-pyridyl)pyrene (tppyr). Inorg Chem 2010, 49(18):8347–8350. 61. Compiling Avogadro on Linux 2012. [ http://avogadro.openmolecules. net/wiki/Compiling on Linux and Mac OS X] 61. Compiling Avogadro on Linux 2012. [ http://avogadro.openmolecules. net/wiki/Compiling on Linux and Mac OS X] 62. Hanwell MD: OpenQube. 2012. [http://www.openchemistry.org] 62. Hanwell MD: OpenQube. 2012. [http://www.openchemistry.org] 37. Fleisher A, Morgan P, Pratt D: High-Resolution Electronic Spectroscopy Studies of meta-Aminobenzoic Acid in the Gas Phase Reveal the Origins of its Solvatochromic Behavior. Chem Phys Chem 2011, 12(10):1808–1815. 63. OpenQube Source 2012. [http://github.com/OpenChemistry/ openqube] 64. Adams S, de Castro, P, Echenique P, Estrada J, Hanwell MD, Murray-Rust P, Sherwood P, Thomas J, Townsend JA: The Quixote project: Collaborative and Open Quantum Chemistry data management in the Internet age. J Cheminformatics 2011, 3:38. 38. Mayorkas N, Malka I, Bar I: Ionization-loss stimulated Raman spectroscopy for conformational probing of ﬂexible molecules. Phys Chem Chem Phys 2011, 13(15):6808–6815. 65. Weininger D: SMILES, a chemical language and information system. 1 Introduction to methodology and encoding rules. J Chem Inf Comp Sci 1988, 28:31–36. 39. Tian H, Bora I, Jiang X, Gabrielsson E, Karlsson KM, Hagfeldt A, Sun L: Modifying organic phenoxazine dyes for eﬃcient dye-sensitized solar cells. J Mater Chem 2011, 21(33):12462. 39. Hanwell et al. Journal of Cheminformatics 2012, 4:17 http://www.jcheminf.com/content/4/1/17 Hanwell et al. Journal of Cheminformatics 2012, 4:17 http://www.jcheminf.com/content/4/1/17 66. James CA, Willighagen EL, Dalke A, Landrum G, Apodaca R, Morley C: OpenSMILES. 2012. [http://opensmiles.org] 67. Schmidt M, Baldridge K, Boatz J, Elbert S, Gordon M, Jensen J, Koseki S, Matsunaga N, Nguyen K, Su S, Windus T, Dupuis M, Montgomery J: General Atomic and Molecular Electronic-Structure System. J Comput Chem 1993, 14(11):1347–1363. 68. Valiev M, Bylaska EJ, Govind N, Kowalski K, Straatsma TP, Van Dam HJJ, Wang D, Nieplocha J, Apra E, Windus TL, de Jong W: NWChem: a comprehensive and scalable open-source solution for large scale molecular simulations. Comput Phys Commun 2010, 181(9):1477–1489 69. Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA, Cheeseman JR, Scalmani G, Barone V, Mennucci B, Petersson GA, Nakatsuji H, Caricato M, Li X, Hratchian HP, Izmaylov AF, Bloino J, Zheng G, Sonnenberg JL, Hada M, Ehara M, Toyota K, Fukuda R, Hasegawa J, Ishida M, Nakajima T, Honda Y, Kitao O, Nakai H, Vreven T, Montgomery JJA, et al.: Gaussian 09 Revision A.1. Wallingford CT: Gaussian Inc.; 2009. g 70. Shao Y, Molnar LF, Jung Y, Kussmann Jr, Ochsenfeld C, Brown ST, Gilbert ATB, Slipchenko LV, Levchenko SV, O Neill DP, DiStasio Jr RA, Lochan RC, Wang T, Beran GJO, Besley NA, Herbert JM, Yeh Lin C, Van Voorhis T, Hung Chien S, Sodt A, Steele RP, Rassolov VA, Maslen PE, Korambath PP, Adamson RD, Austin B, Baker J, Byrd EFC, Dachsel H, Doerksen R J, et al.: Advances in methods and algorithms in a modern quantum chemistry program package. Phys Chem Chem Phys 2006, Hanwell et al. Journal of Cheminformatics 2012, 4:17 http://www.jcheminf.com/content/4/1/17 Tian H, Bora I, Jiang X, Gabrielsson E, Karlsson KM, Hagfeldt A, Sun L: Modifying organic phenoxazine dyes for eﬃcient dye-sensitized solar cells. J Mater Chem 2011, 21(33):12462. Page 17 of 17 y p g 8(27):3172–3191. 71. Werner HJ, Knowles PJ, Knizia G, Manby FR, Sch¨utz M, Celani P, Korona T, Lindh R, Mitrushenkov A, Rauhut G, Shamasundar KR, Adler TB, Amos RD, Bernhardsson A, Berning A, Cooper DL, Deegan MJO, Dobbyn AJ, Eckert F, Goll E, Hampel C, Hesselmann A, Hetzer G, Hrenar T, Jansen G, K¨oppl C, Liu Y, Lloyd AW, Mata RA, May AJ, et al.: MOLPRO, version 2010.1, a package of ab initio programs. Cardiﬀ, UK ; 2010. See http://www.molpro.net 71. Werner HJ, Knowles PJ, Knizia G, Manby FR, Sch¨utz M, Celani P, Korona T, Lindh R, Mitrushenkov A, Rauhut G, Shamasundar KR, Adler TB, Amos RD, Bernhardsson A, Berning A, Cooper DL, Deegan MJO, Dobbyn AJ, Eckert F, Goll E, Hampel C, Hesselmann A, Hetzer G, Hrenar T, Jansen G, K¨oppl C, Liu Y, Lloyd AW, Mata RA, May AJ, et al.: MOLPRO, version 2010.1, a package of ab initio programs. Cardiﬀ, UK ; 2010. See http://www.molpro.net 72. Stewart JJP: MOPAC2009. Internet. USA: Colorado Springs CO; 2008. [http://openmopac.net] 73. Gilbert ATB: QUI - The Q-Chem User Interface 2008. [http://rsc.anu.edu. au/agilbert/gilbertspace/qui.php] 74. Werner HJ, Knowles PJ, Knizia G, Manby FR, Sch¨utz M: Molpro: a general-purpose quantum chemistry program package. Wiley Interdisciplinary Rev: Comput Mol Sci 2012, 2(2):242–253. [http://dx.doi.org/ 10.1002/wcms.82] 74. Werner HJ, Knowles PJ, Knizia G, Manby FR, Sch¨utz M: Molpro: a general-purpose quantum chemistry program package. Wiley Interdisciplinary Rev: Comput Mol Sci 2012, 2(2):242–253. [http://dx.doi.org/ 10.1002/wcms.82] 75. Mart´ınez L, Andrade R, Birgin EG, Mart´ınez JM: Packmol: a package for building initial conﬁgurations for molecular dynamics simulations. J Comp Chem 2009, 30(13):2157–2164. 75. Mart´ınez L, Andrade R, Birgin EG, Mart´ınez JM: Packmol: a package for building initial conﬁgurations for molecular dynamics simulations. J Comp Chem 2009, 30(13):2157–2164. 76. Mart´ınez JM, Mart´ınez L: Packing optimization for automated generation of complex system’s initial conﬁgurations for molecular dynamics and docking. J Comp Chem 2003, 24(7):819–825. 77. Lonie D, Zurek E: XtalOpt. 20 77. Lonie D, Zurek E: XtalOpt. 2011. [http://xtalopt.openmolecules.net] 78. Lonie D, Zurek E: XtalOpt: an Open-Source Evolutionary Algorithm for Crystal Structure Prediction. Comput Phys Commun 2011, 182:372–387. 78. Lonie D, Zurek E: XtalOpt: an Open-Source Evolutionary Algorithm for Crystal Structure Prediction. Comput Phys Commun 2011, 182:372–387. doi:10.1186/1758-2946-4-17 Cite this article as: Hanwell et al.: Avogadro: an advanced semantic chem- ical editor, visualization, and analysis platform. Journal of Cheminformatics 2012 4:17. y p g 8(27):3172–3191. Open access provides opportunities to our colleagues in other parts of the globe, by allowing anyone to view the content free of charge. Publish with ChemistryCentral and every scientist can read your work free of charge W. Jeffery Hurst, The Hershey Company. available free of charge to the entire scientific community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours you keep the copyright Submit your manuscript here: http://www.chemistrycentral.com/manuscript/ Open access provides opportunities to our colleagues in other parts of the globe, by allowing anyone to view the content free of charge. Publish with ChemistryCentral and every scientist can read your work free of charge W. Jeffery Hurst, The Hershey Company. available free of charge to the entire scientific community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours you keep the copyright Submit your manuscript here: http://www.chemistrycentral.com/manuscript/ Open access provides opportunities to our colleagues in other parts of the globe, by allowing anyone to view the content free of charge. Publish with ChemistryCentral and every scientist can read your work free of charge W. Jeffery Hurst, The Hershey Company. available free of charge to the entire scientific community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours you keep the copyright Submit your manuscript here: http://www.chemistrycentral.com/manuscript/ ed Central
https://openalex.org/W4388185370	https://periodicos.uff.br/trabalhonecessario/article/download/53523/32470	Portuguese	null	EDUCAÇÃO PARA O SÉCULO XXI E O CENTRO DE INOVAÇÃO PARA A EDUCAÇÃO BRASILEIRA (CIEB): UMA CONEXÃO ESTRATÉGICA	Trabalho necessário	2,022	cc-by	6,469	Resumo O presente trabalho problematiza o slogan da inovação na educação. A partir de um levantamento bibliográfico, identificamos que a questão remonta ao contexto de agravamento da crise do capital nas décadas de 1960 e 1970, caracterizando uma retomada da teoria do capital humano. O levantamento documental preliminar, no sítio do Ministério da Educação (MEC) e do Centro de Inovação para a Educação Brasileira (CIEB), indica que este tem despontado enquanto agente e referência para o projeto de educação da classe dominante para os trabalhadores do século XXI. p j ç p Palavras-chave: Centro de Inovação para a Educação Brasileira. Políticas públicas. Inova educação. V 20 nº 42 2022 (maio-agosto) ISSN: 1808-799 X ISSN: 1808-799 X Resumen El presente trabajo problematiza la consigna de innovación en educación. Con base en un levantamiento bibliográfico, identificamos que la cuestión se remonta al contexto de agravamiento de la crisis del capital en las décadas de 1960 y 1970, caracterizando una reanudación de la teoría del capital humano. El relevamiento documental preliminar en el sitio web del Ministerio de Educación (MEC) y del Centro de Innovación para la Educación Brasileña (CIEB) indica que ha emergido como agente y referencia para el proyecto de educación de la clase dominante para los trabajadores en el siglo XXI. Palabras clave: Centro de Innovación para la Educación Brasileña. Políticas públicas. Innovación en la educación. EDUCATION FOR THE 21ST CENTURY AND THE INNOVATION CENTER FOR BRAZILIAN EDUCATION (CIEB): A STRATEGIC CONNECTION EDUCATION FOR THE 21ST CENTURY AND THE INNOVATION CENTER FOR BRAZILIAN EDUCATION (CIEB): A STRATEGIC CONNECTION EDUCAÇÃO PARA O SÉCULO XXI E O CENTRO DE INOVAÇÃO PARA A EDUCAÇÃO BRASILEIRA (CIEB): UMA CONEXÃO ESTRATÉGICA1 Patrícia Ferreira Duarte2 Abstract This article analyses the slogan of innovation in education. Based on a bibliographic survey, we identified that the issue goes back to the context of aggravation of the capital crisis in the 1960s and 1970s, characterizing a resumption of the theory of human capital. The preliminary documentary survey on the website of the Ministry of Education (MEC) and the Center for Innovation for Brazilian Education (CIEB) indicates that it has emerged as an agent and reference for the education project of the ruling class for workers in the 21st century. y Keywords: Innovation Center for Brazilian Education. Public Policy. innovation in education. 1Artigo recebido em 19/04/2022. Primeira avaliação em 09/05/2022. Segunda avaliação em 16/05/2022. Aprovado em 07/07/2022. Publicado em 21/07/2022. DOI: https://doi.org/10.22409/tn.v20i42.53523. 2 Doutoranda do Programa de Pós-Graduação em Educação e Demandas Populares (PPGEDUC) da Universidade Federal Rural do Rio de Janeiro (UFRRJ). Professora EBTT dos anos iniciais do ensino fundamental do Colégio de Aplicação João XXIII (UFJF). E-mail: pduarteufjf@gmail.com. Lattes: http://lattes.cnpq.br/4833052154661970. ORCID: http://orcid.org/0000-0002-3468-4549. 1 Introdução As últimas décadas do século XX foram marcadas por mudanças no conteúdo e na forma de organização do trabalho. Segundo Neves e Sant’Anna (2005), essas transformações decorrem da conformação de um novo bloco histórico, preconizada no início do referido século com a passagem do capitalismo concorrencial para o monopolista. Com base nos escritos do pensador sardo Antônio Gramsci, os autores indicam como características do bloco histórico: a reprodução ampliada do capital e a expropriação crescente do trabalho e da mais-valia no plano econômico. No plano político, se estrutura uma ampliação do Estado, que além de exercer as tarefas de comando, governo e domínio, assume a função de dirigir culturalmente a classe dominada, com vias de legitimar o capitalismo como única via histórica possível. Mészaros (2009), analisando as décadas de 1960 e 1970, aponta para a identificação de uma crise estrutural do capital. Esta se diferenciaria das demais, cíclicas, devido seu caráter universal e seu alcance global e consubstancia um aprofundamento de uma perspectiva de produção descolada das reais necessidades humanas, intensificação da destruição da natureza e da precarização do trabalho. O contexto de crise demandou a articulação de novas estratégias de organização da produção, da sociedade política e sociedade civil (GRAMSCI, 2000). Exigiu o realinhamento do modo de ser, pensar e agir na direção do padrão de acumulação flexível (NEVES; SANT’ANNA, 2005). Observando o movimento dos países capitalistas centrais nesse período, Fernandes (2019) retoma o contexto de criação da Organização para a Cooperação e Desenvolvimento Econômico (OCDE) e o fortalecimento das cooperações econômicas, com desígnios políticos em diversas áreas dos governos envolvidos, incluindo a educação. Assim, a tríade produção-capital-trabalho incorporou um elemento suplementar: o capital humano. Noções como “competências” e “habilidades” foram apresentadas como sinônimo de desenvolvimento econômico. No bojo desse processo, é criado, em 1968, o Centro de Pesquisa Inovação Educacional (CERI) da OCDE, financiado pela Fundação Ford, cuja atividade se estende até os dias atuais (FERNANDES, 2019). É interessante observar que o apelo à inovação na educação consubstancia com o período de mundialização da produção, descrito por Neves e Sant’Anna (2005), 2 2 da difusão do paradigma da acumulação flexível, da veloz introdução e difusão da microeletrônica e da informática na organização e no conjunto das relações sociais de produção. 3 3 O conceito nova pedagogia da hegemonia fora cunhado pelo Coletivo de Estudos de Política Educacional da Universidade Federal Fluminense e da Fundação Oswaldo Cruz. Para maior aprofundamento ver Neves e Sant´Anna, 2005. Introdução A política de criação do CERI pela OCDE, já em 1968, indica não ser recente a premissa da inovação em educação como amálgama da formação do novo homem coletivo, do novo modelo de cidadania a ser produzido pela nova pedagogia da hegemonia3. No Brasil, a articulação dos pressupostos da inovação para a educação ganha relevo no processo de elaboração e promulgação do Plano Nacional de Educação (PNE 2014-2024) que é citada em 5 das 20 metas do documento. Em 2015, há a criação da “Iniciativa pela Inovação e Criatividade na Educação Básica” e em 2017 a discussão ganha mais substância com a publicação da Base Nacional Comum Curricular (BNCC), trazendo a inovação entrelaçada ao domínio das Tecnologias de Informação e Comunicação, competência geral 5. Nesse panorama, ocorre a fundação do Centro de Inovação para a Educação Brasileira, o CIEB: um pool de empresas que se apresentam como uma organização sem fins lucrativos com objetivo de impulsionar uma transformação sistêmica na educação brasileira (BRITO e MARINS, 2020). Desde sua criação, tem desenvolvido parcerias técnicas com o Ministério da Educação (MEC), atuando ativamente no desenvolvimento de estudos e plataformas, e apoiando a criação e a execução de “programas de inovação” nas redes de ensino básico para “[...] impulsionar a qualidade, a equidade e a contemporaneidade da educação pública brasileira” (CIEB, 2015, p. 3). O presente artigo é um desdobramento dos primeiros passos na análise das inciativas e pressupostos do Centro de Inovação da Educação Brasileira, pesquisa desenvolvida no curso de doutorado, no âmbito do Programa de Pós-Graduação em Educação e Demandas Populares (PPGEDUC/UFRRJ). O texto tem por objetivo analisar o slogan da inovação na educação e a incidência do mesmo no Brasil, sob a lente do materialismo histórico-dialético. Para tanto, foram elencados objetivos específicos correspondentes a cada uma das seções deste trabalho: mapear o processo de inserção de políticas, na agenda dos governos brasileiros, onde a inovação aparecera como prerrogativa; compilar as iniciativas mais destacadas pelo 3 CIEB, voltadas ao sistema público de educação; e apresentar algumas considerações preliminares sobre a incidência política do CIEB junto ao MEC e às escolas. A partir de um levantamento bibliográfico acerca do tema da inovação e educação, identificamos que a questão remonta ao contexto de agravamento da crise do capital nas décadas de 1960 e 1970, e à retomada da teoria do capital humano, um dos pilares das estratégias capitalistas de dominação (LIMA; MARTINS, 2005). Introdução Na mesma esteira, o levantamento documental preliminar no sítio do Ministério da Educação (MEC) e do Centro de Inovação para a Educação Brasileira (CIEB) indica que este tem despontado enquanto uma das principais agências de referência para a implementação de uma “cultura da inovação” na educação pública, obtendo êxito na promoção do projeto de educação da classe dominante para os trabalhadores do século XXI. A agenda educacional dos governos brasileiros no contexto de contrarreformas Tavares (2019) localiza o aumento de publicações relacionando educação e inovação nas décadas de 1960 e 1970. Nesta última, são publicados inúmeros trabalhos sobre o fenômeno pela Organização das Nações Unidas para a Educação, Ciência e Cultura (UNESCO), com destaque para as obras de Huberman (1973) “Comment s’opbrent les changements en éducation: contribution à l’étude de l’innovation” e Huberman e Havelock (1977) “Solving educational problem: the theory and reality of innovation in developing countries”. As obras apresentam uma preocupação no desenvolvimento de princípios e modelos experimentais. Entretanto, é na década de 1990 que a OCDE estipularia a inovação como um dos eixos estruturantes para a implementação de políticas em diferentes países. Como país que se desenvolveu de forma dependente da economia dos países capitalistas centrais, o Brasil incorporou as orientações de diversos organismos internacionais. Na educação, a “Conferência Mundial de Educação para Todos” foi um dos grandes marcos deste processo. Cento e cinquenta e cinco governos se fizeram presentes e comprometeram-se com a premissa de uma educação básica de qualidade para jovens crianças e adultos (SHIROMA et al., 2007). Os “E9” foram o foco por apresentarem, à época, as maiores taxas de analfabetismo no mundo: Bangladesh, Brasil, Nigéria, Paquistão, Índia, México, China, Egito, Indonésia. Na mesma esteira, se estruturaram os lemas da Comissão 4 Econômica para a América Latina e Caribe (CEPAL) para a educação em 1990. Na concepção cepalina, os resultados para o desempenho no mercado de trabalho e na cidadania tendiam a convergir. A nova educação deveria ensinar para a moderna cidadania e para a competitividade, permitindo a apropriação dos códigos comuns para a modernidade, necessários para a vida pública. Vale também o destaque ao relatório Jacques Delors, onde foi manifesta a necessidade de “conciliar a competição à cooperação e solidariedade” e ajustá-los “às demandas do conhecimento científico e tecnológico” (SHIROMA et al., 2007, p. 55). O slogan da “Educação para Todos” selou a subsunção das finalidades da educação às prerrogativas do capital. Essa tônica fora incorporada no Brasil, ainda que de forma incipiente, pelo governo de José Sarney em 1985 com a publicação do plano “Educação para Todos – Compromisso com a Mudança”. A agenda educacional dos governos brasileiros no contexto de contrarreformas Partindo dos altos índices de analfabetismo no país, evasão e defasagem entre idade escolar e número de matrículas, o plano indicava como objetivo a universalização da educação básica e convocava a mobilização da sociedade em prol da qualidade de ensino (BRASIL, 1985 apud CÊA et al., 2019). O governo Collor de Melo, apesar de ter se tornado signatário da Declaração Mundial sobre Educação para Todos, resultante da reunião de Jontien, não foi capaz de materializá-lo, tendo em vista a não deflagração de seu Plano Decenal. Seria em 1993, no governo de Itamar Franco, que ocorreria a elaboração e a aprovação do Plano Decenal de Educação para Todos (1993-2003), com a chancela de organismos internacionais como a UNICEF e BM. Em meio a presença dos representantes dos nove países mais populosos do chamado Terceiro Mundo (E9) o governo brasileiro incorpora a perspectiva de garantir as necessidades básicas de aprendizagens para todos (CÊA et al., 2019). A vitória de Fernando Henrique Cardoso (1995-2002) garantiu a hegemonia dos setores privatistas e conservadores. Salermo e Kubata (2008) identificam que, desde os anos 2000, iniciativas sistemáticas de apoio à inovação foram implementadas, estando estas em sintonia com as Diretrizes de Política Industrial, Tecnológica e de Comercio Exterior. No plano educacional, várias regulamentações foram aprovadas na direção da reformulação do currículo em diferentes níveis de ensino, bem como da regulamentação do magistério (SHIROMA et al., 2007). 5 5 Ainda que neste período o termo inovação não apareça explicitamente ligado à educação, o alinhamento das práticas educacionais às empresariais foram apresentados como um movimento necessário de modernização, tanto nas políticas educacionais quanto nas práticas pedagógicas. O discurso presente nos documentos do CIEB, mais de uma década depois, também parte do apelo à mudança, pautado na ideia de inovação, com vistas a atingir a “qualidade total” em educação. No entanto, será preciso avaliar no decorrer da pesquisa o que é uma retomada dessa forma de mobilização discursiva, e quais elementos são especificidades geradas pela tríade inovação-tecnologia-educação. Foram implementados fundos e programas que concorriam para uma nova lógica no financiamento da educação e a organização de uma estrutura de monitoramento e avaliação da educação: a implementação do Exame Nacional do Ensino Médio (ENEM), do Sistema de Avaliação da Educação Básica (SAEB), Exame Nacional de Cursos (PROVÃO) e do Censo Escolar (SHIROMA et al., 2007). A agenda educacional dos governos brasileiros no contexto de contrarreformas O governo Lula da Silva, por sua vez, não representou um rompimento em relação às premissas erigidas dos organismos internacionais, solidificando a questão da inovação em diferentes frentes de sua gestão. Oliveira (2011), produz um levantamento do tema na agenda dos governos brasileiros, destacando os planos jurídico, econômico e institucional, de modo que salta aos olhos o aumento do quantitativo de iniciativas nessa direção: No plano jurídico, os principais marcos desse processo foram: a criação dos primeiros fundos setoriais, em 1999; a realização da 2ª Conferência Nacional de Ciência, Tecnologia e Inovação (CNCTI), em 2001;2 a Lei de Inovação, promulgada em 2004; a chamada Lei do Bem, de 2005; e o Plano de Ação em Ciência, Tecnologia e Inovação para o Desenvolvimento Nacional (PACTI), instituído em 2007. No plano econômico, as agências de fomento e órgãos afins dos governos federal e estaduais adotaram inúmeros programas de apoio à pesquisa visando a inovação, e envolvendo o setor empresarial, na forma de convênios, empréstimos subsidiados, financiamentos a fundo perdido, bolsas etc. No plano institucional, criaram-se entidades, tanto no setor público (por exemplo, as "agências de inovação " nas universidades), como no setor privado (na forma de associações empresariais, consultorias, ONGs etc.). Campanhas e concursos são promovidos com o intuito de instilar no público a "cultura da inovação", como, por exemplo, a Olimpíada USP de Inovação (OLIVEIRA, 2011, p. 671-672). A lei de incentivo fiscal à Pesquisa e Desenvolvimento (P&D), promulgada em 2005, possibilitou o subsídio às empresas que apresentassem planos com foco em 6 6 desenvolvimento tecnológico “[...] com programas de financiamento à inovação, com programas de capital empreendedor, e com arcabouço legal propício para a interação universidade/empresa (PIMENTEL, 2020, grifos nossos). Com a sanção da Lei n. 12.193/2010, Luiz Inácio designa o dia 19 de outubro como Dia Nacional da Inovação. Oliveira (2011) também destaca o entusiasmo em torno do inovacionismo à época, no contexto 4ª CNCTI, registrado em documento publicado pelo Ministério da Ciência e Tecnologia: "[...] o Brasil tem uma necessidade gigantesca, urgente, de inocular inovação em todos os poros da economia" (BRASIL, 2010, p. 35 apud OLIVEIRA, 2011 p. 672, grifos nossos). O aprofundamento da noção de inovação na educação se manifesta mais explicitamente com o governo Dilma Roussef (2011-2016) com a aprovação do Plano Nacional de Educação (PNE 2014-2024), onde encontramos a preocupação com a inovação pedagógica e tecnológica em 5 das 20 metas do documento. A agenda educacional dos governos brasileiros no contexto de contrarreformas Em 2015, há a criação da “Iniciativa pela Inovação e Criatividade na Educação Básica” e em 2017 a discussão ganha mais substância com a publicação da Base Nacional Comum Curricular (BNCC), trazendo a inovação entrelaçada ao domínio das Tecnologias de Informação e Comunicação, competência geral 5. Assim, dois marcos importantes da última década na política educacional brasileira – o PNE e a BNCC – trazem traços significativos em relação ao estímulo de uma “cultura da inovação” na educação, ainda que em meio ao intervalo da aprovação de cada documento tenha ocorrido um golpe de Estado, com o impedimento de Dilma Rousseff. Na próxima sessão nos deteremos a descrever e analisar a movimentação e os pressupostos do Centro de Inovação para a Educação Brasileira (CIEB), agência que emerge nesse processo e intervém em prol da materialização do discurso da inovação na educação. O Centro de Inovação para a Educação Brasileira: iniciativas e colaborações na tessitura das políticas públicas Quando sonhamos o Brasil, sonhamos com educação. Quando sonhamos educação, nosso caminho é a inovação. CIEB vação. CIEB Importante movimentação para estimular a “cultura da inovação” nas redes públicas brasileiras ocorre em 2016, com a fundação do Centro de Inovação para a 7 Educação Brasileira (CIEB). Brito e Marins (2020) caracterizam o CIEB como um pool de empresas, ainda que este se apresente como uma organização sem fins lucrativos. Desde sua criação, tem desenvolvido parcerias técnicas com o Ministério da Educação (MEC), atuando ativamente no desenvolvimento de estudos e plataformas, e apoiando a criação e a execução de “programas de inovação” nas redes de ensino básico para “[...] impulsionar a qualidade, a equidade e a contemporaneidade da educação pública brasileira” (CIEB, 2015, p. 3, grifos nossos). O pressuposto central do CIEB, portanto, consiste na defesa da inovação como propulsora da qualidade da educação. Nesse aspecto, cabe destacar a relação do termo inovação com qualidade Em sua primeira nota técnica, seus especialistas advogam “A importância de políticas nacionais e centros de inovação em educação” (CIEB, 2015). O estudo parte da análise do Plano Nacional de Educação (2014-2024), com o objetivo de fomentar o debate sobre a criação de um Centro de Inovação para a Educação no Brasil, e investigar como iniciativas de inovação tecnológica impulsionariam o atingimento das metas do PNE (FIGURA 1): Figura 1- O papel da inovação para implementação das metas do PNE (2014-2024) gura 1- O papel da inovação para implementação das metas do PNE (2014-2024) Figura 1- O papel da inovação para implementação das metas do PNE (201 8 Figura 1 O papel da inovação para implementação das metas do PNE (2014 2024) Fonte: CIEB (2015) Fonte: CIEB (2015) Fonte: CIEB (2015) 8 8 Ao elencar algumas das metas do Plano Nacional de Educação (2014-2024) e caracterizá-las como desafiadoras, o documento apresenta como solução recorrente a “inovação” em diferentes frentes do trabalho educativo. Desde a gestão, passando pela alfabetização e até a formação docente: bastaria às secretarias de educação implementar estratégias adjetivadas como “inovadoras”. Para o CIEB a inovação está fortemente relacionada às Tecnologias da Informação e da Comunicação (TICs), atreladas à uma visão reducionista e gerencialista da educação. Outrossim, o termo inovação é utilizado como incauto e exato, definindo práticas novas – que em aparência superariam as antigas – como se o problema da educação fosse a obsolescência de práticas educativas, dos professores e da escola. 9 5 Para saber mais, consultar os documentos CIEB Notas Técnicas #11: “Contribuições para a inclusão do tema tecnologia na Base Nacional Comum Curricular”; CIEB Notas Técnicas #12: “Conceitos e 4 Sobre a diferença entre educação integral e educação em tempo integral consultar Arco Verde (2003). 9 4 Sobre a diferença entre educação integral e educação em tempo integral consultar Arco Verde (2003). 5 Para saber mais, consultar os documentos CIEB Notas Técnicas #11: “Contribuições para a inclusão do tema tecnologia na Base Nacional Comum Curricular”; CIEB Notas Técnicas #12: “Conceitos e 4 Sobre a diferença entre educação integral e educação em tempo integral consultar Arco Verde (2003). 5 Para saber mais consultar os documentos CIEB Notas Técnicas #11: “Contribuições para a inclusão O Centro de Inovação para a Educação Brasileira: iniciativas e colaborações na tessitura das políticas públicas Questões que se arrastam ao longo da história da educação brasileira, como a erradicação do analfabetismo, seriam facilmente solucionadas com a distribuição de tablets, notebooks e aplicativos gratuitos. Mas, a “democratização” dos chamados objetos digitais seriam capazes por si só de fazer a educação “dar um salto”? A forma com que o CIEB afirma ser o papel da inovação para o cumprimento das metas do PNE, acaba por indicar a inovação na educação como “a meta das metas”. A adoção de equipamentos (hardware), aplicativos e programas (software) permitiria o cumprimento de 8 das 20 metas do PNE em um curto período de tempo! Metas essas que englobam complexas questões como a educação especial, defasagem idade/série, índices em avaliações de larga escala e educação em tempo integral. Em relação à meta 6, inclusive, há um equívoco na concepção de educação integral, que aparece como sinônimo de educação em tempo integral4. Partindo deste equívoco se desenvolve a defesa da implementação da cultura maker, aprendizagem em projetos como meio para promoção do desenvolvimento integral dos estudantes. Por fim, as plataformas EAD e comunidades virtuais correspondem à supressão das dificuldades e questões que assolam a formação de professores, consubstanciando para o cumprimento da meta 15 do PNE. Em 2017, o CIEB não só produziu três notas técnicas sobre a inclusão do tema da tecnologia na Base Nacional Comum Curricular (BNCC)5, como participou das 9 audiências públicas para a discussão da Base. Esta movimentação gerou a incorporação de algumas proposições referentes à tecnologia educacional e pensamento computacional, desenvolvidos pelo Centro. Outra participação de destaque foi a incorporação das dimensões propostas para o uso das tecnologias6 no “Programa de Inovação Educação Conectada”, lançado pelo então Ministério da Educação e Cultura (MEC) no governo Temer e instituído pelo Decreto 9.204/23 de novembro de 2017 (GONSALES, P.; ALLODI, S, 2019). Em 2018, contratou especialistas para elaborar propostas pedagógicas e curriculares para a implementação do eixo transversal referente às tecnologias digitais da BNCC: o “Currículo de Referência em Tecnologia” e “Computação e o Itinerário Formativo Cultura Digital” (CIEB, 2021a). Entretanto, é no ano de 2021 que o Centro de Inovação destaca a ampliação de seu alcance. ç p p p g 6 As dimensões para o uso eficaz de tecnologias na educação propostas pelos intelectuais pela organização em questão consistiam em: a) visão estratégica; b) formação de gestores e professores; c) recursos educacionais digitais e d) infraestrutura (GONSALES, P.; ALLODI, S., 2019). conteúdo de inovação e tecnologia (I&T) na BNCC” e CIEB Notas Técnicas #14: “An contribuições para a proposta da BNCC-EM com foco em tecnologia e computação”. 7 A relação dos parceiros e mantedores do centro está disponível no sítio do centro:<https://cieb.net.br/quem-somos/#parceiros>. O Centro de Inovação para a Educação Brasileira: iniciativas e colaborações na tessitura das políticas públicas Com a reabertura gradual das escolas, ainda em meio a pandemia de COVID-19, o ensino híbrido ganha força, que já vinha sendo defendido nos materiais do CIEB desde 2015, oportunizando ao Centro impulsionar seus pressupostos: A reabertura gradual das escolas em 2021 despertou na comunidade educacional um grande interesse pelo ensino híbrido, abordagem que, na visão do CIEB em suas publicações, utiliza e integra tecnologias digitais em momentos presenciais e remotos para oferecer flexibilidade, diversificação de estratégias pedagógicas, personalização e foco nas crianças e jovens. Não por acaso, a Nota Técnica #18: Ensino híbrido e o uso das tecnologias digitais na educação básica foi a publicação mais baixada no site do CIEB em 2021 (CIEB, 2021b). A ampliação de sua incidência política refletiu no aumento de sua rede. Em 2020, teve como mantenedores: Fundação Leman, Instituto Natura, Instituto Península, Itaú Social, Omidyar Network, Fundação Telefônica. Em 2021, ainda que o site não registre mais a parceria com a Omidyar Network, o Centro contou com importantes reforços: o Instituto General Motors e a Cisco. Também são absolutamente relevantes as agências que estabelecem cooperação técnica com a organização: The Boston Consultin Group, Conselho Nacional de Secretarias de 10 Educação (CONSED), Digital Promise, Empresa Brasileira de Inovação e Pesquisa, Porvir, bem como a representação da UNESCO no Brasil7. A congregação que se formou e ganhou mais robustez ao longo dos últimos anos em torno do CIEB, demonstra não apenas um apoio desinteressado, mas a intencionalidade da classe dominante em relação à ampliação da educação como mercado, além da convergência com os pressupostos do CIEB, na defesa de uma educação pública “inovadora”, como delineado na seção a seguir. 11 p q p 8 Levantamento desenvolvido pela Associação Brasileira de Startups (Abstartups) em parceria com o Centro de Inovação para a Educação Brasileira (CIEB). Considerações preliminares sobre a incidência política do Centro de Inovação para a Educação Brasileira A defesa das Tecnologias de Informação e Comunicação (TICs) como meio para atingir a qualidade na educação não é algo novo no discurso de pesquisadores da área, governos e agências nacionais e internacionais. Todavia, o advento da pandemia de COVID-19, representou um significativo salto da defesa das TICs como “soluções incontestáveis”, devido a adoção do Ensino Remoto Emergencial (ERE). Sob o mote da “mitigação das desigualdades”, o mercado das Edtechs vivenciou uma expansão vertiginosa e uma “calorosa” recepção nos sistemas públicos de educação no Brasil. O “Mapeamento Edtech 2020”8 demonstra o fenômeno: ao final de 2020, o país contava com 566 startups de educação ativas, crescimento de 26% em relação a 2019. Enquanto diversos setores sofreram o impacto da crise com a pandemia, 64% das startups Edtechs mantiveram ou aumentaram o faturamento, 40% aumentaram o quadro de funcionários e 89% não realizaram cortes na folha de pagamento (CIEB; ABSTARTUPS, 2021). Por fim, mas não menos importante, o mesmo estudo também indicou que em 2020, 13% das startups com foco em educação haviam negociado com órgãos públicos. O percentual não foi maior, de acordo com o estudo, devido as dificuldades no acesso às informações sobre os editais de compra, “excesso de burocracia” e a “falta de clareza nas comunicações” (CIEB; ABSTARTUPS, 2021). Tais queixas já 7 A relação dos parceiros e mantedores do centro está disponível no sítio do centro:<https://cieb.net.br/quem-somos/#parceiros>. 11 11 eram apresentadas nos manuais técnicos do Centro, acompanhadas de proposições de políticas públicas. Portanto, não se trata de coincidência a preocupação do CIEB em elaborar, no calor do lançamento da Base Nacional Comum para a Formação Inicial de Professores da Educação Básica (BNC-Formação) (Resolução CNE/CP nº 2, de 20 de dezembro de 2019), uma proposta de componentes curriculares sobre tecnologia para a formação inicial de professores9. O ano de 2021 não foi marcado apenas pela adesão de novos mantenedores ao CIEB. Na verdade, estas adesões indicam forte relação com a incidência política do Centro junto ao MEC. Nesse sentido, é imperioso observar com cautela o Programa de Inovação Educação Conectada (PIEC). Criado pelo Decreto n. 9.204, de 23 de novembro de 2017, tem como objetivo central “[...]a universalização do acesso à internet em alta velocidade e fomentar o uso pedagógico de tecnologias digitais na educação básica” (BRASIL, 2017, p. 1, grifos nossos). 12 9 O documento competências digitais na formação de professores está disponível no sítio do CIEB <https://cieb.net.br/wp-content/uploads/2020/12/Compete%CC%82ncias-Digitais.pdf>. Considerações preliminares sobre a incidência política do Centro de Inovação para a Educação Brasileira Ao ter em seu cerne a promoção da “cultura de inovação” por meio da adoção de tecnologias na educação, o programa demonstra estreito alinhamento com o projeto de educação defendido pelo CIEB, seus mantenedores e parceiros. A esse respeito, é estratégica a localização dessa organização no PIEC, que cumpre a função de assessoria técnica ao programa, tendo sido responsável pela orientação da elaboração dos Planos Locais de Inovação nos estados e municípios. Ademais, Brito e Marins (2020) chamam a atenção para o fato de o CIEB ocupar duas cadeiras no comitê consultivo do PIEC: uma, referente a consultoria técnica e a outra como representante de entidade privada e organizações da sociedade civil (a segunda cadeira desta categoria é ocupada pela Fundação Lemann, uma das mantenedoras do CIEB). Também é curioso observar o item 6.3 do Edital de Seleção n.001/2018, concernente à Chamada Pública “BNDES – Educação Conectada – Implementação e Uso de Tecnologias Digitais na Educação”: O BNDES contará com o suporte técnico do Centro de Inovação para a Educação Brasileira – CIEB, para a execução das atividades da Ch d Públi i t Edit l ti id d d O BNDES contará com o suporte técnico do Centro de Inovação para a Educação Brasileira – CIEB, para a execução das atividades da Chamada Pública previstas nesse Edital; para as atividades de monitoramento e avaliação; e para a implantação dos projetos selecionados, incluindo orientação para a eventual alocação dos a Educação Brasileira – CIEB, para a execução das atividades da Chamada Pública previstas nesse Edital; para as atividades de monitoramento e avaliação; e para a implantação dos projetos selecionados, incluindo orientação para a eventual alocação dos 9 O documento competências digitais na formação de professores está disponível no sítio do CIEB <https://cieb.net.br/wp-content/uploads/2020/12/Compete%CC%82ncias-Digitais.pdf>. 9 O documento competências digitais na formação de professores está disponível no sítio do CIEB <https://cieb.net.br/wp-content/uploads/2020/12/Compete%CC%82ncias-Digitais.pdf>. 12 recursos financeiros, bens e serviços captados junto aos parceiros da iniciativa privada, de acordo com o cronograma a ser definido com as redes de ensino selecionadas (BNDES, 2018, p. 7). recursos financeiros, bens e serviços captados junto aos parceiros da iniciativa privada, de acordo com o cronograma a ser definido com as redes de ensino selecionadas (BNDES, 2018, p. 7). Ao tratar do novo empresariamento na educação, Harvey (1996) apresenta como é estratégica a conexão gerada pelas parcerias público-privadas, onde cabe ao primeiro assumir os riscos e à segunda os benefícios. Considerações preliminares sobre a incidência política do Centro de Inovação para a Educação Brasileira Diante deste panorama, salta aos olhos que uma mesma organização hoje, acumule as funções de prestar assessoria técnica ao principal programa de inovação para a educação do governo federal e realizar o mapeamento e a orientação das Edtechs que se interessem em vender seus produtos para o MEC e para as secretarias estaduais e municipais de educação. Outrossim, é mister a sinalização de Leher e Lamarão (2020) no tocante às tensões entre o público e o privado na educação brasileira: Para os autores, as políticas que possibilitam e/ou direcionam novas “formas de privatização” da educação convergem para uma redefinição do papel do professor. Em outras palavras, os caminhos tomados pelas políticas de financiamento da educação pública no país corroboraram para a desprofissionalização e precarização do trabalho docente. Considerações Finais Este artigo se propôs a reconstituir e mapear o percurso em que a ideia de inovação se insere na agenda política educacional brasileira e a relação deste processo com a criação e fortalecimento do CIEB. Ao realizar esse mapeamento, identificamos que na última década o PNE (2014-2024) e a BNCC (promulgada em 2017) constituíram-se como pilares para o aprofundamento do apelo às TICs na educação. Entre 2016 e 2022, o Brasil sofreu mudanças importantes em relação às agendas dos governos. Vale destacar que o período chega a compreender um golpe de Estado, corroborando com a avaliação de Bianchi (2016), mas mesmo assim, em relação ao fortalecimento da tríade inovação-educação-tecnologia, observa-se uma constância em sua consolidação como modelo a ser buscado, seja na iniciativa pública ou privada. Sob o mote da mitigação das desigualdades educacionais, o ensino híbrido e a entrada cada vez mais ampla de plataformas, aplicativos e outros produtos 13 13 tecnológicos para a educação, vem sendo indicados como sinônimo de “inovação” e de melhoria da qualidade da educação pública. Ou seja, fazer da educação cada vez mais uma bancada de negócios absolutamente lucrativa e um caminho para que os parcos investimentos na área desaguem nas mãos dos investidores das Edtechs. Aqui entendemos que o acesso à tecnologia deve ser sim um direito de todos, que a conectividade nas escolas pode ser uma possibilidade de potencializar o trabalho dos professores e professoras. Quanto mais recursos didáticos na escola, maior a autonomia do professor para desenvolver o trabalho com os conteúdos escolares, e consequentemente, fazer com que os filhos e filhas da classe trabalhadora exerçam o direito do acesso à educação. Mas fica a questão: inovação conectada para quem e para quê? De início, é importante lembrar que, de acordo com a divulgação do próprio governo federal, o PIEC é muito mais que uma ação que pretende levar recursos tecnológicos e conectividade para as escolas. De acordo com o sítio do governo federal: A Política de Inovação Educação Conectada pretende estimular o uso das ferramentas tecnológicas, definir os princípios, assegurar as ações voltadas à inclusão da inovação na prática pedagógica das escolas, capacitar os professores e demais profissionais envolvidos, bem como fomentar a produção de materiais escolares digitais gratuitos. Queremos chamar atenção, primeiramente para o que seria “inovação na prática pedagógica”. Para olhar mais de perto, precisamos lembrar que o termo inovação é importado do léxico empresarial. Considerações Finais Todos os problemas da educação são reduzidos a questões técnicas, e com isso criam-se dispositivos para a melhor gestão dos recursos como a comparação internacional de índices, cujo substrato são avaliações em larga escala. É preciso compreender as diferentes facetas que o apelo à inovação pode apresentar para o trabalho educativo, tendo em vista o conteúdo político que identificamos ao localizar este slogan no percurso da conformação da sociabilidade do capital. Para o CIEB, em 2021, o foco do trabalho pedagógico nas escolas deve ser as competências gerais e socioemocionais descritas na BNCC: 14 É hora de selecionar habilidades e competências por ano e por componente que: ● sejam aprendizagens essenciais para a compreensão de conceitos mais complexos na mesma área; ● sejam habilidades e competências que se conectem com outras áreas e possibilitem melhor compreensão delas; ● estejam relacionadas às competências gerais (socioemocionais e competências do século 21) (CIEB, 2021a, p. 9). O estudo dos diferentes materiais e iniciativas do Centro reforçam a defesa de que conteúdo e forma não de separam e se constituem como um exemplo completo do fenômeno da pedagogia de mercado, e do sequestro da escola (SANTOS, 2012). O mote pela inovação na educação consolida e põe em marcha a punção do fundo público e o esvaziamento da função social da escola. Esvaziamento marcado não somente pela proposição de currículos rarefeitos, mas pela proposição da centralidade do trabalho pedagógico nas competências gerais e socioemocionais. É preciso, corroborando com Accioly e Lamosa (2021), destacar o caráter repressivo/coercitivo do fomento à formação das competências socioemocionais. Em uma conjuntura de aprofundamento da privatização da educação pública, de controle do trabalho, dos corações e mentes dos trabalhadores e trabalhadoras, o desvelamento de slogans como a inovação torna-se imperioso para que se possa intervir na direção da defesa do direito à educação, à liberdade de cátedra e de pensamento. Referências ACCIOLY, I.; LAMOSA, R.. As Competências Socioemocionais na Formação da Juventude: Mecanismos de Coerção e Consenso frente às Transformações no Mundo do Trabalho e os Conflitos Sociais no Brasil. Revista Vértice, Campos dos Goytacazes, v.23, n.3, set./dez. 2021. ARCO-VERDE, Y. F. S. A arquitetura do tempo na cultura escolar: um estudo sobre os Centros de Educação Integral de Curitiba. 2003. 212f. Tese (Doutorado em Educação) – PUC, São Paulo. BRASIL. Presidência da República. Decreto nº 9.204, de 23 de novembro de 2017. Institui o Programa de Inovação Educação Conectada e dá outras providências. Brasília, 2017. Disponível em: http://www.planalto.gov.br/ccivil_03/_Ato2015- 2018/2017/Decreto/D9204.htm. Acesso em 15 de abril de 2020. BRASIL. Presidência da República. Decreto nº 9.204, de 23 de novembro de 2017. Institui o Programa de Inovação Educação Conectada e dá outras providências. Brasília, 2017. Disponível em: http://www.planalto.gov.br/ccivil_03/_Ato2015- 2018/2017/Decreto/D9204.htm. Acesso em 15 de abril de 2020. BANCO NACIONAL DE DESENVOLVIMENTO ECONÔMICO E SOCIAL (BNDES). Edital de Seleção nº 001/2018 (Edital retificado). Rio de Janeiro, 2018. Disponível 15 em:https://www.bndes.gov.br/wps/portal/site/home/onde-atuamos/social/chamada- publicabndes-educacao-conectada. Acesso em 04 de janeiro de 2022. em:https://www.bndes.gov.br/wps/portal/site/home/onde-atuamos/social/chamada- publicabndes-educacao-conectada. Acesso em 04 de janeiro de 2022. em:https://www.bndes.gov.br/wps/portal/site/home/onde-atuamos/social/chamada- publicabndes-educacao-conectada. Acesso em 04 de janeiro de 2022. CÊA, G. S. S..; SILVA, S. R. P. S.; SANTOS, I. M. S. DE “EDUCAÇÃO PARA TODOS” PARA “TODOS PELA EDUCAÇÃO”. RTPS - Revista Trabalho, Política e Sociedade, v. 4, n. 6, 30 jun. 2019. CENTRO DE INOVAÇÃO PARA A EDUCAÇÃO BRASILEIRA E ASSOCIAÇÃO BRASILEIRA DE STARTUPS. Mapeamento Edtech 2020 – investigação sobre as tecnologias educacionais brasileiras. São Paulo: ABSTARTUPS CIEB, 2021. __________. Programa de inovação conectada (PIEC). CIEB, 2021a. Disponível em: <https://cieb.net.br/programa-de-inovacao-educacao-conectada-piec/>. Acesso em 19 de dezembro de 2021. __________. Confira as publicações do CIEB de maior audiência em 2021. CIEB, 2021b. Disponível em: <https://cieb.net.br/confira-as-publicacoes-do-cieb-de-maior- audiencia-em-2021>. Acesso em 04 de janeiro de 2022. __________. CIEB: notas técnicas #1: A importância de políticas nacionais e centros de inovação em educação. São Paulo: CIEB, 2015. E-book em pdf. FERNANDES, E. F. A Política da OCDE para a Educação Básica: das mesas de reuniões internacionais à carteira escolar. 2019. 169f. Dissertação (Mestrado em Educação) – UnB, Brasília. FRIGOTTO, G. A Produtividade da Escola Improdutiva. 7. ed. São Paulo: Cortez, 2006. FRIGOTTO, G. A Produtividade da Escola Improdutiva. 7. ed. São Paulo: Cortez, 2006. GRAMSCI, A. Cadernos do Cárcere. Maquiavel. Notas sobre o Estado e a política. Rio de Janeiro: Civilização Brasileira, 2000. v. 3. GONSALES, P.; ALLODI, S. Design da educação conectada: metodologia para resolução de problemas na implementação do plano de tecnologia educacional. São Paulo: CIEB, 2019. HARVEY, D. Do gerenciamento ao empresariamento: a transformação da administração urbana no capitalismo tardio. Espaços e Debates, v. 16, n. 39, p. 48- 64, 1996. Disponível em: https://edisciplinas.usp.br/pluginfile.php/2078563/mod_resource/content/1/Harvey_D o%20gerenciamento%20ao%20empresariamento%20%281%29.pdf. Acesso em 20 de dezembro de 2021. HAVELOCK, R.; HUBERMAN, M. Solving educational problem: the theory and reality of innovation in developing countries. Paris: UNESCO, 1977. HUBERMAN, M. Comment s’opbrent les changements en éducation: contribution à l’étude de l’innovation. Paris: UNESCO, 1973. 16 LEHER, R..; LAMARÃO, M. V. M. A disputa pelos recursos educacionais e o movimento todos pela educação: antigas e novas formas de privatização. In: VIEIRA, S. V.; LAMOSA, L. (orgs.) Todos pela Educação? Uma década de ofensiva do capital sobre as escolas públicas. Curitiba: Appris, 2020. LIMA, K. R. S.; MARTINS, A. S. Pressupostos, Princípios e Estratégias. In: NEVES, Lúcia Maria Wanderley (org.). A nova pedagogia da hegemonia no Brasil: estratégias do capital para educar o consenso. São Paulo: Xamã, 2005. MENDONÇA, S. R. O Estado Ampliado como Ferramenta Metodológica. Marx e o Marxismo, v.2, n.2, jan/jul., 2014. MÉSZÁROS, I. A Crise Estrutural do Capital. 2ª ed. São Paulo: Boitempo, 2009. ÉSZÁROS, I. A Crise Estrutural do Capital. 2ª ed. São Paulo: Boitempo, 2009 NEVES, L. M. W. e SANT’ANNA, R. S.. Introdução: Gramsci, o Estado educador e a nova pedagogia da hegemonia. In: NEVES, Lúcia Maria Wanderley (org.). A nova pedagogia da hegemonia: estratégias do capital para educar o consenso. São Paulo: Xamã, 2005. OLIVEIRA, M. B. O inovacionismo em questão. Scientiae Studia. 2011, v. 9, n. 3. Disponível em: <https://doi.org/10.1590/S1678-31662011000300011>. Epub 27 Jan 2012. Acesso em 18 de janeiro de 2022. PIMENTEL, N.. M. A inovação como política na educação e na modalidade a distância / Innovation as a policy in education and distance mode. Revista Educação e Políticas em Debate, [S. l.], v. 9, n. 1, 2020. Disponível em: https://seer.ufu.br/index.php/revistaeducaopoliticas/article/view/53333. Acesso em 20 de janeiro de 2022. SALERNO, M. S.; KUBOTA, L. C. Estado e inovação. In: DE NEGRI, J.A.; KUBOTA, L.C. (Org.) Políticas de incentivo à inovação tecnológica. Brasília: Ipea, 2008. SHIROMA, E. O.; MORAES, Maria Célia Marcondes de; EVANGELISTA, Olinda. Política Educacional. Rio de Janeiro: Lamparina, 2007. TAVARES, F. G. O.. O conceito de inovação em educação: uma revisão necessária. Educação, [S. l.], v. 44, p. e4/ 1–19, 2019. Disponível em: https://periodicos.ufsm.br/reveducacao/article/view/32311. Acesso em 20 de janeiro de 2022. TAVARES, F. G. O.. O conceito de inovação em educação: uma revisão necessária. Educação, [S. l.], v. 44, p. e4/ 1–19, 2019. Disponível em: https://periodicos.ufsm.br/reveducacao/article/view/32311. Acesso em 20 de janeiro de 2022. 17
https://openalex.org/W4307952310	https://zenodo.org/records/7321948/files/22%2021073.pdf	English	null	Single parent mating in genetic algorithm for real robotic system identification	IAES International Journal of Artificial Intelligence	2,023	cc-by	5,300	IAES International Journal of Artificial Intelligence (IJ-AI) Vol. 12, No. 1, March 2023, pp. 201~208 ISSN: 2252-8938, DOI: 10.11591/ijai.v12.i1.pp201-208 IAES International Journal of Artificial Intelligence (IJ-AI) Vol. 12, No. 1, March 2023, pp. 201~208 ISSN: 2252-8938, DOI: 10.11591/ijai.v12.i1.pp201-208 IAES International Journal of Artificial Intelligence (IJ-AI) Vol. 12, No. 1, March 2023, pp. 201~208 ISSN: 2252-8938, DOI: 10.11591/ijai.v12.i1.pp201-208  201  201 201 Keywords: Discrete-time system Evolutionary computation Genetic algorithm Mathematical modelling Robotic system System identification Discrete-time system Evolutionary computation Genetic algorithm Mathematical modelling Robotic system System identification This is an open access article under the CC BY-SA license. This is an open access article under the CC BY-SA license. Md Fahmi Abd Samad Faculty of Mechanical Engineering, Universiti Teknikal Malaysia Melaka Hang Tuah Jaya, 76100 Durian Tunggal, Melaka, Malaysia Email: mdfahmi@utem.edu.my Single parent mating in genetic algorithm for real robotic system identification Md Fahmi Abd Samad1,2, Farah Ayiesya Zainuddin1 1Faculty of Mechanical Engineering, Universiti Teknikal Malaysia Melaka, Melaka, Malaysia 2Center for Advanced Computing Technology, Universiti Teknikal Malaysia Melaka, Melaka, Malaysia ABSTRACT System identification (SI) is a method of determining a mathematical model for a system given a set of input-output data. A representation is made using a mathematical model based on certain specified assumptions. In SI, model structure selection is a step where a model structure perceived as an adequate system representation is selected. A typical rule is that the final model must have a good balance between parsimony and accuracy. As a popular search method, genetic algorithm (GA) is used for selecting a model structure. However, the optimality of the final model depends much on the effectiveness of GA operators. This paper presents a mating technique named single parent mating (SPM) in GA for use in a real robotic SI. This technique is based on the chromosome structure of the parents such that a single parent is sufficient in achieving mating that eases the search for the optimal model. The results show that using three different objective functions (Akaike information criterion, Bayesian information criterion and parameter magnitude–based information criterion 2) respectively, GA with the mating technique is able to find more optimal models than without the mating technique. Validations show that the selected models using the mating technique are acceptable. Received Oct 2, 2021 Revised Jul 14, 2022 Accepted Aug 12, 2022 Corresponding Author: Md Fahmi Abd Samad Faculty of Mechanical Engineering, Universiti Teknikal Malaysia Melaka Hang Tuah Jaya, 76100 Durian Tunggal, Melaka, Malaysia Email: mdfahmi@utem.edu.my 1. INTRODUCTION System identification (SI) is known as a field of study where an optimal mathematical model, relating the variables and terms of a system, is determined. This is done by using the input-output data from the system. By developing the model, better control of the system may be achieved [1], [2]. There are two types of system modelling i.e. continuous-time and discrete-time modelling. Noting that data acquisition in industry or laboratory is made by instants of time, discrete-time modelling is commonly seen. There are 4 main steps in SI and these are data acquisition, model structure selection, parameter estimation and, lastly, model validation. The description of an optimal model is one that has adequate accuracy in predicting the response of the system but at the same time, is parsimonious in form. A parsimonious model structure, that contain fewer variables and/or terms, is desirable because the analysis and control of the system becomes easier [3]. To identify an optimal model for a system within a short time and cost, high efficiency in modelling is needed. Therefore, researchers have turned to meta-heuristic methods, including evolutionary computation as a method to allow optimal search for the system’s model [4]–[8]. Evolutionary computation, more specifically genetic algorithm (GA), has proven its strength and endurance, and able to reduce computational Journal homepage: http://ijai.iaescore.com  202 ISSN: 2252-8938 burden. GA has become an interesting area of investigation among researchers for many applications such as wireless sensor network energy optimization [9], control of vehicles [10], [11], modelling of disease severity [12], scheduling in medical field [13], aeronautics and robotics [14]. Although GA has been adopted in SI, its efficiency is still lacking which can be observed from long processing time, premature convergence or non-optimal setting of GA operators that causes loss of computation time and restricted global performance. Premature convergence to local optima is one of the most frequent difficulties that arise when applying GA to complex problem. It occurs when genetic operators can no longer generate offsprings that are fitter than their suboptimal parents. Premature convergence is associated with the loss of diversity in the population. However, too much population diversity can lead to a dramatic deterioration of GA efficiency [15]. Aside from the common operators of GA selection, crossover and mutation and although, relatively rare, researchers have started looking into mating after selection to enhance GA. 1. INTRODUCTION An example include the introduction of a self-adaptive mating based on parent similarity or fitness [15]. Another example introduces gender and kinship to the individuals in GA [16]. The idea is also adopted in [17] and used together with tabu search in [18]. With proper mating technique, the “marriage” of the parents must be able to explore new search space of solution, producing more varied offsprings (hence, in the context of SI, may mean system model) that cannot be achieved by common procedure of GA. This paper aims at introducing and implementing a new mating technique, named single parent mating (SPM), which is simpler yet effective, for a real robotic SI. The technique is shown to be capable of exploring new search space of solutions, thus producing more optimal and valid models according to three different objective functions (OF). The sections are divided as: section 2 explains the method of study including the mechanism of the mating proposal, section 3 reveals the results along with validation analysis and section 4 concludes the findings of the paper. 2.2. Real robotic system The real data comes from a flexible robot arm system available from [22]. The flexible robot arm has 1,024 data points of an input (measured reaction torque of the robotic structure) and an output (the acceleration of the flexible arm). Markovsky et al. [23], [24] selected lag 4 for a linear model identification of the system. Yassin et al. [25], it is identified that the system’s suitable specification is maximum order of input lag=2 and maximum order of output lag=4, also within a linear form. 2.1. GA with mating technique GA is known as an optimization method that takes the metaphor of species evolution [19]. In traditional GA, the search for an optimal model is made through three important processes: selection, crossover and mutation [20], [21]. The selection process copies good chromosomes into a mating pool - some may be copied a number of times. Common crossover operates by the mating of two or more parents taken randomly from the mating pool once the selection process is completed. However, there is a possibility that the 2 selected parents have the same features causing the process to produce similar offsprings to parents and thus suppressing the evolution. In this paper, to speed up the search and avoid premature convergence in the population, the algorithm incorporates a mating technique named SPM. The idea is to have 2 parents of completely different characteristics to be mated in order to try out new offsprings for the next generation. In SPM, once the parents are transferred to the mating pool after the selection process, they are copied and inverted to form a new set of parents. Using a binary representation, all bits 1 are changed to 0 and vice versa. The mating is achieved by pairing the original parent with its inverted self, hence the name SPM. The mating emphasizes how the pair of chromosomes is made, not how their informations are exchanged, as information exchange is carried out through crossover. Figure 1 illustrates that there are 24 possible offsprings with SPM when applied together with uniform crossover, as illustration. By this way, bigger search space is explored, diversity of the population is maintained and higher variability of offsprings is accomplished in the next generation. Figure 2 shows the procedure of GA using the mating technique. Int J Artif Intell, Vol. 12, No. 1, March 2023: 201-208 2.3. Simulation setup In this study, the chromosomes in GA represent specific models for SI. The models, in the form of nonlinear autoregressive with exogenous input (NARX) models, are represented using binary representation where 1 is presence of term and 0 for omission of term. Adopting some literature results, the specification of the search space is as: maximum nonlinearity=2, maximum order of output lag=4, maximum order of input lag=4 and time delay=1. This specification makes up 44 terms to be selected for model structure and the Int J Artif Intell, Vol. 12, No. 1, March 2023: 201-208 203  ISSN: 2252-8938 Int J Artif Intell number of possible models to choose from is 244-1>1013. Once a model structure is selected, the parameters are estimated using the least squares method. Then, they are evaluated based on the minimization of a specific optimality measurement a.k.a. OF in identifying the system. Three different types of OF i.e. Akaike information criteria (AIC), Bayesian information criteria (BIC) and parameter magnitude-based information criteria 2 (PMIC2) are used to evaluate the models throughout evolution. AIC and BIC may be referred from e.g. [26] while PMIC2 is demonstrated in [27] and [28]. The specification of GA is as: population size=200 and 100, maximum generation=300, bit-flip mutation probability=0.01 and single-point crossover probability=0.6. The study uses roulette-wheel selection and the elitism strategy. In the elitism strategy, the chromosome that is evaluated as the best is brought forward, unchanged, into the population of the next iteration (generation). The processes are repeated until termination (maximum generation). Prior study conducted using the mating technique with single-point crossover on simulated data sets revealed that the method found more optimal models by setting its implementation in the range of 10% to 20% of population, inclusively. Hence, in this study, GA is carried out using two specific percentages of the parents in the mating pool where the mating pool is the same size as the population. The percentages tested were 0% and 15%. As an example, for a 15% SPM, 15% of the parents in the mating pool are copied and inverted. This makes another set of parents of size 15% of population. Mating was done between the parents and its inverted selves (making up 30% of a population). The remaining 70% comes from the initial mating pool. With this setting, it may be noted that 0% represents GA without mating. 2.3. Simulation setup For each percentage, 15 runs of GA on the real robot arm system data set are made. Figure 1. Possible offsprings with single parent mating (SPM) Figure 2. GA with SPM Figure 2. GA with SPM Figure 1. Possible offsprings with single parent mating (SPM) Figure 2. GA with SPM 3.1. Model structure selection Finding an optimal model, that has adequate accuracy of prediction yet parsimonious in its structure, is aimed through the minimization of the OF. Figure 3 shows the average OF value of the best chromosome versus generation when using 0% SPM technique (that represents no mating and labelled “0” in legend) and 15% SPM technique (labelled “0.15”) for the real flexible robot arm data with 200 population size. Based on Figure 3(a), the graph of 15% SPM started at a high value but then decreased rapidly until the 100th generation when using AIC. It then settled rather consistently until the 300th generation. The decrement when using 0% SPM was rather slow until the maximum generation. The OF value of 15% SPM is better (lower) than 0% SPM throughout the evolution. Closer investigation revealed that GA with 15% SPM managed to find the same, presumably, the most optimal model in all its runs while with 0% SPM, the very same model is found in only 5 runs. The model has 8 regressors with an OF of -10745. Some of the models in 0% SPM gives higher OF of -10741. The best model from 15% SPM is written as: 𝑦(𝑡) = 3.158𝑦(𝑡−1) −4.439𝑦(𝑡−2) + 3.13𝑦(𝑡−3) −0.982𝑦(𝑡−4) +0.019𝑢(𝑡−1) −0.029𝑢(𝑡−2) + 0.055𝑢(𝑡−3) −0.030𝑢(𝑡−4) (1) 𝑦(𝑡) = 3.158𝑦(𝑡−1) −4.439𝑦(𝑡−2) + 3.13𝑦(𝑡−3) −0.982𝑦(𝑡−4) +0.019𝑢(𝑡−1) −0.029𝑢(𝑡−2) + 0.055𝑢(𝑡−3) −0.030𝑢(𝑡−4) (1) Single parent mating in genetic algorithm for real robotic system identification (Md Fahmi Abd Sama  204 ISSN: 2252-8938 Figures 3(b) to 3(c) show that the results using BIC and PMIC2 have quite the same pattern as graph in Figure 3(a). The graphs show that 15% SPM is better than 0% SPM, noted by achieving lower OF average of the best chromosomes, and, additionally, by shorter time. Similar to the observation when using AIC, with BIC, GA with 15% SPM found the same most optimal model (according to BIC) in all runs with an OF of -10707 whilst 0% SPM found it in 8 runs. Some of the models in 0% SPM gives higher OF of -10694. The model found with BIC has 7 regressors and is: 𝑦(𝑡) = 3.128𝑦(𝑡−1) −4.367𝑦(𝑡−2) + 3.058𝑦(𝑡−3) −0.952𝑦(𝑡−4) +0.004𝑢(𝑡−1) + 0.026𝑢(𝑡−3) −0.015𝑢(𝑡−4) (2) (2) Selecting these linear models as the optimal ones within the space of nonlinear model choices agree with [25]. 3.1. Model structure selection The best model contains several nonlinear terms and the linear ones are 𝑦(𝑡−1) and 𝑦(𝑡−3). This model has a higher OF than the one with 200 population size i.e. 15.583. g p p All the results obtained with 200 and 100 population size indicate that the population in 15% SPM contain more genetic diversity, allowing more optimal models to be found quicker. It indicates that bigger search space was explored and higher variety of offsprings was found in the next generation, compared to no mating. When a population is unable to produce offsprings of higher variability than their parents, an algorithm becomes trapped in local optima. This may have caused 0% SPM to be stuck with the near- optimals. (a) (b) (c) Figure 4. Best chromosome’s OF value for robot arm with 100 population size (a) AIC model, (b) BIC model, and (c) PMIC2 model (a) (b) (b) (a) (a) ( ) (c) Figure 4. Best chromosome’s OF value for robot arm with 100 population size (a) AIC model, (b) BIC (c) (c) Figure 4. Best chromosome’s OF value for robot arm with 100 population size (a) AIC model, (b) BIC model, and (c) PMIC2 model 3.1. Model structure selection When using PMIC2, models with lower OF value is found in 4 runs with 15% SPM than the lowest of 0% SPM. The best model of PMIC2 contains 29 regressors. It contains several nonlinear terms and the linear ones are 𝑦(𝑡−2), 𝑦(𝑡−3) and 𝑦(𝑡−4) with an OF value of 7.032, compared to 21.692 - the lowest when using 0% SPM. (a) (b) (c) Figure 3. Best chromosome’s OF value for robot arm with 200 population size (a) AIC model, (b) BIC model, and (c) PMIC2 model (a) (b) (b) (a) (b) (a) (a) (b) (c) (a) (b) (c) Figure 3. Best chromosome’s OF value for robot arm with 200 population size (a) AIC model, (b) BIC model, and (c) PMIC2 model (c) (c) Figure 3. Best chromosome’s OF value for robot arm with 200 population size (a) AIC model, (b) BIC model, and (c) PMIC2 model Next, another test is made by reducing the population size to 100. This affects GA in a way that less parents are inverted, making the effectiveness of SPM questionable. Figure 4 shows the average results. Based on Figure 4(a), it is seen again that, throughout evolution, the OF of the best model in 15% SPM is lower than that of 0% SPM. The decrement of 15% SPM is rapid until it found a model with a very low OF Int J Artif Intell, Vol. 12, No. 1, March 2023: 201-208 205  ISSN: 2252-8938 Int J Artif Intell whilst the decrement of 0% SPM is slow. 5% SPM found the same model as with 200 population size (1) in all its runs while 0% SPM found the very same, presumably, the most optimal model in 10 runs. Figures 4(b) to 4(c) show the OF average of the best chromosomes when using BIC and PMIC2, respectively. The pattern with PMIC2 is not exactly the same as with AIC and BIC but the outcome is the same. In all the OFs, 15% SPM found models with lower OF than 0% SPM throughout evolutions. With BIC, 0% SPM found, presumably, the most optimal model in 7 runs whilst 15% SPM found the same model in all its runs. This is the same model when using 200 population size (2). When using PMIC2, 8 runs in 15% SPM ended with final models of lower OF than the best model found using 0% SPM in all its runs. 3.2. Model validation Single parent mating in genetic algorithm for real robotic system identification (Md Fahmi Abd Samad) 3.2. Model validation To complement the whole procedure of SI, correlation tests are used as validation stage to ensure that no other significant terms and/or variables are omitted from the model [29]. Only the models selected using AIC and BIC are discussed since the validation of the PMIC2 models do not provide significant result. To complement the whole procedure of SI, correlation tests are used as validation stage to ensure that no other significant terms and/or variables are omitted from the model [29]. Only the models selected using AIC and BIC are discussed since the validation of the PMIC2 models do not provide significant result. Single parent mating in genetic algorithm for real robotic system identification (Md Fahmi Abd Samad)  206 ISSN: 2252-8938 Since the selected models using AIC and BIC are of linear forms, Figure 5 shows the linear correlation tests, consisting of error autocorrelation test (𝜙𝜀𝜀) and input-error cross correlation test (𝜙𝑢𝜀), carried out onto the results of the models, respectively. The dotted horizontal lines resemble the bandwidth such that a valid model with 95% confidence should have the lines within the bandwidth. They both look the same and it can be seen that some points lie outside of the bandwidth. The data reveals that there are small differences between Figures 5(a) and 5(b) where the biggest difference in 𝜙𝜀𝜀 test is at lag -16 while in 𝜙𝑢𝜀 test, the biggest difference is at lag -1. Nonetheless, the result is similar to the ones found in [30] where the authors commented that it is difficult to achieve perfect results for real life cases, and that sufficiently good, as can be seen, is acceptable. These validation deficiencies may be inherent from wrong selection of lag orders or nonlinearity. Including the noise terms will improve the model as carried out in [25]. Since the selected models using AIC and BIC are of linear forms, Figure 5 shows the linear correlation tests, consisting of error autocorrelation test (𝜙𝜀𝜀) and input-error cross correlation test (𝜙𝑢𝜀), carried out onto the results of the models, respectively. The dotted horizontal lines resemble the bandwidth such that a valid model with 95% confidence should have the lines within the bandwidth. They both look the same and it can be seen that some points lie outside of the bandwidth. 3.2. Model validation The data reveals that there are small differences between Figures 5(a) and 5(b) where the biggest difference in 𝜙𝜀𝜀 test is at lag -16 while in 𝜙𝑢𝜀 test, the biggest difference is at lag -1. Nonetheless, the result is similar to the ones found in [30] where the authors commented that it is difficult to achieve perfect results for real life cases, and that sufficiently good, as can be seen, is acceptable. These validation deficiencies may be inherent from wrong selection of lag orders or nonlinearity. Including the noise terms will improve the model as carried out in [25]. (a) (b) Figure 5. Linear correlation tests for best model in 15% SPM (a) AIC model (1) and (b) BIC model (2) (a) (a) (b) (b) (a) Figure 5. Linear correlation tests for best model in 15% SPM (a) AIC model (1) and (b) BIC model (2) Cross validation is also carried out on the results, as shown in Figure 6 and Figure 7, where the predicted output is superimposed to the real output and the error (difference of value between the two) plotted. With the range of real output data from -0.7883 to 0.7891, the highest error is 0.0147 whilst the average error is 0.0043 in Figure 6. From Figure 7, the highest error is 0.0151 whilst the average error is 0.0042. These errors are small and they show that the models selected by GA using AIC and BIC incorporating the mating technique are valid and acceptable. Furthermore, the mean square error of the models are 2.620×10-5 and 2.632×10-5 for AIC and BIC models, respectively. These data are comparable to the ones from [30] who obtained 2.69×10-5 and 2.72×10-5 for training data set and testing data set, respectively. I J A if I ll V l 12 N 1 M h 2023 201 208 Figure 6. Superimposition of predicted output of model using AIC with 15% SPM onto real output Figure 7. Superimposition of predicted output of model using AIC with 15% SPM onto real output Figure 6. Superimposition of predicted output of model using AIC with 15% SPM onto real output Figure 7. Superimposition of predicted output of model using AIC with 15% SPM onto real output Int J Artif Intell, Vol. 12, No. 1, March 2023: 201-208 207  ISSN: 2252-8938 Int J Artif Intell REFERENCES [1] K. J. Keesman, System identification: An introduction. London: Springer, 2011. p g [2] L. Ljung, System identification - theory for the user, 2 nd Editi. Prentice Hall PTR, 1999. [3] M. F. A. Samad and A. R. M. Nasir, “Comparison of information criterion on identification of discrete-time dynamic system,” Journal of Engineering and Applied Sciences, vol. 12, no. Special Issue 1, pp. 5660–5665, 2017, doi: 10.3923/jeasci.2017.5660.5665. [4] J. Nowaková and M. Pokorný, “System identification using genetic algorithms,” in Proceedings of the Fifth International Conference on Innovations in Bio-Inspired Computing and Applications IBICA 2014, vol. 303, Springer International Publishing, 2014, pp. 413–418. pp [5] M. I. A. Zamri, M. S. Hadi, A. N. S. Shaari, and N. M. Mokhtar, “Proportional integral derivative controller based on ant colony optimization for vibration cancellation of horizontal flexible plate structure,” International Journal of Electrical and Computer Engineering, vol. 12, no. 4, pp. 3583–3594, Aug. 2022, doi: 10.11591/ijece.v12i4.pp3583-3594. g g pp g j pp [6] R. Zhang and J. Tao, “A nonlinear fuzzy neural network modeling approach using an improved genetic algorithm,” IEEE Transactions on Industrial Electronics, vol. 65, no. 7, pp. 5882–5892, Jul. 2017, doi: 10.1109/TIE.2017.2777415. pp [7] B. Keshanchi, A. Souri, and N. J. Navimipour, “An improved genetic algorithm for task scheduling in the cloud environments using the priority queues: formal verification, simulation, and statistical testing,” Journal of Systems and Software, vol. 124, pp. 1–21, Feb. 2017, doi: 10.1016/j.jss.2016.07.006. pp , , j j [8] Y. Huang et al., “An application of evolutionary system identification algorithm in modelling of energy production system,” Measurement: Journal of the International Measurement Confederation, vol. 114, pp. 122–131, Jan. 2018, doi: 10.1016/j.measurement.2017.09.009. j [9] D. L. Shanthi and K. Prasanna, “Energy efficient intelligent routing in WSN using dominant genetic algorithm,” International Journal of Electrical and Computer Engineering (IJECE), vol. 10, no. 1, pp. 500–511, Feb. 2020, doi: 10.11591/ijece.v10i1.pp500-511. j pp [10] I. K. Mohammed and A. I. Abdulla, “Balancing a Segway robot using LQR controller based on genetic and bacteria foraging optimization algorithms,” Telkomnika (Telecommunication Computing Electronics and Control), vol. 18, no. 5, pp. 2642–2653, Oct. 2020, doi: 10.12928/TELKOMNIKA.v18i5.14717. [11] A. J. Moshayedi, M. Gheibollahi, and L. Liao, “The quadrotor dynamic modeling and study of meta-heuristic algorithms performance on optimization of PID controller index to control angles and tracking the route,” IAES International Journal of Robotics and Automation (IJRA), vol. 9, no. 4, pp. 256–270, Dec. 4. CONCLUSION The aim of this paper is to show the effectiveness of SPM for GA in optimization of a real discrete- time SI. Three types of OFs are used and according to all OFs, GA with SPM is able to find more optimal models, and additionally, quicker than without mating. In all runs, the models found using the mating technique have either lower or equal OF value than the runs without mating, indicating that the selected models are parsimonious yet with adequate accuracy. In validating the models, correlation tests are done to the selected models. Although the correlation tests show that the models do not fulfill the 95% confidence bandwidth completely, which is expected to be due to inherent noise of real data, superimposition of the predicted output from the models with the real output value provide convincing validation. Even when using a small population size, the technique is shown to be capable of reaching into untested territory of the search space. Using the mating technique, new points are able to be explored thus enabling the search for optimal models to become more promising than the traditional procedure of GA. From application perspective, future work may focus on changing the phenotype-to-genotype conversion for a more effective search of optimal models while from the method’s perspective, other crossover and selection types may be tested together with SPM to see whether faster convergence may be achieved. ACKNOWLEDGEMENTS The authors would like to express gratitude to Ministry of Higher Education Malaysia and Universiti Teknikal Malaysia Melaka (UTeM) for their financial support through FRGS/1/2018/TK03/UTEM/02/13 grant, technical support and facility. Single parent mating in genetic algorithm for real robotic system identification (Md Fahmi Abd Sama 8] C. K. Ting and H. K. Büning, “A mating strategy for multi-parent genetic algorithms by integrating tabu search,” in 20 Congress on Evolutionary Computation, CEC 2003 - Proceedings, 2003, vol. 2, pp. 1259–126 doi: 10.1109/CEC.2003.1299813. REFERENCES Voon, “Correlation based model validity tests for non-linear models,” International Journal of Control, vol. 44, no. 1, pp. 235–244, Jul. 1986, doi: 10.1080/00207178608933593. [30] A. Zabidi, N. M. Tahir, I. M. Yassin, and Z. I. Rizman, “The performance of binary artificial bee colony (BABC) in structure selection of polynomial NARX and NARMAX models,” International Journal on Advanced Science, Engineering and Information Technology, vol. 7, no. 2, pp. 373–379, Apr. 2017, doi: 10.18517/ijaseit.7.2.1387. REFERENCES Golberg, Genetic algorithms in search, optimization, and machine learning, 13th editi. Addison-Wesley Professional, 1989. [20] S. N. Sivanandam and S. N. Deepa, Introduction to genetic algorithms. Berlin, Heidelberg: Springer Berlin Heidelberg, 2008. g g p g y [20] S. N. Sivanandam and S. N. Deepa, Introduction to genetic algorithms. Berlin, Heidelberg: Springer Berlin Heidelberg, 2008. p g g g p g g 1] S. Mirjalili, J. Song Dong, A. S. Sadiq, and H. Faris, “Genetic algorithm: Theory, literature review, and application in ima reconstruction,” Nature-inspired optimizers, pp. 69–85, 2020. , p p , pp , [22] D. M. B.L.R., “DaISy: database for the identification of systems,” Department of Electrical Engineering, ESAT/STADIUS, KU Leuven, Belgium. Accessed: Aug. 15, 2021. [Online]. Available: http://homes.esat.kuleuven.be/$~$smc/daisy/. [23] I. Markovsky, J. C. Willems, S. Van Huffel, B. De Moor, and R. Pintelon, “Application of structured total least squares for system identification,” in Proceedings of the IEEE Conference on Decision and Control, 2004, vol. 4, pp. 3382–3387, doi: 10.1109/cdc.2004.1429229. [24] I. Markovsky, J. C. Willems, B. De Moor, K. U. Leuven, K. Arenberg, and B.- Leuven, “Comparison of identification algorithms on the database DAISY,” in Proceedings of the 17th International Symposium on Mathematical Theory of Networks and Systems (MTNS 2006), 2006, pp. 2858–2869. [25] I. M. Yassin, A. Zabidi, M. S. A. M. Ali, N. M. Tahir, H. Z. Abidin, and Z. I. Rizman, “Binary particle swarm optimization structure selection of nonlinear autoregressive moving average with exogenous inputs (NARMAX) model of a flexible robot arm,” International Journal on Advanced Science, Engineering and Information Technology, vol. 6, no. 5, pp. 630–637, 2016, doi: 10.18517/ijaseit.6.5.919. j [26] S. Hosseini, S. M. Saberali, and M. F. Sabahi, “Model order selection based on different information criteria for PDF estimation using maximum entropy method and application in cognitive radio systems,” in 7’th International Symposium on Telecommunications (IST’2014), Sep. 2014, pp. 1239–1244, doi: 10.1109/ISTEL.2014.7000893. [27] M. F. Abd Samad and A. R. Mohd Nasir, “Parameter magnitude-based information criterion in identification of discrete-time dynamic system,” Journal of Mechanical Engineering, vol. SI 4, no. 1, pp. 119–128, 2017. y y pp 8] M. F. A. Samad and A. R. M. Nasir, “Implementation of parameter magnitude-based information criterion in identification o real system,” Defence Science and Technical Bulletin, vol. 11, no. 1, pp. 99–106, 2018. [29] S. A. Billings and W. S. F. REFERENCES 2020, doi: 10.11591/ijra.v9i4.pp256-270. [12] N. A. M. Aseri et al., “Comparison of meta-heuristic algorithms for fuzzy modelling of COVID-19 illness’ severity classification,” IAES International Journal of Artificial Intelligence, vol. 11, no. 1, pp. 50–64, Mar. 2022, doi: 10.11591/ijai.v11.i1.pp50-64. j pp [13] M. El Midaoui, M. Qbadou, and K. Mansouri, “A fuzzy-based prediction approach for blood delivery using machine learning and genetic algorithm,” International Journal of Electrical and Computer Engineering (IJECE), vol. 12, no. 1, pp. 1056–1068, Feb. 2022, doi: 10.11591/ijece.v12i1.pp1056-1068. [14] F. A. Zainuddin and M. F. A. Samad, “A review of crossover methods and problem representation of genetic algorithm in recent engineering applications,” International Journal of Advanced Science and Technology, vol. 29, no. 6, pp. 759–769, 2020. [15] S. F. Galán, O. J. Mengshoel, and R. Pinter, “A novel mating approach for genetic algorithms,” Evolutionary Computation, vol. 21, no. 2, pp. 197–229, May 2013, doi: 10.1162/EVCO_a_00067. engineering applications, International Journal of Advanced Science and Technology, vol. 29, no. 6, pp. 759–769, 2020. [15] S. F. Galán, O. J. Mengshoel, and R. Pinter, “A novel mating approach for genetic algorithms,” Evolutionary Computation, vol. 21, no. 2, pp. 197–229, May 2013, doi: 10.1162/EVCO_a_00067. vo . , o. , pp. 97 9, ay 0 3, do : 0. 6 / VCO_a_00067. [16] A. C. Rizal and S. Suyanto, “Human-like constrained-mating to make genetic algorithm more explorative,” Jun. 2020, doi: 10.1109/ICoICT49345.2020.9166387. J. A. Ventura and S. H. Yoon, “A new genetic algorithm for lot-streaming flow shop scheduling with limited capacity buffers,” Journal of Intelligent Manufacturing, vol. 24, no. 6, pp. 1185–1196, May 2013, doi: 10.1007/s10845-012-0650-9. [ 7] J. . Ve tu a a d S. . oo , ew ge et c a go t o ot st ea g ow s op sc edu g w t ted capac ty bu e s, Journal of Intelligent Manufacturing, vol. 24, no. 6, pp. 1185–1196, May 2013, doi: 10.1007/s10845-012-0650-9. [18] C. K. Ting and H. K. Büning, “A mating strategy for multi-parent genetic algorithms by integrating tabu search,” in 2003 Congress on Evolutionary Computation, CEC 2003 - Proceedings, 2003, vol. 2, pp. 1259–1266, doi: 10.1109/CEC.2003.1299813. pp y [18] C. K. Ting and H. K. Büning, “A mating strategy for multi-parent genetic algorithms by integrating tabu search,” in 2003 Congress on Evolutionary Computation, CEC 2003 - Proceedings, 2003, vol. 2, pp. 1259–1266, doi: 10.1109/CEC.2003.1299813.  208 ISSN: 2252-8938 [19] D. E. Int J Artif Intell, Vol. 12, No. 1, March 2023: 201-208 BIOGRAPHIES OF AUTHORS Md Fahmi Abd Samad has earned his Ph.D. degree in mechanical engineering from Universiti Teknologi Malaysia and currently serving as an associate professor in Faculty of Mechanical Engineering, Universiti Teknikal Malaysia Melaka. His research interests are in system identification, control engineering and evolutionary computation. He is also a Chartered Engineer. He can be contacted at email: mdfahmi@utem.edu.my. Farah Ayiesya Zainuddin has earned Bachelor of Mechanical Engineering (Thermal-Fluids) and Master of Science in Mechanical Engineering from Universiti Teknikal Malaysia Melaka. Her research areas include genetic algorithm and aeronautics. She is currently pursuing PhD degree in mechanical engineering. She can be contacted at email: p041910001@student.utem.edu.my. Int J Artif Intell, Vol. 12, No. 1, March 2023: 201-208
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u=}QH "CU= CN=wvm} lU}O K]U s=tD QO w OwW\|t pY=L CmQL xR=Ov= Ci= C}=yv QO "CU= xOW xOQw; 1 pmW QO \|O=@ u}@QwD QwDwQ \|xLiY R= xOvQPo %OQm G=QNDU= 1 \|x]@=Q j@=]t =Q u=QW}B \|wQ}v '\|rwvQ@ uwv=k R= xO=iDU= =@ u=wD\|t T = 1 2AU2 1 [4a(1 a)] (1) \|x}Q_v QO 'Qorta lU}O swyit hqNQ@ "CU= \|QwLt \|}=kr= ?}Q[ a u; QO xm CU= \|WNQJ Qorta lU}O \UwD xOW O=H}= \|}x}w=R CmQL xR=Ov= 'CmQL xR=Ov= VNQJ CyH hr=Nt xm R}v =Q QwDwQ CWB u=}QH "OwW\|t xDio Qw=DWo u; x@ xm VNQJCyHR=}vOQwt\|W@vH\|SQv=Qo="Ovt=v\|txr=@vOVNQJ'ONQJ\|tQwDwQ "O@=}\|t Vy=m O}it Q=m TB 'OwW sm Qorta lU}O R= xOt; CUO x@ \|SQv= R= xr=@vO u=tr= l} QO CU=QD "OQw;\|t OwHw x@ =Q \|U=tt \|xir-wt Qorta lU}O VNQJ "O=O u=Wv 2 \|xrO=at \UwD u=wD\|t =Q \|wkrL dT = 1 2 2r2 4a0(1 + a0) (2rdr) (2) "OwW\|t h}QaD a0 = !2 CQwYx@ w xOw@ \|U=tt \|}=kr= ?}Q[ 'a0 u; QO xm =@ "CU= u=}QH VNQJ \|}x}w=R CaQU w QwDwQ VNQJ \|}x}w=R CaQU ! xtOkt "1 VN@ R= \|HwQN u=}QH ,=twO 'Ovm\|t u}t-=D Tmwrwv}= \|OwQw VN@ QO =Q u=}QH w \|}qa \UwD Tmwrwv}= swyit "OW=@ nvy=ty u; 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TBU w xOQm \|L=Q] \|Oa@xU \|x_iLt \|=Q@ j}kLD u}= QO "OvO=O Q=Qk \|UQQ@ OQwt \|OOa w \|r}rLD CQwYx@ \|x}L=v l} O=H}= Cra x@ pR=v \|OwQw CtUk QO xm OW xOy=Wt Ovv=t pR=v pwUt xOvU}wv "1400 12 24 VQ}PB '1400 10 28 x}LqY= '1400 8 25 Ci=}QO %M}Q=D DOI:10.24200/J40.2020.59289.1626 sm=L CqO=at "2 BIM \|x}Q_v Q@ sm=L CqO=at "1"2 w xQB u=tr= \|x}Q_v VwQ wO j}irD R= BIM \|x}Q_v wvax@ \|O=@ u}@QwD \|Oa@l} swDvtwt \|x}Q_v QO "CU= wL u=}QH "OwW\|t xDiQo Q_v QO xQB \|OwOLt=v O=OaD =@ _v QO C@=F Qorta lU}O uwJ w xOw@ =}=B w Q}PB=v sm=QD "OQ=Ov OwHw u=}QH xr=@vO QO \|WNQJ CaQU u}}=B QO u=}QH Q=}at sHL OwW\|t ZQi u}vJty =N \|wQ}v I}y 'u; Q@ xwqa "CU= xDi=} VQDUo QwDwQ \|m}D=DU= Q=Wi TB OwW\|tv p=ta= u=}QH CUOu}}=B wDwQ \|xLiY QO xOW O}rwD \|wQ}v =yvD "OUQ\|t O=R; U}O K]U s=tD QO w OwW\|t pY=L CmQL xR=Ov= Ci= ; 1 pmW QO \|O=@ u}@QwD QwDwQ \|xLiY R= xOvQPo ]t =Q u=QW}B \|wQ}v '\|rwvQ@ uwv=k R= xO=iDU= =@ u=wD\|t a)] (1) wyit hqNQ@ "CU= \|QwLt \|}=kr= ?}Q[ a u; QO xm O \UwD xOW O=H}= \|}x}w=R CmQL xR=Ov= 'CmQL xR=Ov= }v =Q QwDwQ CWB u=}QH "OwW\|t xDio Qw=DWo u; x@ xm W@vH\|SQv=Qo="Ovt=v\|txr=@vOVNQJ'ONQJ\|tQwDwQ 'OwW sm Qorta lU}O R= xOt; CUO x@ \|SQv= R= xr=@vO \|t OwHw x@ =Q \|U=tt \|xir-wt Qorta lU}O VNQJ "O=O u=Wv 2 \|xrO=at \UwD u=wD\|t =Q \|wkrL + a0) (2rdr) (2) CQwYx@ w xOw@ \|U=tt \|}=kr= ?}Q[ 'a0 u; QO xm }x}w=R CaQU w QwDwQ VNQJ \|}x}w=R CaQU ! xtOkt "1 CQwYx@ u=wD\|t =Q \|O}rwD u=wD w QwDwQ Qw=DWo '\|}x}w=R xv=mD \|=k@ uwv=k R= xO=iDU= "OQw; CUO x@ Q}R dQ = 4U1a0(1 a) r3dr (3) \|v=wNty \|y=oW}=tR; G}=Dv =@ xm CU= xOW Vy=m Q=JO u=}QH CaQU 'Q=W xOQm \|L=Q] QDlJwm O=a@= QO =Q Tmwrwv}= sDU}U l} [6]Vv=Q=mty w p}D=B " CN=U \|=Q@ ?U=vt \|xO=t l} ?=NDv= w \|UQQ@ u=v; \|rY= hOy "OvDN= Tmwrwv}= \|xvO@ Qo= xm OvO}UQ xH}Dv u}= x@ =yu; "CU= Tmwrwv}= sDU}U \| Q=Okt u}QDW}@ sDU}U \|HwQN u=wD OwW xDN=U xOW C}wkD \|m}DUqB \|=yQ@} Cr=L QO \|HwQN u=wD uOW QDsm OwHw =@ '?}=at \|NQ@ p}rO x@ \|rw Ow@ Oy sQv Oqwi R= xOW C}wkD \|m}DUqB \|=yQ@}i \|=Hx@ u=wD\|t 'sQv Oqwi R= xO=i "OQm xO=i \|@=}DUO Qw_vt x@ =Q Tmwrwv}= \|L=Q] QO QF-wt \|=yQDt=Q=B [7]xv}=D=@ w Qwm; =@ w \|OOa CQwYx@ \|QwDvw VN@ QO CaQU V}=Ri= \|=Q@ xv}y@ \|L=Q] l \|}xv}y@ \|L=Q] C}=yv QO w xO=O Q=Qk p}rLD w \|UQQ@ OQwt \|Oa@xU \|R=U VN@ QO CaQU u}QDW}@ xm OyO\|t u=Wv G}=Dv "Ov=xOQm x=Q= sDU}U u}= u}@ \|}x}w=R O=@ CyH =@ Tmwrwv}= sDU}U \|xvO@ xm OwW\|t pY=L \|v=tR \|Q \|QwDvw VN@ Q]k Vy=m =@ [8]Vv=Q=mty w C=y@ "OW=@ xDW=O xHQO 45 =D w OQmrta \|UQQ@ x@ '\|QwDvw VN@ pN=O u}@QwD \|xQB xU R= xO=iDU= w Tmw "OvDN=OQB \|Oa@xU \|R=UpOt R= xO=iDU= =@ w \|OOa VwQ x@ sDU}U \|HwQN Q=Okt l} x@ =Q OwHwt Tmwrwv}= sDU}U \|QwDvw VN@ Q]k Qo= xm OvDi=}QO = \|HwQNu=wDxH}DvQOwswUwswO\|=yu}@QwDQOu=}QHCaQU'OvyOVy=mx "O@=}\|t V}=Ri= =yu}@QwD swUQt \|O}rwD `@=vt =@ xv}Ry T=U= Q@ CU}=@\|t u=vJty O=@ \|SQv= CavY Vy=m \|Q}otWJ RQ] x@ xDWPo xyO OvJ QO O=@ \|SQv= \|xv}Ry xJQo= "Ovm C \|=Q@ xv}Ry u}QDsm =@ \|O=YDk= Q_v R= Ovv=wD@ O}=@ \|O=@ \|=yxSwQB =t= CU= x s=Hv= \|QDW}@ CkO =@ \|O=@ \|=yu}@QwD \|R=UpOt xJQy "Ovvm C@=kQ jQ@ O \|x}Q_v \|O=@ \|=yu}@QwD \|R=UpOt T=U= "Ow@ Oy=wN QDOvtOwU C@=kQ u}= 'O "CU= xOW x=Q= [9]CQ;wro \UwD xm CU= B =Q \|ki= QwLt \|O=@ \|=yu}@QwD R= hrDNt pOt xU [10]Vv=Q=mty w w=}Uy \|x}w=R w QDw `}RwD =@ \|}=yxQB =@ O=@ u}@QwD xm OvDi=}QO w OvO=O Q=Qk \|UQQ@ O [1311]Vv=Q=mtywu=}t}Qm"Ovm\|tO}rwD=Qu=wD?}Q[u}QDW}@xOWxv}y@V \|O=@ \|=yu}@QwD OQmrta \|R=UpOt w \|UQQ@ x@ BIM \|x}Q_v R= xO=iDU \|}=v=wD BIM \|x}Q_v xm OvO=O u=Wv w OvDN=OQB =}=B=v w =}=B \}=QW QO hr \|x}Q_v R= xO=iDU= =@ [14]Vv=Q=mty w \|v=L] "OQ=O XwYN u}= QO \|yHwD 60 xm OvDiQo xH}Dv 'VJ}B \|x}w=R w QDw `}RwD \|R=U\|]N R= TB w B sm=L CqO=at "2 BIM \|x}Q_v Q@ sm=L CqO=at "1"2 xOt; CUO x@ xv=mD \|x}Q_v w xQB u=tr= \|x}Q_v VwQ wO j}irD R= BIM \|x}Q_v \|va} lQLt lU}O l} u=wvax@ \|O=@ u}@QwD \|Oa@l} swDvtwt \|x}Q_v QO "CU= 'uoty Qorta lU}O u}= pwL u=}QH "OwW\|t xDiQo Q_v QO xQB \|OwOLt=v O=OaD =@ xir-wt TB 'OwW\|t xDiQo Q_v QO C@=F Qorta lU}O uwJ w xOw@ =}=B w Q}PB=v sm=QD "OQ=Ov OwHw u=}QH xr=@vO QO \|WNQJ CaQU \|xLiY CUOq=@ w CUOu}}=B QO u=}QH Q=}at sHL OwW\|t ZQi u}vJty w CUOq=@ QO p=}U Q@ \|HQ=N \|wQ}v I}y 'u; Q@ xwqa "CU= xDi=} VQDUo QwDwQ u=}QH Q=Wi x@ xOW \|@=}R=@ \|m}D=DU= Q=Wi TB OwW\|tv p=ta= u=}QH CUOu}}=B R= xm CU= xv=QW}B \|wQ}v 'QwDwQ \|xLiY QO xOW O}rwD \|wQ}v =yvD "OUQ\|t O=R; u=}QH "CU= CN=wvm} lU}O K]U s=tD QO w OwW\|t pY=L CmQL xR=Ov= Ci= C}=yv QO "CU= xOW xOQw; 1 pmW QO \|O=@ u}@QwD QwDwQ \|xLiY R= xOvQPo %OQm G=QNDU= 1 \|x]@=Q j@=]t =Q u=QW}B \|wQ}v '\|rwvQ@ uwv=k R= xO=iDU= =@ u=wD\|t T = 1 2AU2 1 [4a(1 a)] (1) \|x}Q_v QO 'Qorta lU}O swyit hqNQ@ "CU= \|QwLt \|}=kr= ?}Q[ a u; QO xm CU= \|WNQJ Qorta lU}O \UwD xOW O=H}= \|}x}w=R CmQL xR=Ov= 'CmQL xR=Ov= VNQJ CyH hr=Nt xm R}v =Q QwDwQ CWB u=}QH "OwW\|t xDio Qw=DWo u; x@ xm VNQJCyHR=}vOQwt\|W@vH\|SQv=Qo="Ovt=v\|txr=@vOVNQJ'ONQJ\|tQwDwQ "O@=}\|t Vy=m O}it Q=m TB 'OwW sm Qorta lU}O R= xOt; CUO x@ \|SQv= R= xr=@vO u=tr= l} QO CU=QD "OQw;\|t OwHw x@ =Q \|U=tt \|xir-wt Qorta lU}O VNQJ "O=O u=Wv 2 \|xrO=at \UwD u=wD\|t =Q \|wkrL dT = 1 2 2r2 4a0(1 + a0) (2rdr) (2) "OwW\|t h}QaD a0 = !2 CQwYx@ w xOw@ \|U=tt \|}=kr= ?}Q[ 'a0 u; QO xm \|v=wNty \|y=oW}=tR; G}=Dv =@ xOQm \|L=Q] QDlJwm O=a@= QO =Q CN=U \|=Q@ ?U=vt \|xO=t l Tmwrwv}= \|xvO@ Qo= xm OvO}UQ Q=Okt u}QDW}@ sDU}U \|HwQN Cr=L QO \|HwQN u=wD uOW QD sQv Oqwi R= xOW C}wkD \|m}DU \|@=}DUO Qw_vt x@ =Q Tmwrwv}= =@ w \|OOa CQwYx@ \|QwDvw V \|}xv}y@ \|L=Q] C}=yv QO w xO VN@ QO CaQU u}QDW}@ xm O u}@ \|}x}w=R O=@ CyH =@ Tmwrw \|QwDvw VN@ Q]k Vy=m =@ [8] w OQmrta \|UQQ@ x@ '\|QwDvw V "OvDN=OQB \|Oa@xU \|R=UpOt R Q=Okt l} x@ =Q OwHwt Tmwrwv} \|HwQNu=wDxH}DvQOwswUws swUQt \|O}rwD `@=vt =@ xv}Ry T Vy=m \|Q}otWJ RQ] x@ xDWPo \|=Q@ xv}Ry u}QDsm =@ \|O=YDk= s=Hv= \|QDW}@ CkO =@ \|O=@ \| \|x}Q_v \|O=@ \|=yu}@QwD \|R=U =Q \|ki= QwLt \|O=@ \|=yu}@QwD \|x}w=R w QDw `}RwD =@ \|}=yxQB =@ [1311] = =m = m m \|v=wNty \|y=oW}=tR; G}=Dv =@ xm CU= xOW Vy=m Q=JO u=}QH CaQU 'Q=WiQB xOQm \|L=Q] QDlJwm O=a@= QO =Q Tmwrwv}= sDU}U l} [6]Vv=Q=mty w p}D=B "OQ=O CN=U \|=Q@ ?U=vt \|xO=t l} ?=NDv= w \|UQQ@ u=v; \|rY= hOy "OvDN=U w Tmwrwv}= \|xvO@ Qo= xm OvO}UQ xH}Dv u}= x@ =yu; "CU= Tmwrwv}= sDU}U \|xvO@ Q=Okt u}QDW}@ sDU}U \|HwQN u=wD OwW xDN=U xOW C}wkD \|m}DUqB \|=yQ@}i R= Cr=L QO \|HwQN u=wD uOW QDsm OwHw =@ '?}=at \|NQ@ p}rO x@ \|rw Ow@ Oy=wN sQv Oqwi R= xOW C}wkD \|m}DUqB \|=yQ@}i \|=Hx@ u=wD\|t 'sQv Oqwi R= xO=iDU= "OQm xO=iDU= \|@=}DUO Qw_vt x@ =Q Tmwrwv}= \|L=Q] QO QF-wt \|=yQDt=Q=B [7]xv}=D=@ w Qwm; =@ w \|OOa CQwYx@ \|QwDvw VN@ QO CaQU V}=Ri= \|=Q@ xv}y@ \|L=Q] l} x@ \|}xv}y@ \|L=Q] C}=yv QO w xO=O Q=Qk p}rLD w \|UQQ@ OQwt \|Oa@xU \|R=UpOt VN@ QO CaQU u}QDW}@ xm OyO\|t u=Wv G}=Dv "Ov=xOQm x=Q= sDU}U u}= \|=Q@ u}@ \|}x}w=R O=@ CyH =@ Tmwrwv}= sDU}U \|xvO@ xm OwW\|t pY=L \|v=tR \|QwDvw \|QwDvw VN@ Q]k Vy=m =@ [8]Vv=Q=mty w C=y@ "OW=@ xDW=O xHQO 45 =D 45 w OQmrta \|UQQ@ x@ '\|QwDvw VN@ pN=O u}@QwD \|xQB xU R= xO=iDU= w Tmwrwv}= "OvDN=OQB \|Oa@xU \|R=UpOt R= xO=iDU= =@ w \|OOa VwQ x@ sDU}U \|HwQN u=wD Q=Okt l} x@ =Q OwHwt Tmwrwv}= sDU}U \|QwDvw VN@ Q]k Qo= xm OvDi=}QO =yu; \|HwQNu=wDxH}DvQOwswUwswO\|=yu}@QwDQOu=}QHCaQU'OvyOVy=mxv}y@ "O@=}\|t V}=Ri= =yu}@QwD u}= (2) x@ '\|r}rLD w \|OOa QDU@ wO Qy R= xO=iDU= Q@ O}m-=D =@ Q[=L VywSB QO u}@QwD R}v w Tmwrwv}= \|O=@ \|SQv= ?P=H sDU}U \|QDt=Q=B \|UQQ@ w \|R=UpOt \|OOa QDU@ wO ?}mQD "CU= xOW xDN=OQB u; \|QwDvw VN@ QO xOW x}@aD \|O=@ u}O@ "CU= xDiQo CQwY u}qi; \N R= GQ=N CQwYx@ xar=]t u}= QO \|r}rLD w pL xr}Uwx@ u; R= \|Qw@a u=}QH w Tmwrwv}= sDU}U xUOvy =OD@= QO xm \|vat \|r}rLDxt}v Om \|=y\|OwQw u=wvax@ pL u}= \|=y\|HwQN w xOW pOt \|OOa R= pY=L G}=Dv R= xO=iDU= =@ TBU "Ov=xOW xDiQo Q_v QO BIM \|x}Q_v C=Q}}eD w BIM \|x}Q_v KqY= hrDNt ?}=Q[ Q}F-=D \|UQQ@ x@ QwmPt \|x}Q_v xOW xDN=OQB xQB \|=DU=Q QO R}v w xQB lwv CaQU ?ULQ@ \|m}t=v}OwQ}; \|=yQDt=Q=B "CU= 38 2 \|xQ=tW '38 3 \|xQwO '14 1 R}}=B h}QW l}v=mt \|UOvyt "xDiW; xr=@vO K}LYD \|=Q@ uwDQ@ w CQ;wro \|=ypOt "1 pwOH xOW KqY= u=QW}B \|wQ}v ?}Q[ \|v=QL@ \|QwLt \|}=kr= ?}Q[ pOt 4a(1 (53a)a 4 )F 0 2 [9]CQ;wro 1 816 4 p1 816 1 (1 a) 0 3262 [17]u=Q=mty w uwDQ@ u=wD\|tp=L'OvOWK}QWDpt=mQw]x@xQBu=tr=wxv=mD\|=yx}Q_vxmu}=R=TB x@ u=QW}B \|wQ}v xm ?}DQD u}= x@ "OQm G=QNDU= =Q R=}v OQwt \@=wQ w ?}mQD =Q =yu; xv=mD \|x}Q_v \UwD xQB \|=DU=Q QO \|wkrL \|=yu=tr= R= s=Om Qy \|=Q@ xOt; CUO =D 8 \@=wQ R= xO=iDU= =@ u=wD\|t p=L "OvQ}o\|t Q=Qk Q@=Q@ sy =@ xQB u=tr= \|x}Q_v w %CN=OQB u=wD w Qw=DWo 'u=QW}B \|wQ}v \|x@U=Lt x@ 10 T = B N X i=1 Fni (8) Q = B N X i=1 Ftiri (9) P = B N X i=1 Ftiri = Q (10) Tmwrwv}= sDU}U Q@ sm=L CqO=at "2"2 \|=yu}@QwDQOxJv;=@xrUtl}R}iTmwrwv}=O=@\|SQv=?P=HsDU}UQOxm=Hu;R= sDU}U u}= \|UQQ@ QO CU= sRq u}=Q@=v@ 'OQ=O Cw=iD OwW\|t QwYDt pw=ODt \|O=@ CqO=at w \|SQv= w sQH \|=k@ CqO=at =Pr "OwW xi=[= \|Q_v C=_Lqt \|NQ@ Q}PB=vsm=QD'GRrQ}ep=}Uu=O}t\|=Q@pQDvmsHLl}\|wQQ@\|}x}w=Rw\|]NswDvtwt [42]"Ow@ Ovy=wN Q}R CQwYx@ w \|r=QoDv= sQi x@ uQ=kDt w I A V:dA = 0 (11) I A uzV:dA = T I A PdA:ez (12) I A V:dA = 0 (13) [16]"xQB Q@ OQ=w \|m}t=v}OwQ}; \|=ywQ}v C=}RH "2 pmW xQB lwv CaQU C@Uv = R U1 w \|rLt xQB lwv CaQU C@Uv r = r U1 xm "OvDUy OwW\|txO=iDU=xQBu=tr=\|x}Q_vR='xQB\|UOvyC=YNWtuDiQoQ_vQO\|=Q@ w OwW\|t s}UkD p=@ xv=yO \|=DU=Q QO u=tr= \|v}at O=OaD x@ QwDwQ xQB u; QO xm "OW=@ xDW=Ov OwHw =yu=tr= u}@ \|m}t=v}OwQ; VvmsyQ@ I}y xm CU= u}= Q@ ZQi x@ =Q xQB Qy Q@ OQ=w pm u=tt w wQ}v u=wD\|t xQB \|=DU=Q QO \|Q}op=QoDv= =@ C}=yv QO "CU= xOW xOQw; 2 pmW QO xQB Q@ OQ=w \|m}t=v}OwQ}; \|=ywQ}v p}rLD "OQw; CUO Qy Q@ OQ=w Qw=DWo w u=QW}B \|wQ}v 'xQB Qy \|wQ u=tr= \|YNWt O=OaD ZQi =@ %OW Oy=wN x@U=Lt u}vJ R}v u=tr= dQ = (1 a)2U2 1 sin2 (CL(sin ) Cd(cos )) rdr (5) 2 \|xQ=tW '383 \|xQwO '1401 R}}=B h}QW l}v=mt \|UOvyt "xDiW; xr=@vO K}LYD \|=Q@ uwDQ@ w CQ;wro \|=ypOt "1 pwOH T = B N X i=1 Fni (8) Q = B N X i=1 Ftiri (9) P = B N X i=1 Ftiri = Q (10) [16]"xQB Q@ OQ=w \|m}t=v}OwQ}; \|=ywQ}v C=}RH "2 pmW xQB lwv CaQU C@Uv = R U1 w \|rLt xQB lwv CaQU C@Uv r = r U1 xm "OvDUy Tmwrwv}= sDU}U Q@ sm=L CqO=at "2"2 \|=yu}@QwDQOxJv;=@xrUtl}R}iTmwrwv}=O=@\|SQv=?P=HsDU}UQOxm=Hu;R= sDU}U u}= \|UQQ@ QO CU= sRq u}=Q@=v@ 'OQ=O Cw=iD OwW\|t QwYDt pw=ODt \|O=@ CqO=at w \|SQv= w sQH \|=k@ CqO=at =Pr "OwW xi=[= \|Q_v C=_Lqt \|NQ@ Q}PB=vsm=QD'GRrQ}ep=}Uu=O}t\|=Q@pQDvmsHLl}\|wQQ@\|}x}w=Rw\|]NswDvtwt [42]"Ow@ Ovy=wN Q}R CQwYx@ w \|r=QoDv= sQi x@ uQ=kDt w I A V:dA = 0 (11) I A uzV:dA = T I A PdA:ez (12) [16]"xQB Q@ OQ=w \|m}t=v}OwQ}; \|=ywQ}v C=}RH "2 pmW xQB lwv CaQU C@Uv = R U1 w \|rLt xQB lwv CaQU C@Uv r = r U1 xm "OvDUy OwW\|txO=iDU=xQBu=tr=\|x}Q_vR='xQB\|UOvyC=YNWtuDiQoQ_vQO\|=Q@ w OwW\|t s}UkD p=@ xv=yO \|=DU=Q QO u=tr= \|v}at O=OaD x@ QwDwQ xQB u; QO xm "OW=@ xDW=Ov OwHw =yu=tr= u}@ \|m}t=v}OwQ; VvmsyQ@ I}y xm CU= u}= Q@ ZQi x@ =Q xQB Qy Q@ OQ=w pm u=tt w wQ}v u=wD\|t xQB \|=DU=Q QO \|Q}op=QoDv= =@ C}=yv QO "CU= xOW xOQw; 2 pmW QO xQB Q@ OQ=w \|m}t=v}OwQ}; \|=ywQ}v p}rLD "OQw; CUO Qy Q@ OQ=w Qw=DWo w u=QW}B \|wQ}v 'xQB Qy \|wQ u=tr= \|YNWt O=OaD ZQi =@ %OW Oy=wN x@U=Lt u}vJ R}v u=tr= Tmwrwv}= sDU}U Q@ sm=L CqO=at "2"2 \|=yu}@QwDQOxJv;=@xrUtl}R}iTmwrwv}=O=@\|SQv=?P=HsDU}UQOxm=Hu;R= sDU}U u}= \|UQQ@ QO CU= sRq u}=Q@=v@ 'OQ=O Cw=iD OwW\|t QwYDt pw=ODt \|O=@ CqO=at w \|SQv= w sQH \|=k@ CqO=at =Pr "OwW xi=[= \|Q_v C=_Lqt \|NQ@ Q}PB=vsm=QD'GRrQ}ep=}Uu=O}t\|=Q@pQDvmsHLl}\|wQQ@\|}x}w=Rw\|]NswDvtwt [42]"Ow@ Ovy=wN Q}R CQwYx@ w \|r=QoDv= sQi x@ uQ=kDt w A I A V:dA = 0 (13) I A ruV:dA = QT (14) I A p + 1 2 V2 V:dA = P (15) dQ = (1 a)2U2 1 sin2 (CL(sin ) Cd(cos )) rdr (5) dT = (1 a)2U2 1 sin2 (CL(cos ) + Cd(sin )) rdr (6) QwDwQ l} R= Cw=iDt 'OwOLt xQB O=OaD =@ QwDwQ l} \|=Q@ p=@ lwv \|=yx@=OQo Qw[L p=ta= w OwOLt xQB O=OaD uDiQo Q_v QO =@ "CU= C}=yv\|@ xQB O=OaD =@ (13) (14) dQ = (1 a)2U2 1 sin2 (CL(sin ) Cd(cos )) rdr (5) dT = (1 a)2U2 1 sin2 (CL(cos ) + Cd(sin )) rdr (6) (5) (6) QwDwQ l} R= Cw=iDt 'OwOLt xQB O=OaD =@ QwDwQ l} \|=Q@ p=@ lwv \|=yx@=OQo Qw[L p=ta= w OwOLt xQB O=OaD uDiQo Q_v QO =@ "CU= C}=yv\|@ xQB O=OaD =@ QF= pDv=QB KqY= `k=w QO "OvwW p}OaD O}=@ Qw=DWo w wQ}v 'xQB Qy lwv \|=yx@=OQo Q}R CQwYx@ pDv=QB KqY= ?}Q[ "Ovm\|t ^=Lr =Q u=QwO lU}O \|oDUw}B sOa %OwW\|t h}QaD QwDwQ l} R= Cw=iDt 'OwOLt xQB O=OaD =@ QwDwQ l} \|=Q@ p=@ lwv \|=yx@=OQo Qw[L p=ta= w OwOLt xQB O=OaD uDiQo Q_v QO =@ "CU= C}=yv\|@ xQB O=OaD =@ QF= pDv=QB KqY= `k=w QO "OvwW p}OaD O}=@ Qw=DWo w wQ}v 'xQB Qy lwv \|=yx@=OQo Q}R CQwYx@ pDv=QB KqY= ?}Q[ "Ovm\|t ^=Lr =Q u=QwO lU}O \|oDUw}B sOa %OwW\|t h}QaD F = 2 cos1 e B2 Rr r sin() 2 cos1 e B2 rRhub r sin() (7) xOW Q=@Da=\|@ xv=mD \|x}Q_v 0 5 R= QDnQR@ \|QwLt \|}=kr= ?}Q[ Q}O=kt \|=Q@ "OyO x=Q= O=@ u}@QwD u=QW}B \|wQ}v ?}Q[ R= \|DUQO \|v}@V}B Ov=wD\|tv w u}= \|=Q@ "OwW p=ta= xOt; CUO x@ \@=wQ x@ \|D=LqY= xm CU= R=}v u}=Q@=v@ xOW xOQw; 1 pwOH QO xm xOW x=Q= [17]uwDQ@ w [9]CQ;wro \UwD \|]@=wQ Qw_vt "CU= CL=Ut Q=OQ@ A '`=aW r 'CaQU Q=OQ@ V = (ur; u; uz) CqO=at u}= QO xm '\|QwLt \|wQ}v T 'Q=Wi p '=yz QwLt \|xm} Q=OQ@ ez '=wy \|r=oJ 'pQDvm K]U \|Oa@ l} pOt 3 pmW QO "CU= xQB \UwD xOW p=YLDU= u=wD P w Qw=DWo QT j}kO pLt w Tmwrwv}= O=@ \|SQv= ?P=H sDU}U \|QwDvw VN@ R= \|Qw@a u=}QH "CU= xOW xO=O u=Wv sDU}U R= VN@ u}= QO u}@QwD \|Q}oQ=Qk \|xrO=atx@xHwD=@'OwWxDiQoQ_vQO U1 Q@=Q@xQBR=p@kO=@\UwDtCaQUQo= \|k=@ Q}}eD uwO@ CaQU 'C@=F `]kt K]U =@ p=v=m R= u=}QH Qw@a =@ sQH \|=k@ "Ov=t\|t e = h p + 12 V2 i = P0 Q@=Q@ O=R; u=}QH \|tQH \|@O \|Br=Dv; xm =Hu; R= u=}QH =Pr 'CU= P0 = p + 12V 2 Q@=Q@ w pm Q=Wi P0 u; QO xm OwW\|t u=}@ CL=Ut Q=OQ@ A '`=aW r 'CaQU Q=OQ@ V = (ur; u; uz) CqO=at u}= QO xm '\|QwLt \|wQ}v T 'Q=Wi p '=yz QwLt \|xm} Q=OQ@ ez '=wy \|r=oJ 'pQDvm K]U \|Oa@ l} pOt 3 pmW QO "CU= xQB \UwD xOW p=YLDU= u=wD P w Qw=DWo QT j}kO pLt w Tmwrwv}= O=@ \|SQv= ?P=H sDU}U \|QwDvw VN@ R= \|Qw@a u=}QH "CU= xOW xO=O u=Wv sDU}U R= VN@ u}= QO u}@QwD \|Q}oQ=Qk F = 2 cos1 e B2 Rr r sin() 2 cos1 e B2 rRhub r sin() (7) u s } R V @ u} Q u}@Qw \|Q} QQ \|xrO=atx@xHwD=@'OwWxDiQoQ_vQO U1 Q@=Q@xQBR=p@kO=@\UwDtCaQUQo= \|k=@ Q}}eD uwO@ CaQU 'C@=F `]kt K]U =@ p=v=m R= u=}QH Qw@a =@ sQH \|=k@ "Ov=t\|t h i e = h p + 12 V2 i = P0 Q@=Q@ O=R; u=}QH \|tQH \|@O \|Br=Dv; xm =Hu; R= u=}QH =Pr 'CU= P0 = p + 12V 2 Q@=Q@ w pm Q=Wi P0 u; QO xm OwW\|t u=}@ 39 Tmwrwv}= O=@ \|SQv= ?P=H sDU}U l} \|R=UpOt \|QwDvw VN@ R= \|Qw@a u=}QH w u}@QwD \|Q}oQ=Qk pLt \|Oa@ l} pOt "3 pmW [3]"Tmwrwv}= "Tmwrwv}= sDU}U O=a@= "4 pmW "Tmwrwv}= O=@ \|SQv= ?P=H sDU}U \|UOvy C=YNWt "2 pwOH QDt O=a@= O=tv QDt=Q=B 12 19 DF unnel;Upper \|i}k \|}q=@ Q]k 21 34 H pm `=iDQ= 10 67 HF unnel \|i}k `=iDQ= 1 83 DF unnel;Lower \|i}k \|v}}=B Q]k 6 1 HInlet \|OwQw `=iDQ= 1 83 DV enturi \|QwDvw Q]k 3 05 DOutlet \|HwQN Q]k "OvDUy QDt x@ O=a@= xrUt pL \|xvt=O "5 pmW \|QDW}@ Q=@Da= R= \|OOa pL =D OW=@ QDlJwm =yxQ=w}O \|m}ORv QO xOW O}rwD xm@W O=H}= \|xm@W R= \|}=tv 6 pmW QO w xrUt pL \|xvt=O 5 pmW QO "OwW Q=OQwNQ "CU= xOW x=Q= xOW xm@W R= pqkDU= \|UQQ@ R= TB \|Ov@xm@W u}QD?U=vt 'QwmPt xUOvy \|=Q@ "Tmwrwv}= sDU}U O=a@= "4 pmW "Tmwrwv}= O=@ \|SQv= ?P=H sDU}U \|UOvy C=YNWt "2 pwOH QDt O=a@= O=tv QDt=Q=B 12 19 DF unnel;Upper \|i}k \|}q=@ Q]k 21 34 H pm `=iDQ= 10 67 HF unnel \|i}k `=iDQ= 1 83 DF unnel;Lower \|i}k \|v}}=B Q]k 6 1 HInlet \|OwQw `=iDQ= 1 83 DV enturi \|QwDvw Q]k 3 05 DOutlet \|HwQN Q]k "OvDUy QDt x@ O=a@= xrUt pL \|xvt=O "5 pmW \|QDW}@ Q=@Da= R= \|OOa pL =D OW=@ QDlJwm =yxQ=w}O \|m}ORv QO xOW O}rwD xm@W O=H}= \|xm@W R= \|}=tv 6 pmW QO w xrUt pL \|xvt=O 5 pmW QO "OwW Q=OQwNQ "CU= xOW x=Q= xOW xm@W R= pqkDU= \|UQQ@ R= TB \|Ov@xm@W u}QD?U=vt 'QwmPt xUOvy \|=Q@ xm@W u}= ?=NDv= =@ "OW=@ 1018037 Q@=Q@ =ypwrU O=OaD xm OyO\|t MQ \|Dr=L Q "OwW\|t xv}tm xH}Dv u}QD?U=vt x@ \|@=}DUO \|=Q@ \|OOa pL \|xv}Ry Q@=Q@ \|HwQN Q=Wi Q=Wi \|RQt \QW '\|OwQw QO CaQU \|RQt \QW u}vJty \|=yVN@ Q}=U \|=Q@ Q=w}O \|RQt \QW w CU= xOW xDiQo Q_v QO QiUtD= Q=W = i o _ m r = = = =] """ Tmwrwv}= O=@ \|SQv= ?P=H sDU}U l} \|R=UpOt \|QwDvw VN@ R= \|Qw@a u=}QH w u}@QwD \|Q}oQ=Qk pLt \|Oa@ l} pOt "3 pmW [3]"Tmwrwv}= "Tmwrwv}= sDU}U O=a@= "4 pmW "Tmwrwv}= O=@ \|SQv= ?P=H sDU}U \|UOvy C=YNWt "2 pwOH QDt O=a@= O=tv QDt=Q=B 12 19 DF unnel;Upper \|i}k \|}q=@ Q]k 21 34 H pm `=iDQ= 10 67 HF unnel \|i}k `=iDQ= 1 83 DF unnel;Lower \|i}k \|v}}=B Q]k 6 1 HInlet \|OwQw `=iDQ= 1 83 DV enturi \|QwDvw Q]k 3 05 DOutlet \|HwQN Q]k """ Tmwrwv}= O=@ \|SQv= ?P=H sDU}U l} \|R=UpOt "Tmwrwv}= sDU}U O=a@= "4 pmW sy p}Uv=DB \|SQv= pt=W xmr@ \|W@vH \|SQv= pt=W =yvD xv '\|SQv= `@vt u=wvax@ O=@ QO xm 'Ci= \|Q=Okt =@ 'sDU}U x@ OwQw R= TB u=}QH sQH OL=w \|SQv= u}= "CUy u=}QH "OUQ\|t xQB u=}QH CUOq=@ C}akwt x@ 'OQm Q_v hQY u; R= u=wD\|t x_Lr u=}QHu=wvax@Ov=wD\|t'QRw}i}Ow\|QwDvw'xOvvmRmQtDtVN@xUpt=W'p=v=mpN=O CU= \|}=H ,=k}kO pR=v x=owro u; QO xm OwW xDiQo Q_v QO QRw}i}O pR=v l} pN=O CL=Ut AT &CU= A = A1 AT Q@=Q@ u; `]kt K]U w OQ=O Q=Qk xQB xm "CU= u=}QH CyH Q@ Owta \|xLiY \|wQ =yxQB \|xOW Q}wYD "Tmwrwv}= O=@ \|SQv= ?P=H sDU}U \|UOvy C=YNWt "2 pwOH QDt O=a@= O=tv QDt=Q=B 12 19 DF unnel;Upper \|i}k \|}q=@ Q]k 21 34 H pm `=iDQ= 10 67 HF unnel \|i}k `=iDQ= 1 83 DF unnel;Lower \|i}k \|v}}=B Q]k 6 1 HInlet \|OwQw `=iDQ= 1 83 DV enturi \|QwDvw Q]k 3 05 DOutlet \|HwQN Q]k u=}@ P = H A h P0 i V:dA \|xrO=at =@ 'xQB \UwD xOt; CUO x@ u=wD \|x]@=Q \UwD A1 `]kt K]U =@ \|r=v=m pw] QO P0 \UwDt Q=Okt "OwW\|t \|tQH \|@O _m u; QO xm OwW\|t XNWt h P0 i A1 = 1 _m H A h P0 i V:dA j@=]t u=wD Q=Okt xH}Dv QO "CU= Tmwrwv}= O=@ p=kDv= sDU}U pw] QO u=}QH %O};\|t CUO x@ 16 \|xrO=at "OvDUy QDt x@ O=a@= xrUt pL \|xvt=O "5 pmW P = P0 1 P0 2 _m (16) P = P0 1 P0 2 _m (16) \|OOa pL \|R=UxO=}B "3 Tmwrwv}= sDU}U xUOvy \|iQat "1"3 u=Wv =Q xar=]t OQwt Tmwrwv}= \|O=@ u}@QwD sDU}U \|Oa@xU xUOvy O=a@= 4 pmW QO xm xvwou=ty "CU= xOW xOQw; 2 pwOH QO R}v xUOvy \|ak=w O=a@= "OyO\|t CU= xOW \|L=Q] \|Qw] \|Umwrwv}= \|O=@ u}@QwD sDU}U 'OwW\|t x_Lqt 4 pmW Ovm\|t ?PH '\|}xHQO 360 \|xvt=O l} QO \|va} 'C=yH s=tD R= =Q O=@ u=}QH xm CtUx@ u}@QwD \|=yxQB CyH s}kDUtQ}e =} w s}kDUt s}_vD x@ \|R=}v u}=Q@=v@ w sDU}U \|OwQw QO xOvvmC}=Oy \|xQB Q=yJ 'u}= Q@ xwqa "CU}v O=@ VRw CyH O=@ u=}QH C}=Oy w pQDvm =yu; xi}_w xm OQ=O OwHw Tmwrwv}= O=@ \|SQv= ?P=H \|SQv= ?P=H sDU}U \|OwQw \UwD xOW ?PH O=@ u=}QH "CU= sDU}U x@ \|OwQw VN@ QO '\|}wv=R l} R= Qw@a R= TB w xOW pkDvt u}}=B CtU x@ 'Tmwrwv}= O=@ p=v=m pN=O w u}tR K]U QO xOW x}@aD \|=yu}@QwD =} u}@QwD \|=yxQB x@ \|QwDvw "OwW\|t xO}@=D 'Tmwrwv}= \|O=@ u}@QwD sDU}U \|ki= "OvDUy QDt x@ O=a@= xrUt pL \|xvt=O "5 pmW "OvDUy QDt x@ O=a@= xrUt pL \|xvt=O "5 pmW \|QDW}@ Q=@Da= R= \|OOa pL =D OW=@ QDlJwm =yxQ=w}O \|m}ORv QO xOW O}rwD xm@W O=H}= \|xm@W R= \|}=tv 6 pmW QO w xrUt pL \|xvt=O 5 pmW QO "OwW Q=OQwNQ@ "CU= xOW x=Q= xOW xm@W R= pqkDU= \|UQQ@ R= TB \|Ov@xm@W u}QD?U=vt 'QwmPt xUOvy \|=Q@ xm@W u}= ?=NDv= =@ "OW=@ 1018037 Q@=Q@ =ypwrU O=OaD xm OyO\|t MQ \|Dr=L QO "OwW\|t xv}tm xH}Dv u}QD?U=vt x@ \|@=}DUO \|=Q@ \|OOa pL \|xv}Ry Q@=Q@ \|HwQN Q=Wi Q=Wi \|RQt \QW '\|OwQw QO CaQU \|RQt \QW u}vJty \|=yVN@ Q}=U \|=Q@ Q=w}O \|RQt \QW w CU= xOW xDiQo Q_v QO QiUtD= Q=Wi QO "CU= xOW xDiQo Q_v QO Tmwrwv}= \|O=@ u}@QwD sDU}U \|=Q@ xOW \|L=Q] xUOvy 'Tmwrwv}=O=@\|SQv=?P=H sDU}Uh=Q]=\|=yQ=w}O\|xrY=ixOW\|L=Q]\|xUOvy "OvW=@ xDW=Ov O=@ u=}QH Q@ \|Q}F-=D xm xOW ?=NDv= \|}xvwox@ '\|v}=B Q=w}O RH x@ \|RQt \}=QW w xm@W R= pqkDU= 'xrUt \|Ov@xm@W "2"3 uwO@ \|Ov@xm@W R= 'xUOvy uOw@ \|Oa@xU w \|oO}J}B p}rO x@ Q[=L \|xar=]t QO \|UQQ@ w \|OOa \|R=UpOt CkO V}=Ri= Qw_vt x@ "CU= xOW xO=iDU= Q=DN=U xm CU= xOW \|aU 'u; C=QF= w =yxQ=w}O \|m}ORv QO u=}QH V}=OH QDj}kO xJQy 40 "x}v=F Q@ QDt 6 71 \|OwQw u=}QH CaQU \|=Q@ CaQU QwDv=m "7 pmW [10]"OwHwt G}=Dv =@ Q[=L Q=m \|=Q@ =O@tq ?ULQ@ u=wD ?}Q[ \|xU}=kt "8 pmW O=@ \|SQv= ?P=H sDU}U pN=O O=@ u=}QH `k=w QO "OQ@ s=v \|O=@ \|=yu}@QwD =O}B V}=Ri= VDaQU w OQ}o\|t ?=DW 'Ovv=t \|QwDvw VN@ l} QO w Tmwrwv}= sDU}U pN=O xOW x}@aD u}@QwD \|=yxQB \UwD xm \|O=@ u=}QH u}=Q@=v@ "Ovm\|t O=R; u=}QH x@ C@Uv \|QDW}@ CaQU 'OwW\|t TL Tmwrwv}= O=@ \|SQv= ?P=H \|SQv= ?P=H sDU}U \|yOR=@ xm OwW\|t ?@U Qt= u}ty w CW=O Oy=wN O=@ sDU}U u}= R= xH}Dv QO "OW=@ QDW}@ \|RwQt= \|=yu}@QwD x@ C@Uv Tmwrwv}= O=@ xO=iDU= 'CU= u}}=B =yu; QO O=@ VRw CaQU xm \|k]=vt QO \|DL u=wD\|t "OQm \|r}rLDxt}v Om \|R=UxO=}B "4 \|r}rLDxt}v Om \|HvUQ=@Da= "1"4 pY=L \|r}rLD w \|@QHD G}=Dv w xUOvy R= 'BIM ?rDt Om \|HvUQ=@Da= Qw_vtx@ "CU= xOW xO=iDU= [10]u}W}B u=kkLt \|UQQ@ R= pY=L BIM \|x}Q_v R= TvQiQ \UwD xOW xv}y@ u}@QwD 'Qw_vt u}= \|=Q@ xOW xDiQo Q_v QO xUOvy =O@tq w C=w 50 \|t=v u=wD 'xQB 3 'QDt 0 72 Q]k \|=Q=O xm CU= QwmPt ?ULQ@ u=wD ?}Q[ G}=Dv \|xU}=kt 8 pmW QO "CU= \|L=Q] x]kv QO 5 Q_v QO =@ 'Q[=L Q=m QO xOW \|L=Q] BIM Om R= pY=L xQB lwv CaQU G}=Dv =@ 'CQ;wro K}LYD R}v w pDv=QB \|BwD w xQB lwv KqY= ?}=Q[ Q}F-=D uDiQo G}=Dv \|x_Lqt p@=k ji=wD \|xOvyOu=Wv xm xOW x=Q= [10]OwHwt \|r}rLD w \|@QHD "CU= "Tmwrwv}= O=@ \|SQv= ?P=H sDU}U \|=Q@ xOW O=H}= xm@W R= \|}wQ@wQ \|=tv "6 pm "QorL \|HvUQ=@Da= Qw_vt x@ [3w2]xr=kt =@ Q[=L Q=m G}=Dv xU}=kt "3 pwOH Q[=L Q=m [3w2]TwrwBwQOv= w \|}qa \|r=a \|r=a xm@W C}i}m 6 71 6 71 (m=s) \|OwQw O=@ CaQU 10 845 10 6 (m=s) \|QwDvw QO CaQU 1 6162 1 58 CaQU C@Uv 2 31 \|@Uv \|=]N OYQO \|OOa pL \|HvUQ=@Da= "3 u}@QwD sDU}U QO u=}QH \|OOa \|R=Ux}@W Qw_vt x@ xm \|QopL \|HvUQ=@Da= \| xJv; j@=]t xrUt \}=QW =OD@= QO 'CU= xDiQo Q=Qk xO=iDU= OQwt Tmwrwv}= \| u}= QO "OW xDiQo Q_v QO 'CU= xOW xOQw; [3w2]TwrwBwQOv= w \|}qa xr=kt QwLt C@Ft CyH hqN w x}v=F Q@ QDt 6 71 Q@=Q@ w C@=F O=@ u=}QH CaQU xr Tmwrwv}= O=@ \|SQv= ?P=H sDU}U \|UOvy O=a@= w CU= xOW xDiQo Q_v QO pY=L G}=Dv \|xU}=kt "CU= u=Um} xOW xOQw; Q[=L Q=m QO xJv; =@ xOW \|L CaQU u}ov=}t \|=Q@ [3w2]'xr=kt w \|Ov@xm@W Cr=L u}QDy@ QO Q[=L Q=m xOW xOQw; 3 pwOH QO CaQU C@Uv u}vJty w \|QwDvw VN@ QO O=@ u=} "C =@ w \|}q=@ Q=}U@ CkO =@ Q[=L Q=m R= pY=L G}=Dv 'OwW\|t x_Lqt xm u=vJ "OQ=O Ck@=]t [3w2]\|r=a C}i}m =@ pOt R= pY=L G}=Dv =@ =]N OYQO 2 Ow \|OOa pL G}=Dv "4 xrUt Q@ sm=L \|RQt \}=QW w Tmwrwv}= O=@ \|SQv= ?P=H sDU}U xUOvy xm p =@ u=wD\|t 'OW s=Hv= R}v \|OOa QopL \|HvUQ=@Da= w OW XNWt pt=m Qw] \|=yCaQU \|=Q@ =Q \|QwDvw pLt QO u=}QH CaQU '\|OOa pL QDU@ R= xO=iD R= \|r}rLD xt}v Om \|OwQw QDt=Q=B u=wvax@ w xOQm G=QNDU= hrDNt \|OwQw CaQU \UwDt 'x}v=F Q@ QDt 6 71 O=R; u=}QH CaQU ZQi =@ "CiQo xQy@ QO u=}QH CaQU C@Uv w x}v=F Q@ QDt 10 845 Q@=Q@ \|QwDvw VN@ RmQt QO u=} CaQU QwDv=m "O};\|t CUO x@ 1 661 Q@=Q@ \|OwQw QO u=}QH CaQU x@ \|Qw "CU= xOW xOQw; 7 pmW QO x}v=F Q@ QDt 6 71 \|OwQw u=}QH CaQU \| xOW O=}R \|Q}otWJ Qw] x@ \|QwDvw VN@ QO u=}QH CaQU xm OwW\|t x_L xm OwW\|t Ea=@ '=wy O=R; u=}QH CaQU x@ C@Uv CaQU V}=Ri= u}= "C \|xRwL QO QF-wt \|Qw;wv l} u=wvax@ u=wD@ =Q Tmwrwv}= O=@ \|SQv= ?P=H sDU 2 \|xQ=tW '383 \|xQwO '1401 R}}=B h}QW l}v=mt \|UOvyt "x}v=F Q@ QDt 6 71 \|OwQw u=}QH CaQU \|=Q@ CaQU QwDv=m "7 pmW "x}v=F Q@ QDt 6 71 \|OwQw u=}QH CaQU \|=Q@ CaQU QwDv=m "7 pmW "x}v=F Q@ QDt 6 71 \|OwQw u=}QH CaQU \|=Q@ CaQU QwDv=m "7 pmW "x}v=F Q@ QDt 6 71 \|OwQw u=}QH CaQU \|=Q@ CaQU QwDv=m "7 pmW \|OOa pL \|HvUQ=@Da= "3"3 u}@QwD sDU}U QO u=}QH \|OOa \|R=Ux}@W Qw_vt x@ xm \|QopL \|HvUQ=@Da= \|=Q@ xJv; j@=]t xrUt \}=QW =OD@= QO 'CU= xDiQo Q=Qk xO=iDU= OQwt Tmwrwv}= \|O=@ u}= QO "OW xDiQo Q_v QO 'CU= xOW xOQw; [3w2]TwrwBwQOv= w \|}qa xr=kt QO QwLt C@Ft CyH hqN w x}v=F Q@ QDt 6 71 Q@=Q@ w C@=F O=@ u=}QH CaQU xr=kt Tmwrwv}= O=@ \|SQv= ?P=H sDU}U \|UOvy O=a@= w CU= xOW xDiQo Q_v QO Z pY=L G}=Dv \|xU}=kt "CU= u=Um} xOW xOQw; Q[=L Q=m QO xJv; =@ xOW \|L=Q] CaQU u}ov=}t \|=Q@ [3w2]'xr=kt w \|Ov@xm@W Cr=L u}QDy@ QO Q[=L Q=m R= xOW xOQw; 3 pwOH QO CaQU C@Uv u}vJty w \|QwDvw VN@ QO O=@ u=}QH "CU= \|OOa pL G}=Dv "4"3 \|r}rLDxt}v Om \|R=UxO=}B "4 \|r}rLDxt}v Om \|HvUQ=@Da= "1"4 pY=L \|r}rLD w \|@QHD G}=Dv w xUOvy R= 'BIM ?rDt Om \|HvUQ=@Da= Qw_vtx@ "CU= xOW xO=iDU= [10]u}W}B u=kkLt \|UQQ@ R= pY=L BIM \|x}Q_v R= TvQiQ \UwD xOW xv}y@ u}@QwD 'Qw_vt u}= \|=Q@ xOW xDiQo Q_v QO xUOvy =O@tq w C=w 50 \|t=v u=wD 'xQB 3 'QDt 0 72 Q]k \|=Q=O xm CU= QwmPt ?ULQ@ u=wD ?}Q[ G}=Dv \|xU}=kt 8 pmW QO "CU= \|L=Q] x]kv QO 5 Q_v QO =@ 'Q[=L Q=m QO xOW \|L=Q] BIM Om R= pY=L xQB lwv CaQU G}=Dv =@ 'CQ;wro K}LYD R}v w pDv=QB \|BwD w xQB lwv KqY= ?}=Q[ Q}F-=D uDiQo G}=Dv \|x_Lqt p@=k ji=wD \|xOvyOu=Wv xm xOW x=Q= [10]OwHwt \|r}rLD w \|@QHD "CU= G xrUt Q@ sm=L \|RQt \}=QW w Tmwrwv}= O=@ \|SQv= ?P=H sDU}U xUOvy xm p=L =@ u=wD\|t 'OW s=Hv= R}v \|OOa QopL \|HvUQ=@Da= w OW XNWt pt=m Qw]x@ \|=yCaQU \|=Q@ =Q \|QwDvw pLt QO u=}QH CaQU '\|OOa pL QDU@ R= xO=iDU= R= \|r}rLD xt}v Om \|OwQw QDt=Q=B u=wvax@ w xOQm G=QNDU= hrDNt \|OwQw O=@ CaQU \UwDt 'x}v=F Q@ QDt 6 71 O=R; u=}QH CaQU ZQi =@ "CiQo xQy@ u; QO u=}QH CaQU C@Uv w x}v=F Q@ QDt 10 845 Q@=Q@ \|QwDvw VN@ RmQt QO u=}QH CaQU QwDv=m "O};\|t CUO x@ 1 661 Q@=Q@ \|OwQw QO u=}QH CaQU x@ \|QwDvw "CU= xOW xOQw; 7 pmW QO x}v=F Q@ QDt 6 71 \|OwQw u=}QH CaQU \|=Q@ xOW O=}R \|Q}otWJ Qw] x@ \|QwDvw VN@ QO u=}QH CaQU xm OwW\|t x_Lqt xm OwW\|t Ea=@ '=wy O=R; u=}QH CaQU x@ C@Uv CaQU V}=Ri= u}= "CU= \|xRwL QO QF-wt \|Qw;wv l} u=wvax@ u=wD@ =Q Tmwrwv}= O=@ \|SQv= ?P=H sDU}U 41 m r v= = S v= P= D l = pO ?=UDL= =@ w uwDQ@ w CQ;wro K}LYD \|=Q@ =O@tq ?ULQ@ u=wD ?}Q[ "10 pmW "pDv=QB xQB lwv KqY= ?}Q[ ZQi =@ w uwDQ@ w CQ;wro K}LYD \|=Q@ =O@tq ?ULQ@ u=wD ?}Q[ "11 pmW "pDv=QB \|BwD w xQB lwv KqY= ?}Q[ =O@tq ?ULQ@ \|QDt=Q=B \|xar=]t "3"4 TB '=O@tq ?ULQ@ O=@ u}@QwD QO p}NO \|m}t=v}OwQ}= \|=yQDt=Q=B p}rLD Qw_vtx@ QO "CU= xOW ZQi xQB Qy \|wQ u=tr= 100 'xm@W R= pqkDU= Ov}=Qi s=Hv= R= Cr=L wO \|=Q@ =O@tq ?ULQ@ u=wD ?}Q[ C=Q}}eD \|xU}=kt R= \|Q=Owtv 10 pmW xQB lwv KqY= ?}Q[ uDiQo Q_v QO =@ w uwDQ@ w CQ;wro xDiW; xr=@vO K}LYD \w@Qt Q=Owtv OwW\|t x_Lqt xm u=vJ "CU= xOW xOQw; Cr=L wO Qy \|=Q@ pDv=QB xr=@vO K}LYD x@ \w@Qt Q=Owtv u}}=B xQ=wty uwDQ@ \|xDiW; \|xr=@vO K}LYD x@ C@Uv =Q \|QDsm G}=Dv uwDQ@ K}LYD xm CU=vat u=O@ u}= w CU= CQ;wro xDiW; QO u=wD ?}Q[ xv}W}@ "Ovm\|t \|v}@V}B u=wD ?}Q[ \|=Q@ CQ;wro K}LYD x@ xm \|DQwY QO "OyO\|t MQ 7 929 =O@tq QO w xOw@ 0 457 Q@=Q@ CQ;wro K}LYD j=iD= 7 194 =O@tq QO w xOw@ 0 393 Q@=Q@ uwDQ@ K}LYD QO u=wD ?}Q[ xv}W}@ "ODi=\|t K}LYD wO \|=Q@ =O@tq ?ULQ@ u=wD ?}Q[ C=Q}}eD Q=Owtv 11 pmW QO \|=Q@ pDv=QB \|BwD w xQB lwv KqY= ?}Q[ uDiQo Q_v QO =@ w uwDQ@ w CQ;wro ?}Q[ xv}W}@ Q=Okt 'OwW\|t x_Lqt xm u=vJ "CU= xOW xOQw; Cr=L wO Qy x@ 'CU= xOW xO=O QF= xQB lwv KqY= ?}Q[ \ki xm \|Dr=L x@ C@Uv u=wD u}= QO "CU= xDi=} Vy=m \|BwD VN@ KqY= ?}Q[ p=ta= R= \|W=v Ci= Cra QO w xOw@ 0 3813 Q@=Q@ CQ;wro K}LYD Cr=L QO u=wD ?}Q[ xv}W}@ Cr=L uwDQ@ K}LYD QO u=wD ?}Q[ xv}W}@ xm \|DQwY QO "OyO\|t MQ 7 876 =O@tq [18]"`HQt u}@QwD x@ \w@Qt C=aq]= "4 pwOH Q=Okt xYNWt 3 xQB O=OaD QDt 0 8192 xQB Qy pw] QDt 1 7336 QwDwQ Q]k QDt 0 0952 \|BwD Q]k [20]"10e5 ROrwv}Q OOa QO NREL S822 p}wiQ}= C=aq]= "5 pwOH Q=Okt xYNWt 9 Ncrit 94 7 (CL=CD)max 5=075 opt 0 8582 CL;opt 0 00906 CD;opt "xOW \|L=Q] xQB \|rm \|=tW w xv}y@ VJ}B \|x}w=R w QDw `}RwD Q=Owtv "9 pmW u}@QwD \|=yxQB \|xUOvy "2"4 \|QwDvw VN@ QO \|Q}oxQy@ Qw_vt x@ xOW \|L=Q] u}@QwD x@ \w@Qt C=YNWt R= xm [18]'Vv=Q=mty w MwtUO \UwD xOW x=Q= G}=Dv x@ xHwD =@ Tmwrwv}= sDU}U QO 'OvOw@ xDN=OQB Tmwrwv}= sD}U \|=Q@ u}@QwD \|L=Q] x@ \|OOa pL QDU@ j}Q] "Ov=xOW VQ=Ro 4 pwOH QO C=aq]= u}= "CU= xOW xDiQo Q_v NREL S822 p}wiQ}= 'QDt 5 Q}R pw] =@ \|=yxQB \|=Q@ [19]RQtwU w Q}Hv=D s=tD \|=Q@ p}wiQ}= u}ty R= xr=kt u}= QO =yxQB sm pw] Crax@ xm Ov=xOQm O=yvW}B =Q xDiQo xQy@ xOW xO=iDU= u; \|=Q@ \|wQ}=O p}wiQ}= R= xm \|BwD VN@ RHx@ xQB `]=kt OOa QO [20]NREL S822 p}wiQ}= x@ \w@Qt \|=yxO=O 5 pwOH QO "CU= xOW u=}QH CaQU x@ xHwD =@ Q[=L Q=m u}@QwD \|OQmrta \|xOwOLt xm '10e5 ROrwv}Q "Ov=xOW xOQw; 'CU= =yxQB xUOvy w O=@ \|=yxQB \|rm \|=tW R}v w xv}y@ VJ}B \|x}w=R w QDw `}RwD Q=Owtv C}=yv QO "O};\|t CUO x@ 9 pmW CQwYx@ xOW \|L=Q] ?=UDL= =@ w uwDQ@ w CQ;wro K}LYD \|=Q@ =O@tq ?ULQ@ u=wD ?}Q[ "10 pmW "pDv=QB xQB lwv KqY= ?}Q[ ZQi =@ w uwDQ@ w CQ;wro K}LYD \|=Q@ =O@tq ?ULQ@ u=wD ?}Q[ "11 pmW "pDv=QB \|BwD w xQB lwv KqY= ?}Q[ [18]"`HQt u}@QwD x@ \w@Qt C=aq]= "4 pwOH Q=Okt xYNWt 3 xQB O=OaD QDt 0 8192 xQB Qy pw] QDt 1 7336 QwDwQ Q]k QDt 0 0952 \|BwD Q]k [20]"10e5 ROrwv}Q OOa QO NREL S822 p}wiQ}= C=aq]= "5 pwOH Q=Okt xYNWt 9 Ncrit 94 7 (CL=CD)max 5=075 opt 0 8582 CL;opt 0 00906 CD;opt "xOW \|L=Q] xQB \|rm \|=tW w xv}y@ VJ}B \|x}w=R w QDw `}RwD Q=Owtv "9 pmW [18]"`HQt u}@QwD x@ \w@Qt C=aq]= "4 pwOH Q=Okt xYNWt 3 xQB O=OaD QDt 0 8192 xQB Qy pw] QDt 1 7336 QwDwQ Q]k QDt 0 0952 \|BwD Q]k [20]"10e5 ROrwv}Q OOa QO NREL S822 p}wiQ}= C=aq]= "5 pwOH Q=Okt xYNWt 9 Ncrit 94 7 (CL=CD)max 5=075 opt 0 8582 CL;opt 0 00906 CD;opt ?=UDL= =@ w uwDQ@ w CQ;wro K}LYD \|=Q@ =O@tq ?ULQ@ u=wD ?}Q[ "10 pmW "pDv=QB xQB lwv KqY= ?}Q[ =O@tq ?ULQ@ \|QDt=Q=B \|xar=]t "3"4 TB '=O@tq ?ULQ@ O=@ u}@QwD QO p}NO \|m}t=v}OwQ}= \|=yQDt=Q=B p}rLD Qw_vtx@ QO "CU= xOW ZQi xQB Qy \|wQ u=tr= 100 'xm@W R= pqkDU= Ov}=Qi s=Hv= R= Cr=L wO \|=Q@ =O@tq ?ULQ@ u=wD ?}Q[ C=Q}}eD \|xU}=kt R= \|Q=Owtv 10 pmW xQB lwv KqY= ?}Q[ uDiQo Q_v QO =@ w uwDQ@ w CQ;wro xDiW; xr=@vO K}LYD \w@Qt Q=Owtv OwW\|t x_Lqt xm u=vJ "CU= xOW xOQw; Cr=L wO Qy \|=Q@ pDv=QB xr=@vO K}LYD x@ \w@Qt Q=Owtv u}}=B xQ=wty uwDQ@ \|xDiW; \|xr=@vO K}LYD x@ C@Uv =Q \|QDsm G}=Dv uwDQ@ K}LYD xm CU=vat u=O@ u}= w CU= CQ;wro xDiW; QO u=wD ?}Q[ xv}W}@ "Ovm\|t \|v}@V}B u=wD ?}Q[ \|=Q@ CQ;wro K}LYD x@ xm \|DQwY QO "OyO\|t MQ 7 929 =O@tq QO w xOw@ 0 457 Q@=Q@ CQ;wro K}LYD j=iD= 7 194 =O@tq QO w xOw@ 0 393 Q@=Q@ uwDQ@ K}LYD QO u=wD ?}Q[ xv}W}@ "ODi=\|t "xOW \|L=Q] xQB \|rm \|=tW w xv}y@ VJ}B \|x}w=R w QDw `}RwD Q=Owtv "9 pmW u}@QwD \|=yxQB \|xUOvy "2"4 \|QwDvw VN@ QO \|Q}oxQy@ Qw_vt x@ xOW \|L=Q] u}@QwD x@ \w@Qt C=YNWt R= xm [18]'Vv=Q=mty w MwtUO \UwD xOW x=Q= G}=Dv x@ xHwD =@ Tmwrwv}= sDU}U QO 'OvOw@ xDN=OQB Tmwrwv}= sD}U \|=Q@ u}@QwD \|L=Q] x@ \|OOa pL QDU@ j}Q] "Ov=xOW VQ=Ro 4 pwOH QO C=aq]= u}= "CU= xOW xDiQo Q_v NREL S822 p}wiQ}= 'QDt 5 Q}R pw] =@ \|=yxQB \|=Q@ [19]RQtwU w Q}Hv=D s=tD \|=Q@ p}wiQ}= u}ty R= xr=kt u}= QO =yxQB sm pw] Crax@ xm Ov=xOQm O=yvW}B =Q xDiQo xQy@ xOW xO=iDU= u; 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QO xm 16 pmW QO \|wQ}v '=O@tq V}=Ri= =@ xm OwW\|t x_Lqt 'CU= xOW xOQw; hrDNt \|=y=O@tq QO =UB \|wQ}v xm CU= x_Lqt p@=k Kw[w x@ ,=vt[ "OwW\|t Vy=m Q=JO =UB 'xQB \|}=OD@= \=kv QO xvW}@ x]kv l} x@ uO}UQ R= TB w xOw@ \|OwaY xQB \|=OD@= QDw pw] V}=Ri= Cra x@ `k=w QO \|}=OD@= \=kv QO =UB \|wQ}v V}=Ri= "OwW\|t \|rwRv \|xOwOLt QO xQB \|}=OD@= \=kv uwJ u}vJty "CU= \|L=wv u}= QO 9 pmW j@=]t QDW}@ xQB \|}=yDv= \=kv x@ C@Uv \=kv u}= QO =UB \|wQ}v u}=Q@=v@ 'OvQ=O Q=Qk p=DU= "CU= xQB Oa@\|@ pw] ?ULQ@ \|QDt=Q=B \|xar=]t "4"4 QO \|yHwD p@=k \|}=v=wD Tmwrwv}= O=@ \|SQv= ?P=H sDU}U 'OW KQ]t xm u=vJ OYQO 66 ,=OwOL Ov=wD\|t w OQ=O O=@ u=}QH R= \|SQv= ?PH u=tOv=Q V}=Ri= \|=yxQB x@ xm \|O=@ u=}QH CaQU V}=Ri= =@ "OyO V}=Ri= =Q O=R; O=@ u=}QH CaQU Q}}eD VwNDUO O=@ u}@QwD \|m}t=v}OwQ}; \|=yQDt=Q=B Q}O=kt 'OwW\|t xO}@=D u}@QwD Q}otWJ CaQU V}=Ri= R= \|W=v xm '=yQDt=Q=B u}= C=Q}}eD \|UQQ@ \|=Q@ "OW Oy=wN XNWt \|=O@tq l} l CU= sRq 'CU= Tmwrwv}= sDU}U \UwD O=@ u=}QH XNWt xQB Oa@\|@ pw] O=ODt= QO hrDNt \|m}t=v}OwQ}; \|=yQDt=Q=B \|UQQ@ \|=Q@ OW=@ xv}y@ \|L=Q] x]kv x@ \w@Qt \|=O@tq u=ty =O@tq u}= xm CU= QDy@ "OwW 9 pwOH "O=O Q=Qk QDj}kO \|UQQ@ OQwt \|i}m Q_v R= u=wD@ =Q \|HwQN G}=Dv =D u=Wv =Q Q[=L Q=m QO xOW \|L=Q] \|O=@ u}@QwD \|L=Q] x]kv x@ \w@Qt C=aq]= "OyO\|t QO hrDNt \|=yQDt=Q=B Q=Owtv u=wD\|t '\|L=Q] \|xv}y@ \|x]kv uOw@ XNWt =@ xO}O hrDNt \|=y=O@tq \|=Q@ xQB \|=DU=Q QO u=wD ?}Q[ Q=Owtv 17 pmW QO xDi=} V}=Ri= \|rLt u=wD ?}Q[ xv}W}@ Q=Okt '=O@tq V}=Ri= =@ =OD@= QO "OwW\|t C=Q}}eD OvwQ XNWt \|=O@tq l} x@ uO}UQ =@ =t= "OwW\|t QwO xQB lwv R= w R}v =O@tq ?ULQ@ u=wD ?}Q[ C=Q}}eD XwYNQO OvwQ u}ty "OwW\|t Tma TB TBU w xOW QDW}@ =OD@= QO u=wD ?}Q[ Q=Okt '=O@tq V}=Ri= =@ "CW=O OwHw \=kv xm u}= x@ xHwD =@ u}vJty "O@=}\|t Vy=m '\|a[wt xv}W}@ l} R= Qw@a R= u=wD O}rwD QO \|QDsm syU u}=Q@=v@ 'OvQ=O Q=Qk p=DU= ZQat QO QDW}@ xQB \|}=OD@= xQB \|}=yDv= \=kv QO u=wD ?}Q[ xv}W}@ xm OwQ\|t Q=_Dv= xH}Dv QO "CW=O Ovy=wN "ODi}@ j=iD= = 18 pm r = = q = = = ; = xOy=Wt 18 pmW QO hrDNt \|=y=O@tq \|=Q@ xQB \|=DU=Q QO ;Q@ \|wQ}v Q=Owtv 44 "hrDNt \|=y=O@tq \|=Q@ xQB \|=DU=Q QO ;Q@ \|wQ}v "18 pmW "hrDNt \|=y=O@tq \|=Q@ xQB \|=DU=Q QO u=QW}B \|wQ}v "19 pmW "hrDNt \|=y=O@tq \|=Q@ xQB \|=DU=Q QO ;Q@ \|wQ}v "18 pmW "hrDNt \|=y=O@tq \|=Q@ xQB \|=DU=Q QO ;Q@ \|wQ}v "18 pmW "hrDNt \|=y=O@tq \|=Q@ xQB \|=DU=Q QO ;Q@ \|wQ}v "18 pmW K}LYD ?}Q[ R= BIM \|x}Q_v Om \|HvUQ=@Da= \|=Q@ w \|r}rLD VN@ QO K}LYD ?}=Q[ Q}=U \|xU}=kt Qw_vt x@ "xOW xO=iDU= CQ;wro xDiW; xr=@vO u=}t `t=H \|}xU}=kt '=yu; CkO u=Q}t \|UQQ@ w CQ;wro K}LYD ?}Q[ =@ K}LYD xm OyO\|t u=Wv G}=Dv "CiQo CQwY uwDQ@ w CQ;wro K}LYD ?}=Q[ u; R= \|QDsm G}=Dv w xOQm pta QDxv=Q=m x_i=Lt CQ;wro K}LYD C@Uv x@ uwDQ@ \}=QW QO uwDQ@ K}LYD xm CQwY u}= x@ &O};\|t CUO x@ u=wD ?}Q[ \|=Q@ QDsm Q@=Q@ 0 913 w 0 878 ?}DQD x@ =Q u; Q_=vDt \|=O@tq w u=wD ?}Q[ x@=Wt "Ovm\|t \|v}@V}B "hrDNt \|=y=O@tq \|=Q@ xQB \|=DU=Q QO u=QW}B \|wQ}v "19 pmW Q=_Dv= R= QwO u}=Q@=v@ 'OvQ=O Q=Qk p=DU= \|xOwOLt QO xQB \|}=OD@= \=kv "OwW\|t xOy=Wt "OW=@ QDsm ;Q@ \|wQ}v xQB \|}=yDv= \=kv x@ C@Uv \=kv u}= QO xm CU}v 0 1445 `=aW QO Q=Owtv xm u}= =D CU= \|OwaY Q=Owtv OvwQ =OD@= QO xm OwW\|t uwDw}v 2 29 Q@=Q@ x]kv u}= QO ;Q@ \|wQ}v Q=Okt "OUQ\|t \|a[wt \|xv}W}@ x@ QDt `wkw p=tDL= V}=Ri= w QDw C=Q}}eD OvwQ QO Q}}eD Crax@ ;Q@ \|wQ}v TBU "CU= xm "OUQ\|t OwN Q=Okt u}QDsm x@ 0 2797 `=aW QO w xDi=} Vy=m 'p=DU= x@ \|OwaY OvwQ Q=Owtv xt=O= QO "CU= uwDw}v 2 165 Q@=Q@ ;Q@ \|wQ}v u; QO uwDw}v 2 552 \|va} OwN Q=Okt xv}W}@ x@ QDt 0 7537 `=aW QO w xDiQo OwN 'OvW=@ xDW=O Q=Qk p=DU= xOwOLt QO xQB \|}=OD@= \=kv xm OwQ\|t Q=_Dv= "OUQ\|t QDsm ;Q@ \|wQ}v w QDW}@ =UB \|wQ}v xQB \|}=yDv= \=kv x@ C@Uv \=kv u}= QO u}=Q@=v@ "CU= =yO=tv &`@Qt QDt CL=Ut %A &pDv=QB hqD= ?}Q[ %F &\|QwLt \|}=kr= ?}Q[ %a &\|U=tt \|}=kr= ?}Q[ %a0 &;Q@ ?}Q[ %CL &=UB ?}Q[ %CD &xv}W}@ \|=UB x@ ;Q@ ?}Q[ C@Uv %(CL=CD)max &u=wD ?}Q[ %CP &uDw}v ;Q@ \|wQ}v %L &uDw}v =UB \|wQ}v %D &C=w u=wD %P &uDw}v \|QwLt \|wQ}v %T &QDt uDw}v Qw=DWo %Q &x}v=F Q@ QDt p=}U CaQU %U &QDt QwDwQ `=aW %R &QDt \|rLt `=aW %r &QDt xQB \|=DU=Q QO QDw `}RwD %C(r) References `@=vt 11. Karimian Aliabadi, S. and Rasekh, S. \Eect of platform disturbance on the performance of oshore wind turbine under pitch control", Wind Energy, 23, pp. 1210-1230 (2020). 1. Rezaey, S. \Numerical investigation of a globe con- trol valve and estimating its loss coecient at dif- ferent opening states", European Journal of Compu- tational Mechanics, 29(4-6), pp. 549-576 (2021). DOI: https://doi.org/10.13052/ejcm1779-7179.294610. 12. Karimian, S. and Rasekh, S. \Power and noise perfor- mance assessment of a variable pitch vertical axis dar- rieus type wind turbine", Journal of the Brazilian So- ciety of Mechanical Sciences and Engineering, 43, pp. 1-21 (2021). 2. Allaei, D. and Andreopoulos, Y. \INVELOX: a new con- cept in wind Energy harvesting, Proceeding of ASME 2013 7th International Conference on energy Sustain- ability & 11th Fuel Cell Science, Engineering and Tech- nology Conference ES-Fuel Cell, pp. 14-19 (2013). 13. Asghari, M., Karimian Aliabadi, S. and Hashemi, M. \Study the eect of suction ow control on the aerody- namic performance of a wind turbine using 2D section numerical results", Sharif Journal of Mechanical Engi- neering, 37 pp. 79-89 (2021). 3. Allaei, D. and Andreopoulos, Y. \INVELOX: description of a new concept in wind power and its performance evaluation", Energy, 69, pp. 336-344 (2014). 4. Allaei, D., Tarnowski, D. and Andreopoulos, Y. \IN- VELOX with multiple wind turbine generator systems", Energy, 93, pp. 1030-1040 (2015). 14. Tahani, M., Kavari, G., Masdari, M. and et al. \Aero- dynamic design of horizontal axis wind turbine with in- novative local linearization of chord and twist distribu- tions", Energy, 131, pp. 78-91 (2017). 5. Sedaghat, A., Waked, A., Assad, H. and et al. Anahysis of accelerating devices for enclosure wind turbines. Int. J. Astronaut. Aeronaut. Eng, 2(9), pp.9-23 (2017). 15. Branlard, E., Wind Turbine Aerodynamics and Vorticity-Based Methods: Fundamentals and Recent Applications, Springer (2017). 6. Patil, M.N, Ghadage, S.M., Gaikwad, O.R. and et al. De- sign and Fabrication of Invelox, International Research Journal of Engineering and Technology (IRJET), 6(6), pp.765-768 (2019). 16. Manwell, J.F., McGowan, J.G. and Rogers, A.L., Wind Energy Explained: Theory, Design and Application, John Wiley & Sons (2010). 7. Akour, S.N. and Bataineh, H.O. \Design considerations of wind funnel concentrator for low wind speed regions", AIMS Energy, 7, pp. 728-742 (2019). 17. Burton, T., Jenkins, N., Sharpe, D. and et al., Wind Energy Handbook, John Wiley & Sons (2011). 8. Solanki, A.L., Kayasth, B.D. and Bhatt, H. =yO=tv '4 hrDNt \|=y=O@tq \|=Q@ xQB \|=DU=Q QO =Q u=QW}B \|wQ}v Q=Owtv 19 pmW u}= w CU= \|OwaY u=QW}B \|wQ}v xm OwW\|t xO}O =OD@= QO "Ovm\|t x=Q= 8 w 6 \=kv QO QDw pw] V}=Ri= p}rO x@ xm OQ}o\|t CQwY \|OvD ,=D@Uv ?}W =@ OwaY `=aW QO uwDw}v 0 01 Q=Okt x@ uO}UQ R= TB u=QW}B \|wQ}v "CU= xQB Qy \|}=OD@= Q}}eD x@ u=wD\|t =Q u=QW}B \|wQ}v OvwQ Q}}eD "OwW\|t Vy=m Q=JO '0 5056 Oa@\|@ u=wD\|t 8 \|x]@=Q R= \|Q}op=QoDv= =@ "O=O C@Uv u; uOW \|Wy=m w QDw `}RwD OvwQ \|xv}y@ \|x]kv QO,qFt "OQm x@U=Lt \|}=O@tq Qy QO =Q QwDwQ u=QW}B \|wQ}v pm Q=Okt uwDw}v21 912'xQB3ZQi=@xOW\|L=Q]O=@u}@QwDu=QW}B\|wQ}vQ=Okt'\|L=Q] "O};\|t CUO x@ 45 """ Tmwrwv}= O=@ \|SQv= ?P=H sDU}U l} \|R=UpOt &xQB lwv CaQU C@Uv % &QDt \|BwD `=aW %Rhub &ROrwv}Q OOa %Re &xQB O=OaD %B &xHQO xrtL \|x}w=R % &xHQO VJ}B \|x}w=R % &xHQO xv}y@ VJ}B \|x}w=R %opt &xHQO u=}QH x}w=R % &?amt QDt Q@ sQowr}m \|r=oJ % &xOR=@ % &C}@rY =} s=mLDU= ?}Q[ % "xk}kO Q@ QwO QwDwQ \|v=QwO CaQU %! (HAWT) blade shapes using experimental and numerical methods", Energies, 6 pp. 2784-2803 (2013). References `@=vt References `@=vt \Design modication & analysis for venturi section of INVELOX system to maximize power using multiple wind turbine", Int J Innovat Res Sci Technol, 3, pp. 125-127 (2017). 18. SnehalNarendrabhai, P. and Desmukh, T. \Numerical simulation of ow through INVELOX wind turbine sys- tem", International Journal of Renewable Energy Re- search, 8, pp. 291-301 (2018). 9. Glauert, H. \The analysis of experimental results in the windmill brake and vortex ring states of an airscrew", HM Stationery Oce (1926). 19. Tangler, J.L. and Somers, D.M. \NREL airfoil families for HAWTs", National Renewable Energy Lab., Golden, CO (United States) (1995). 10. Hsiao, F.-B., Bai, C.-J. and Chong, W.-T. \The perfor- mance test of three dierent horizontal axis wind turbine 20. http://airfoiltools.com/airfoil/details. 46 46
https://openalex.org/W4246514263	https://ora.ox.ac.uk/objects/uuid:c2ca694d-a23a-4411-9aba-711dc3e55033/files/mf851b4737db199abec2bc0b8926e4125	English	null	Quantum correlations between single telecom photons and a multimode on-demand solid-state quantum memory	null	2,017	cc-by	6,981	Quantum Correlations between Single Telecom Photons and a Multimode On-Demand Solid-State Quantum Memory Alessandro Seri,1 Andreas Lenhard,1 Daniel Rieländer,1 Mustafa Gündoğan,1,† Patrick M. Ledingham,1 Margherita Mazzera,1,* and Hugues de Riedmatten1,2 1ICFO-Institut de Ciences Fotoniques, The Barcelona Institute of Science and Technology, Mediterranean Technology Park, 08860 Castelldefels (Barcelona), Spain 2ICREA-Institució Catalana de Recerca i Estudis Avançats, 08015 Barcelona, Spain (Received 9 December 2016; revised manuscript received 30 March 2017; published 24 May 2017) Alessandro Seri,1 Andreas Lenhard,1 Daniel Rieländer,1 Mustafa Gündoğan,1,† Patrick M. Ledingham Margherita Mazzera,1,* and Hugues de Riedmatten1,2 1ICFO-Institut de Ciences Fotoniques, The Barcelona Institute of Science and Technology, Mediterranean Technology Park, 08860 Castelldefels (Barcelona), Spain 2ICREA-Institució Catalana de Recerca i Estudis Avançats, 08015 Barcelona, Spain (Received 9 December 2016; revised manuscript received 30 March 2017; published 24 May 2017) Alessandro Seri,1 Andreas Lenhard,1 Daniel Rieländer,1 Mustafa Gündoğan,1,† Patrick M. Ledingham,1 Margherita Mazzera,1,* and Hugues de Riedmatten1,2 1ICFO-Institut de Ciences Fotoniques, The Barcelona Institute of Science and Technology, Mediterranean Technology Park, 08860 Castelldefels (Barcelona), Spain 2ICREA-Institució Catalana de Recerca i Estudis Avançats, 08015 Barcelona, Spain (Received 9 December 2016; revised manuscript received 30 March 2017; published 24 May 2017) Quantum correlations between long-lived quantum memories and telecom photons that can propagate with low loss in optical fibers are an essential resource for the realization of large-scale quantum information networks. Significant progress has been realized in this direction with atomic and solid-state systems. Here, we demonstrate quantum correlations between a telecom photon and a multimode on- demand solid state quantum memory. This is achieved by mapping a correlated single photon onto a spin collective excitation in a Pr3þ∶Y2SiO5 crystal for a controllable time. The stored single photons are generated by cavity-enhanced spontaneous parametric down-conversion and heralded by their partner photons at telecom wavelength. These results represent the first demonstration of a multimode on-demand solid state quantum memory for external quantum states of light. They provide an important resource for quantum repeaters and pave the way for the implementation of quantum information networks with distant solid state quantum nodes. Subject Areas: Photonics, Quantum Physics, Quantum Information *margherita.mazzera@icfo.es †Present address: Cavendish Laboratory, University of Cambridge, J.J. Thomson Avenue, Cambridge CB3 0HE, United Kingdom. ‡Present address: Department of Physics, Clarendon Laboratory, University of Oxford, Oxford OX1 3PU, United Kingdom. Selected for a Viewpoint in Physics PHYSICAL REVIEW X 7, 021028 (2017) Selected for a Viewpoint in Physics PHYSICAL REVIEW X 7, 021028 (2017) DOI: 10.1103/PhysRevX.7.021028 Published by the American Physical Society II. EXPERIMENTAL DETAILS y p g Our cavity-enhanced SPDC source produces ultra- narrow-band photon pairs, where one photon, the idler, is in the telecom E-band at 1436 nm, and the other, the signal, is resonant with the Pr3þ optical transition at 606 nm, specifi- cally with the transition where the AFC is prepared [28,35]. The pump laser wavelength is 426.2 nm, and the average power for the measurements presented in this paper is 3.3 0.5 mW. The probability to obtain a single signal photon in front of the quantum memory conditioned on a detection in the idler SPD (i.e., the heralding efficiency) is ηH ¼ ð20.9 0.5Þ%. We also switch off the SPDC pump after the detection of the idler photons for 30 to 40 μs (depending on the experiment), thus interrupting the creation of photon pairs during the detection of the stored and FIG. 1. (a) The experimental setup. Photon pairs are created by cavity-enhanced spontaneous parametric down-conversion (SPDC) in a periodically poled lithium niobate (PPLN) crystal. At the output of the cavity, they are separated by a dichroic mirror (DM). The idler photon is further filtered with a cavity (FCav) to select a single frequency mode, before being coupled in an optical fiber and detected with an InGaAs single-photon detector (SPD). The signal photon is spectrally filtered with a band-pass filter (BPF) and an etalon, before being coupled in a single-mode optical fiber and directed towards the quantum memory (QM), where it is stored using the AFC scheme. Control pulses (CPs) are used to transfer optical excitations to spin waves and back; they are sent at an angle and counterpropagating with respect to the input photons. The retrieved single photon is spectrally filtered using a filter crystal (FC) before being detected by a silicon SPD. Temporal filtering is achieved with acousto-optic modulators, placed after the memory crystal, opened only when we expect the SW echo. This prevents additional hole burning in the filter crystal and the SPD to be blinded by eventual leakage of the control pulses. During the preparation of the memory crystal via optical pumping, the SPDs of both idler and signal arms are gated off. After each AFC preparation, the gates are opened, and we detect the arrival time of both photons of the pair during a measurement time of about 100 ms, leading to a duty cycle of 0.14. II. EXPERIMENTAL DETAILS “write-read” quantum memory that can store an externally prepared quantum optical state sharing a quantum corre- lation with a telecom band photon. To date, this has not been achieved with an on-demand spin-wave solid state quantum memory. Its successful realization with single photons requires an efficient quantum light source match- ing the spectral properties of the quantum memory [35,36] and the quasisuppression of the noise generated by the strong control pulses. These tasks are challenging because of the small spectral separation between the hyperfine states of the optically active ions (a few MHz in our system). Figure 1 depicts the experimental setup (a) and the relevant energy-level scheme of Pr3þ∶Y2SiO5 (b), where the chosen Λ system is indicated by arrows. The atomic frequency comb is prepared, following the spectral hole-burning procedure described in Ref. [37], at the frequency of the 1=2g −3=2e transition, and the control pulses drive the coherence from the 3=2e to the empty 3=2g storage state and back. The narrow-band spectral filtering of the noise resulting from the control pulses is accomplished with a second Pr3þ∶Y2SiO5 crystal, where a narrow trans- parency window (around 5.5 MHz) is burned at the frequency of the AFC [31]. An example of AFC prepared in the memory crystal overlapped with the narrow spectral hole burned in the filter crystal is reported in Fig. 2(a). Here, we demonstrate the spin-wave storage with on-demand retrieval of heralded single photons in a Pr3þ∶ Y2SiO5 crystal using the full atomic frequency comb (AFC) scheme. This is achieved by generating pairs of nondegenerate single photons where one photon is reso- nant with the optical transition of Pr3þ∶Y2SiO5 while the other photon is at telecom wavelength. The telecom photon is used to herald the presence of the other photon, which is stored as a SW in the crystal and retrieved on demand after a controllable time. We measure second- order cross-correlation values between the heralding and the retrieved photons, which exceed the classical bound fixed by the Cauchy-Schwarz inequality for storage times longer than 30 μs, effectively demonstrating quantum correlations between telecom photons and single spin waves in a solid. Moreover, we demonstrate that our memory can store spin waves in multiple independent temporal modes. I. INTRODUCTION [19,20] or optomechanical systems [10]. However, the most efficient and long-lived storage systems up to date are working at wavelengths far from the telecom window, leading to a large loss in optical fibers. Possible solutions to overcome this problem include quantum frequency con- version [3,21–25] or nondegenerate photon pair sources to establish entanglement between quantum memories and telecom photons [15,26–29]. The latter approach has been demonstrated using the atomic frequency comb scheme [30] in rare-earth-doped single crystals or waveguides [15,27,28], but the storage of photonic entanglement has only been performed so far in the excited state for short and predetermined storage times. Photonic quantum memories [1] are essential elements for quantum information networks [2], providing efficient and on-demandinterfacingbetweensinglephotonsandstationary qubits, e.g., atomic gases [3–6], electronic spins in diamonds [7,8], or phonons [9,10]. Besides featuring efficiencies and storage times comparable to or even surpassing those of atomic gases [11–14], solid state photonic memories based on rare-earth-doped crystals are becoming increasingly important as they offer prospects for scalability and inte- grability [15–18]. Most protocols for long-distance quan- tum communication require quantum memories connected to communication channels through optical fibers. One possible direction to achieve this goal is to use telecom quantum memories, e.g., based on erbium-doped solids Longer and programmable storage times can be obtained by transferring the optical atomic excitations to long-lived spin collective excitations [spin waves (SWs)] thanks to control laser pulses [30]. Recently, spin-wave storage of weak coherent states at the single-photon level [31,32], including qubit storage [31,33], has been demonstrated with rare-earth-doped crystals. The generation and storage of continuous variable entanglement between a multimode solid state quantum memory and a light field have also been reported recently [34]. In this experiment, light- matter entanglement is created within the memory between spontaneously emitted light and spin waves, the matter part then being converted into a light field. This is a “read- only” quantum memory [1], with the generated light fields being resonant and, for this demonstration, outside the telecommunication band. This motivates the need for a Published by the American Physical Society under the terms of the Creative Commons Attribution 4.0 International license. Further distribution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI. 021028-1 Published by the American Physical Society Published by the American Physical Society 2160-3308=17=7(2)=021028(7) ALESSANDRO SERI et al. PHYS. REV. X 7, 021028 (2017) II. EXPERIMENTAL DETAILS (b) Hyperfine splitting of the first sublevels of the ground 3H4 and the excited 1D2 manifold of Pr3þ in Y2SiO5. The arrows highlight the Λ system chosen for the storage. FIG. 1. (a) The experimental setup. Photon pairs are created by cavity-enhanced spontaneous parametric down-conversion (SPDC) in a periodically poled lithium niobate (PPLN) crystal. At the output of the cavity, they are separated by a dichroic mirror (DM). The idler photon is further filtered with a cavity (FCav) to select a single frequency mode, before being coupled in an optical fiber and detected with an InGaAs single-photon detector (SPD). The signal photon is spectrally filtered with a band-pass filter (BPF) and an etalon, before being coupled in a single-mode optical fiber and directed towards the quantum memory (QM), where it is stored using the AFC scheme. Control pulses (CPs) are used to transfer optical excitations to spin waves and back; they are sent at an angle and counterpropagating with respect to the input photons. The retrieved single photon is spectrally filtered using a filter crystal (FC) before being detected by a silicon SPD. Temporal filtering is achieved with acousto-optic modulators, placed after the memory crystal, opened only when we expect the SW echo. This prevents additional hole burning in the filter crystal and the SPD to be blinded by eventual leakage of the control pulses. During the preparation of the memory crystal via optical pumping, the SPDs of both idler and signal arms are gated off. After each AFC preparation, the gates are opened, and we detect the arrival time of both photons of the pair during a measurement time of about 100 ms, leading to a duty cycle of 0.14. (b) Hyperfine splitting of the first sublevels of the ground 3H4 and the excited 1D2 manifold of Pr3þ in Y2SiO5. The arrows highlight the Λ system chosen for the storage. 021028-2 021028-2 IV. SPIN-WAVE STORAGE OF HERALDED SINGLE PHOTONS To access the spectrum of the heralded photons to be stored, we compare two measurements. First, we measure the temporal distribution of coincidences between the signal and idler photons of the source alone [36]. This correlation function has a width of 78 ns (FWHM), which is the correlation time of the photons. From this value, we calculate a biphoton bandwidth of 2.8 MHz. In a second experiment, we employ the Pr3þ∶Y2SiO5 memory crystal as a tunable frequency filter [31,39,40]. We prepare a 800-kHz- wide spectral hole and record coincidence histograms when the central frequency is swept by about 10 MHz around the frequency of the signal photons. In this experiment, the photons passing through the transparency window are directly steered to the APD for detection, bypassing the temporal and spectral filtering stages. The coincidence rate as a function of the hole position [blue diamonds overlapped with the AFC in Fig. 2(a)] gives the spectral distribution of the heralded single photons at 606 nm. The result of this measurement agrees with the spectrum extrapolated from the signal-idler coincidence histogram measured immedi- ately after the SPDC source [36]. This is confirmed by the good overlap between the diamonds and the blue dotted line, which represents the convolution of a Lorentzian curve of width 2.8 MHz and the trace of the 800-kHz-wide spectral We first measure the coincidence histogram when the signal photons are sent through a 18-MHz-wide transparency window prepared in the memory crystal [input trace at 0 μs in Fig. 2(b)]. The correlation time between the signal and idler photons is τc ¼ ð89 4Þ ns, leading to a heralded photon line width of ð2.5 0.1Þ MHz [36]. The correlation between the two photons is inferred by measuring the normalized second-order cross-correlation function gð2Þ s;i ¼ Ps;i=ðPs · PiÞ, where Ps;i is the probability to detect a coincidence between idler and signal photons in a time window ΔTd ¼ 320 ns, while Pi and Ps are the uncondi- tional probabilities to detect each photon. We find a gð2Þ s;i value of 96 32. Then, we prepare an AFC with periodicity Δ and storethesinglephotonasacollectiveopticalatomicexcitation jψei ¼ ð1= ﬃﬃﬃﬃ N p Þ PN j¼1 eixj·kinjg1…ej…gNi, where kin is the wave vector of the incoming photon [see level scheme in Fig. 1(b)]. 021028-2 QUANTUM CORRELATIONS BETWEEN SINGLE TELECOM … PHYS. REV. X 7, 021028 (2017) FIG. 2. (a) Example of an atomic frequency comb prepared for τ ¼ 7.3 μs (red trace). The light violet trace is the transparency window that we burn in the filter crystal. The diamonds are signal-idler coincidence rates taken after preparing a single 800-kHz broad transparency window in the memory crystal and moving its frequency along the input photons. The error is smaller than the data points. The dotted blue line is a simulation of a Lorentzian peak with FWHM ¼ 2.8 MHz convoluted with a 800-kHz-wide spectral hole. (b) Time histograms of the input photons (black trace), the AFC echo at τ ¼ 7.3 μs (red trace), and the SW echo (blue trace) acquired over an integration time of 343 min. We construct the coincidence histogram by taking the detection of the idler photons as a start and the detection of a signal photon after the memory as a stop. The coincidence count rate in the AFC echo is 4= min and in the SW echo ≈1= min. The control pulses, detected with a reference photodetector, are displayed as plain pulses and are separated by Ts ¼ 6 μs. They are modulated in amplitude and frequency with Gaussian and hyperbolic tangent waveforms, respectively, as described in Ref. [38]. The peak power is 21 mW. The dashed vertical lines indicate the integration window for the signal (ΔTd ¼ 320 ns), while the dashed horizontal line represents the noise floor. (c) Coincidence counts for the SW echo at T ¼ τ þ Ts ¼ 13.3 μs normalized by the average noise level, along with its fit to a double exponential function, to account for the Lorentzian spectral shape of the SPDC photons. FIG. 2. (a) Example of an atomic frequency comb prepared for τ ¼ 7.3 μs (red trace). The light violet trace is the transparency window that we burn in the filter crystal. The diamonds are signal-idler coincidence rates taken after preparing a single 800-kHz broad transparency window in the memory crystal and moving its frequency along the input photons. The error is smaller than the data points. The dotted blue line is a simulation of a Lorentzian peak with FWHM ¼ 2.8 MHz convoluted with a 800-kHz-wide spectral hole. 021028-2 (b) Time histograms of the input photons (black trace), the AFC echo at τ ¼ 7.3 μs (red trace), and the SW echo (blue trace) acquired over an integration time of 343 min. We construct the coincidence histogram by taking the detection of the idler photons as a start and the detection of a signal photon after the memory as a stop. The coincidence count rate in the AFC echo is 4= min and in the SW echo ≈1= min. The control pulses, detected with a reference photodetector, are displayed as plain pulses and are separated by Ts ¼ 6 μs. They are modulated in amplitude and frequency with Gaussian and hyperbolic tangent waveforms, respectively, as described in Ref. [38]. The peak power is 21 mW. The dashed vertical lines indicate the integration window for the signal (ΔTd ¼ 320 ns), while the dashed horizontal line represents the noise floor. (c) Coincidence counts for the SW echo at T ¼ τ þ Ts ¼ 13.3 μs normalized by the average noise level, along with its fit to a double exponential function, to account for the Lorentzian spectral shape of the SPDC photons. retrieved photons. Further details about the experimental setup and the preparation of the atomic frequency comb can be found in Ref. [38]. hole. We estimate the spectral overlap between the heralded single photons and the AFC to be about 70%, which currently limits the AFC storage efficiency. IV. SPIN-WAVE STORAGE OF HERALDED SINGLE PHOTONS We obtain, for a preprogrammed storage time τ ¼ ð1=ΔÞ ¼ 7.3 μs, an efficiency ηAFC ¼ ð11.0 0.5Þ% and gð2Þ AFC;i ¼ 130 31 [see Fig. 2(b)]. This result represents an improvement in terms of gð2Þ AFC;i of more than 1 order of magnitude compared to the state of the art in the same system an improvement in terms of gð2Þ AFC;i of more than 1 order of magnitude compared to the state of the art in the same system 021028-3 PHYS. REV. X 7, 021028 (2017) ALESSANDRO SERI et al. [28]. The gð2Þ value increases after the AFC storage due to the fact that the stored photons are transferred to a temporal mode free of noise [28]. After the retrieval, we measure τc ¼ ð147 7Þ ns, larger than the value measured before storage. We attribute this temporal stretching to spectral mismatch between the input photons before the memory (FWHM ¼ 2.8 MHz) and the atomic frequency comb (total width 4 MHz), as evidenced in Fig. 2(a). [28]. The gð2Þ value increases after the AFC storage due to the fact that the stored photons are transferred to a temporal mode free of noise [28]. After the retrieval, we measure τc ¼ ð147 7Þ ns, larger than the value measured before storage. We attribute this temporal stretching to spectral mismatch between the input photons before the memory (FWHM ¼ 2.8 MHz) and the atomic frequency comb (total width 4 MHz), as evidenced in Fig. 2(a). infer gð2Þ swe;i. With our filtering strategy, we reach a noise floor of ð2.0 0.1Þ × 10−3 photons per storage trial at the memory crystal (horizontal dashed line in the SW echo temporal mode). The correlation time of τc ¼ ð200 40Þ ns exceeds the one after storage in the excited state. This further increase after the SW storage is attributed to the limited chirp of the control pulses (see Ref. [38]). We then perform spin-wave storage experiments by sending pairs of strong control pulses after the detection of each heralding photon. The first control pulse with wave vector kC transfers the collective optical excitation jψei to a collective spin excitation (a spin wave), which can be written as jψswi ¼ ð1= ﬃﬃﬃﬃ N p Þ PN j¼1 eixj·ðkin−kCÞjg1…sj…gNi. V. QUANTUM CORRELATION BETWEEN SINGLE TELECOM PHOTONS AND SINGLE SPIN WAVES To investigate the nonclassical nature of the photon correlation after the SW storage, we measure gð2Þ swe;iðΔTdÞ and compare it to the unconditional autocorrelation of the idler [gð2Þ i;i ðΔTdÞ] and retrieved signal [gð2Þ swe;sweðΔTdÞ] fields, respectively. To access these quantities, we correlate photon detections from different unconditional storage iterations [see Fig. 3(a)]. The time between two consecutive storage trials is 190 μs. In each trial (500 per comb preparation), we maintain the gate of the idler SPD open during 4.5 μs before sending the control pulses. We find gð2Þ swe;i ¼ 6.1 0.7. We note that our gð2Þ swe;i is limited by the spin-wave read-out efficiency, which we estimate to be approximately FIG. 3. (a) Unconditional cross-correlation between the idler photons and the retrieved signal photons. The gð2Þ swe;iðΔTd ¼ 320 nsÞ value for this measurement is 6.1 0.7. The classical bound given by the Cauchy-Schwarz inequality is reported as a horizontal line. The error bars are calculated considering Poissonian statistics. (b) Scheme representing the analysis of the cross-correlation measurement to evidence the multimodality. Both idler and retrieved signal integration windows are divided into smaller intervals (640 ns), and the correlation is calculated between intervals separated by different storage time. (c) Cross-correlation value between idler and retrieved signal photons detected in small temporal modes separated by different storage times. For this analysis, we also set ΔTd ¼ 640 ns for better statistics. The gð2Þ swe;iðΔTd ¼ 640 nsÞ exceeds the classical threshold for ΔTd ¼ 640 ns (reported in the color bar) only for windows separated by the total storage time T ¼ 13.3 μs. Violations outside these windows are not statistically significant because of a low number of counts. (d) Cross- correlation value between the idler photons and the retrieved signal photons (full circles) and the coincidence counts in the SWecho (empty squares) as a function of the detection window for the idler photons. Both the signal detection window and the coincidence window remain constant at 4.5 μs and 320 ns, respectively. The classical threshold is also reported as a horizontal line. The integration time for this measurement is 38.5 h. The error bars are calculated considering Poissonian statistics. FIG. 3. (a) Unconditional cross-correlation between the idler photons and the retrieved signal photons. The gð2Þ swe;iðΔTd ¼ 320 nsÞ value for this measurement is 6.1 0.7. IV. SPIN-WAVE STORAGE OF HERALDED SINGLE PHOTONS For a storage time in the ground state of Ts ¼ 6 μs (thus, a total storage time T ¼ Ts þ τ ¼ 13.3 μs), we detect the retrieved photons, i.e., the spin-wave echo (swe), with an efficiency ηsw ¼ ð3.6 0.2Þ%. Note that ηsw is inferred with a coincidence window of ΔTd ¼ 320 ns, containing 80% of the signal, and it includes the loss in the filter crystal. Figure 2(c) shows a magnification of the SW echo mode, normalized by the average value of the noise measured outside the peak, leading directly to the signal-to-noise ratio (SNR) of the stored and retrieved photons. We observe a maximum SNR of around 5. This curve can also be used to V. QUANTUM CORRELATION BETWEEN SINGLE TELECOM PHOTONS AND SINGLE SPIN WAVES The classical bound given by the Cauchy-Schwarz inequality is reported as a horizontal line. The error bars are calculated considering Poissonian statistics. (b) Scheme representing the analysis of the cross-correlation measurement to evidence the multimodality. Both idler and retrieved signal integration windows are divided into smaller intervals (640 ns), and the correlation is calculated between intervals separated by different storage time. (c) Cross-correlation value between idler and retrieved signal photons detected in small temporal modes separated by different storage times. For this analysis, we also set ΔTd ¼ 640 ns for better statistics. The gð2Þ swe;iðΔTd ¼ 640 nsÞ exceeds the classical threshold for ΔTd ¼ 640 ns (reported in the color bar) only for windows separated by the total storage time T ¼ 13.3 μs. Violations outside these windows are not statistically significant because of a low number of counts. (d) Cross- correlation value between the idler photons and the retrieved signal photons (full circles) and the coincidence counts in the SWecho (empty squares) as a function of the detection window for the idler photons. Both the signal detection window and the coincidence window remain constant at 4.5 μs and 320 ns, respectively. The classical threshold is also reported as a horizontal line. The integration time for this measurement is 38.5 h. The error bars are calculated considering Poissonian statistics. 021028-4 021028-4 021028-4 QUANTUM CORRELATIONS BETWEEN SINGLE TELECOM … PHYS. REV. X 7, 021028 (2017) ηR ¼ 24%, while the write efficiency is ηW ¼ 31% (see Ref. [38] for a detailed discussion). With this unconditional sequence, the measured noise floor [ð1.3 0.1Þ × 10−3 photons per storage trial] is lower than with the conditional one (Sec. IV). We attribute this result to the fact that the number of control pulse pairs per comb is larger in the unconditional sequence.Thisprobablycontributes to further emptying of the spin storage state. The classical bound from the Cauchy-Schwarz inequality gð2Þ swe;i < ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ gð2Þ swe;swe · gð2Þ i;i q is indicated in Fig. 3(a) as a horizontal line. The measured unconditional autocorrelations are (also for ΔTd ¼ 320 ns) gð2Þ i;i ¼ 1.32 0.04 [36] and gð2Þ swe;swe ¼ 1.0 0.4 (see Ref. [38]). The Cauchy-Schwarz parameter R ¼ ððgð2Þ swe;iÞ2= gð2Þ swe;swe · gð2Þ i;i Þ ¼ 28 12 exceeds the classical limit of R ¼ 1 by more than 2 standard deviations. The confidence level for violating the Cauchy-Schwarz inequality, i.e., for observing a nonclassical correlation between the telecom heralding photon and the single spin wave stored in the crystal, is 98.8%. If a larger coincidence window is con- sidered, ΔTd ¼ 1 μs, the R value is reduced to 8.3 2.3 because of a bigger contribution of noise in the gð2Þ swe;i. On the other hand, because of a reduction of the statistical error, the confidence level for the demonstration of nonclassical correlation rises up to 99.92% (see Ref. [38]). boundoverthewholerange.Definingthenumberoftemporal modes as Nm ¼ Δti=ΔTd, we confirm nonclassical storage of a maximum of seven temporal modes with a ΔTd ¼ 640 ns that contains 94% of the coincidence peak. However, considering ΔTd ¼ 320 ns, which still contains 80% of the SW echo, a 4.5 μs-wide gate can accommodate up to 14 independent temporal modes. ηR ¼ 24%, while the write efficiency is ηW ¼ 31% (see Ref. [38] for a detailed discussion). With this unconditional sequence, the measured noise floor [ð1.3 0.1Þ × 10−3 photons per storage trial] is lower than with the conditional one (Sec. IV). We attribute this result to the fact that the number of control pulse pairs per comb is larger in the unconditional sequence.Thisprobablycontributes to further emptying of the spin storage state. 021028-4 The classical bound from Finally, to illustrate the ability to read out the stored spin wave ondemand, we perform storageexperiments at different SW storage times. For these measurements, we implement a semiconditional storage sequence in order to obtain good statistics with shorter integration time. We wait for heralding photons and, after each detection, we send 15 pairs of control pulses (each pair beingseparated fromtheneighboringoneby 190 μs), which we exploit to correlate the idler and the retrieved signal (see Ref. [38]). The measured storage and retrieval efficiencies are reported in Fig. 4(a), together with a Gaussian fit, which accounts for the inhomogeneous broad- ening of the spin state. As a fitting parameter, we obtain the spin inhomogeneous line width γinh ¼ ð20 3Þ kHz, in good agreement with that measured in different experiments on the same crystal [18,31]. This result further confirms that the photons are stored as spin waves. The second-order cross- correlation function for increasing SW storage times Ts is shown in Fig. 4(b). The red dashed curve is calculated by consideringtheGaussian fit ofthe signal decay [solid curve in panel (a)], normalized by the source heralding efficiency ηH and theaveragenoise floor of ð1.9 0.2Þ × 10−3 photonsper trial (the average being over the different Ts). We measure The main advantage of the full AFC protocol is the possibility to store multiple distinguishable temporal modes while maintaining their coherence and quantum correlation [30]. This ability is crucial for applications in quantum information protocols, e.g., to enable temporally multiplexed quantum repeater protocols with high communication speed [26] and storage of time-bin qubits robust against decoherence in optical fibers. To test this aspect, we perform experiments with detection gates much longer than the photon duration. We divide both the idler and the retrieved signal detection windows, Δti and Δts, respectively, into smaller temporal modes of width 640 ns, as sketched in Fig. 3(b). For this analysis, we also consider ΔTd ¼ 640 ns in order to have better statistics. We then check that we have nonclassical correlations between modes separated by the total storage time T ¼ Ts þ τ ¼ 13.3 μs and classical cor- relations between modes separated by T ≠13.3 μs, as shown in Fig. 3(c). Contrary to other temporally multimode storage protocols [11,34,41], in the full AFC protocol, the total storage time is maintained for the different temporal modes. VI. DISCUSSION AND CONCLUSION The demonstrated quantum correlation between a tele- com photon and a spin wave in a solid is an essential resource to generate entanglement between remote solid state quantum memories [26]. The measured value of gð2Þ swe;i after spin-wave storage is currently limited by the signal-to- noise ratio of the retrieved photon, which is, in turn, mostly limited by the low storage and retrieval efficiency. This value could be greatly improved by using higher optical depth [11] with higher-quality combs, or crystals in impedance matched cavities [42,43]. The storage time is currently limited by the spin inhomogeneous broadening and could be increased using spin-echo and dynamical decoupling techniques [32], with prospects for achieving values up to one minute [13] in our crystal, while even longer storage times (of order of hours) may be available in Eu3þ∶Y2SiO5 [14]. Finally, our experiment could be extended to the storage of entangled qubits, e.g., using time-bin encoding [31]. [5] S.-J. Yang, X.-J. Wang, X.-H. Bao, and J.-W. Pan, An Efficient Quantum Light-Matter Interface with Sub-Second Lifetime, Nat. Photonics 10, 381384 (2016). [6] Y.-W. Cho, G. T. Campbell, J. L. Everett, J. Bernu, D. B. Higginbottom, M. T. Cao, J. Geng, N. P. Robins, P. K. Lam, and B. C. Buchler, Highly Efficient Optical Quantum Memory with Long Coherence Time in Cold Atoms, Optica 3, 100 (2016). [7] B. Hensen, H. Bernien, A. E. Dreau, A. Reiserer, N. Kalb, M. S. Blok, J. Ruitenberg, R. F. L. Vermeulen, R. N. Schouten, C. Abellan et al., Loophole-Free Bell Inequality Violation Using Electron Spins Separated by 1.3 Kilometres, Nature (London) 526, 682 (2015). [8] S. Yang et al., High-Fidelity Transfer and Storage of Photon States in a Single Nuclear Spin, Nat. Photonics 10, 507 (2016). [9] D. G. England, K. A. G. Fisher, J.-P. W. MacLean, P. J. Bustard, R. Lausten, K. J. Resch, and B. J. Sussman, Storage and Retrieval of THz-Bandwidth Single Photons Using a Room-Temperature Diamond Quantum Memory, Phys. Rev. Lett. 114, 053602 (2015). In conclusion, we have reported the first demonstration of quantum storage of heralded single photons in an on-demand solid state quantum memory. We have shown that the nonclassical correlations between the heralding and the stored photons are maintained after the retrieval, thus demonstrating nonclassical correlations between single tele- com photons and single collective spin excitations in a solid. 021028-4 Toadditionallydemonstratethat themultimodecapacitydoes not decrease the correlations, we compute gð2Þ swe;iðΔTd ¼ 320 nsÞ for idler detection windows Δti varying from 320 ns to 4.5 μs, as shown in Fig. 3(d) (full circles), together with the coincidence counts measured in the center peak for each window size (empty squares). As expected, the latter increases linearly with Δti, but the gð2Þ swe;i value remains constant, within the error bar, and well above the classical FIG. 4. (a) Spin-wave storage efficiency as a function of the storage time Ts. The solid line is a fit of the experimental data, taking into account a Gaussian inhomogeneous broadening of the spin state. The spin inhomogeneous broadening extracted from the fit is γinh ¼ ð20 3Þ kHz. (b) The gð2Þ swe;ið320 nsÞ value as a function of the storage time. The classical threshold is reported as a horizontal line. In both panels, the error bars are calculated by considering Poissonian statistics, and the circled data points refer to the measurement reported in Fig. 2. FIG. 4. (a) Spin-wave storage efficiency as a function of the storage time Ts. The solid line is a fit of the experimental data, taking into account a Gaussian inhomogeneous broadening of the spin state. The spin inhomogeneous broadening extracted from the fit is γinh ¼ ð20 3Þ kHz. (b) The gð2Þ swe;ið320 nsÞ value as a function of the storage time. The classical threshold is reported as a horizontal line. In both panels, the error bars are calculated by considering Poissonian statistics, and the circled data points refer to the measurement reported in Fig. 2. 021028-5 021028-5 PHYS. REV. X 7, 021028 (2017) ALESSANDRO SERI et al. nonclassical correlations between the idler and the retrieved signal photons up to a total storage time T ¼ τ þ Ts ¼ 32.3 μs. Note that, while we measure the Cauchy-Schwarz parameter R with unconditional measurements at T ¼ 13.3 μs, to assess the nonclassicality for longer storage times, we make the assumption that the retrieved signal autocorre- lation does not increase for semiconditional measurements at different storage times. This is a conservative estimate since the gð2Þ swe;swe value is mainly determined by the noise in the read-out, which we verify to be constant over thewhole range of storage times investigated and not bunched (see Ref. [38]). 021028-4 Under this hypothesis, the Cauchy-Schwarz inequality is violated at T ¼ 32.3 μs with a confidence of 94% for ΔTd ¼ 320 ns. MINECO Severo Ochoa through Grant No. SEV-2015- 0522 and through the Ph.D. Fellowship Program (for A. S.), by AGAUR via 2014 SGR 1554, by Fundació Cellex, and by CERCA Programme/Generalitat de Catalunya. MINECO Severo Ochoa through Grant No. SEV-2015- 0522 and through the Ph.D. Fellowship Program (for A. S.), by AGAUR via 2014 SGR 1554, by Fundació Cellex, and by CERCA Programme/Generalitat de Catalunya. [1] M. Afzelius, N. Gisin, and H. de Riedmatten, Quantum Memory for Photons, Phys. Today 68, No. 12, 42 (2015). [2] H. J. Kimble, The Quantum Internet, Nature (London) 453, 1023 (2008). [3] A. G. Radnaev, Y. O. Dudin, R. Zhao, H. H. Jen, S. D. Jenkins, A. Kuzmich, and T. A. B. Kennedy, A Quantum Memory with Telecom-Wavelength Conversion, Nat. Phys. 6, 894 (2010). [4] K. S. Choi, H. Deng, J. Laurat, and H. J. Kimble, Mapping Photonic Entanglement into and out of a Quantum Memory, Nature (London) 452, 67 (2008). VI. DISCUSSION AND CONCLUSION Finally, we showed that the full atomic frequency comb protocol employed allows one to store a single photon in multiple independent temporal modes. These results re- present a fundamental step towards the implementation of quantum communication networks where solid state quan- tum memories are interfaced with the current fiber networks operating in the telecom window [26]. [10] R. Riedinger, S. Hong, R. A. Norte, J. A. Slater, J. Shang, A. G. Krause, V. Anant, M. Aspelmeyer, and S. Gröblacher, Non-classical Correlations between Single Photons and Phonons from a Mechanical Oscillator, Nature (London) 530, 313 (2016). [11] M. P. Hedges, J. J. Longdell, Y. Li, and M. J. Sellars, Efficient Quantum Memory for Light, Nature (London) 465, 1052 (2010). [12] D. Schraft, M. Hain, N. Lorenz, and T. Halfmann, Stopped Light at High Storage Efficiency in a Pr3þ∶Y2SiO5 Crystal, Phys. Rev. Lett. 116, 073602 (2016). [13] G. Heinze, C. Hubrich, and T. Halfmann, Stopped Light and Image Storage by Electromagnetically Induced Transpar- ency up to the Regime of One Minute, Phys. Rev. Lett. 111, 033601 (2013). QUANTUM CORRELATIONS BETWEEN SINGLE TELECOM … PHYS. REV. X 7, 021028 (2017) Broadband Waveguide Quantum Memory for Entangled Photons, Nature (London) 469, 512 (2011). Broadband Waveguide Quantum Memory for Entangled Photons, Nature (London) 469, 512 (2011). [30] M. Afzelius, C. Simon, H. de Riedmatten, and N. Gisin, Multimode Quantum Memory Based on Atomic Frequency Combs, Phys. Rev. A 79, 052329 (2009). [16] T. Zhong, J. M. Kindem, E. Miyazono, and A. Faraon, Nanophotonic Coherent Light-Matter Interfaces Based on Rare-Earth-Doped Crystals, Nat. Commun. 6, 8206 (2015). [31] M. Gündoğan, P. M. Ledingham, K. Kutluer, M. Mazzera, and H. de Riedmatten, Solid State Spin-Wave Quantum Memory for Time-Bin Qubits, Phys. Rev. Lett. 114, 230501 (2015). [17] S. Marzban, J. G. Bartholomew, S. Madden, K. Vu, and M. J. Sellars, Observation of Photon Echoes from Evanescently Coupled Rare-Earth Ions in a Planar Waveguide, Phys. Rev. Lett. 115, 013601 (2015). [32] P. Jobez, C. Laplane, N. Timoney, N. Gisin, A. Ferrier, P. Goldner, and M. Afzelius, Coherent Spin Control at the Quantum Level in an Ensemble-Based Optical Memory, Phys. Rev. Lett. 114, 230502 (2015). [18] G. Corrielli, A. Seri, M. Mazzera, R. Osellame, and H. de Riedmatten, Integrated Optical Memory Based on Laser- Written Waveguides, Phys. Rev. Applied 5, 054013 (2016). [33] C. Laplane, P. Jobez, J. Etesse, N. Timoney, N. Gisin, and M. Afzelius, Multiplexed On-Demand Storage of Polari- zation Qubits in a Crystal, New J. Phys. 18, 013006 (2016). [19] B. Lauritzen, J. Minar, H. de Riedmatten, M. Afzelius, N. Sangouard, C. Simon, and N. Gisin, Telecommunication- Wavelength Solid-State Memory at the Single Photon Level, Phys. Rev. Lett. 104, 080502 (2010). [34] K. R. Ferguson, S. E. Beavan, J. J. Longdell, and M. J. Sellars, Generation of Light with Multimode Time-Delayed Entanglement Using Storage in a Solid-State Spin-Wave Quantum Memory, Phys. Rev. Lett. 117, 020501 (2016). [20] E. Saglamyurek, J. Jin, V. B. Verma, M. D. Shaw, F. Marsili, S. W. Nam, D. Oblak, and W. Tittel, Quantum Storage of Entangled Telecom-Wavelength Photons in an Erbium- Doped Optical Fibre, Nat. Photonics 9, 83 (2015). [35] J. Fekete, D. Rieländer, M. Cristiani, and H. de Riedmatten, Ultranarrow-Band Photon-Pair Source Compatible with Solid State Quantum Memories and Telecommunication Networks, Phys. Rev. Lett. 110, 220502 (2013). [21] K. De Greve et al., Quantum-Dot Spin-Photon Entangle- ment via Frequency Downconversion to Telecom Wave- length, Nature (London) 491, 421 (2012). [36] D.Rieländer, A.Lenhard, M. QUANTUM CORRELATIONS BETWEEN SINGLE TELECOM … Mazzera, and H.de Riedmatten, Cavity Enhanced Telecom Heralded Single Photons for Spin-Wave Solid State Quantum Memories, New J. Phys. 18, 123013 (2016). [22] B. Albrecht, P. Farrera, X. Fernandez-Gonzalvo, M. Cristiani, and H. de Riedmatten, A Waveguide Frequency Converter Connecting Rubidium-Based Quantum Memo- ries to the Telecom C-Band, Nat. Commun. 5, 3376 (2014). [37] P. Jobez, N. Timoney, C. Laplane, J. Etesse, A. Ferrier, P. Goldner, N. Gisin, and M. Afzelius, Towards Highly Multimode Optical Quantum Memory for Quantum Repeaters, Phys. Rev. A 93, 032327 (2016). [23] N. Maring, K. Kutluer, J. Cohen, M. Cristiani, M. Mazzera, P. M. Ledingham, and H. de Riedmatten, Storage of Up- Converted Telecom Photon in a Doped Crystal, New J. Phys. 16, 113021 (2014). [38] See Supplemental Material at http://link.aps.org/ supplemental/10.1103/PhysRevX.7.021028 for details about the experimental setup, the preparation and charac- terization of the AFC, the storage sequences and the discussion of the results. [24] P. Farrera, N. Maring, B. Albrecht, G. Heinze, and H. de Riedmatten, Nonclassical Correlations between a C-Band Telecom Photon and a Stored Spin-Wave, Optica 3, 1019 (2016). [25] R. Ikuta, T. Kobayashi, K. Matsuki, S. Miki, T. Yamashita, H. Terai, T. Yamamoto, M. Koashi, T. Mukai, and N. Imoto, Heralded Single Excitation of Atomic Ensemble via Solid- State-Based TelecomPhoton Detection,Optica3,1279(2016). [39] H. Zhang, M. Sabooni, L. Rippe, C. Kim, S. Kröll, L. V. Wang, and P. R. Hemmer, Slow Light for Deep Tissue Imaging with Ultrasound Modulation, Appl. Phys. Lett. 100, 131102 (2012). [26] C. Simon, H. de Riedmatten, M. Afzelius, N. Sangouard, H. Zbinden, and N. Gisin, Quantum Repeaters with Photon Pair Sources and Multimode Memories, Phys. Rev. Lett. 98, 190503 (2007). [40] S. E. Beavan, E. A. Goldschmidt, and M. J. Sellars, Demonstration of a Dynamic Bandpass Frequency Filter in a Rare-Earth Ion-Doped Crystal, J. Opt. Soc. Am. B 30, 1173 (2013). [27] C. Clausen, I. Usmani, F. Bussières, N. Sangouard, M. Afzelius, H. de Riedmatten, and N. Gisin, Quantum Storage of Photonic Entanglement in a Crystal, Nature (London) 469, 508 (2011). [41] P. Sekatski, N. Sangouard, N. Gisin, H. de Riedmatten, and M. Afzelius, Photon-Pair Source with Controllable Delay Based on Shaped Inhomogeneous Broadening of Rare-Earth-Metal-Doped Solids, Phys. Rev. A 83, 053840 (2011). [28] D. Rieländer, K. Kutluer, P. M. Ledingham, M. Gündoğan, J. Fekete, M. Mazzera, and H. de Riedmatten, Quantum Storage of Heralded Single Photons in a Praseodymium- Doped Crystal, Phys. Rev. Lett. ACKNOWLEDGMENTS [14] M. Zhong, M. P. Hedges, R. L. Ahlefeldt, J. G. Bartholomew, S. E. Beavan, S. M. Wittig, J. J. Longdell, and M. J. Sellars, Optically Addressable Nuclear Spins in a Solid with a Six- Hour Coherence Time, Nature (London) 517, 177 (2015). We acknowledge financial support by the ERC Starting Grant QuLIMA, by the Spanish Ministry of Economy and Competitiveness (MINECO) and Fondo Europeo de Desarrollo Regional (FEDER) (FIS2015-69535-R), by [15] E. Saglamyurek, N. Sinclair, J. Jin, J. A. Slater, D. Oblak, F. Bussières, M. George, R. Ricken, W. Sohler, and W. Tittel, 021028-6 QUANTUM CORRELATIONS BETWEEN SINGLE TELECOM … QUANTUM CORRELATIONS BETWEEN SINGLE TELECOM … 112, 040504 (2014). [42] M. Sabooni, Q. Li, S. Kröll, and L. Rippe, Efficient Quantum Memory Using a Weakly Absorbing Sample, Phys. Rev. Lett. 110, 133604 (2013). [29] W. Zhang, D.-S. Ding, S. Shi, Y. Li, Z.-Y. Zhou, B.-S. Shi, and G.-C. Guo, Storing a Single Photon as a Spin Wave Entangled with a Flying Photon in the Telecommunication Bandwidth, Phys. Rev. A 93, 022316 (2016). [43] P. Jobez, I. Usmani, N. Timoney, C. Laplane, N. Gisin, and M. Afzelius, Cavity-Enhanced Storage in an Optical Spin-Wave Memory, New J. Phys. 16, 083005 (2014). 021028-7
https://openalex.org/W2809178129	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0199549&type=printable	English	null	Characterization of normative hand movements during two functional upper limb tasks	PloS one	2,018	cc-by	12,786	Editor: Antoine Nordez, Universite de Nantes, FRANCE Received: December 21, 2017 Accepted: June 8, 2018 Published: June 21, 2018 RESEARCH ARTICLE Methods Twenty non-disabled participants were recruited to perform two tasks: the Pasta Box Task and Cup Transfer Task. A 12-camera motion capture system was used to collect kinematic data from which hand movement and grip aperture measures were calculated. Measures reported for reach-grasp and transport-release segments were hand distance travelled, hand trajectory variability, movement time, peak and percent-to-peak hand velocity, number of movement units, peak and percent-to-peak grip aperture, and percent-to-peak hand deceleration. A between-session repeatability analysis was conducted on 10 participants. Copyright: © 2018 Valevicius et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files (S3 Data_Supplementary Data File). Funding: This work was sponsored by the Defense Advanced Research Projects Agency (DARPA) BTO under the auspices of Dr. Doug Weber and Dr. Al Emondi through the DARPA Contracts Management Office; Grant/Contract No. N66001- 15-C-4015 (JSH). Partial funding support was also received through Discovery Grants from the Natural Sciences and Engineering Research Characterization of normative hand movements during two functional upper limb tasks Aïda M. Valevicius1, Quinn A. Boser1, Ewen B. Lavoie2, Glyn S. Murgatroyd3, Patrick M. Pilarski4, Craig S. Chapman2, Albert H. Vette1,3,5, Jacqueline S. Hebert1,3,4* 1 Department of Biomedical Engineering, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada, 2 Faculty of Kinesiology, Sport, and Recreation, University of Alberta, Edmonton, Alberta, Canada, 3 Glenrose Rehabilitation Hospital, Alberta Health Services, Edmonton, Alberta, Canada, 4 Department of Medicine, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada, 5 Department of Mechanical Engineering, Faculty of Engineering, University of Alberta, Edmonton, Alberta, Canada a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * jhebert@ualberta.ca Background Citation: Valevicius AM, Boser QA, Lavoie EB, Murgatroyd GS, Pilarski PM, Chapman CS, et al. (2018) Characterization of normative hand movements during two functional upper limb tasks. PLoS ONE 13(6): e0199549. https://doi.org/ 10.1371/journal.pone.0199549 Dexterous hand function is crucial for completing activities of daily living (ADLs), which typi- cally require precise hand-object interactions. Kinematic analyses of hand trajectory, hand velocity, and grip aperture provide valuable mechanistic insights into task performance, but there is a need for standardized tasks representative of ADLs that are amenable to motion capture and show consistent performance in non-disabled individuals. Our objective was to develop two standardized functional upper limb tasks and to quantitatively characterize the kinematics of normative hand movement. Editor: Antoine Nordez, Universite de Nantes, FRANCE * jhebert@ualberta.ca Introduction Dexterous hand function is essential for successfully performing many activities of daily living (ADLs). Neurological or musculoskeletal impairments such as stroke [1], spinal cord injury [2], and upper limb amputation [3,4] result in deficiencies in hand and upper limb function, such that alternate control strategies and compensations must be used to accomplish ADLs. A key aspect of ADLs are hand-object interactions, where successfully reaching, grasping, and transferring an object is crucial for task completion. Quantifying these hand-object interac- tions in non-disabled populations to allow comparison to strategies used by impaired individ- uals could provide a valuable tool for assessing hand function. p g One method of examining hand-object interactions is through kinematic analysis using optical motion capture, either to measure joint angles of the full upper body kinematic chain to infer hand function, or to directly quantify hand function through specific features of how the hand moves [5]. However, the design of the object interaction task becomes crucial in developing a standard assessment protocol that is repeatable and reliable for comparison within a normative population and across impaired populations. Specifically, hand movement during object interactions is influenced by an object’s extrinsic (location and orientation) and intrinsic parameters (size, color, shape, mass, and texture) [6,7]. When reaching for objects, normative adult behaviour will show typical hand trajectories, hand velocities, and grip aper- ture motions [7–9]. Reaching is influenced by the object’s extrinsic parameters, and typically characterized by a straight or gently curved hand trajectory path from an initial hand position to the object [8], with a smooth bell-shaped velocity profile with one velocity peak occurring approximately halfway through the movement [9], and with greater peak hand velocities observed for targets that are further away [10]. Grasp is primarily influenced by intrinsic parameters [6] and characterized by hand pre-shaping at hand movement onset [7], with grip aperture (the distance between the thumb and index finger) reaching a maximum at approxi- mately 60 to 70% of the reaching phase, followed by hand closing around the object [9]. Grip aperture is also a function of object size, where a larger grip aperture is required for larger objects [6,9]. Given the importance of object interactions in ADLs and the influence of an object’s intrin- sic and extrinsic proprieties on hand movement, task selection for kinematic analysis should include ecologically valid tasks for clinical assessment. Discussion Competing interests: The authors have declared that no competing interests exist. Competing interests: The authors have declared that no competing interests exist. Normative hand kinematics for two standardized functional tasks challenging various aspects of hand-object interactions important for ADLs showed excellent repeatability. The consistency in normative task performance across a variety of task demands shows prom- ise as a potential outcome assessment for populations with upper limb impairment. movements. Movements requiring the clearing of an obstacle while transporting an object displayed a double velocity peak and typically a longer deceleration phase. Council of Canada (RGPIN-2014-04666; RGPIN- 2014-05248) (AHV). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Results Movement times were longer for transport-release compared to reach-grasp for every movement. Hand and grip aperture measures had low variability, with 55 out of 63 measures showing good repeatability (ICC > 0.75). Cross-body movements in the Pasta Box Task had longer movement times and reduced percent-to-peak hand velocity values. The Cup Trans- fer Task showed decoupling of peak grip aperture and peak hand deceleration for all 1 / 21 PLOS ONE \| https://doi.org/10.1371/journal.pone.0199549 June 21, 2018 Normative hand movements during two functional upper limb tasks movements. Movements requiring the clearing of an obstacle while transporting an object displayed a double velocity peak and typically a longer deceleration phase. PLOS ONE \| https://doi.org/10.1371/journal.pone.0199549 June 21, 2018 Normative hand movements during two functional upper limb tasks asymmetries in hand velocity profile and decoupling of reach and grasp in those with hemipar- esis [1,16,17], spinal cord injury [2] and with use of a prosthesis [18]. These studies have used a variety of objects for grasping such as a cup [16], ball [1], or cylinder [1,17,18]. However, it has been shown that not only the object characteristics but also the goal of the object interaction will affect grasp kinematics [19]. Therefore, a standardized functional task protocol for kine- matic assessment with applicability to clinical populations would ideally involve reaching and grasping real objects, with specific movement goals, in order to most accurately mimic typical daily tasks. Clinical assessments currently exist that evaluate hand function involving hand-object interactions. Performance tests, such as the Jebsen Test of Hand Function [20], the Box and Blocks Test [21], and Standardized Object Test [22], as well as subjective rater tests, such as the Action Research Arm Test [23] and Assessment of Capacity of Myoelectric Control (ACMC) [24] are commonly administered in a clinical environment. Although these tests involve hand- object interactions and provide a global outcome measurement of function, they do not allow for precise quantitative assessment of grasp, dexterity, movement quality, and efficiency. Ide- ally, functional assessment tasks used for kinematic analysis would utilize elements of these current clinical assessments, such as moving the arm in different positions; lateral reaches; crossing the body’s midline; adjusting hand opening and closing; varying grasp patterns; mod- ulating force when gripping; and grasping and releasing objects. The objectives of this study were to develop two standardized functional upper limb tasks with hand-object interactions that result in consistent and repeatable performance in non- disabled individuals, and to use kinematic analysis to quantitatively characterize hand move- ment for those two tasks. The hypothesis was that the performance of the tasks in non-disabled individuals would result in consistent performance (low variability) within and across per- formers and good between-session repeatability due to the standardized sequencing of the task movements. PLOS ONE \| https://doi.org/10.1371/journal.pone.0199549 June 21, 2018 Introduction Although some upper limb kinematic assessment protocols for motion capture mimic functional movements, such as hand to head (e.g., for combing hair), hand to shoulder (e.g., for dressing, applying deodorant), and hand to back pocket (e.g., for reaching for wallet, perineal care) [11–15], they provide limited informa- tion on hand function during real object interactions. In clinical populations, reaching and grasping tasks using real objects have shown alterations in hand kinematics such as PLOS ONE \| https://doi.org/10.1371/journal.pone.0199549 June 21, 2018 2 / 21 Functional task development The tasks were developed through iteration and consensus by a team involving a movement neuroscientist, kinesiologist, physiatrist, and occupational therapist. Current best-practice out- come measures for upper limb function were explored for commonalities in task requirements [20,21,24–27]. Functional tasks incorporated elements that would be challenging, but not impossible for clinical populations to complete, while being representative of ‘real-world’ tasks that might be performed in anyone’s daily environment. A key feature of the tasks was that they needed to be amenable to motion capture with standardized, discrete movement sequences to optimize kinematic analysis of hand movement characteristics across performers. Specifically, standardization of order of task execution allows kinematic data segmentation into specific movement sequences that can be averaged across trials and individuals to isolate characteristic movement strategies. Two standardized functional tasks were developed, the Pasta Box Task and Cup Transfer Task (Fig 1). Full task set up and descriptions are available in the supplementary materials. The Pasta Box Task (S1 Text) was designed to mimic moving objects from a counter to a cup- board, between cupboards at different heights, and across the body’s midline. The Pasta Box Task consisted of three movements, during which the performer moved a box of pasta from a lower side shelf on their right (height: 30 inches) to a shelf in front of them (height: 43 inches); then to a second shelf at a higher height across the body (height: 48 inches); and then back to the starting position. The performer was required to return the hand to a “home” PLOS ONE \| https://doi.org/10.1371/journal.pone.0199549 June 21, 2018 3 / 21 Normative hand movements during two functional upper limb tasks Fig 1. Functional tasks. Sequence of the Pasta Box Task (A) and Cup Transfer Task (B). During the Pasta Box Task, the participant moved the box of pasta from a lower side table to two shelves of different heights in front of them (Movements 1 and 2) and back again to the start position (Movement 3). Tasks were completed using three distinct movements, with standardized placement positions, including returning the hand to a standard “home” position at the end of each movement. During the Cup Transfer Task, the participant moved two compliant cups filled with therapeutic Fig 1. Functional tasks. Sequence of the Pasta Box Task (A) and Cup Transfer Task (B). Functional task development During the Pasta Box Task, the participant moved the box of pasta from a lower side table to two shelves of different heights in front of them (Movements 1 and 2) and back again to the start position (Movement 3). Tasks were completed using three distinct movements, with standardized placement positions, including returning the hand to a standard “home” position at the end of each movement. During the Cup Transfer Task, the participant moved two compliant cups filled with therapeutic Fig 1. Functional tasks. Sequence of the Pasta Box Task (A) and Cup Transfer Task (B). During the Pasta Box Task, the participant moved the box of pasta from a lower side table to two shelves of different heights in front of them (Movements 1 and 2) and back again to the start position (Movement 3). Tasks were completed using three distinct movements, with standardized placement positions, including returning the hand to a standard “home” position at the end of each movement. During the Cup Transfer Task, the participant moved two compliant cups filled with therapeutic PLOS ONE \| https://doi.org/10.1371/journal.pone.0199549 June 21, 2018 4 / 21 Normative hand movements during two functional upper limb tasks beads over a partition to a target location using a top grasp for the first cup and a side grasp for the second cup (Movements 1 and 2). These movements were followed by a return of the hand to the “home” position, and then by moving the cups back to their initial positions using respective types of grasps (Movements 3 and 4). https://doi.org/10.1371/journal.pone.0199549.g001 beads over a partition to a target location using a top grasp for the first cup and a side grasp for the second cup (Movements 1 and 2). These movements were followed by a return of the hand to the “home” position, and then by moving the cups back to their initial positions using respective types of grasps (Movements 3 and 4). https://doi.org/10.1371/journal.pone.0199549.g001 https://doi.org/10.1371/journal.pone.0199549.g001 position after each specified movement. The Cup Transfer Task (S2 Text) was designed to involve greater risk by using compliant cups with content that can be spilled, and requiring careful placement around barriers such as would be encountered at a sink or countertop. Functional task development The Cup Transfer Task consisted of four movements, where the performer moved two compliant cups filled with therapeutic beads from an initial position on the right side of a box to specific target positions on the left side of the box, and then back again to the start locations, while hav- ing to clear a middle partition. The box was placed at a standard counter height of 36 inches to recreate a real-world environment. The performer was required to return the hand to a “home” position after the first two movements and at the end of the task. In order to challenge grasp capabilities which might be difficult for impaired populations, two types of grasps, linked to the placement of the cups in the box, were required: a top grasp for the near cup and a side grasp for the far cup. Experimental setup and procedures A 12-camera Vicon Bonita motion capture system (Vicon Motion Systems Ltd, Oxford, UK) with an accuracy of 0.5 mm and 0.5 degrees was used to collect three-dimensional marker tra- jectories at 120 Hz. A rigid plate with three 11.1 mm reflective markers was attached to the dorsal side of the right hand using double-sided tape. Two 14 mm single reflective markers were attached to the middle phalange of the index finger and the distal phalange of the thumb. Additional markers and marker clusters were attached to upper body segments (pelvis; trunk; upper arms; forearms; head), but were not used in the current analysis. Prior to data collection, each participant received verbal instructions, a demonstration, and a practice trial to be familiarized with the tasks. Each participant performed a minimum of 20 successful trials for both tasks. If there was an error in performance of the task (i.e., dropping the object), that trial was marked as an “error trial” and the trial was repeated until a total of 20 error-free trials were recorded. The order of the tasks was block-randomized, with ten partici- pants starting with the Pasta Box Task and ten with the Cup Transfer Task. Ten participants returned on a separate day for repeatability testing after the initial testing session (7.5 months ± 11 days), with the identical set up. The same two testers ran all data collection sessions. Study participants Twenty non-disabled individuals (11 male; 18 right-handed; age: 25.8 ± 7.2 years; height: 173.8 ± 8.3 cm; mean ± standard deviation) were recruited to participate in the study. They had no upper body pathology or history of neurological or musculoskeletal injuries within the past two years. The study followed the Declaration of Helsinki guidelines and was approved by the University of Alberta Health Research Ethics Board (Pro00054011), the Department of the Navy Human Research Protection Program (DON-HRPP), and the SSC-Pacific Human Research Protection Office (SSCPAC HRPO). The individual in Fig 1 has provided written consent to publish the photographs. Normative hand movements during two functional upper limb tasks Table 1. Segmentation and phase definitions. Phase Name Start/End Definition Reach Start: Hand leaves the home position First occurrence of the hand exceeding the ‘Hand Velocity Threshold’ OR first occurrence of the hand exceeding the ‘Target Distance Threshold’, whichever happens first Grasp Start: Closing of grip aperture First occurrence of the hand falling below the ‘Grasp Distance Threshold’ Transport Start: Start of object movement First occurrence of the object exceeding the ‘Object Velocity Threshold’ OR first occurrence of the object exceeding the ‘Target Distance Threshold’, whichever happens first Release Start: End of object movement First occurrence of the object falling below the ‘Object Velocity Threshold’ OR first occurrence of the object distance falling below the ‘Target Distance Threshold’, whichever happens last End: End of grip aperture opening Last occurrence of the hand before exceeding the ‘Release Distance Threshold’ Table 1. Segmentation and phase definitions. Phase start and end definitions for the two functional tasks. Each task movement is separated into four phases: reach, grasp, transport, and release. The start and end of the phases are based on kinematic variables of hand velocity, object velocity, grip aperture, and hand-to-target or object-to-target distance. ‘Hand Velocity Threshold’ was defined as 5% of the peak hand velocity during the trial. ‘Object Velocity Threshold’ was defined as 5% of the peak object velocity during the trial. ‘Grasp Distance Threshold’ and ‘Release Distance Threshold’ were based on average occurrence of peak grip aperture prior to and following object movement, respectively. ‘Target Distance Threshold’ was defined as the location of the hand or object during a transition phase with respect to the target plus a tolerable distance of 70 mm. https://doi.org/10.1371/journal.pone.0199549.t001 velocity, object velocity, and grip aperture (Table 1). The Pasta Box Task was divided into three movements, and the Cup Transfer Task was divided into four movements. Each task had a standard starting position for the hand (labelled ‘home’). Participants had to bring their hand to the “home” position after each movement for the Pasta Box Task and after the first two movements of the Cup Transfer Task to allow for task segmentation and standardization. Each movement was composed of four phases: reach, grasp, transport, and release. PLOS ONE \| https://doi.org/10.1371/journal.pone.0199549 June 21, 2018 Experimental data analysis Raw marker trajectory data were filtered using a second-order, low-pass Butterworth filter with a cut-off frequency of 6 Hz [28]. Motion capture data were segmented based on hand 5 / 21 PLOS ONE \| https://doi.org/10.1371/journal.pone.0199549 June 21, 2018 For the quantitative kinematic analysis, due to the short duration of grasp and release phases, and to interpret the results in light of functional hand movement sequences, the reach and grasp phases were combined into a reach-grasp segment, and the transport and release phases into a transport-release segment. Kinematic measures were selected based on commonly reported measures in assessments of individuals with upper limb impairments [5,29]. Hand movement was calculated using the average position of the three markers on the hand plate. Grip aperture was defined as the dis- tance between the markers attached to the index and thumb. Analysed measures were move- ment time, hand distance travelled, hand trajectory variability, peak hand velocity, percent-to- peak hand velocity, number of movement units, peak grip aperture, percent-to-peak grip aper- ture, and percent-to-peak hand deceleration. Percent-to-peak measures were defined as the percent of time elapsed before the peak for a specific reach-grasp or transport-release segment. Hand trajectory variability was quantified as the maximum of the mean three-dimensional standard deviation at each time-normalized point. Number of movement units was defined as a local maximum velocity, or velocity peak [30–32] of the hand and was calculated by finding the zero-crossings in the hand acceleration profile where the signal switches from positive to negative. The hand trajectory, hand velocity, and grip aperture time series were time-normal- ized by segment (resampled to have 100 points per segment), averaged across trials and 6 / 21 PLOS ONE \| https://doi.org/10.1371/journal.pone.0199549 June 21, 2018 Normative hand movements during two functional upper limb tasks participants for each segment, and resampled across segments based on average segment length (with 1,000 points per overall trial). Statistical analysis Statistical analysis was completed using the SPSS software (IBM Corporation, Armonk, NY, USA). For each task, hand trajectory variability, movement time, peak hand velocity and per- cent-to-peak hand velocity were analyzed using a two-way repeated-measures analysis of vari- ance (ANOVA) examining effects of movement (3 for Pasta, 4 for Cups) and segment (reach- grasp, transport-release). Significant interactions (p < 0.05) were examined by conducting simple main effect one-way repeated-measures ANOVA’s of movement at each level of seg- ment. Significant main or simple main effects (p < 0.05) were followed up by conducting all pairwise comparisons with Bonferroni correction. Normality was assessed using the Kolmogo- rov-Smirnov Test and sphericity was assessed through a Mauchly’s Test of Sphericity. In cases where the assumption of sphericity was not met, a Greenhouse-Geisser Correction was applied and reported. For peak grip aperture, percent-to-peak grip aperture, and percent-to-peak hand deceleration a one-way repeated-measures ANOVA, with Bonferroni corrected pairwise comparisons where significant (p < 0.05), was conducted to assess potential differences between movements for the reach-grasp segment only. A between-session repeatability analysis was performed by calculating the intra-class corre- lation (ICC) for model (2,k), the standard error of measurement (SEM), and the minimal detectable change (MDC) [33] between the first and second session for ten participants. SEM was calculated based on the ICC analysis scores. The equation for SEM was: SEM ¼ SD ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 ICC p ð1Þ ð1Þ where SD is the standard deviation of all the participants in the first session. The MDC was cal- culated based on the SEM values and the 95% confidence interval. The equation for MDC was: MDC ¼ SEM 1:96 ﬃﬃﬃ 2 p ð2Þ ð2Þ MDC ¼ SEM 1:96 ﬃﬃﬃ 2 p where 1.96 is the z score associated with the 95% confidence interval [34]. SEM and MDC scores were also represented as a percentage of the absolute average measurement value to indicate relative error. ICC, SEM, and MDC values were calculated for movement time, peak hand velocity, percent-to-peak hand velocity, peak grip aperture, percent-to-peak grip aper- ture, and percent-to-peak hand deceleration. ICC values above 0.90 were considered to indi- cate reasonable reliability for clinical measurements, above 0.75 indicated good repeatability, and below 0.75 indicated poor to moderate repeatability [33]. Task performance Overall performance time (from start to finish) for the Pasta Box Task was 8.84 ± 0.34 seconds, and for the Cup Transfer Task 10.60 ± 0.49 seconds. Error trials occurred at a rate of 4% for the Pasta Box Task, and 11% for the Cups Transfer Task. The most common errors were sequence hesitation (Pasta Box Task: 38% of errors, Cup Transfer Task: 54% of errors), hitting a partition/obstacle (Pasta Box Task: 31% of errors, Cup Transfer Task: 25% of errors), and incorrect grasp of the object (Pasta Box Task: 12% of errors, Cup Transfer Task: 16% of errors). Movement times, hand distance travelled, and hand trajectory variability for each move- ment are listed in Tables 2 and 3 for the Pasta Box Task and Cup Transfer Task, respectively. PLOS ONE \| https://doi.org/10.1371/journal.pone.0199549 June 21, 2018 7 / 21 Pasta Box Task measures for hand distance travelled, hand trajectory variability, movement time, peak hand velocity, percent-to-peak hand velocity, number of movement units, peak grip aperture, percent-to-peak grip aperture, and percent-to-peak hand deceleration. Data are presented, for movements and segments separately, as group means and across-participant standard deviations (SD). Average within-participant variability (WPV) is also presented for each measure. Movements are: Movement 1 (Mvmt 1), Movement 2 (Mvmt 2), and Movement 3 (Mvmt 3); segments are: reach-grasp (RG) and transport-release (TR). PLOS ONE \| https://doi.org/10.1371/journal.pone.0199549 June 21, 2018 Normative hand movements during two functional upper limb tasks Table 2. Pasta Box Task kinematic measures. Hand distance travelled (mm) Hand trajectory variability (mm) Movement time (sec) Peak hand velocity (mm/s) Percent-to-peak hand velocity (%) Mean ± SD WPV Mean ± SD Mean ± SD WPV Mean ± SD WPV Mean ± SD WPV Mvmt 1 RG 464 ± 25 21 17 ± 5 0.97 ± 0.15 0.07 1007 ± 125 71 42.5 ± 4.0 2.9 TR 850 ± 27 14 19 ± 3 1.36 ± 0.16 0.07 1330 ± 140 69 33.1 ± 3.7 4.0 Mvmt 2 RG 478 ± 21 12 13 ± 3 0.68 ± 0.11 0.05 1204 ± 151 54 43.2 ± 6.5 3.8 TR 740 ± 72 23 19 ± 4 1.45 ± 0.18 0.08 1035 ± 114 52 45.8 ± 5.7 8.5 Mvmt 3 RG 701 ± 21 14 19 ± 4 0.84 ± 0.15 0.05 1466 ± 197 74 38.8 ± 5.2 4.9 TR 1069 ± 22 16 32 ± 6 1.68 ± 0.24 0.10 1470 ± 164 94 32.5 ± 3.8 3.6 Number of movement units Peak grip aperture (mm) Percent-to-peak grip aperture (%) Percent-to-peak hand deceleration (%) Mean ± SD WPV Mean ± SD WPV Mean ± SD WPV Mean ± SD WPV Mvmt 1 RG 1.2 ± 0.3 0.3 117 ± 7 3 71.6 ± 5.3 3.1 58.2 ± 8.5 11.0 TR 1.3 ± 0.3 0.5 - - - - - - Mvmt 2 RG 1.1 ± 0.1 0.2 107 ± 7 3 78.0 ± 4.3 3.1 74.4 ± 8.0 5.3 TR 2.3 ± 0.3 0.4 - - - - - - Mvmt 3 RG 1.2 ± 0.2 0.3 110 ± 6 3 79.0 ± 4.1 3.3 72.8 ± 7.8 4.9 TR 1.8 ± 0.5 0.9 - - - - - - Table 2. Pasta Box Task kinematic measures. Pasta Box Task measures for hand distance travelled, hand trajectory variability, movement time, peak hand velocity, percent-to-peak hand velocity, number of movement units, peak grip aperture, percent-to-peak grip aperture, and percent-to-peak hand deceleration. Data are presented, for movements and segments separately, as group means and across-participant standard deviations (SD). Average within-participant variability (WPV) is also presented for each measure. Movements are: Movement 1 (Mvmt 1), Movement 2 (Mvmt 2), and Movement 3 (Mvmt 3); segments are: reach-grasp (RG) and transport-release (TR). Pasta Box Task measures for hand distance travelled, hand trajectory variability, movement time, peak hand velocity, percent-to-peak hand velocity, number of movement units, peak grip aperture, percent-to-peak grip aperture, and percent-to-peak hand deceleration. Data are presented, for movements and segments separately, as group means and across-participant standard deviations (SD). Average within-participant variability (WPV) is also presented for each measure. Movements are: Movement 1 (Mvmt 1), Movement 2 (Mvmt 2), and Movement 3 (Mvmt 3); segments are: reach-grasp (RG) and transport-release (TR). Statistical results including post-hoc pairwise comparisons are presented in Tables 4 and 5 for the Pasta Box Task and Cup Transfer Task, respectively, and discussed below in the relevant sections per task. Movement times for reach-grasp segments were significantly smaller than for transport- release segments (p < 0.001), even in the single case where transport distance was shorter than reach distance during Movement 3 in the Cup Transfer Task (Table 3). For the Pasta Box Task, post-hoc pairwise comparisons revealed that movement time for both reach-grasp and transport-release segments was significantly different between all individual movements (p < 0.001). For the Cup Transfer Task, post-hoc pairwise comparisons revealed that move- ment time for both reach-grasp and transport-release segments was significantly different between individual movements (p < 0.001), except for the transport-release segment of Move- ment 1 compared to Movement 4 (p = 0.079) and Movement 2 compared to Movement 3 (p = 1.000). Hand trajectory graphs displaying the average hand trajectories and across-participant stan- dard deviations are shown in Figs 2 and 3. Maximum hand trajectory variability was overall small for both tasks, ranging from 13 mm to 32 mm for the Pasta Box Task (Table 2) and 16 mm to 27 mm for the Cup Transfer Task (Table 3). For the Pasta Box Task (Table 4), during reach-grasp, Movement 2 was significantly less variable than Movement 1 and 3 (p < 0.001) and, during transport-release, Movement 3 was significantly more variable than Movement 1 and 2 (p < 0.001). For the Cup Transfer Task (Table 5), during reach-grasp, Movement 1 was significantly less variable than Movement 2, 3, and 4 (p < 0.001) and, during transport-release, Movement 1 was significantly less variable than Movement 3 (p < 0.05). 8 / 21 PLOS ONE \| https://doi.org/10.1371/journal.pone.0199549 June 21, 2018 Cup Transfer Task measures for hand distance travelled, hand trajectory variability, movement time, peak hand velocity, percent-to-peak hand velocity, number of movement units, peak grip aperture, percent-to-peak grip aperture, and percent-to-peak hand deceleration. Data are presented, for movements and segments separately, as group means and across-participant standard deviations (SD). Average within-participant variability (WPV) is also presented for each measure. Movements are: Movement 1 (Mvmt 1), Movement 2 (Mvmt 2), Movement 3 (Mvmt 3), and Movement 4 (Mvmt 4); segments are: reach-grasp (RG) and transport-release (TR). https://doi.org/10.1371/journal.pone.0199549.t003 Normative hand movements during two functional upper limb tasks Table 3. Cup Transfer Task kinematic measures. Hand distance travelled (mm) Hand trajectory variability (mm) Movement time (sec) Peak hand velocity (mm/s) Percent-to-peak hand velocity (%) Mean ± SD WPV Mean ± SD Mean ± SD WPV Mean ± SD WPV Mean ± SD WPV Mvmt 1 RG 380 ± 44 22 16 ± 3 0.86 ± 0.13 0.07 818 ± 117 60 43.7 ± 7.7 6.0 TR 608 ± 36 21 16 ± 2 1.32 ± 0.15 0.10 970 ± 79 47 21.1 ± 3.8 4.2 Mvmt 2 RG 445 ± 45 30 27 ± 7 0.75 ± 0.15 0.07 1050 ± 104 62 25.9 ± 7.7 6.7 TR 635 ± 46 26 17 ± 4 1.47 ± 0.15 0.10 898 ± 73 46 39.9 ± 7.7 6.3 Mvmt 3 RG 851 ± 35 25 26 ± 6 1.10 ± 0.17 0.07 1435 ± 157 75 42.5 ± 5.6 6.3 TR 673 ± 42 28 18 ± 3 1.46 ± 0.17 0.10 976 ± 53 48 25.1 ± 2.1 2.3 Mvmt 4 RG 426 ± 43 25 24 ± 6 0.63 ± 0.09 0.06 1041 ± 113 59 25.0 ± 7.2 7.5 TR 622 ± 42 25 17 ± 3 1.36 ± 0.17 0.10 956 ± 66 47 23.8 ± 5.3 7.1 Number of movement units Peak grip aperture (mm) Percent-to-peak grip aperture (%) Percent-to-peak hand deceleration (%) Mean ± SD WPV Mean ± SD WPV Mean ± SD WPV Mean ± SD WPV Mvmt 1 RG 1.3 ± 0.3 0.4 99 ± 5 3 80.9 ± 5.1 4.5 69.7 ± 7.9 9.2 TR 2.5 ± 0.4 0.8 - - - - - Mvmt 2 RG 1.3 ± 0.3 0.4 114 ± 6 3 71.9 ± 5.9 6.0 49.8 ± 8.2 8.7 TR 2.5 ± 0.5 0.8 - - - - - - Mvmt 3 RG 1.4 ± 0.3 0.4 113 ± 7 2 81.0 ± 3.7 4.5 60.6 ± 5.7 5.4 TR 2.2 ± 0.7 1.0 - - - - - - Mvmt 4 RG 1.3 ± 0.3 0.4 118 ± 7 3 78.1 ± 6.9 5.1 68.4 ± 13.5 10.7 TR 2.5 ± 0.4 0.7 - - - - - - Table 3. Cup Transfer Task kinematic measures. PLOS ONE \| https://doi.org/10.1371/journal.pone.0199549 June 21, 2018 Pasta Box Task For the Pasta Box Task (Table 2), movements involving the side table location affected several kinematic parameters compared to other movements. Movement 1, where participants had to turn their body and reach to the side table to pick up the box, had the lowest peak hand veloc- ity for the reach-grasp segment (Fig 4A), which was significantly different from Movements 2 and 3 (p < 0.001). Peak grip aperture (Fig 5A) was also greatest for the first reach-grasp seg- ment, which was significantly different from the reach-grasp segment of Movements 2 and 3. The peak grip aperture during the first reach-grasp segment also occurred significantly earlier than for the following two movements (p < 0.001) and did not align with the percent-to-peak hand deceleration. The transport-release segments of Movement 1 and 3, both involving mov- ing the box from or to the side cart, had similar percent-to-peak hand velocities (p = 1.000). They were significantly lower than for Movement 2 (p < 0.001). The distinct feature of Movement 2 was that the participant had to transfer the box of pasta from the first shelf to the second shelf by moving around the middle cart barrier, which served as an obstacle. The transport-release segment of this movement had a significantly lower peak hand velocity than the other transport-release segments (p < 0.001), displayed two velocity peaks (indicated by the number of movement units in Table 2 and as seen in Fig 4A), and exhibited the greatest within-participant variability in percent-to-peak hand velocity. Movement 3 had the longest distances for both reach and transport segments. Although the magnitude and timing of peak grip aperture for the reach-grasp segment was statistically PLOS ONE \| https://doi.org/10.1371/journal.pone.0199549 June 21, 2018 9 / 21 indicates that the F-statistic was significant at p < 0.001; indicates that the F-statistic was significant at p < 0.05; << indicates that the pairwise comparison was significantly smaller at p < 0.001; < indicates that the pairwise comparison was significantly smaller at p < 0.05. Pasta Box Task results of the two-factor and one-factor repeated-measures analysis of variance (ANOVA). The results for the interaction of Movement and Segment for the two-factor repeated-measures ANOVA is reported for hand trajectory variability, movement time, peak hand velocity, and percent-to-peak hand velocity. The effect of movement for the one-way repeated measures ANOVA is reported for peak grip aperture, percent-to-peak grip aperture, and percent-to-peak-grip hand deceleration. The simple main effects ANOVA is reported for hand trajectory variability, movement time, peak hand velocity, and percent-to-peak hand velocity. indicates that the F-statistic was significant at p < 0.001; i di t th t th F t ti ti i ifi t t < 0 05 << i di t th t th i i i i ifi tl ll t < 0 001 < i di t th t th i i g p ; indicates that the F-statistic was significant at p < 0.05; << indicates that the pairwise comparison was significantly smaller at p < 0.001; < indicates that the pairwise comparison was significantly smaller at p < 0.05. different for each movement, the absolute values were very close. Reach-grasp of Movement 3 had significantly higher peak hand velocities than for reach-grasp in the other movements (p < 0.001), and the peak hand velocity in reach-grasp occurred significantly earlier than that of Movement 1 (p = 0.028) and Movement 2 (p < 0.001). Peak hand velocity during transport- release of Movement 3 was similarly significantly higher than that of Movement 1 and 2 (p < 0.001), but with a lower percent-to-peak hand velocity compared to Movement 2 (p < 0.001) (but not significantly different from Movement 1 (p = 1.000)). Pasta Box Task results of the two-factor and one-factor repeated-measures analysis of variance (ANOVA). The results for the interaction of Movement and Segment for the two-factor repeated-measures ANOVA is reported for hand trajectory variability, movement time, peak hand velocity, and percent-to-peak hand velocity. The effect of movement for the one-way repeated measures ANOVA is reported for peak grip aperture, percent-to-peak grip aperture, and percent-to-peak-grip hand deceleration. The simple main effects ANOVA is reported for hand trajectory variability, movement time, peak hand velocity, and percent-to-peak hand velocity. indicates that the F-statistic was significant at p < 0.001; indicates that the F-statistic was significant at p < 0.05; << indicates that the pairwise comparison was significantly smaller at p < 0.001; < indicates that the pairwise comparison was significantly smaller at p < 0.05. Normative hand movements during two functional upper limb tasks Table 4. Pasta Box Task statistical analysis results. Movement time (s) Interaction: Mvmt x Segment F(2, 38) = 238.5 Mvmt 1 Mvmt2 Mvmt 3 F (Mvmt effect) Pairwise Reach-grasp 0.97 0.68 0.84 (2, 38) = 255.9 2 << 3 << 1 Transport-release 1.36 1.45 1.68 (2, 38) = 128.5 1 << 2 << 3 Hand trajectory variability (mm) Interaction: Mvmt x Segment F(2, 38) = 22.6 Mvmt 1 Mvmt 2 Mvmt 3 F (Mvmt effect) Pairwise Reach-grasp 17 13 19 (1.4, 27.5) = 23.6 2 << 1,3 Transport-release 19 19 32 (1.5, 28.7) = 63.1 1 << 3; 2 << 3 Peak hand velocity (mm/s) Interaction: Mvmt x Segment F(2, 38) = 154.3 Mvmt 1 Mvmt 2 Mvmt 3 F (Mvmt effect) Pairwise Reach-grasp 1008 1204 1466 (1.4, 26.4) = 183.1 1 << 2 << 3 Transport-release 1330 1035 1470 (2, 38) = 199.1 2 << 1 << 3 Percent-to-peak hand velocity (%) Interaction: Mvmt x Segment F(2, 38) = 22.2 Mvmt 1 Mvmt 2 Mvmt 3 F (Mvmt effect) Pairwise Reach-grasp 42.4 43.2 38.8 (1.4, 26.2) = 6.9 3 < 1; 3 << 2 Transport-release 33.1 45.8 32.5 (2, 38) = 80.4 1 << 2; 3 << 2 Peak grip aperture (mm) Mvmt 1 Mvmt 2 Mvmt 3 F (Mvmt effect) Pairwise Reach-grasp 117 107 109 (1.3, 25.2) = 92.7 2 << 3 << 1 Percent-to-peak grip aperture (%) Mvmt 1 Mvmt 2 Mvmt 3 F (Mvmt effect) Pairwise Reach-grasp 71.6 77.9 79.0 (1.2, 23.5) = 61.3 1 << 2,3; 2 < 3 Percent-to-peak hand deceleration (%) Mvmt 1 Mvmt 2 Mvmt 3 F (Mvmt effect) Pairwise Reach-grasp 58.2 74.4 72.8 (1.1, 20.9) = 58.0 1 << 2,3; 3 < 2 Table 4. Pasta Box Task statistical analysis results. Pasta Box Task results of the two-factor and one-factor repeated-measures analysis of variance (ANOVA). The results for the interaction of Movement and Segment for the two-factor repeated-measures ANOVA is reported for hand trajectory variability, movement time, peak hand velocity, and percent-to-peak hand velocity. The effect of movement for the one-way repeated measures ANOVA is reported for peak grip aperture, percent-to-peak grip aperture, and percent-to-peak-grip hand deceleration. The simple main effects ANOVA is reported for hand trajectory variability, movement time, peak hand velocity, and percent-to-peak hand velocity. Normative hand movements during two functional upper limb tasks Table 5. Cup Transfer Task statistical analysis results. Movement time (s) Interaction: Mvmt x Segment F(3, 57) = 167.1 Mvmt 1 Mvmt2 Mvmt 3 Mvmt 4 F (Mvmt effect) Pairwise Reach-grasp 0.86 0.75 1.10 0.63 (3, 57) = 273.2 4 << 2 << 1 << 3 Transport-release 1.32 1.48 1.47 1.36 (2.5, 48.1) = 55.2 1 << 2,3; 4 << 2,3 Hand trajectory variability (mm) Interaction: Mvmt x Segment F(3, 57) = 10.2 Mvmt 1 Mvmt2 Mvmt 3 Mvmt 4 F (Mvmt effect) Pairwise Reach-grasp 16 27 26 24 (3, 57) = 19.5 1 << 2,3,4 Transport-release 16 17 18 17 (3, 57) = 4.42 1 < 3 Peak hand velocity (mm/s) Interaction: Mvmt x Segment F(3, 57) = 130.3 Mvmt 1 Mvmt2 Mvmt 3 Mvmt 4 F (Mvmt effect) Pairwise Reach-grasp 818 1050 1435 1041 (3, 57) = 176.3 1 << 2,3,4; 2 << 3; 4 << 3 Transport-release 970 897 976 956 (3, 57) = 18.5 2 < 1; 2 << 3,4 Percent-to-peak hand velocity (%) Interaction: Mvmt x Segment F(3, 57) = 132.0 Mvmt 1 Mvmt2 Mvmt 3 Mvmt 4 F (Mvmt effect) Pairwise Reach-grasp 43.7 25.9 42.5 25.0 (3, 57) = 54.1 2 << 1,3; 4 << 1,3 Transport-release 21.1 39.9 25.1 23.8 (1.8, 35.0) = 70.0 1 << 2,3; 3 << 2; 4 << 2 Peak grip aperture (mm) Mvmt 1 Mvmt2 Mvmt 3 Mvmt 4 F (Mvmt effect) Pairwise Reach-grasp 99 114 113 118 (3, 57) = 102.1 1 << 2,3,4; 2 < 4; 3 < 4 Percent-to-peak grip aperture (%) Mvmt 1 Mvmt2 Mvmt 3 Mvmt 4 F (Mvmt effect) Pairwise Reach-grasp 80.9 71.9 80.9 78.1 (3, 57) = 24.9 2 << 1,3; 2 < 4 Percent-to-peak hand deceleration (%) Mvmt 1 Mvmt2 Mvmt 3 Mvmt 4 F (Mvmt effect) Pairwise Reach-grasp 69.7 49.2 60.6 68.4 (2.1, 40.4) = 45.4 2 << 1,3,4; 3 << 1; 3 < 4 Cup Transfer Task results of the two-factor and one-factor repeated-measures analysis of variance (ANOVA). The results for the interaction of Movement and Segment for the two-factor repeated-measures ANOVA is reported for hand trajectory variability, movement time, peak hand velocity, and percent-to-peak hand velocity. The effect of movement for the one-way repeated measures ANOVA is reported for peak grip aperture, percent-to-peak grip aperture, and percent-to-peak-grip hand deceleration. The simple main effects ANOVA is reported for hand trajectory variability, movement time, peak hand velocity, and percent-to-peak hand velocity. indicates that the F-statistic was significant at p < 0.001; indicates that the F-statistic was significant at p < 0.05; << indicates that the pairwise comparison was significantly smaller at p < 0.001; < indicates that the pairwise comparison was significantly smaller at p < 0.05. Table 5. Cup Transfer Task statistical analysis results. Cup Transfer Task results of the two-factor and one-factor repeated-measures analysis of variance (ANOVA). The results for the interaction of Movement and Segment for the two-factor repeated-measures ANOVA is reported for hand trajectory variability, movement time, peak hand velocity, and percent-to-peak hand velocity. The effect of movement for the one-way repeated measures ANOVA is reported for peak grip aperture, percent-to-peak grip aperture, and percent-to-peak-grip hand deceleration. The simple main effects ANOVA is reported for hand trajectory variability, movement time, peak hand velocity, and percent-to-peak hand velocity. indicates that the F-statistic was significant at p < 0.001; indicates that the F-statistic was significant at p < 0.05; << indicates that the pairwise comparison was significantly smaller at p < 0.001; < indicates that the pairwise comparison was significantly smaller at p < 0.05. Cup Transfer Task results of the two-factor and one-factor repeated-measures analysis of variance (ANOVA). The results for the interaction of Movement and Segment for the two-factor repeated-measures ANOVA is reported for hand trajectory variability, movement time, peak hand velocity, and percent-to-peak hand velocity. The effect of movement for the one-way repeated measures ANOVA is reported for peak grip aperture, percent-to-peak grip aperture, and percent-to-peak-grip hand deceleration. The simple main effects ANOVA is reported for hand trajectory variability, movement time, peak hand velocity, and percent-to-peak hand velocity. indicates that the F-statistic was significant at p < 0.001; indicates that the F-statistic was significant at p < 0.05; << indicates that the pairwise comparison was significantly smaller at p < 0.001; < indicates that the pairwise indicates that the F-statistic was significant at p < 0.05; << indicates that the pairwise comparison was significantly smaller at p < 0.001; < indicates that the pairwise comparison was significantly smaller at p < 0.05. https://doi.org/10.1371/journal.pone.0199549.t005 transport-release segments displayed small, double hand velocity peaks, reflecting a conse- quence of transporting the cup over an obstacle in the vertical plane. Cup Transfer Task Overall, peak velocities for the Cup Transfer Task (Table 3) were significantly higher for reach-grasp segments than transport-release segments (p < 0.001). Slower movement during transport was expected given the risk of spilling the compliant cups filled with beads. As evi- denced by the hand velocity graph (Fig 4B) and the number of movement units (Table 3), all PLOS ONE \| https://doi.org/10.1371/journal.pone.0199549 June 21, 2018 10 / 21 PLOS ONE \| https://doi.org/10.1371/journal.pone.0199549 June 21, 2018 Cup Transfer Task results of the two-factor and one-factor repeated-measures analysis of variance (ANOVA). The results for the interaction of Movement and Segment for the two-factor repeated-measures ANOVA is reported for hand trajectory variability, movement time, peak hand velocity, and percent-to-peak hand velocity. The effect of movement for the one-way repeated measures ANOVA is reported for peak grip aperture, percent-to-peak grip aperture, and percent-to-peak-grip hand deceleration. The simple main effects ANOVA is reported for hand trajectory variability, movement time, peak hand velocity, and percent-to-peak hand velocity. indicates that the F-statistic was significant at p < 0.001; indicates that the F-statistic was significant at p < 0.05; << indicates that the pairwise comparison was significantly smaller at p < 0.001; < indicates that the pairwise comparison was significantly smaller at p < 0.05. The Cup Transfer Task was unique in that it required two different grasp patterns, which we hypothesized would affect grip aperture and velocity of movements based on confidence in modulating grip patterns to not crush the cup and spill the contents. Movement 1 had the shortest distance to reach and the lowest peak velocity (Fig 4B) compared to all the other seg- ments (p < 0.001). Movement 1 also had the lowest peak grip aperture (Fig 5B) compared to the other three movements (p < 0.001). The transport velocity for Movement 1 was similar to that of Movements 3 and 4 (p = 1.000). Movement 2 required a change in grasp to a side grasp of the cup, and displayed a similar peak grip aperture to Movement 3 also requiring a side grasp (p = 0.959), but was significantly different from the top grasps (p < 0.05). Although Movements 2 and 3 had similar grip aper- tures, there were differences in the velocity profiles. Movement 2 had a lower and earlier peak hand velocity than Movement 3, and the earliest deceleration peak of all the reach-grasp PLOS ONE \| https://doi.org/10.1371/journal.pone.0199549 June 21, 2018 11 / 21 Normative hand movements during two functional upper limb tasks Fig 2. Pasta Box Task hand trajectory. Hand trajectories for the Pasta Box Task. The group average hand trajectory is plotted as a dark line, and the standard deviation of participant means as three-dimensional shading. Movement 1 (A), Movement 2 (B), and Movement 3 (C) are segmented into reach (red), grasp (orange), transport (blue), and release (green) phases. The maximum of the mean three-dimensional standard deviation was calculated for reach-grasp and transport-release segments in each movement to quantify variability, reported in Table 2. https://doi.org/10.1371/journal.pone.0199549.g002 Fig 2. Pasta Box Task hand trajectory. Hand trajectories for the Pasta Box Task. The group average hand trajectory is plotted as a dark line, and the standard deviation of participant means as three-dimensional shading. Movement 1 (A), Movement 2 (B), and Movement 3 (C) are segmented into reach (red), grasp (orange), transport (blue), and release (green) phases. The maximum of the mean three-dimensional standard deviation was calculated for reach-grasp and transport-release segments in each movement to quantify variability, reported in Table 2. https://doi.org/10.1371/journal.pone.0199549.g002 https://doi.org/10.1371/journal.pone.0199549.g002 https://doi.org/10.1371/journal.pone.0199549.g002 segments. Between-session repeatability Both the Pasta Box Task and Cup Transfer Task presented mostly good repeatability (ICC > 0.75) for movement time, peak hand velocity, percent-to-peak hand velocity, peak grip aper- ture, percent-to-peak grip aperture, and percent-to-peak hand deceleration. For the Pasta Box Task (Table 6), poor to moderate repeatability (ICC values of below 0.75) was found for only two measures: peak hand velocity and percent-to-peak hand velocity for the transport-release segment of Movement 1. For the Cup Transfer Task (Table 7), poor to moderate repeatability (ICC values of below 0.75) was found for six of the 36 measures: peak hand velocity for the transport-release segment of Movement 1, Movement 3, and Movement 4, as well as for percent-to-peak hand velocity, percent-to-peak grip aperture, and percent-to- peak hand deceleration for the reach-grasp segment of Movement 1. For the Pasta Box Task (Table 6), SEM values ranged from 1 to 7% of the average absolute measurement value across measures, and MDC ranged from 3 to 20% across measures. For the Cup Transfer Task (Table 7), SEM values ranged from 1 to 15% across measures and MDC ranged from 2 to 42% across measures. This slowed movement was also seen in the transport-release segment of Movement 2, which had the lowest peak hand velocity of all the transport-release segments. The percent- to-peak grip aperture occurred significantly earlier in the reach-grasp segment of Movement 2 compared to the other 3 movements (p < 0.01). In contrast, the reach-grasp segment in Movement 3 had the highest peak hand velocity. The hand velocity for the transport-release segment of Movement 3 was not significantly dif- ferent from Movement 1 (p = 1.000) and 4 (p = 0.137). Movement 4 had the largest peak grip aperture (p < 0.05). It otherwise showed similar characteristics in percent-to-peak grip aper- ture and percent-to-peak hand deceleration as Movement 1, which had the same grasp. PLOS ONE \| https://doi.org/10.1371/journal.pone.0199549 June 21, 2018 Discussion The purpose of developing new standardized functional tasks representative of real-world ADLs was to create a meaningful assessment metric for clinical populations with upper limb impairments, that specifically focused on quantifying hand kinematics. Reach-grasp tasks have 12 / 21 PLOS ONE \| https://doi.org/10.1371/journal.pone.0199549 June 21, 2018 Normative hand movements during two functional upper limb tasks Fig 3. Cup Transfer Task hand trajectory. Hand trajectories for the Cup Transfer Task. The group average hand trajectory is plotted as a dark line, and the standard deviation of participant means as three-dimensional shading. Movement 1 (A), Movement 2 (B), Movement 3 (C), and Movement 4 (D) are segmented into reach (red), grasp (orange), transport (blue), and release (green) phases. The maximum of the mean three-dimensional standard deviation was calculated for reach-grasp and transport-release segments for each movement to quantify variability, reported in Table 3. https://doi.org/10.1371/journal.pone.0199549.g003 Fig 3. Cup Transfer Task hand trajectory. Hand trajectories for the Cup Transfer Task. The group average hand trajectory is plotted as a dark line, and the standard deviation of participant means as three-dimensional shading. Movement 1 (A), Movement 2 (B), Movement 3 (C), and Movement 4 (D) are segmented into reach (red), grasp (orange), transport (blue), and release (green) phases. The maximum of the mean three-dimensional standard deviation was calculated for reach-grasp and transport-release segments for each movement to quantify variability, reported in Table 3. https://doi.org/10.1371/journal.pone.0199549.g003 https://doi.org/10.1371/journal.pone.0199549.g003 been shown to provide insights into altered motor control strategies in populations with impaired upper limb function [1,16,30,35–37]. The importance of involving objects in goal- directed tasks with a functional context has been previously demonstrated as resulting in smoother, faster, more preplanned movement compared to non-goal directed movement through space [38]. Using natural objects for completing a task and providing functional infor- mation on the objects, rather than simulated devices, is important to enhance functional per- formance in both normative and impaired populations [39]. Our tasks, designed to be consistent with these parameters and also the requirements of known clinical upper limb assessments [20,23–26,40,41], were relatively easy for non-disabled individuals to perform. However, since errors were made by participants, the tasks required some level of attention and concentration. This may be valuable for assessing clinical populations with not only motor difficulties but also motor planning impairments. PLOS ONE \| https://doi.org/10.1371/journal.pone.0199549 June 21, 2018 13 / 21 Normative hand movements during two functional upper limb tasks Fig 4. Hand velocity. Hand velocity graphs for Pasta Box Task (A) and Cup Transfer Task (B). The solid line represents the group average, and grey shading the standard deviation of participant means. The task is segmented into reach (red), grasp (orange), transport (blue), and release (green) phases for each movement, with light grey representing the return to “home” phase. Kinematics of the reach-grasp segment and the transport-release segment were analyzed together. Fig 4. Hand velocity. Hand velocity graphs for Pasta Box Task (A) and Cup Transfer Task (B). The solid line represents the group average, and grey shading the standard deviation of participant means. The task is segmented into reach (red), grasp (orange), transport (blue), and release (green) phases for each movement, with light grey representing the return to “home” phase. Kinematics of the reach-grasp segment and the transport-release segment were analyzed together. https://doi.org/10.1371/journal.pone.0199549.g004 https://doi.org/10.1371/journal.pone.0199549.g004 Fig 5. Grip aperture. Grip aperture graphs for Pasta Box Task (A) and Cup Transfer Task (B). The solid line represents the group average, and grey shading represents the standard deviation of participant means. The task is segmented into reach (red), grasp (orange), transport (blue), and release (green) phases for each movement, with light grey representing the return to “home” phase. Kinematics of the reach-grasp segment and the transport- release segment were analyzed together. Fig 5. Grip aperture. Grip aperture graphs for Pasta Box Task (A) and Cup Transfer Task (B). The solid line represents the group average, and grey shading represents the standard deviation of participant means. The task is segmented into reach (red), grasp (orange), transport (blue), and release (green) phases for each movement, with light grey representing the return to “home” phase. Kinematics of the reach-grasp segment and the transport- release segment were analyzed together. https://doi.org/10.1371/journal.pone.0199549.g005 PLOS ONE \| https://doi.org/10.1371/journal.pone.0199549 June 21, 2018 14 / 21 Cup Transfer Task repeatability results. Repeatability analysis was performed, for movements and segments separately, on movement time, peak hand velocity, percent- to-peak hand velocity, peak grip aperture, percent-to-peak grip aperture, and percent-to-peak hand deceleration. Repeatability measures include intra-class correlation (ICC) with corresponding 95% confidence intervals, standard error of measurement (SEM), and minimal detectable change (MDC). ICC values above 0.90 are presented in bold. ICC values below 0.75 are presented in italics. ICC values that failed the F-test (p > 0.05) are presented with an asterisks (), indicating the validity of the ICC may be compromised for this result. Movements are: Movement 1 (Mvmt 1), Movement 2 (Mvmt 2), Movement 3 (Mvmt 3), and Movement 4 (Mvmt 4); segments are: reach-grasp (RG) and transport-release (TR). Normative hand movements during two functional upper limb tasks Table 6. Pasta Box Task repeatability. Movement time (sec) Peak hand velocity (mm/s) Percent-to-peak hand velocity (%) ICC SEM MDC ICC SEM MDC ICC SEM MDC Mvmt 1 RG 0.84 (0.37–0.96) 0.035 0.098 0.89 (0.54–0.97) 38 106 0.90 (0.61–0.98) 1.4 3.8 TR 0.81 (0.25–0.95) 0.046 0.128 0.56 (-0.77–0.89) 56 156 0.74 (-0.06–0.93) 2.4 6.6 Mvmt 2 RG 0.88 (0.50–0.97) 0.030 0.083 0.79 (0.15–0.95) 64 177 0.95 (0.79–0.99) 1.6 4.4 TR 0.86 (0.45–0.97) 0.057 0.159 0.91 (0.64–0.98) 30 84 0.83 (0.30–0.96) 2.0 5.5 Mvmt 3 RG 0.93 (0.70–0.98) 0.029 0.081 0.93 (0.72–0.98) 47 130 0.89 (0.57–0.97) 2.1 5.7 TR 0.89 (0.56–0.97) 0.052 0.145 0.78 (0.10–0.94) 40 112 0.86 (0.45–0.97) 1.6 4.4 Peak grip aperture (mm) Percent-to-peak grip aperture (%) Percent-to-peak hand deceleration (%) ICC SEM MDC ICC SEM MDC ICC SEM MDC Mvmt 1 RG 0.95 (0.78–0.99) 2 4 0.88 (0.50–0.97) 1.8 4.9 0.84 (0.35–0.96) 3.4 9.5 Mvmt 2 RG 0.92 (0.68–0.98) 2 4 0.95 (0.81–0.99) 1.0 2.7 0.80 (0.20–0.95) 2.6 7.2 Mvmt 3 RG 0.95 (0.79–0.99) 1 4 0.87 (0.47–0.97) 1.3 3.7 0.75 (0.01–0.94) 2.4 6.6 Table 6. Pasta Box Task repeatability. Pasta Box Task repeatability results. Repeatability analysis was performed for movements and segments separately, on movement time, peak hand velocity, percent-to- peak hand velocity, peak grip aperture, percent-to-peak grip aperture, and percent-to-peak hand deceleration. Repeatability measures include intra-class correlation (ICC) with corresponding 95% confidence intervals, standard error of measurement (SEM), and minimal detectable change (MDC). ICC values above 0.90 are presented in bold. ICC values below 0.75 are presented in italics. ICC values that failed the F-test (p > 0.05) are presented with an asterisks (), indicating the validity of the ICC may be compromised for this result. Movements are: Movement 1 (Mvmt 1), Movement 2 (Mvmt 2), and Movement 3 (Mvmt 3); segments are: reach-grasp (RG) and transport-release (TR). Pasta Box Task repeatability results. Repeatability analysis was performed for movements and segments separately, on movement time, peak hand velocity, percent-to- peak hand velocity, peak grip aperture, percent-to-peak grip aperture, and percent-to-peak hand deceleration. Repeatability measures include intra-class correlation (ICC) with corresponding 95% confidence intervals, standard error of measurement (SEM), and minimal detectable change (MDC). ICC values above 0.90 are presented in bold. ICC values below 0.75 are presented in italics. Pasta Box Task repeatability results. Repeatability analysis was performed for movements and segments separately, on movement time, peak hand velocity, percent-to- peak hand velocity, peak grip aperture, percent-to-peak grip aperture, and percent-to-peak hand deceleration. Repeatability measures include intra-class correlation (ICC) with corresponding 95% confidence intervals, standard error of measurement (SEM), and minimal detectable change (MDC). ICC values above 0.90 are presented in bold. ICC values below 0.75 are presented in italics. ICC values that failed the F-test (p > 0.05) are presented with an asterisks (), indicating the validity of the ICC may be compromised for this result. Movements are: Movement 1 (Mvmt 1), Movement 2 (Mvmt 2), and Movement 3 (Mvmt 3); segments are: reach-grasp (RG) and transport-release (TR). Normative hand movements during two functional upper limb tasks The tasks had specific movement sequences that were standardized, repeatable, of short duration, and consistently performed by individual participants. Other tasks used in literature have shown low within-participant variability [12,13,35], however typically using more con- strained tasks not as representative of real-world object interactions. Within-participant vari- ability is an important factor to assess as, for some clinical populations, increased variability in motor performance is a key indicator of poor motor skill, and may indicate the adoption of various strategies for accomplishing a task rather than converging on one strategy. For exam- ple, prosthesis users have been shown to have increased variability in upper limb angular kinematics, as reflected by increased average standard deviation [36], whereas more skilled prosthesis users have less deviation in end-point kinematic profiles from non-disabled move- ment patterns [42]. The measurement of variability may play an important role given that it has been shown in the occupational literature that both kinematic compensation and motor variability are associated with musculoskeletal pain [43]. The inclusion of mean participant standard deviation as a measure of within-participant variability in this normative data set will allow comparison in future study of impaired populations. The between-session repeatability of the task was also found to be good for 55 out of 63 parameters, which is a prerequisite prior to investigating sensitivity to change in clinical populations. The task design with specific sequencing allowed for segmentation of movements into the crucial phases of reaching and grasping, and transporting and releasing objects within the same task. This allows examination of discrete characteristics of hand movement pertaining to hand trajectory, hand velocity, and grip aperture for each of these phases. This is important as many clinical populations will have impaired dexterity impacting grasp, which has been exten- sively investigated [30,44]; however, valuable information can also be obtained by examining control of the hand during transport, such as grip modulation. In addition, grasp features are known to be affected by the task goal and setting [19]; therefore, it is most ecologically valid to use tasks that not only reach and grasp but involve a logical next step of movement and place- ment of the object. The influence of an object’s intrinsic and extrinsic parameters on hand kinematics was con- sistent with prior literature. PLOS ONE \| https://doi.org/10.1371/journal.pone.0199549 June 21, 2018 ICC values that failed the F-test (p > 0.05) are presented with an asterisks (), indicating the validity of the ICC may be compromised for this result. Movements are: Movement 1 (Mvmt 1), Movement 2 (Mvmt 2), and Movement 3 (Mvmt 3); segments are: reach-grasp (RG) and transport-release (TR). https://doi.org/10.1371/journal.pone.0199549.t006 Table 7. Cup Transfer Task repeatability. Movement time (sec) Peak hand velocity (mm/s) Percent-to-peak hand velocity (%) ICC SEM MDC ICC SEM MDC ICC SEM MDC Mvmt 1 RG 0.83 (0.33–0.96) 0.032 0.089 0.77 (0.07–0.94) 64 176 0.54 (-0.84–0.89) 4.6 12.6 TR 0.85 (0.41–0.96) 0.042 0.116 0.65 (-0.43–0.91) 50 139 0.82 (0.25–0.95) 0.9 2.5 Mvmt 2 RG 0.85 (0.40–0.96) 0.033 0.092 0.96 (0.83–0.99) 26 72 0.93 (0.71–0.98) 2.1 5.9 TR 0.81 (0.22–0.95) 0.053 0.147 0.76 (0.03–0.94) 29 81 0.91 (0.63–0.98) 2.8 7.8 Mvmt 3 RG 0.85 (0.41–0.96) 0.045 0.125 0.76 (0.05–0.94) 60 166 0.91 (0.62–0.98) 2.1 5.9 TR 0.76 (0.05–0.94) 0.068 0.189 0.52 (-0.93–0.88) 25 68 0.85 (0.38–0.96) 0.8 2.1 Mvmt 4 RG 0.75 (0.01–0.94) 0.031 0.086 0.84 (0.35–0.96) 50 139 0.78 (0.10–0.95) 3.8 10.5 TR 0.85 (0.38–0.96) 0.058 0.162 0.64 (-0.44–0.91) 29 80 0.99 (0.96–1.00) 0.7 2.0 Peak grip aperture (mm) Percent-to-peak grip aperture (%) Percent-to-peak hand deceleration (%) ICC SEM MDC ICC SEM MDC ICC SEM MDC Mvmt 1 RG 0.97 (0.87–0.99) 1 3 0.60 (-0.61–0.90) 1.8 5.0 0.68 (-0.30–0.92) 4.1 11.4 Mvmt 2 RG 0.97 (0.86–0.99) 1 3 0.79 (0.14–0.95) 2.1 5.9 0.86 (0.43–0.97) 2.8 7.9 Mvmt 3 RG 0.97 (0.86–0.99) 1 2 0.81 (0.24–0.95) 1.6 4.3 0.95 (0.78–0.99) 1.5 4.3 Mvmt 4 RG 0.96 (0.83–0.99) 2 4 0.86 (0.45–0.97) 2.2 6.0 0.84 (0.36–0.96) 5.2 14.5 Table 7. Cup Transfer Task repeatability. https://doi.org/10.1371/journal.pone.0199549.t007 PLOS ONE \| https://doi.org/10.1371/journal.pone.0199549 June 21, 2018 15 / 21 Normative hand movements during two functional upper limb tasks velocities occurring no later than 25% of the movement, indicating that movement of the com- pliant cup with risk of spillage was potentially challenging. This is consistent with Butler et al. who found lower percent-to-peak hand velocity values for the segment of their task where the performer had to bring a cup to their mouth, suggesting that this movement was riskier and required more conservative control strategies [35]. Both tasks involved obstacle avoidance; in the vertical plane for every movement of the cups, and in the horizontal plane for the second movement of the box of pasta. As previously shown by Chapman & Goodale, obstacles change the spatiotemporal characteristics of hand movement by increasing movement time and decreasing peak hand velocity [50]. This effect is even greater when obstacles are closer to the performer and on the side of the reaching arm [50]. These differences in spatiotemporal characteristics may have been amplified in our task since these trends were observed during the transport-release segment as opposed to the reach-grasp segment, where moving the object adds a further level of uncertainty. The challenges presented by these varied intrinsic and extrinsic properties is expected to result in significant performance differences in conditions with impaired hand sensation and impaired upper limb function. Reaching and grasping are functionally linked to the specific task, with the characteristics of the object determining the relative timing of peak grip aperture and peak hand deceleration [51]. Abnormalities in these features resulting in decoupling of reach and grasp have been shown in prosthesis users [18], cerebellar lesions [37], spinal cord injury [2] and in stroke populations [52], suggesting that this type of kinematic analysis could have applicability to multiple populations with upper limb impairment. Limitations and future work The assessment of normative hand movement characteristics demonstrated consistent trends across varying task challenges. The limitations of the current study include the assessment of only between-session repeatability, not the repeatability among different test administrators and study sites. Further study of inter-rater repeatability will assist with determining reproduc- ibility of the task assessment. Considering that the presented normative data set establishes an ideal young adult performance standard, further work may focus on establishing differences between the sexes or with aging cohorts to obtain a fully comparative data set for populations with impairment. Finally, future work will also test the application of this methodology in pop- ulations with upper limb impairments, and validate the measures against other clinically vali- dated hand outcome assessments in these populations. PLOS ONE \| https://doi.org/10.1371/journal.pone.0199549 June 21, 2018 Location of the object influenced several parameters, particularly the first movement of the Pasta Box Task where the box of pasta was not within the direct field of view and required a turn of the body and the head for the grasp. This misalignment of the body space to the visual space has been shown to increase the latency of the movement towards a target and decrease accuracy [45,46]. This first movement also required the arm to move multiple degrees of freedom across several planes (i.e., sagittal, transverse, and coronal planes) to complete the movement accurately. Therefore, a greater deceleration phase was necessary, evidenced by the hand velocity peaks occurring earlier than for other movements. This aligns with previous research by Fisk & Goodale who found hand velocity peaks to occur roughly around one third of the movement for lateral reaches [45], compared to studies that restricted reaching tasks to single plane movement and reported more symmetrical hand velocity profiles [47]. The location of the cups in the task space and the required grasp conformation also influ- enced reaching strategies. The first reach-grasp of the Cup Transfer Task showed the smallest grip aperture, suggesting confidence with the upcoming grasp of the top of the cup, but a slo- wed velocity of the reach likely due to the short distance. The two cylindrical side grasps showed similar grip apertures, but were different in movement strategies in that the first cylin- drical side grasp showed several features suggesting it was the reach with the highest perceived risk, with lower, earlier peak velocity and the earliest deceleration. This is consistent with pre- vious studies where hand velocity was lower during the reach when the task following the approach required precision [48,49]. Three of the transport release segments showed peak PLOS ONE \| https://doi.org/10.1371/journal.pone.0199549 June 21, 2018 16 / 21 Acknowledgments We thank Elizabeth Crockett, Ognen Kovic, Brody Kalwajtys, and Thomas R. Dawson for their contributions to task design, data collection and data processing. We thank Elizabeth Crockett, Ognen Kovic, Brody Kalwajtys, and Thomas R. Dawson for their contributions to task design, data collection and data processing. Author Contributions Conceptualization: Patrick M. Pilarski, Craig S. Chapman, Albert H. Vette, Jacqueline S. Hebert. Conceptualization: Patrick M. Pilarski, Craig S. Chapman, Albert H. Vette, Jacqueline S. Hebert. Data curation: Aïda M. Valevicius, Quinn A. Boser, Ewen B. Lavoie. Formal analysis: Aïda M. Valevicius, Quinn A. Boser, Craig S. Chapman. Funding acquisition: Craig S. Chapman, Jacqueline S. Hebert. Investigation: Aïda M. Valevicius, Quinn A. Boser, Ewen B. Lavoie. Investigation: Aïda M. Valevicius, Quinn A. Boser, Ewen B. Lavoie. Methodology: Glyn S. Murgatroyd, Craig S. Chapman, Albert H. Vette, Jacqueline S. Hebert. Project administration: Jacqueline S. Hebert. Methodology: Glyn S. Murgatroyd, Craig S. Chapman, Albert H. Vette, Jacqueline S. Hebert. Project administration: Jacqueline S. Hebert. Resources: Craig S. Chapman, Albert H. Vette, Jacqueline S. Hebert. Software: Aïda M. Valevicius, Quinn A. Boser, Patrick M. Pilarski, Craig S. Chapman. Supervision: Albert H. Vette, Jacqueline S. Hebert. Validation: Craig S. Chapman, Albert H. Vette, Jacqueline S. Hebert. Visualization: Aïda M. Valevicius, Craig S. Chapman, Albert H. Vette. Writing – original draft: Aïda M. Valevicius, Albert H. Vette, Jacqueline S. Hebert. Writing – review & editing: Patrick M. Pilarski, Craig S. Chapman, Albert H. Vette, Jacque- line S. Hebert. Conclusion Standardized upper limb functional tasks which mimic ADLs and incorporate elements of risk and accuracy, lateral reaches, reaches crossing the body’s midline, objects of different shapes and sizes, and different grasp patterns to assess hand movements were developed. A normative dataset for hand movement was created based on non-disabled performance characterizing hand trajectory, hand velocity, and grip aperture features for reach-grasp and transport-release segments of the movements. These features verified that the tasks challenged a variety of motor control strategies, and these unique movement characteristics were reflected in the quantitative results while being highly consistent within-performers. In addition to the low within-participant and between-participant variability for these complex tasks, a repeatability analysis showed that this novel assessment approach has good between-session repeatability. This assessment promises to be a valuable tool for future research in populations with upper limb impairments. PLOS ONE \| https://doi.org/10.1371/journal.pone.0199549 June 21, 2018 17 / 21 Normative hand movements during two functional upper limb tasks References 1. Lang CE, Wagner JM, Bastian AJ, Hu Q, Edwards DF, Sahrmann SA, et al. Deficits in grasp versus reach during acute hemiparesis. Exp Brain Res. 2005; 166(1):126–36. https://doi.org/10.1007/s00221- 005-2350-6 PMID: 16021431 2. Mateo S, Roby-Brami A, Reilly KT, Rossetti Y, Collet C, Rode G. Upper limb kinematics after cervical spinal cord injury: a review. J Neuroeng Rehabil. 2015; 12(1):9. 3. Metzger AJ, Dromerick AW, Holley RJ, Lum PS. Characterization of compensatory trunk movements during prosthetic upper limb reaching tasks. Arch Phys Med Rehabil. 2012; 93(11):2029–34. https://doi. org/10.1016/j.apmr.2012.03.011 PMID: 22449551 4. Hebert JS, Lewicke J. Case report of modified Box and Blocks test with motion capture to measure prosthetic function. J Rehabil Res Dev. 2012; 49(8):1163–74. PMID: 23341309 PLOS ONE \| https://doi.org/10.1371/journal.pone.0199549 June 21, 2018 18 / 21 Normative hand movements during two functional upper limb tasks 5. De Los Reyes-Guzma´n A, Dimbwadyo-Terrer I, Trincado-Alonso F, Monasterio-Huelin F, Torricelli D, Gil-Agudo A. Quantitative assessment based on kinematic measures of functional impairments during upper extremity movements: a review. Clin Biomech. 2014; 29(7):719–27. 6. Smeets JB, Brenner E. A new view on grasping. Motor Control. 1999; 3(3):237–71. PMID: 10409797 7. Cuijpers RH. On the relation between object shape and grasping kinematics. J Neurophysiol. 2004; 91(6):2598–606. https://doi.org/10.1152/jn.00644.2003 PMID: 14749319 8. Abend WBE, Morasso P. Human arm movement trajectory formation. Brain. 1982; 105:331–48. PMID: 7082993 9. Jeannerod M. The timing of natural prehension movements. J Mot Behav. 1984; 16(3):235–54. PMID: 15151851 10. Gordon J, Ghilardi MF, Cooper SE, Ghez C. Accuracy of planar reaching movements. Exp Brain Res. 1994; 99(1):112–30. PMID: 7925785 11. Gates DH, Walters LS, Cowley J, Wilken JM, Resnik L. Range of motion requirements for upper-limb activities of daily living. Am J Occup Ther. 2016; 70(1):7001350010p1–7001350010p10. https://doi.org/ 10.5014/ajot.2016.015487 PMID: 26709433 12. Jaspers E, Feys H, Bruyninckx H, Harlaar J, Molenaers G, Desloovere K. Upper limb kinematics: Devel- opment and reliability of a clinical protocol for children. Gait Posture. 2011; 33(2):279–85. https://doi. org/10.1016/j.gaitpost.2010.11.021 PMID: 21196120 13. Vanezis A, Robinson MA, Darras N. The reliability of the ELEPAP clinical protocol for the 3D kinematic evaluation of upper limb function. Gait Posture. 2015; 41(2):431–9. https://doi.org/10.1016/j.gaitpost. 2014.11.007 PMID: 25534948 14. Petuskey K, Bagley A, Abdala E, James MA, Rab G. Upper extremity kinematics during functional activ- ities: Three-dimensional studies in a normal pediatric population. Gait Posture. 2007; 25(4):573–9. https://doi.org/10.1016/j.gaitpost.2006.06.006 PMID: 16875821 15. van Andel CJ, Wolterbeek N, Doorenbosch CAM, Veeger D(H. EJ), Harlaar J. References J Rehabil Res Dev. 2014; 51(6):919–32. 28. Winter DA. Biomechanics of Human Movement. In: Biomechanics of Human Movement. John Wiley & Sons, Inc.; 1979. p. 32–5. 29. Alt Murphy M, Ha¨ger CK. Kinematic analysis of the upper extremity after stroke–how far have we reached and what have we grasped? Phys Ther Rev. 2015; 20(3):137–55. 30. Alt Murphy M, Wille´n C, Sunnerhagen KS. Kinematic variables quantifying upper-extremity perfor- mance after stroke during reaching and drinking from a glass. Neurorehabil Neural Repair. 2011; 25(1):71–80. https://doi.org/10.1177/1545968310370748 PMID: 20829411 31. Cirstea MC, Mitnitski AB, Feldman AG, Levin MF. Interjoint coordination dynamics during reaching in stroke. Exp Brain Res. 2003; 151(3):289–300. https://doi.org/10.1007/s00221-003-1438-0 PMID: 12819841 32. Schneiberg S, Mckinley P, Gisel E, Sveistrup H, Levin MF. Reliability of kinematic measures of func- tional reaching in children with cerebral palsy. Dev Med Child Neurol. 2010; 52(7):167–73. 33. Portney LG, Watkins MP. Foundations of clinical research : applications to practice. [Internet]. Upper Saddle River, N.J. : Pearson/Prentice Hall, c2009.; 2009. 34. Weir JP. Quantifying test-retest reliability using the intraclass correlation coefficient and the SEM. J Strength Cond Res. 2005; 19(1):231–40. PMID: 15705040 35. Butler EE, Ladd AL, LaMont LE, Rose J. Temporal-spatial parameters of the upper limb during a Reach & Grasp Cycle for children. Gait Posture. 2010; 32(3):301–6. https://doi.org/10.1016/j.gaitpost.2010.05. 013 PMID: 20558067 36. Major MJ, Stine RL, Heckathorne CW, Fatone S, Gard S a. Comparison of range-of-motion and variabil- ity in upper body movements between transradial prosthesis users and able-bodied controls when exe- cuting goal-oriented tasks. J Neuroeng Rehabil. 2014; 11(1):132. 37. Zackowski K, Thach W, Bastian A. Cerebellar subjects show impaired coupling of reach and grasp movements. Exp Brain Res. 2002; 146(4):511–22. https://doi.org/10.1007/s00221-002-1191-9 PMID: 12355280 38. Wu C-Y, Trombly CA, Lin K-C, Tickle-Degnen L. Effects of Object Affordances on Movement Perfor- mance: A Meta-Analysis. Vol. 5, Scandinavian Journal of Occupational Therapy. 1998 [cited]. p. 83– 92. 39. Wu C-Y, Trombly CA, Lin K-C, Tickle-Degnen L. Effects of Object Affordances on Reaching Perfor- mance in Persons with and Without Cerebrovascular Accident. Am J Occup Ther. 1998; 52(6):447–56. PMID: 9612495 40. Mathiowetz V, Volland G, Kashman N, Weber K. Adult Norms for the Box and Block Test of Manual Dexterity. Am J Ocupational Ther. 1985; 39(6):387–91. 41. Light CM, Chappell PH, Kyberd PJ. Establishing a standardized clinical assessment tool of pathologic and prosthetic hand function: Normative data, reliability, and validity. Arch Phys Med Rehabil. 2002; 83(6):776–83. References Complete 3D kinematics of upper extremity functional tasks. Gait Posture. 2008; 27(1):120–7. https://doi.org/10.1016/j.gaitpost. 2007.03.002 PMID: 17459709 16. van Vliet PM, Sheridan MR. Coordination between reaching and grasping in patients with hemiparesis and healthy subjects. Arch Phys Med Rehabil. 2007; 88(10):1325–31. https://doi.org/10.1016/j.apmr. 2007.06.769 PMID: 17908577 17. Schaefer SY, DeJong SL, Cherry KM, Lang CE. Grip type and task goal modify reach-to-grasp perfor- mance in post-stroke hemiparesis. Motor Control. 2012; 16(2):245–64. PMID: 22357103 18. Bouwsema H, van der Sluis CK, Bongers RM. Movement characteristics of upper extremity prostheses during basic goal-directed tasks. Clin Biomech. 2010; 25(6):523–9. 19. Valyear KF, Chapman CS, Gallivan JP, Mark RS, Culham JC. To use or to move: Goal-set modulates priming when grasping real tools. Exp Brain Res. 2011; 212(1):125–42. https://doi.org/10.1007/s00221- 011-2705-0 PMID: 21584628 20. Jebsen RH, Taylor N, Trieschmann RB, Trotter MJ, Howard LA. An objective and standardized test of hand function. Arch Phys Med Rehabil. 1969 Jun; 50(6):311–9. PMID: 5788487 21. Jongbloed-Pereboom M, Nijhuis-Van Der Sanden MWG, Steenbergen B. Norm scores of the box and block test for children ages 3–10 years. Am J Occup Ther. 2013 May 1; 67(3):312–8. https://doi.org/10. 5014/ajot.2013.006643 PMID: 23597689 22. Thrope G, Stroh K, Baco A. A Standardized Object Test for quantitative assessment of hand grasp func- tion using the C.W.R.U. upper extremity neuroprosthesis. Presperin J, editor. New Orleans: RESNA Press; 1989. 481 p. 23. Lyle RC. A performance test for assessment of upper limb function in physical rehabilitation. Int J Reha- bil Res. 1981; 4(4):483–92. PMID: 7333761 24. Lindner HYN, Linacre JM, Norling Hermansson LM. Assessment of capacity for myoelectric control: evaluation of construct and rating scale. J Rehabil Med. 2009; 41(6):467–74. https://doi.org/10.2340/ 16501977-0361 PMID: 19479160 25. Hudak PL, Amadio PC, Bombardier C, Beaton D, Cole D, Davis A, et al. Development of an upper extremity outcome measure: The DASH (disabilities of the arm, shoulder, and head). Am J Ind Med. 1996 Jun; 29(6):602–8. https://doi.org/10.1002/(SICI)1097-0274(199606)29:6<602::AID-AJIM4>3.0. CO;2-L PMID: 8773720 26. Resnik L, Adams L, Borgia M, Delikat J, Disla R, Ebner C, et al. Development and evaluation of the activities measure for upper limb amputees. Arch Phys Med Rehabil. 2013; 94(3):488–494.e4. https:// doi.org/10.1016/j.apmr.2012.10.004 PMID: 23085376 19 / 21 PLOS ONE \| https://doi.org/10.1371/journal.pone.0199549 June 21, 2018 Normative hand movements during two functional upper limb tasks 27. Hebert JS, Lewicke J, Williams TR, Vette AH. Normative data for modified Box and Blocks test measur- ing upper-limb function via motion capture. 50. Chapman CS, Goodale MA. Missing in action: The effect of obstacle position and size on avoidance while reaching. Exp Brain Res. 2008; 191(1):83–97. https://doi.org/10.1007/s00221-008-1499-1 PMID: 18716765 51. Marteniuk G, Leavitt JL, Mackenzie L. Functional relationship between grasp and transport components in a prehension task. 1990; 9:149–76. 52. Nowak DA. The impact of stroke on the performance of grasping: Usefulness of kinetic and kinematic motion analysis. Vol. 32, Neuroscience and Biobehavioral Reviews. 2008 [cited]. p. 1439–50. PLOS ONE \| https://doi.org/10.1371/journal.pone.0199549 June 21, 2018 References PMID: 12048655 42. Bouwsema H, Kyberd PJ, Hill W, van der Sluis CK, Bongers RM. Determining skill level in myoelectric prosthesis use with multiple outcome measures. J Rehabil Res Dev. 2012; 49(9):1331. PMID: 23408215 43. Madeleine P. On functional motor adaptations: From the quantification of motor strategies to the pre- vention of musculoskeletal disorders in the neck-shoulder region. Acta Physiol. 2010; 199(SUPPL. 679):1–46. 44. Alt Murphy M, Sunnerhagen KS, Johnels B, Wille´n C. Three-dimensional kinematic motion analysis of a daily activity drinking from a glass: a pilot study. J Neuroeng Rehabil. 2006; 3:18. https://doi.org/10. 1186/1743-0003-3-18 PMID: 16914057 45. Fisk JD, Goodale MA. The organization of eye and limb movements during unrestricted reaching to tar- gets in contralateral and ipsilateral visual space. Exp Brain Res. 1985; 60(1):159–78. PMID: 4043274 46. Prablanc C, Echallier JE, Jeannerod M, Komilis E. Optimal response of eye and hand motor systems in pointing at a visual target. II. Static and dynamic visual cues in the control of hand movement. Biol Cybern. 1979; 35(3):183–7. PMID: 518938 47. Morasso P. Spatial control of arm movements. Exp Brain Res. 1981; 42(2):223–7. PMID: 7262217 48. Marteniuk RC, Mackenzie CL. Constraints on Human Arm Movement Trajectories. Can J Psychol. 1987; 41(3). 49. Claxton LJ, Keen R, McCarty ME. Evidence of motor planning in infant reaching behavior. Psychol Sci. 2003; 14(4):354–6. https://doi.org/10.1111/1467-9280.24421 PMID: 12807409 20 / 21 PLOS ONE \| https://doi.org/10.1371/journal.pone.0199549 June 21, 2018 Normative hand movements during two functional upper limb tasks 21 / 21
https://openalex.org/W4394757617	https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2024.1355069/pdf	English	null	Risk factors of Carbapenem-resistant Enterobacterales intestinal colonization for subsequent infections in hematological patients: a retrospective case-control study	Frontiers in microbiology	2,024	cc-by	7,383	Risk factors of Carbapenem-resistant Enterobacterales intestinal colonization for subsequent infections in hematological patients: a retrospective case-control study OPEN ACCESS EDITED BY Silpak Biswas, Calcutta School of Tropical Medicine, India REVIEWED BY Anna Giammanco, University of Palermo, Italy Martina Barchitta, University of Catania, Italy CORRESPONDENCE Yan Jin sdjinyan@163.com RECEIVED 13 December 2023 ACCEPTED 01 April 2024 PUBLISHED 12 April 2024 CITATION Wang Z, Shao C, Shao J, Hao Y and Jin Y (2024) Risk factors of Carbapenem-resistant Enterobacterales intestinal colonization for subsequent infections in hematological patients: a retrospective case-control study. Front. Microbiol. 15:1355069. doi: 10.3389/fmicb.2024.1355069 Zihan Wang, Chunhong Shao, Jing Shao, Yingying Hao and Yan Jin Department of Clinical Laboratory, Shandong Provincial Hospital Afliated to Shandong First Medical University, Jinan, China Objective: Infections caused by Carbapenem-resistant Enterobacterales (CRE) have high treatment costs, high mortality and few efective therapeutic agents. This study aimed to determine the risk factors for progression from intestinal colonization to infection in hematological patients and the risk factors for 30-day mortality in infected patients. COPYRIGHT © 2024 Wang, Shao, Shao, Hao and Jin. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Methods: A retrospective case-control study was conducted in the Department of Hematology at Shandong Provincial Hospital afliated to Shandong First Medical University from April 2018 to April 2022. Patients who developed subsequent infections were identiﬁed as the case group by electronic medical record query of patients with a positive rectal screen for CRE colonization, and patients who did not develop subsequent infections were identiﬁed as the control group by stratiﬁed random sampling. Univariate analysis and logistic regression analysis determined risk factors for developing CRE infection and risk factors for mortality in CRE-infected patients. Results: Eleven hematological patients in the study developed subsequent infections. The overall 30-day mortality rate for the 44 hematological patients in the case-control study was 11.4% (5/44). Mortality was higher in the case group than in the control group (36.5 vs. 3.0%, P = 0.0026), and septic shock was an independent risk factor for death (P = 0.024). Univariate analysis showed that risk factors for developing infections were non-steroidal immunosuppressants, serum albumin levels, and days of hospitalization. TYPE Original Research PUBLISHED 12 April 2024 DOI 10.3389/fmicb.2024.1355069 TYPE Original Research PUBLISHED 12 April 2024 DOI 10.3389/fmicb.2024.1355069 Carbapenem-resistant Enterobacterales, colonization, infections, risk factors, hematological patients Risk factors of Carbapenem-resistant Enterobacterales intestinal colonization for subsequent infections in hematological patients: a retrospective case-control study In multivariable logistic regression analysis, immunosuppressants [odds ratio (OR), 19.132; 95% conﬁdence interval (CI), 1.349–271.420; P = 0.029] and serum albumin levels (OR, 0.817; 95% CI, 0.668–0.999; P = 0.049) were independent risk factors for developing infections. Conclusion: Our ﬁndings suggest that septic shock increases mortality in CRE- infected hematological patients. Hematological patients with CRE colonization using immunosuppressive agents and reduced serum albumin are more likely to progress to CRE infection. This study may help clinicians prevent the onset of infection early and take measures to reduce mortality rates. 1 Introduction University. This hospital has 3,889 beds, 90 of which belong to the hematology department. This study was approved by the Shandong Provincial Hospital Ethics Committee (NO. SWYX2023-610). University. This hospital has 3,889 beds, 90 of which belong to the hematology department. This study was approved by the Shandong Provincial Hospital Ethics Committee (NO. SWYX2023-610). Carbapenem-resistant Enterobacterales (CRE) infections pose a signiﬁcant threat to global public health due to their high treatment costs, high mortality, and limited availability of eﬀective therapeutic agents (Xu et al., 2017; European Centre for Disease Prevention and Control, 2018; Tacconelli et al., 2018; Brolund et al., 2019; Centers for Disease Control and Prevention, 2019). A report published in 2023 by the European Center for Disease Prevention and Control (ECDC) and the World Health Organization (WHO) revealed that the prevalence of carbapenem resistance in Klebsiella pneumoniae isolates rose by 0, 8, 31, and 20% between 2017 and 2021 (European Centre for Disease Prevention and Control and World Health Organization, 2023). Based on data from the China Antimicrobial Resistance Surveillance System (CARSS), the detection rate of carbapenem-resistant Klebsiella pneumoniae (CR-KPN) increased from 10.9% in 2020 to 11.3% in 2021, marking a rise from 6.4% in 2014. Additionally, the national average of Escherichia coli resistance to carbapenems remained at 1.6%, the same as in 2020 (China Antimicrobial Resistance Surveillance System, 2023). Patients with hematological disorders are at a higher risk of contracting CRE infections due to their compromised immune systems, low levels of neutrophils, extended hospital stays, undergoing hematopoietic stem cell transplantation, receiving chemotherapy, taking immunosuppressant medications, and frequent use of broad-spectrum antibiotics (Lalaoui et al., 2020; Cao et al., 2021). CRE rectal colonization was deﬁned as a positive CRE stool screen and the absence of invasive infection. CRE rectal infection was deﬁned as the presence of clinical signs and symptoms of infection and the detection of CRE in specimens taken from the site of infection. Infections such as bacteremia, pneumonia, urinary tract infections, and perianal infections are deﬁned according to guidelines issued by Centers for Disease Control and Prevention (2023). Patients with CRE colonization who were hospitalized for <3 days and discharged without identifying the colonizing strain were excluded, and patients with positive rectal screening for CRE in the hematology department from April 2018 to April 2022 were further studied. KEYWORDS Carbapenem-resistant Enterobacterales, colonization, infections, risk factors, hematological patients Frontiers in Microbiology 01 frontiersin.org Wang et al. Wang et al. 10.3389/fmicb.2024.1355069 1 Introduction The case group consisted of patients colonized with CRE and subsequently developed CRE infections caused by the same strain (Figure 1). The control group comprised patients selected in a 3:1 ratio relative to the 11 patients in the case group. To enhance the comparability between the case and control groups, we employed frequency matching based on age and sex to mitigate the inﬂuence of confounding factors. First, we determined the proportions of age and sex in the case group. Then, we used statistical software to conduct a stratiﬁed random sample of colonized patients who did not develop subsequent infections. The control and case groups were compared using clinical data variables to examine the risk factors associated with subsequent CRE infection. We conducted a 30-day follow-up study on the patients, with death as the end event. The initial event for the control group was the detection of positive test results for CRE colonization samples, while the case group involved detecting positive test results for CRE infection samples. Subsequently, CRE-infected patients were further divided into survival and non-survival groups to analyze risk factors for 30-day mortality. Furthermore, a time-to-event study was conducted to evaluate the characteristics of CRE colonization and infection in CRE- infected patients. As a reservoir for secondary infections, intestinal colonization correlates signiﬁcantly and independently with CRE infection (Gorrie et al., 2017; Cao et al., 2022). Multiple clinical studies have found that CRE colonization is associated with an increased risk of CRE infection and mortality in patients (Giannella et al., 2014; McConville et al., 2017; Lin et al., 2021; Gomides et al., 2022; Zhu et al., 2022). According to the guidelines from the ECDC, it is advisable to actively screen for CRE and apply eﬀective infection prevention and control strategies to stop the spread of CRE (Magiorakos et al., 2017). Hence, promptly identifying the risk factors contributing to the transition from CRE colonization to subsequent infection will signiﬁcantly decrease the incidence of CRE infections and mortality. 2.2 Data collection Fewer studies have been conducted regarding secondary CRE infections in hematological patients with intestinal colonization of CRE. Consequently, this study aimed to identify the factors that increase the likelihood of hematological patients with intestinal CRE colonization progressing to infection, as well as the factors that contribute to mortality in hematological patients already infected with CRE. It provides hematology clinicians with a reference for early identiﬁcation of high-risk hospitalized patients, allowing them to implement timely preventative measures against CRE infection and mortality. Between April 2018 and April 2022, we obtained stool sample data from 1,390 individuals with hematological conditions. Of these, 131 patients had CRE colonization, and we examined their clinical data. Clinical data is obtained from the patient’s electronic medical record and encompasses various factors such as demographic characteristics (age and gender), length of hospitalization, hematological diseases (such as acute myeloid leukemia, acute lymphoblastic leukemia, multiple myeloma, myelodysplastic syndrome, lymphoma, etc.), comorbidities (such as diabetes, hypertension, chronic liver disease, gastrointestinal disease, graft vs. host disease, hemorrhagic cystitis, mucositis, pneumonia, diarrhea, and shock), previous invasive procedures (such as deep venous catheterization, urinary catheterization, hematopoietic stem cell transplantation), exposure to drugs (such as chemotherapy, glucocorticoids, non-steroidal immunosuppressants, proton pump inhibitors, carbapenems, cephalosporins, ﬂuoroquinolones, aminoglycosides, glycopeptide, Frontiers in Microbiology 2.1 Study design This retrospective case-control study was conducted at the Shandong Provincial Hospital Aﬃliated to Shandong First Medical Frontiers in Microbiology 02 frontiersin.org Wang et al. 10.3389/fmicb.2024.1355069 FIGURE 1 Flowchart of the study design. penicillins, tigecycline), laboratory examinations (neutrophil count and serum albumin levels), CRE isolates (such as Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, and others), and outcomes (mortality at 30 days). Furthermore, we collected in vitro susceptibility data, carbapenemase phenotypes (serine carbapenemase and metal β-lactamases), sensitive antibiotic treatments, and the timing of colonization and infection in patients infected with CRE. system determined antimicrobial susceptibility tests. Tigecycline susceptibility was determined using the US Food and Drugs Administration (FDA) interpretive criteria (US Food Drug and Administration, 2023). Colistin using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoint (European Committee on Antimicrobial Susceptibility Testing, 2023), and the remaining susceptibility results were interpreted using the Clinical and Laboratory Standards Institute (CLSI) documentation standards (Clinical and Laboratory Standards Institute, 2022). The Modiﬁed Carbapenem Inactivation Method (mCIM) and Modiﬁed EDTA-Carbapenem Inactivation Method (eCIM) were employed to detect carbapenemase phenotypes (Pierce et al., 2017). 2.3 Microbiology Bacterial isolates for this study were identiﬁed using the Vitek 2 automatic system (bioMérieux, France). The stool samples were inoculated on MacConkey agar plates (ThermoFisher, USA), followed by carbapenem antimicrobial susceptibility testing using the disk diﬀusion method to conﬁrm the presence of carbapenem-resistant Enterobacteriaceae (CRE). CRE refers to Enterobacterales that exhibit resistance to at least one carbapenem antibiotic, namely ertapenem, imipenem, or meropenem. The broth microdilution method or the Vitek 2 Frontiers in Microbiology frontiersin.org 3.1 Patient characteristics A total of 131 patients were identiﬁed as positive for CRE colonization in this study (131/1,390, 9.4%), and the colonization frequency has been seen to increase annually (Supplementary Figure 1). After applying the exclusion criteria, 116 patients with CRE colonization were selected for further study. Eleven patients with CRE colonization subsequently acquired CRE infection (11/116, 9.5%). A control group of 33 out of the 105 patients colonized with CRE but did not acquire subsequent infection was selected using stratiﬁed random sampling. This control group was included in the case-control research alongside the group of infected patients (Figure 1). There was a higher proportion of males than females in the case and control groups (both 63.6%). The age of the case group was 35.36 ± 13.44 years, and the age of the control group was 36.33 ± 12.78 years. Acute myeloid leukemia was the most prevalent hematological condition in the case group (5/11, 45.5%), with acute lymphoblastic leukemia being the second most common (4/11, 36.4%). The control group primarily comprised individuals with acute myeloid leukemia (15/33, 45.5%), lymphoma (9/33, 27.3%), and acute lymphoblastic leukemia (3/33, 9.1%). Furthermore, we analyzed the time characteristics of 11 CRE-infected patients from admission to colonization and subsequent infection. The median duration from admission to colonization was 3 days (2–23 days). Similarly, the median duration from colonization to infection was 4 days (2–11 days; Figure 2). overall increasing trend from year to year. Escherichia coli was the dominant strain among the colonizers in the hematology department each year (Figure 4). Based on the CLSI and FDA breakpoints, the infected group showed the greatest susceptibility to colistin (100%) among CRE, followed by tigecycline and amikacin (both 81.8%) and gentamicin (63.6%). 72.7% of CRE exhibited resistance to aztreonam, while 72.7% of CRE showed resistance or intermediate susceptibility to tobramycin. All CRE found in infected patients exhibited resistance to a wide range of antibiotics, including ampicillin, ampicillin-sulbactam, piperacillin-tazobactam, cefoperazone- sulbactam, cefazolin, cefuroxime, ceftazidime, ceftriaxone, cefepime, cefoxitin, cefotetan, ertapenem, imipenem, meropenem, ciproﬂoxacin, levoﬂoxacin, and trimethoprim/sulfamethoxazole (Supplementary Table 1). In addition, the carbapenemase phenotypes of CRE-infected patients were characterized. Of these, seven strains produced metal β-lactamases (7/11, 63.6%), and four strains produced serine carbapenemases (4/11, 36.4%; Supplementary Table 2). 3.3 Analysis of risk factors for progression to CRE infection Table 1 displays the ﬁndings of the risk factor analysis for the progression from CRE colonization to CRE infection. No signiﬁcant diﬀerences were observed between the case and control groups regarding the prevalence of hematological conditions, comorbidities, invasive operations, and CRE strains (all P > 0.05). The median duration of hospitalization in the case group was 29 days (21–47 days), signiﬁcantly longer than the control group’s median duration of 11 days (6–26 days). Additionally, the mean albumin level in the case group was 28.11 ± 3.61 g/L, lower than the mean albumin level of 34.64 ± 6.92 g/L in the control group. In univariate analyses, variables associated with progression to infection included length of hospitalization, application of non-steroidal immunosuppressants, and serum albumin level. In 2.4 Statistical analysis All statistical analyses were performed using IBM SPSS Statistics version 25.0 software. We ﬁrst applied the Shapiro- Wilk test for normality to the continuous variables. Continuous variables that were normally distributed with variance equivalence 03 frontiersin.org Wang et al. 10.3389/fmicb.2024.1355069 were denoted as mean ± standard deviation (SD) and analyzed using independent samples t-test. While continuous variables that were not normally distributed or had non-equivalent variances were denoted as median with interquartile ranges (IQR) and analyzed using the Mann-Whitney U-Test. Categorical variables were analyzed using the Pearson Chi-square test, Continuity correction test, or Fisher’s Exact Test. Variables with P < 0.05 in the univariate analysis were included in the logistic regression analysis after Collinearity diagnostics and Variance inﬂation factor (VIF) checks to exclude multicollinearity. Kaplan-Meier curves and the log-rank test for the CRE colonization group vs. the infection group performed survival analysis. All tests were two-tailed and P < 0.05 indicated statistical signiﬁcance. FIGURE 2 Time characteristics of colonization and infection in 11 CRE-infected patients. The 11 CRE-infected patients are denoted as P01-P11. Frontiers in Microbiology 3.2 Microbiological characteristics Of the 44 CRE-colonized patients, 20 were colonized with Escherichia coli (20/44, 45.5%), 16 with Klebsiella pneumoniae (16/44, 36.4%), 6 with Enterobacter cloacae (6/44, 13.6%), and two with others (2/44, 4.5%). Escherichia coli was the most prevalent among patients with secondary CRE infections (5/11, 45.5%), followed by Klebsiella pneumoniae (4/11, 36.4%; Figure 3). Out of the 11 cases, 72.7% (8/11) were caused by infectious strains originating from bacteremia, while perianal infections, pneumonia, and urinary tract infections each accounted for 9% (1/11). In addition, we found that CRE colonization showed an 04 frontiersin.org Wang et al. 10.3389/fmicb.2024.1355069 FIGURE 3 Microbial characterization of CRE strains isolated from 44 CRE-colonized patients (A); Microbial characterization of CRE strains isolated from 11 CRE-infected patients (B). Escherichia coli was the most common strain for CRE colonization and infection, followed by Klebsiella pneumoniae. FIGURE 4 Trends of CRE colonization and infection in case-control studies, annual distribution of CRE-colonized strains. The annual trend of CRE colonization was upward, and the Escherichia coli counts were signiﬁcantly diferent from the other strains. FIGURE 3 Microbial characterization of CRE strains isolated from 44 CRE-colonized patients (A); Microbial characterization of CRE strains isolated from 11 CRE-infected patients (B). Escherichia coli was the most common strain for CRE colonization and infection, followed by Klebsiella pneumoniae. FIGURE 3 Microbial characterization of CRE strains isolated from 44 CRE-colonized patients (A); Microbial characterization of CRE strains isolated from 11 CRE-infected patients (B). Escherichia coli was the most common strain for CRE colonization and infection, followed by Klebsiella pneumoniae. FIGURE 3 Microbial characterization of CRE strains isolated from 44 CRE-colonized patients (A); Microbial characterization of CRE strains iso CRE-infected patients (B). Escherichia coli was the most common strain for CRE colonization and infection, followed by Klebsiella FIGURE 4 Trends of CRE colonization and infection in case-control studies, annual distribution of CRE-colonized strains. The annual trend of CRE colonization was upward, and the Escherichia coli counts were signiﬁcantly diferent from the other strains. FIGURE 4 Trends of CRE colonization and infection in case-control studies, annual distribution of CRE-colonized strains. The annual trend of CRE colonization was upward, and the Escherichia coli counts were signiﬁcantly diferent from the other strains. FIGURE 4 Trends of CRE colonization and infection in case-control studies, annual distribution of CRE-colonized strains. The annual trend of CRE colonization was upward, and the Escherichia coli counts were signiﬁcantly diferent from the other strains. 3.2 Microbiological characteristics and carbapenemase phenotypes (Supplementary Table 2). Four patients were treated with monotherapy (tigecycline, polymyxin, or aminoglycoside), whereas seven other patients were treated with combination therapy (all combined with tigecycline). Carbapenems were administered in combination to nine patients. Univariate analysis revealed that patients with CRE infection complicated by septic shock had a higher likelihood of mortality. Furthermore, no statistically signiﬁcant distinction was observed between monotherapy and combination therapy. multivariate logistic regression analysis, immunosuppressants (OR, 19.132; 95% CI, 1.349–271.420; P = 0.029) and albumin level (OR, 0.817; 95% CI, 0.668–0.999; P = 0.049) were identiﬁed as independent risk factors. 3.4 Analysis of risk factors for 30-day mortality in CRE-infected patients A total of ﬁve patients in both the case and control groups died during the 30-day follow-up period (5/44, 11.4%). The case group’s mortality rate was signiﬁcantly higher than the control group (36.5 vs. 3.0%, P = 0.0026; Figure 5). Within 30 days, four patients infected with CRE died of bacteremia. In order to determine mortality risk factors among hematological patients infected with CRE, we conducted a comparative analysis of the survival and non-survival groups using the same variables outlined in Table 1. Furthermore, we considered combined carbapenems, in vitro sensitive antibiotic treatment, Frontiers in Microbiology frontiersin.org 4 Discussion Controlling the spread of CRE has become a critical public health concern on a global scale, necessitating the implementation of infection prevention and control measures (Magiorakos et al., 2017; Zeng et al., 2023). Intensive Care Unit (ICU) admission and hematological malignancies are identiﬁed as risk factors associated with CRE infection (Tian et al., 2016; Chen et al., 2022; Zhang 05 frontiersin.org Wang et al. 10.3389/fmicb.2024.1355069 Univariate analysis and multivariate logistic regression analysis of risk factors for progression of CRE colonization to CRE infection TABLE 1 Univariate analysis and multivariate logistic regression analysis of risk factors for progression of CRE colonization to CRE infection. Characteristics Univariable analysis Multivariate analysis Case Control P-value OR (95% CI) P-value n = 11 (%) n = 33 (%) Sex-male 7 (63.6) 21 (63.6) Age (years), mean ± SD 35.36 ± 13.44 36.33 ± 12.78 Hospital stay (days), median (IQR) 29 (21.47) 11 (6.26) 0.004∗ 1.062 (0.996–1.132) 0.066 Hematological disease Acute myeloid leukemia 5 (45.5) 15 (45.5) 1.000 Acute lymphoblastic leukemia 4 (36.4) 3 (9.1) 0.096 Lymphoma 0 (0.0) 9 (27.3) 0.131 Myelodysplastic syndrome 1 (9.1) 2 (6.1) 1.000 Multiple myeloma 0 (0.0) 2 (6.1) 1.000 Others 1 (9.1) 2 (6.1) 1.000 Comorbidities Diabetes 1 (9.1) 2 (6.1) 1.000 Hypertension 0 (0.0) 3 (9.1) 0.561 Chronic liver disease 0 (0.0) 4 (12.1) 0.545 Gastrointestinal disease 3 (27.3) 5 (15.2) 0.652 GVHD 2 (18.2) 3 (9.1) 0.784 Hemorrhagic cystitis 1 (9.1) 3 (9.1) 1.000 Mucositis 7 (63.6) 10 (30.3) 0.108 Pneumonia 9 (81.8) 25 (75.8) 1.000 Diarrhea 5 (45.5) 6 (18.2) 0.159 Shock 0 (0.0) 1 (3.0) 1.000 Previous invasive procedures Deep venous catheterization 7 (63.6) 10 (30.3) 0.108 Urinary catheterization 1 (9.1) 1 (3.0) 0.442 HSCT 3 (27.3) 3 (9.1) 0.310 Exposure to drug Carbapenems (≤90 days) 10 (90.9) 23 (69.7) 0.315 Cephalosporins (≤90 days) 8 (72.7) 19 (57.6) 0.592 Fluoroquinolones (≤90 days) 3 (27.3) 13 (39.4) 0.717 Aminoglycosides (≤90 days) 2 (18.2) 9 (27.3) 0.841 Glycopeptides (≤90 days) 2 (18.2) 10 (30.3) 0.696 Penicillins (≤90 days) 2 (18.2) 10 (30.3) 0.696 Tigecycline (≤90 days) 5 (45.5) 9 (27.3) 0.455 Chemotherapy (≤30 days) 9 (81.8) 22 (66.7) 0.567 Glucocorticoids (≤30 days) 9 (81.8) 19 (57.6) 0.278 Non-steroidal immunosuppressants (≤30 days) 10 (90.9) 12 (36.4) 0.002∗ 19.132 (1.349–271.420) 0.029∗ PPIs (≤30 days) 6 (54.5) 21 (63.6) 0.858 (Continued) 06 Frontiers in Microbiology frontiersin.org Wang et al. 4 Discussion 10.3389/fmicb.2024.1355069 Characteristics Univariable analysis Multivariate analysis Case Control P-value OR (95% CI) P-value n = 11 (%) n = 33 (%) Laboratory examinations Neutrophils (×109/L), median (IQR) 0.56 (0.03, 3.03) 2.41 (0.93, 3.94) 0.080 Albumin (g/L), mean ± SD 28.11 ± 3.61 34.64 ± 6.92 0.005∗ 0.817 (0.668–0.999) 0.049∗ CRE isolates Escherichia coli 5 (45.5) 15 (45.5) 1.000 Klebsiella pneumoniae 4 (36.4) 12 (36.4) 1.000 Enterobacter cloacae 2 (18.2) 4 (12.1) 1.000 Other Enterobacterales 0 (0.0) 2 (6.1) 1.000 ∗Signiﬁcant statistical diﬀerence (P < 0.05). P, test signiﬁcance; OR, odds ratio; CI, conﬁdence interval; SD, standard deviation; IQR, Interquartile range; GVHD, graft vs. host disease; HSCT, hematopoietic stem cell transplantation; PPIS, proton pump inhibitors. ∗Signiﬁcant statistical diﬀerence (P < 0.05). Signiﬁcant statistical diﬀerence (P < 0.05). P, test signiﬁcance; OR, odds ratio; CI, conﬁdence interval; SD, standard deviation; IQR, Interquartile range; GVHD, graft vs. host disease; HSCT, hematopoietic stem cell transplantation; PPIS, p t p p i hibit ence (P < 0.05). s ratio; CI, conﬁdence interval; SD, standard deviation; IQR, Interquartile range; GVHD, graft vs. host disease; HSCT, hematopoietic stem cell transplantation; PPIS Signiﬁcant statistical diﬀerence (P < 0.05). P, test signiﬁcance; OR, odds ratio; CI, conﬁdence interval; SD, standard deviation; IQR, Interquartile range; GVHD, graft vs. host disease; HSCT, hematopoietic stem cell transplantation; PPIS, proton pump inhibitors. factor in CRE infection, which does not occur in all patients with colonization (Gorrie et al., 2017; Qin et al., 2020). Only a limited number of studies have been conducted concerning the risk factors that contribute to CRE infection in patients with hematological diseases (Zhang et al., 2019). Our study aims to identify the risk variables associated with the progression from colonization to infection in hematological patients and the risk factors for mortality in infected patients. These could provide clinicians with recommendations for preventing and controlling CRE infections. FIGURE 5 Survival curves of the case and control groups. The 30-day mortality rate was considerably higher in CRE-infected patients than in CRE-colonized patients (36.5 vs. 3.0%, P = 0.0026). The two studies on hematological malignancies had CRE colonization rates of 6.56 and 10.3%, respectively (Zhu et al., 2022; Chen et al., 2023). The CRE colonization rate in studies conducted in the ICU ranged from 15.5 to 45.4% (McConville et al., 2017; Kontopoulou et al., 2019; Gomides et al., 2022). frontiersin.org Frontiers in Microbiology 4 Discussion Our ﬁndings indicate that individuals with clinically severe disease had a higher likelihood of being prescribed combination therapies. Furthermore, the sample size of patients receiving combination therapy is small. Therefore, the eﬀect of combination therapy needs to be evaluated in randomized controlled trials. Chemotherapy, invasive operations, ICU admission, prolonged hospitalization, and exposure to carbapenem antibiotics are common risk factors for acquiring infections in patients with CRE colonization (Schechner et al., 2013; McConville et al., 2017; Collingwood et al., 2020; Chu et al., 2022; Chen et al., 2023). In our univariate analysis, prolonged hospitalization was identiﬁed as a risk factor for developing infections in patients harboring CRE colonization. Nevertheless, when doing multivariate analyses, the duration of hospitalization was not shown to be statistically signiﬁcant. Instead, non- steroidal immunosuppressants and albumin levels were identiﬁed as independent risk factors for the progression of infections. This study has some limitations. First, the study was single- center, and the results may not be generalizable to other departments and regions. Second, the study was retrospective and could not determine that the deaths were caused entirely by the CRE, thus making it impossible to analyze the attributable mortality rates. Third, the small sample size of infection and bias in treatment selection had limited ability to analyze risk factors for death. Therefore, multicenter prospective studies are necessary to address these issues. While prior use of carbapenems was a common culprit, and chemotherapy as well as proton pump inhibitors (PPIs) have also been found to be associated with infections (Chen et al., 2023), our analysis of previous drug exposures showed that immunosuppressant use signiﬁcantly increased the risk of infection in patients (OR, 19.132; 95% CI, 1.349– 271.420; p = 0.029). Moreover, a recent international matched case-control-control study found that immunosuppressive drugs in an inpatient population were risk factors for CRE infection (Perez-Galera et al., 2023). Many individuals with weakened immune systems due to hematological malignancies and hematopoietic stem cell transplants are admitted to hematology departments. These patients are regularly exposed to chemotherapy and immunosuppressive medications (Bar- Yoseph et al., 2019). Myelosuppression, an adverse eﬀect, can be experienced with any immunosuppressive drug and may result in agranulocytosis, hence increasing vulnerability to infections (Fraiser et al., 1991; Lee et al., 2016; Toksvang et al., 2022). Furthermore, reducing albumin levels ampliﬁes the susceptibility to infection in patients with CRE colonization (Rao et al., 2020; Liu et al., 2022; Qian et al., 2023). 4 Discussion administering immunosuppressive medicines and promptly address hypoalbuminemia in patients with CRE colonization to decrease CRE infections eﬀectively. Wang et al.’s (2018) longitudinal large-scale CRE data revealed that the predominant strain of clinical CRE isolates was Klebsiella pneumoniae, which exhibited a yearly upward trend. Escherichia coli was the primary strain that colonized the hematology department of our hospital, both in terms of total colonization and colonization on an annual basis. The major strain of secondary CRE infection was Escherichia coli, followed by Klebsiella pneumoniae, consistent with a prior study conducted in the Department of Hematology (Zhang et al., 2019). The infected strains mainly produced metal β- lactamases, and the choice of antibiotics varied for diﬀerent carbapenemases (Wang et al., 2018). KPC is a serine enzyme that hydrolyzes aztreonam but can be inhibited by the novel enzyme inhibitors avibactam and vebobactam, whereas NDM is a metalloenzyme that does not hydrolyze aztreonam but is not inhibited by avibactam and vebobactam. Hence, it is critical to identify the carbapenemase phenotype for subsequent antimicrobial treatment. In the present study, the 30-day mortality rate of patients with CRE infection was 36.4%, similar to that in studies on patients with hematological diseases (Liu et al., 2019; Zhang et al., 2019; Chen et al., 2023). The independent risk factor for death was septic shock, a variable that has been found to be associated with high mortality in several previously conducted studies (Tumbarello et al., 2012; Daikos et al., 2014; Chen et al., 2022, 2023). Septic shock is a sign of severe infection, and critically ill patients are more likely to die after the onset of the disease. Our study found that combination therapy did not reduce patient mortality, which is inconsistent with previous studies (Daikos et al., 2014; Tumbarello et al., 2015; Trecarichi et al., 2016; Chen et al., 2022). However, Paul et al. propose that patients infected with pathogens that exhibit limited susceptibility to antibiotics in vitro are more likely to be prescribed monotherapy. Conversely, patients infected with microorganisms more susceptible to in vitro susceptibility antibiotics are more likely to receive combination therapy. Patients with multiple antibiotic resistance may be more severely ill at baseline than patients with less antibiotic resistance. Consequently, the comparisons made between monotherapy and combination therapy may be inﬂuenced by selection bias (Paul et al., 2014). 4 Discussion The CRE colonization rate in this study was 9.4% (131/1390), similar to the studies on hematological malignancies and lower than the ICU colonization rate. A prospective study also suggests intestinal CRE colonization is more prevalent in the ICU, with widespread rapid spread (Chu et al., 2022). Furthermore, Cao et al. (2022) observed that the CRE colonization rate among recipients of allogeneic hematopoietic stem cell transplantation would be marginally elevated at 23.8%. The colonization rate exhibits variation within diﬀerent departments and may be associated with pharmacological therapies, patient groups, and clinical settings. FIGURE 5 Survival curves of the case and control groups. The 30-day mortality rate was considerably higher in CRE-infected patients than in CRE-colonized patients (36.5 vs. 3.0%, P = 0.0026). et al., 2022). Multiple studies have investigated the factors that increase the risk of infection and mortality from CRE in patients with either ICU or hematological malignancies (McConville et al., 2017; Kontopoulou et al., 2019; Lin et al., 2021; Chen et al., 2023). The hematology department admits many immunocompromised patients with hematological disorders who are more vulnerable to CRE infections. Our study’s infection rate in patients with CRE colonization was 9.5% (11/116). In patients colonized with CRE, the prevalence of CRE infection varied between 9.1 and 86% (Borer et al., 2012; Schechner et al., 2013; Dickstein et al., 2016; Tischendorf et al., 2016; Gomides et al., 2022). Due to the diﬀerent types of diseases and treatments administered to patients in each department, the results may be inconsistent; therefore, it is essential to examine hematology departments separately for CRE infection risk factors. Additionally, CRE colonization is a critical Our study also observed an overall trend of increasing CRE colonization in hematological patients throughout the years, aligning with the ﬁndings of a multicenter investigation (Fasciana et al., 2023). Nevertheless, the growth rate in 2020– 2022 is considerably diminished compared to 2018–2020. This can be ascribed to the proactive implementation of the national policy regarding the rational utilization of antimicrobial drugs in clinical practice and the enhanced surveillance of hospital infections by medical institutions in recent years (China Antimicrobial Resistance Surveillance System, 2023). Medical institutions must prioritize improving the appropriate utilization of antimicrobial drugs, minimizing the excessive use of broad-spectrum antimicrobial medicines like carbapenems, and Frontiers in Microbiology 07 frontiersin.org Wang et al. 10.3389/fmicb.2024.1355069 eﬀectively implementing hospital infection control measures to contain the widespread transmission of drug-resistant bacteria. 5 Conclusion In summary, the results of our study suggest that careful use of non-steroidal immunosuppressive agents and prompt correction of reduced albumin levels in hematological patients with CRE colonization can help reduce the incidence of CRE infections. Septic shock leads to a signiﬁcant increase in mortality in patients with CRE infection. These ﬁndings may help clinicians take appropriate precautions to reduce the incidence of CRE infections and decrease mortality in such patients. 4 Discussion Critically sick patients often exhibit hypoalbuminemia, which is characterized by a gradual depletion of vital protein components in the body due to inﬂammation caused by infection (McMillan et al., 2001). Hence, it is imperative to exercise caution in References China Antimicrobial Resistance Surveillance System (2023). Antimicrobial Resistance Surveillance in China 2023–2021 Data. Available online at: http://carss.cn/ Report/Details/862 (accessed June 15, 2023). Bar-Yoseph, H., Cohen, N., Korytny, A., Andrawus, E. R., Even Dar, R., Geﬀen, Y., et al. (2019). Risk factors for mortality among carbapenem-resistant Enterobacteriaceae carriers with focus on immunosuppression. J. Infect. 78, 101–105. doi: 10.1016/j.jinf.2018.10.003 Bar-Yoseph, H., Cohen, N., Korytny, A., Andrawus, E. R., Even Dar, R., Geﬀen, Y., et al. (2019). Risk factors for mortality among carbapenem-resistant Enterobacteriaceae carriers with focus on immunosuppression. J. Infect. 78, 101–105. doi: 10.1016/j.jinf.2018.10.003 Chu, W., Hang, X., Li, X., Ye, N., Tang, W., Zhang, Y., et al. (2022). Bloodstream infections in patients with rectal colonization by carbapenem-resistant Enterobacteriaceae: a prospective cohort study. Infect. Drug Resist. 15, 6051–6063. doi: 10.2147/IDR.S383688 Borer, A., Saidel-Odes, L., Eskira, S., Nativ, R., Riesenberg, K., Livshiz- Riven, I., et al. (2012). Risk factors for developing clinical infection with carbapenem-resistant Klebsiella pneumoniae in hospital patients initially only colonized with carbapenem-resistant K. pneumoniae. Am. J. Infect. Contr. 40, 421–425. doi: 10.1016/j.ajic.2011.05.022 Clinical and Laboratory Standards Institute (2022). M100-Performance Standards for Antimicrobial Susceptibility Testing, 32nd Edn. Malvern, PA: Clinical and Laboratory Standards Institute. Brolund, A., Lagerqvist, N., Byfors, S., Struelens, M. J., Monnet, D. L., Albiger, B., et al. (2019). Worsening epidemiological situation of carbapenemase-producing Enterobacteriaceae in Europe, assessment by national experts from 37 countries, July 2018. Eurosurveillance 24:1900123. doi: 10.2807/1560-7917.ES.2019.24.9.1900123 Collingwood, A., Blostein, F., Seekatz, A. M., Wobus, C. E., Woods, R. J., Foxman, B., et al. (2020). Epidemiological and microbiome associations between Klebsiella pneumoniae and vancomycin-resistant enterococcus colonization in intensive care unit patients. Open For. Infect. Dis. 7:ofaa012. doi: 10.1093/oﬁd/ofaa012 Cao, W., Guan, L., Li, X., Zhang, R., Li, L., Zhang, S., et al. (2021). Clinical analysis of bloodstream infections during agranulocytosis after allogeneic hematopoietic stem cell transplantation. Infect. Drug Resist. 14, 185–192. doi: 10.2147/IDR.S280869 Daikos, G. L., Tsaousi, S., Tzouvelekis, L. S., Anyfantis, I., Psichogiou, M., Argyropoulou, A., et al. (2014). Carbapenemase-producing Klebsiella pneumoniae bloodstream infections: lowering mortality by antibiotic combination schemes and the role of carbapenems. Antimicrob. Agents Chemother. 58, 2322–2328. doi: 10.1128/AAC.02166-13 Cao, W., Zhang, J., Bian, Z., Li, L., Zhang, S., Qin, Y., et al. (2022). Active screening of intestinal colonization of carbapenem-resistant Enterobacteriaceae for subsequent bloodstream infection in allogeneic hematopoietic stem cell transplantation. Infect. Drug Resist. 15, 5993–6006. doi: 10.2147/IDR.S387615 Dickstein, Y., Edelman, R., Dror, T., Hussein, K., Bar-Lavie, Y., and Paul, M. Funding The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2024. 1355069/full#supplementary-material The author(s) declare ﬁnancial support was received for the research, authorship, and/or publication of this article. Data availability statement The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author. Frontiers in Microbiology 08 frontiersin.org Wang et al. Wang et al. 10.3389/fmicb.2024.1355069 10.3389/fmicb.2024.1355069 Ethics statement This study was funded by Shandong Provincial Natural Science Foundation (ZR2021MH214). The studies involving humans were approved by Biomedical Research Ethic Committee of Shandong Provincial Hospital. The studies were conducted in accordance with the local legislation and institutional requirements. The human samples used in this study were acquired from primarily isolated as part of your previous study for which ethical approval was obtained. Written informed consent for participation was not required from the participants or the participants’ legal guardians/next of kin in accordance with the national legislation and institutional requirements. Conﬂict of interest The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Author contributions All claims expressed in this article are solely those of the authors and do not necessarily represent those of their aﬃliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. ZW: Writing – original draft, Software, Methodology, Investigation, Formal analysis, Data curation. CS: Writing – review & editing, Supervision, Resources. JS: Writing – review & editing, Supervision, Resources. YH: Writing – review & editing, Supervision, Resources. YJ: Resources, Writing – review & editing, Supervision, Funding acquisition, Conceptualization. References (2014). Risk factors for carbapenem-resistant Klebsiella pneumoniae bloodstream infection among rectal carriers: a prospective observational multicentre study. Clin. Microbiol. Infect. 20, 1357–1362. doi: 10.1111/1469-0691.12747 Schechner, V., Kotlovsky, T., Kazma, M., Mishali, H., Schwartz, D., Navon-Venezia, S., et al. (2013). Asymptomatic rectal carriage of blaKPC producing carbapenem- resistant Enterobacteriaceae: who is prone to become clinically infected? Clin. Microbiol. Infect. 19, 451–456. doi: 10.1111/j.1469-0691.2012.03888.x Gomides, M. D. A., Fontes, A. M. S., Silveira, A., Matoso, D. C., Ferreira, A. L., and Sadoyama, G. (2022). The importance of active surveillance of carbapenem- resistant Enterobacterales (CRE) in colonization rates in critically ill patients. PLoS ONE 17:e0262554. doi: 10.1371/journal.pone.0262554 Tacconelli, E., Carrara, E., Savoldi, A., Harbarth, S., Mendelson, M., Monnet, D. L., et al. (2018). Discovery, research, and development of new antibiotics: the WHO priority list of antibiotic-resistant bacteria and tuberculosis. Lancet Infect. Dis. 18, 318–327. doi: 10.1016/S1473-3099(17)30753-3 Gorrie, C. L., Mirˇceta, M., Wick, R. R., Edwards, D. J., Thomson, N. R., Strugnell, R. A., et al. (2017). Gastrointestinal carriage is a major reservoir of Klebsiella pneumoniae infection in intensive care patients. Clin. Infect. Dis. 65, 208–215. doi: 10.1093/cid/cix270 Tian, L., Tan, R., Chen, Y., Sun, J., Liu, J., Qu, H., et al. (2016). Epidemiology of Klebsiella pneumoniae bloodstream infections in a teaching hospital: factors related to the carbapenem resistance and patient mortality. Antimicrob. Resist. Infect. Contr. 5:145. doi: 10.1186/s13756-016-0145-0 Kontopoulou, K., Iosiﬁdis, E., Antoniadou, E., Tasioudis, P., Petinaki, E., Malli, E., et al. (2019). The clinical signiﬁcance of carbapenem-resistant Klebsiella pneumoniae rectal colonization in critically ill patients: from colonization to bloodstream infection. J. Med. Microbiol. 68, 326–335. doi: 10.1099/jmm.0.000921 Tischendorf, J., de Avila, R. A., and Safdar, N. (2016). Risk of infection following colonization with carbapenem-resistant Enterobactericeae: a systematic review. Am. J. Infect. Contr. 44, 539–543. doi: 10.1016/j.ajic.2015.12.005 Lalaoui, R., Javelle, E., Bakour, S., Ubeda, C., and Rolain, J. M. (2020). Infections due to carbapenem-resistant bacteria in patients with hematologic malignancies. Front. Microbiol. 11:1422. doi: 10.3389/fmicb.2020.01422 Toksvang, L. N., Lee, S. H. R., Yang, J. J., and Schmiegelow, K. (2022). Maintenance therapy for acute lymphoblastic leukemia: basic science and clinical translations. Leukemia 36, 1749–1758. doi: 10.1038/s41375-022-01591-4 Lee, J. Y., Kang, C. I., Ko, J. H., Lee, W. J., Seok, H. R., Park, G. E., et al. (2016). Clinical features and risk factors for development of breakthrough gram- negative bacteremia during carbapenem therapy. Antimicrobial. Agents Chemother. 60, 6673–6678. doi: 10.1128/AAC.00984-16 Trecarichi, E. References (2016). Carbapenem-resistant Enterobacteriaceae colonization and infection in critically ill patients: a retrospective matched cohort comparison with non-carriers. J. Hosp. Infect. 94, 54–59. doi: 10.1016/j.jhin.2016.05.018 Centers for Disease Control and Prevention (2019). Antibiotic Resistance Threats in the United States, 2019. Available online at: https://www.cdc.gov/drugresistance/ biggest-threats.html (accessed August 21, 2023). European Centre for Disease Prevention and Control (2018). Rapid Risk Assessment: Carbapenem-Resistant Enterobacteriaceae. Available online at: https://www.ecdc. europa.eu/en/publications-data/rapid-risk-assessment-carbapenem-resistant- enterobacteriaceae-ﬁrst-update (accessed August 21, 2023). Centers for Disease Control and Prevention (2023). CDC/NHSN Surveillance Deﬁnitions for Speciﬁc Types of Infections. Available online at: https://stacks.cdc.gov/ view/cdc/127235 (accessed June 17, 2023). Chen, J., Ma, H., Huang, X., Cui, Y., Peng, W., Zhu, F., et al. (2022). Risk factors and mortality of carbapenem-resistant Klebsiella pneumoniae bloodstream infection in a tertiary-care hospital in China: an eight-year retrospective study. Antimicrob. Resist. Infect. Control 11:161. doi: 10.1186/s13756-022-01204-w European Centre for Disease Prevention and Control and World Health Organization (2023). Antimicrobial Resistance Surveillance in Europe 2023–2021 Data. Available online at: https://www.ecdc.europa.eu/en/publications-data/antimicrobial- resistance-surveillance-europe-2023-2021-data (accessed August 22, 2023). Chen, X., Wen, X., Jiang, Z., and Yan, Q. (2023). Prevalence and factors associated with carbapenem-resistant Enterobacterales (CRE) infection among hematological malignancies patients with CRE intestinal colonization. Ann. Clin. Microbiol. Antimicrob. 22:3. doi: 10.1186/s12941-023-00554-6 European Committee on Antimicrobial Susceptibility Testing (2023). Clinical Breakpoints and Dosing of Antibiotics. Available online at: http://www.eucast.org/ clinical_breakpoints/ (accessed February 20, 2023). Frontiers in Microbiology 09 frontiersin.org Wang et al. Wang et al. 10.3389/fmicb.2024.1355069 Fasciana, T., Antonelli, A., Bianco, G., Lombardo, D., Codda, G., Roscetto, E., et al. (2023). Multicenter study on the prevalence of colonization due to carbapenem- resistant Enterobacterales strains before and during the ﬁrst year of COVID-19, Italy 2018-2020. Front. Public Health 11:1270924. doi: 10.3389/fpubh.2023.1270924 with carbapenem-resistant organisms bloodstream infection. Infect. Drug Resist. 16, 201–215. doi: 10.2147/IDR.S393932 Qin, X., Wu, S., Hao, M., Zhu, J., Ding, B., Yang, Y., et al. (2020). The colonization of carbapenem-resistant Klebsiella pneumoniae: epidemiology, resistance mechanisms, and risk factors in patients admitted to intensive care units in China. J. Infect. Dis. 221(Suppl.2), S206–S214. doi: 10.1093/infdis/jiz622 Fraiser, L. H., Kanekal, S., and Kehrer, J. P. (1991). Cyclophosphamide toxicity. Characterising and avoiding the problem. Drugs 42, 781–795. doi: 10.2165/00003495-199142050-00005 Rao, K., Seekatz, A., Bassis, C., Sun, Y., Mantlo, E., and Bachman, M. A. (2020). Enterobacterales infection after intestinal dominance in hospitalized patients. mSphere 5:20. doi: 10.1128/mSphere.00450-20 Giannella, M., Trecarichi, E. M., De Rosa, F. G., Del Bono, V., Bassetti, M., Lewis, R. E., et al. References M., Pagano, L., Martino, B., Candoni, A., Di Blasi, R., Nadali, G., et al. (2016). Bloodstream infections caused by Klebsiella pneumoniae in onco-hematological patients: clinical impact of carbapenem resistance in a multicentre prospective survey. Am. J. Hematol. 91, 1076–1081. doi: 10.1002/ajh.24489 Lin, Q., Wang, Y., Yu, J., Li, S., Zhang, Y., Wang, H., et al. (2021). Bacterial characteristics of carbapenem-resistant Enterobacteriaceae (CRE) colonized strains and their correlation with subsequent infection. BMC Infect. Dis. 21:638. doi: 10.1186/s12879-021-06315-0 Tumbarello, M., Trecarichi, E. M., De Rosa, F. G., Giannella, M., Giacobbe, D. R., Bassetti, M., et al. (2015). Infections caused by KPC-producing Klebsiella pneumoniae: diﬀerences in therapy and mortality in a multicentre study. J. Antimicrob. Chemother. 70, 2133–2143. doi: 10.1093/jac/dkv086 Liu, J., Wang, H., Huang, Z., Tao, X., Li, J., Hu, Y., et al. (2019). Risk factors and outcomes for carbapenem-resistant Klebsiella pneumoniae bacteremia in onco- hematological patients. J. Infect. Dev. Ctries 13, 357–364. doi: 10.3855/jidc.11189 Tumbarello, M., Viale, P., Viscoli, C., Trecarichi, E. M., Tumietto, F., Marchese, A., et al. (2012). Predictors of mortality in bloodstream infections caused by Klebsiella pneumoniae carbapenemase-producing K. pneumoniae: importance of combination therapy. Clin. Infect. Dis. 55, 943–950. doi: 10.1093/cid/c is,588 Liu, P., Mai, Y., Yuan, W., Xie, L., Ma, W., Liu, J., et al. (2022). Risk factors for mortality and antimicrobial regimens in pediatric intensive care unit patients with carbapenem-resistant Enterobacteriaceae infections: a six-year retrospective study. Infect. Drug Resist. 15, 7307–7316. doi: 10.2147/IDR.S394283 US Food and Drug Administration (2023). Tigecycline—Injection Products. Available online at: https://www.fda.gov/drugs/development-resources/tigecycline- injection-products (accessed February 15, 2023). Magiorakos, A. P., Burns, K., Rodríguez Baño, J., Borg, M., Daikos, G., Dumpis, U., et al. (2017). Infection prevention and control measures and tools for the prevention of entry of carbapenem-resistant Enterobacteriaceae into healthcare settings: guidance from the European Centre for Disease Prevention and Control. Antimicrob. Resist. Infect. Contr. 6:259. doi: 10.1186/s13756-017-0259-z Wang, Q., Wang, X., Wang, J., Ouyang, P., Jin, C., Wang, R., et al. (2018). Phenotypic and genotypic characterization of Carbapenem-resistant Enterobacteriaceae: data from a longitudinal large-scale CRE study in China (2012–2016). Clin. Infect. Dis. 67(Suppl.2), S196–S205. doi: 10.1093/cid/ciy660 McConville, T. H., Sullivan, S. B., Gomez-Simmonds, A., Whittier, S., and Uhlemann, A. C. (2017). Carbapenem-resistant Enterobacteriaceae colonization (CRE) and subsequent risk of infection and 90-day mortality in critically ill patients, an observational study. PLoS ONE 12:e0186195. doi: 10.1371/journal.pone.0186195 Xu, L., Sun, X., and Ma, X. (2017). References Systematic review and meta-analysis of mortality of patients infected with carbapenem-resistant Klebsiella pneumoniae. Ann. Clin. Microbiol. Antimicrob. 16:3. doi: 10.1186/s12941-017-0191-3 McMillan, D. C., Watson, W. S., O’Gorman, P., Preston, T., Scott, H. R., and McArdle, C. S. (2001). Albumin concentrations are primarily determined by the body cell mass and the systemic inﬂammatory response in cancer patients with weight loss. Nutr. Cancer 39, 210–213. doi: 10.1207/S15327914nc392_8 Zeng, M., Xia, J., Zong, Z., Shi, Y., Ni, Y., Hu, F., et al. (2023). Guidelines for the diagnosis, treatment, prevention and control of infections caused by carbapenem-resistant gram-negative bacilli. J. Microbiol. Immunol. Infect. 56, 653–671. doi: 10.1016/j.jmii.2023.01.017 Paul, M., Carmeli, Y., Durante-Mangoni, E., Mouton, J. W., Tacconelli, E., Theuretzbacher, U., et al. (2014). Combination therapy for carbapenem- resistant Gram-negative bacteria. J. Antimicrob. Chemother. 69, 2305–2309. doi: 10.1093/jac/dku168 Zhang, H., Hu, S., Xu, D., Shen, H., Jin, H., Yang, J., et al. (2022). Risk factors for carbapenem resistant gram negative bacteria (CR-GNB) carriage upon admission to the gastroenterology department in a tertiary ﬁrst class hospital of China: development and assessment of a new predictive nomogram. Infect. Drug Resist. 15, 7761–7775. doi: 10.2147/IDR.S396596 Perez-Galera, S., Bravo-Ferrer, J. M., Paniagua, M., Kostyanev, T., de Kraker, M. E. A., Feifel, J., et al. (2023). Risk factors for infections caused by carbapenem-resistant Enterobacterales: an international matched case-control-control study (EURECA). EClinicalMedicine 57:101871. doi: 10.1016/j.eclinm.2023.101871 Zhang, L., Zhai, W., Lin, Q., Zhu, X., Xiao, Z., Yang, R., et al. (2019). Carbapenem- resistant Enterobacteriaceae in hematological patients: outcome of patients with Carbapenem-resistant Enterobacteriaceae infection and risk factors for progression to infection after rectal colonization. Int. J. Antimicrob. Agents 54, 527–529. doi: 10.1016/j.ijantimicag.2019.06.023 Pierce, V. M., Simner, P. J., Lonsway, D. R., Roe-Carpenter, D. E., Johnson, J. K., Brasso, W. B., et al. (2017). Modiﬁed carbapenem inactivation method for phenotypic detection of carbapenemase production among Enterobacteriaceae. J. Clin. Microbiol. 55, 2321–2333. doi: 10.1128/JCM.00193-17 Zhu, R., Xu, X., Lian, S., Cai, M., Zhang, H., Chen, X., et al. (2022). Intestinal colonization with Carbapenem-resistant Enterobacteriaceae in acute leukemia patients: risk factors and molecular characteristics. Infect. Drug Resist. 15, 4275–4283. doi: 10.2147/IDR.S376413 Qian, C., Wu, Q., Ruan, Z., Liu, F., Li, W., Shi, W., et al. (2023). A visualized mortality prediction score model in hematological malignancies patients 10 frontiersin.org Frontiers in Microbiology
https://openalex.org/W2883557846	http://www.revistas.usp.br/espinosanos/article/download/145925/141532	Portuguese	null	JOHN LOCKE E AS TEORIAS DO DIREITO DE RESISTÊNCIA DE MATRIZ LUTERANA	Cadernos Espinosanos/Cadernos espinosanos	2,018	cc-by	5,718	imagem Detalhe do retrato The Honourable Algernon Sidney gravado por J. Cochran em torno de 1800. Cadernos Espinosanos estudos sobre o século xvii 2018 n. 38 issn 1413-6651 jan-jun estudos sobre o século xvii 2018 n. 38 issn 1413-6651 jan-jun estudos sobre o século xvii imagem Detalhe do retrato The Honourable Algernon Sidney gravado por J. Cochran em torno de 1800. JOHN LOCKE E AS TEORIAS DO DIREITO DE RESISTÊNCIA DE MATRIZ LUTERANA Silvio Gabriel Serrano Nunes Doutor em Filosofia, Universidade de São Paulo, São Paulo, Brasil serrano.nunes@gmail.com Silvio Gabriel Serrano Nunes Doutor em Filosofia, Universidade de São Paulo, São Paulo, Brasil serrano.nunes@gmail.com resumo: Pretende-se abordar como os argumentos luteranos – de na- tureza constitucional das “magistraturas inferiores” e de direito privado – acerca do direito de resistência, desenvolvidos no final da década de 1520 e início de 1530, foram recepcionados no Segundo Tratado Sobre o Governo Civil, de John Locke, escrito no século xvii. O argumento de direito privado compreende que todo governante que abandona as boas ações e se dedica a cometer atos tirânicos se despoja de sua autoridade e, consequentemente, deve ser tratado como uma pessoa privada, por- tanto, sujeita ao lícito revide, o que configura uma espécie de legítima defesa. O argumento constitucional das “magistraturas inferiores” afirma a possibilidade de órgãos intermediários (agentes políticos como corpos legislativos e judiciais) entre o governante e o povo estarem legitimados para oferecer resistência às tiranias. Em John Locke, ambos os argumen- tos objetivam a solução do problema da tirania para se obter liberdade política e religiosa. palavras-chave: Direito de Resistência, Teorias Luteranas, Argumento Constitucional, Argumento de Direito Privado, John Locke. Silvio Gabriel Serrano Nunes p. 189 - 205 189 O presente texto objetiva abordar como os argumentos luteranos – de natureza constitucional das “magistraturas inferiores” e de direito privado – acerca do direito de resistência, desenvolvidos no final da dé- cada de 1520 e início de 1530, foram recepcionados no Segundo Tratado Sobre o Governo Civil, de John Locke, escrito no século xvii. O argumento de direito privado afirma que todo governante que abandona as boas ações e se dedica a cometer atos tirânicos perde sua condição de autoridade e deve ser tratado como uma pessoa privada, portanto, sujeita ao revide, o que configura uma espécie de legítima de- fesa. O argumento constitucional das “magistraturas inferiores” afirma a possibilidade de órgãos intermediários (agentes políticos como corpos legislativos e judiciais) entre o governante e o povo estarem legitimados para oferecer resistência às tiranias. Em John Locke, ambos os argumen- tos recepcionados têm como objetivo a solução do problema da tirania para se obter liberdade política. John Locke, em seu Segundo Tratado sobre o Governo Civil, no Ca- pítulo xiii, “Da subordinação dos poderes da sociedade política”, recep- cionando o argumento constitucional de origem luterana, descreve o legislativo como o poder supremo nos seguintes termos: Em todos os casos, enquanto subsistir o governo, o legislativo é o poder supremo. 90 Cadernos Espinosanos São Paulo n.38 jan-jun 2018 Silvio Gabriel Serrano Nunes Doutor em Filosofia, Universidade de São Paulo, São Paulo, Brasil serrano.nunes@gmail.com Pois o que pode legislar para outrem deve por força ser-lhe superior; e como o legislativo o é tão somente pelo direito de elaborar leis para todas as partes e para cada membro da sociedade, prescreven- do regras para as suas ações e concedendo poder de execução onde quer que sejam transgredidas, deve por força ser o poder supremo, e todos os demais poderes depositados em quaisquer membros ou partes da sociedade devem derivar dele ou servir-lhe subordinados (locke, 2005, p. 519). No Capítulo xviii – “Da tirania” – deste clássico da filosofia polí- tica, Locke, após definir a tirania como o exercício do poder que exorbita o direito, bem como o uso do poder para benefício privado em detri- mento do interesse público, afirma que essa prática ensejaria conferir ao Cadernos Espinosanos São Paulo n.38 jan-jun 2018 190 tirano o tratamento não mais de uma autoridade constituída, mas de um particular merecedor de resposta imediata ao mal provocado: Onde termina a lei, começa a tirania, se a lei for transgredida para preju- ízo de outrem. E todo aquele que, investido de autoridade, exceda o poder que lhe é conferido por lei e faça uso da força que tem sob seu comando para impor ao súdito o que a lei não permite, deixa, com isso, de ser magistrado e, agindo sem autoridade, pode ser combatido, como qualquer outro homem que pela força invade o direito alheio. Tal é reconhecido nos magistrados subalternos. Aquele que tem auto- ridade para prender a minha pessoa na rua pode ser combatido como ladrão e assaltante, se tentar invadir-me a casa para executar um man- dato, não obstante eu saiba ter ele permissão e autoridade legal para prender-me fora de casa (locke, 2005, p. 563-564). Antes de aprofundar a análise da teoria do argumento constitu- cional das magistraturas inferiores, desenvolvida pelos luteranos no sé- culo xvi, considerando-se o clássico estudo de Richard Roy Benert, Inferior Magistrates in Sixteenth – Century Political and Legal Thought, con- vém destacar, no âmbito do pensamento político medieval, o esforço em se afirmar a legitimidade do combate às tiranias em nome de uma uni- versalidade ou corporação conferida genericamente a agentes da aristo- cracia, o que constituiu um precedente de grande importância para que os protestantes desenvolvessem seu pensamento de resistência. Para Benert (1967, p. 192 Cadernos Espinosanos São Paulo n.38 jan-jun 2018 Silvio Gabriel Serrano Nunes Doutor em Filosofia, Universidade de São Paulo, São Paulo, Brasil serrano.nunes@gmail.com 80-81), a partir do século xii ocorreu um reavivamento do direito romano, sendo que a influência das ideias dos canonistas quanto ao conceito de corporação levou os autores medievais a formular que o povo constituía uma verdadeira universitas ou corpora- ção, apta a exercer sua autoridade política contra o supremo magistrado que a governasse, como um rei ou o imperador. Segundo vários autores medievais, uma corporação deveria agir como unidade e, no advento de uma tirania, combatê-la em nome do “povo como um todo” (benert, 1967, p. 81). Silvio Gabriel Serrano Nunes p. 189 - 205 191 Na Idade Média, o traço essencial da corporação não seria sim- plesmente “uma pluralidade de pessoas reunidas em um só corpo” num determinado momento, mas consistiria em algo mais complexo, pois essa pluralidade deve ser compreendida em sucessão no tempo e mediante a ação deste. Consequentemente, [...] seria errôneo considerar a universitas corporativista meramente como os simul cohabitantes, aqueles vivendo juntos no mesmo momen- to; pois, na linguagem de Tomás de Aquino, seriam semelhantes apenas no corpo físico humano, cujos os membros estavam presentes “todos de uma vez”, mas não constituíam o corpus mysticum genuíno, tal como Tomás de Aquino o definira. A pluralidade em sucessão, portanto, ou a pluralidade no tempo, era o fator essencial costurando a universitas na continuidade e tornando-a imortal (kantorowicz, 1998, p. 190). Em linhas gerais, E. H. Kantorowicz, em seu clássico estudo Os Dois Corpos do Rei, mostra que no período medieval já se manifestava a distinção entre o corpo físico do rei e o corpo político (um binômio razoavelmente análogo ao da pessoa de Cristo e seu corpo místico, a Igreja), o primeiro perecível e o segundo perene. A distinção entre um ente concreto e outro abstrato se manifestava visivelmente nos funerais dos reis, uma vez que os principais magistrados do reino, membros do corpo político, estavam dispensados de usar roupas de luto, de modo a atestar a continuidade do corpo político. Esses mesmos magistrados, cuja autoridade se associava mais ao corpo político do que ao próprio rei, passariam a ter legitimidade para o exercício do direito de resistência contra a tirania. Kantorowicz (1998, p. 193) explica que os jurisconsultos, ao inter- pretarem o povo como uma universitas “que nunca morre”, elaboraram conceitualmente a ideia de uma “perpetuidade tanto da totalidade do corpo político (cabeça e membros em conjunto) como de cada um dos membros constituintes”. Silvio Gabriel Serrano Nunes Doutor em Filosofia, Universidade de São Paulo, São Paulo, Brasil serrano.nunes@gmail.com O princípio de resistência por parte de corpos representativos perenes das comunidades políticas encontrou várias formas de se expres- O princípio de resistência por parte de corpos representativos perenes das comunidades políticas encontrou várias formas de se expres- Cadernos Espinosanos São Paulo n.38 jan-jun 2018 sar na pena dos pensadores políticos medievais. A título de exemplo, São Tomás de Aquino afirma ser lícito à autoridade pública depor um tirano graças ao “direito da multidão”; Guilherme de Occam reconheceria em determinadas circunstâncias a superioridade do “reino”, de modo que lhe seria facultado depor o rei; Jean Gerson evoca a autoridade de Aristó- teles (A Política, Livro v) para alegar que “a comunidade como um todo” estaria apta a corrigir a situação e remover o tirano; Coluccio Salutati, versando sobre as repúblicas independentes, afirma que a resistência de- pende de um decreto do “povo”, que exerce a autoridade política, e, uma vez que toda punição criminal deveria decorrer de um ato público da autoridade da comunidade, desta mesma forma deveria se proceder para destronar os tiranos (benert, 1967, p. 81-82). Richard Roy Benert ainda explica que, apesar de desenvolverem habilmente o argumento da legitimidade da autoridade da comunidade para o exercício do direito de resistência, esses autores medievais “foram menos bem-sucedidos em identificar os instrumentos pelos quais a auto- ridade pública age [deveria agir]”. Em regra, faziam genéricas referências aos “tradicionais líderes aristocráticos que na ocasião agiam individual- mente contra o rei, mas a partir de então podiam clamar que agiam cor- porativamente pelo [bem do] reino como um todo” (benert, 1967, p. 82). Algumas vezes, esses pensadores chegavam a indicar os nobres que poderiam perpetrar a resistência, mencionando genericamente, porém, um grupo de “líderes naturais do reino”. A título exemplificativo, João de Paris apontava os “barões e pares do reino”; Egídio de Roma mencio- nava os “gentil-homens e grandes homens da nação”; Marsílio de Pádua, os homens que compunham os “valentior pars” que atuavam em nome de todo o povo; Nicolau de Cusa, no século xv, asseverava que o combate ao tirano era responsabilidade daqueles que governavam em nome de todo o império, como os “praesides” e “rectors” (benert, 1967, p. 83). Os autores medievais que interpretavam a lex regia da mesma for- ma que Hugolino e Azo tinham em mente que “a revogabilidade da au- toridade do imperador era uma garantia do povo romano”, cabendo-lhes Silvio Gabriel Serrano Nunes p. Cadernos Espinosanos São Paulo n.38 jan-jun 2018 Silvio Gabriel Serrano Nunes Doutor em Filosofia, Universidade de São Paulo, São Paulo, Brasil serrano.nunes@gmail.com 189 - 205 193 então a tarefa de identificar a quem competiria revogar a autoridade do imperador, o que, segundo Benert, resultou em vagas menções, como no caso de Guilherme de Occam que, sem tecer maiores considerações, ape- nas alegou que o imperador poderia ser removido por “aqueles a quem os romanos conferissem tal poder”. Lupold von Bebenberg entendia que os agentes públicos aptos a destronar o imperador seriam “os eleitores [do Sacro Império Romano-Germânico, ou seja, o “povo romano” à época], príncipes, condes e barões do império” (benert, 1967, p. 83-84). Ainda segundo Richard Benert (1967, p. 84), indicava-se a no- breza como os agentes políticos competentes para a solução da tirania, numa concretização dos anseios da comunidade política sob seu jugo, tendo em vista a “ausência de instituições regulares do poder público” nesse contexto. Um exemplo prático e histórico das teorias que defendiam o empoderamento da nobreza data de 1464, a formação da Ligue du Bien Public contra Luís XI, composta por um apanhado aleatório de nobres franceses que justificavam sua atuação em nome do bem público (benert, 1967, p. 84-86). De acordo com Benert (1967, p. 85): [...] os huguenotes continuariam a operar sob essa combinação de re- sistência feudal e pública, porém alocariam muito maior ênfase na au- toridade pública pela qual os nobres atuavam, particularmente porque em geral viam os nobres como membros dos Estados-Gerais [abrindo a perspectiva de afirmação das prerrogativas do parlamento oponíveis aos reis], e não meramente indivíduos poderosos. Na Era Moderna, a Reforma Protestante se aproveitaria da tradi- ção medieval e contribuiria com contornos mais precisos para a teoria das magistraturas inferiores de afinidade constitucional. Quentin Skinner consigna que, em 1529, quando Carlos v convocou a Dieta Imperial (o parlamento do Sacro Império Romano-Germânico), foi imposta a obri- gação de se revogarem todas as concessões obtidas pelos luteranos até Cadernos Espinosanos São Paulo n.38 jan-jun 2018 194 então, tendo a maioria católica elaborado uma resolução determinando, mesmo que à força, a imediata entrada em vigor do Édito de Worms, que instituía a ilegalidade da “heresia” luterana. Silvio Gabriel Serrano Nunes Doutor em Filosofia, Universidade de São Paulo, São Paulo, Brasil serrano.nunes@gmail.com O protesto formal dos luteranos diante desse contexto originou a expressão “protestantismo”, que passou a designar a fé dissidente de Roma na Europa Ocidental desde então, realizado “em nome de seis príncipes e catorze cidades, inspirados por João da Saxônia, Jorge de Bradenburgo -Ansbach e o jovem Filipe de Hesse, que nessa ocasião se destacou como o mais brilhante militante entre os príncipes luteranos” (skinner, 2000, p. 471), além de ter sido o principal articulador para a gênese da teoria das magistraturas inferiores. O Landegrave da região de Hesse abriu o debate com os juristas de sua corte sobre a licitude ou não de se efetivar a resistência ativa con- tra Carlos v. Concluíram pela licitude da resistência por meio de uma [...] engenhosa reafirmação da teoria feudal e particularista da consti- tuição imperial – com base na qual [...] os eleitores já haviam resistido ao imperador Wezel, depondo-o em 1400. Tal como a interpretavam os juristas de Hesse, essa leitura da constituição permitia-lhes desem- penhar duas tarefas ideológicas vitais: legitimava a ideia de resistência armada contra o imperador, porém ao mesmo tempo conseguia sus- tentar a tese luterana fundamental segundo a qual todas as autoridades constituídas são estabelecidas por Deus (skinner, 2000, p. 471). [...] engenhosa reafirmação da teoria feudal e particularista da consti- tuição imperial – com base na qual [...] os eleitores já haviam resistido ao imperador Wezel, depondo-o em 1400. Tal como a interpretavam os juristas de Hesse, essa leitura da constituição permitia-lhes desem- penhar duas tarefas ideológicas vitais: legitimava a ideia de resistência armada contra o imperador, porém ao mesmo tempo conseguia sus- tentar a tese luterana fundamental segundo a qual todas as autoridades constituídas são estabelecidas por Deus (skinner, 2000, p. 471). Ainda que Skinner não especifique o documento considerado como Constituição Imperial, assim como também não o fez Gottfried G. Krodel – cuja introdução à Carta de Lutero ao Eleitor João da Saxô- nia, de 24 de Dezembro de 1529, muito contribuiu para explicar a gênese da teoria das magistraturas inferiores entre os protestantes -, entendemos com base em Política Metodicamente Apresentada e Ilustrada com Exemplos Sagrados e Profanos, de Johannes Althusius (2003, p. 231), que a referida Constituição Imperial é o Áureo Édito do Imperador Carlos IV, institu- ído em 1356, que estabeleceu as regras para as eleições e coroações dos Silvio Gabriel Serrano Nunes p. Cadernos Espinosanos São Paulo n.38 jan-jun 2018 Silvio Gabriel Serrano Nunes Doutor em Filosofia, Universidade de São Paulo, São Paulo, Brasil serrano.nunes@gmail.com 189 - 205 195 imperadores do Sacro Império Romano-Germânico, bem como as obri- gações e prerrogativas dos eleitores (althusius, 2003, p. 231). Filipe de Hesse, em cartas ao Eleitor da Saxônia e ao Margrave de Bradenburgo-Anbach, escritas em Dezembro de 1529, afirma que a cor- reta exegese da exortação de Paulo no capítulo 13 da Carta aos Romanos seria a de que o apóstolo se referia a todos os soberanos territoriais que exercem jurisdição sobre um império ou reino. Afirma também que os poderes foram ordenados com a finalidade de “desempenhar uma deter- minada função, na qual se inclui o dever de observar algumas obrigações legais mútuas, bem como o de garantir o bem-estar e a salvação de seus próprios súditos” (skinner, 2000, p. 471-472). Segundo Krodel, essa correspondência mostra o esforço do Lan- degrave Filipe de circunscrever a possível aplicação da exortação de Paulo aos Romanos (Capítulo 13) à situação de perigo para a causa protestan- te alemã, enfatizando que os soberanos dos territórios dos principados alemães “eram as autoridades governamentais de que Paulo falava nesse capítulo, e que o imperador era somente primus inter pares com base em pactos [tratados] com os soberanos, um arranjo contratual que o impera- dor quebraria agora ao fazer a guerra contra os protestantes em razão de sua fé” (lutero, 1972, p. 254). Em outras palavras, para Filipe de Hesse, a relação político-jurídi- ca entre o imperador e os demais príncipes não poderia ser reconhecida no binômio governante-governado, implicava antes uma relação alheia a essa lógica de subordinação, com competências coordenadas entre au- toridades detentoras de imperium em seus respectivos domínios e que, como tais, podiam em plena licitude se opor aos rompimentos de tra- tados firmados com autoridades também soberanas como o imperador. Skinner (2000, p. 472) entende perseverar nesse contexto a ótica de que “se o imperador ultrapassar os limites de seu cargo perseguindo o Evangelho ou tratando com violência qualquer um dos príncipes, estará violando as obrigações a ele impostas por ocasião de sua eleição, sendo, portanto, legítimo combatê-lo”. Cadernos Espinosanos São Paulo n.38 jan-jun 2018 196 Acerca do conteúdo da correspondência de Hesse, o Eleitor da Saxônia acionou Lutero para se manifestar sobre suas teses, que entendeu ilegítimo o direito de resistência. Silvio Gabriel Serrano Nunes Doutor em Filosofia, Universidade de São Paulo, São Paulo, Brasil serrano.nunes@gmail.com Em carta ao Eleitor da Saxônia, de 1529, Lutero assevera que, mesmo que a intenção do imperador fosse proceder contra o Evangelho com força militar sem ouvir o partido protestante e sem um concílio geral, a ninguém seria dado o direito de colocar as tropas no campo de batalha, por três razões. A primeira: naquele momento de dificuldade os protestantes de- veriam enviar uma resposta humilde ao imperador, uma vez que a Es- critura em 1 Pedro 2:17 é categórica quanto ao dever de submissão à autoridade suprema constituída: “Honrai a todos, amai os irmãos, temei a Deus, tributai honra ao rei”. A segunda razão consistia em que mesmo sendo a atitude do imperador injusta e também contra a lei natural, “trazer as tropas para o campo de batalha e desferir a resistência deveriam ser evitados, a não ser que existisse violência iminente ou um constrangimento inevitável. Esse movimento precipitado para a guerra e essa determinação para resistir não devem ser considerados legítima defesa, mas um desafio e um ato de insolência contra quem ainda está em posição de calmaria e não agiu [contra os protestantes]” (lutero, 1972, p. 257-258). Lutero, nutrindo a esperança de que a providência divina se ma- nifestasse e resolvesse a situação, argumenta que mesmo se sua majestade imperial expedisse mandados contra os príncipes eleitores, ou mesmo se esses mandados já tivessem sido expedidos, isso ainda não significaria uma proibição aos protestantes sob a tutela dos príncipes que aderiram à Reforma de professarem a sua fé. Afirma ser censurável a resistên- cia mesmo se hipoteticamente o imperador fosse considerado um igual aos demais dirigentes. Em suas palavras, “durante esse tempo muita água pode rolar rio abaixo, e Deus pode facilmente encontrar muitos meios em direção à paz e talvez estabeleça a paz [...]. Por conseguinte, em busca da acima mencionada paz, ninguém pode começar a guerra ou trazer as Silvio Gabriel Serrano Nunes p. 189 - 205 197 tropas ao campo de batalha, mesmo se o imperador fosse um soberano em pé de igualdade [com os eleitores]” (lutero, 1972, p. 258). Alocando o imperador no topo da organização político-adminis- trativa do império, Lutero enfatiza o dever de negar a tese constitucio- nalista elaborada por Filipe de Hesse e seus juristas, afirmando que “o imperador, claramente, é o senhor e governador superior desses sobera- nos. Cadernos Espinosanos São Paulo n.38 jan-jun 2018 Silvio Gabriel Serrano Nunes Doutor em Filosofia, Universidade de São Paulo, São Paulo, Brasil serrano.nunes@gmail.com De fato, nenhum desses senhores gostaria de ter seus próprios sú- ditos colocados em tal condição precipitada de resistência a ele próprio, como nesse caso seria feito contra o imperador; então toda a questão seria realmente uma rebelião e desobediência” (lutero, 1972, p. 259). Para Gottfried G. Krodel (lutero, 1972, p. 259), a posição de Lu- tero contra o argumento constitucional legitimador do direito de resis- tência pode ser vista como uma possível falha de sua compreensão sobre essa teoria: “é uma questão em aberto o quão claramente Lutero aqui entendeu a base constitucional do cargo imperial e a administração de fato [os feitos concretos] dessa função em seu tempo”, quando Carlos v governava. Por fim, Lutero levanta um terceiro argumento contrário à resis- tência armada: ela representaria uma injustiça, uma vez que fatalmente se atacariam outros soberanos católicos e as respectivas populações civis dos principados que não aderiram à Reforma e que fossem aliados do imperador. Além disso, teria chegado a Lutero a informação de que o im- perador formalmente declarara estar empenhado pela paz com os estados do império e, “consequentemente, se apesar disso, [...] [os protestantes atacassem o imperador preventivamente] então Deus e o mundo se tor- nariam imensamente furiosos e com toda justiça seríamos condenados. Eles poderiam então com toda justiça e excelentes argumentos apresen- tarem todas essas questões como prova de sua inocência, e nos cobrir de desgraça e vergonha” (lutero, 1972, p. 260). Apesar do tom de extrema cautela de Lutero, em 1530 os proble- mas se agravariam ainda mais para os protestantes, depois da Dieta em que Carlos V mandou refutar as teses teológicas apresentadas por Melan- 198 chton na Confissão de Augsburgo, que inclusive aspirava a uma concórdia com os católicos. A maioria católica presente na Dieta impôs o dever de todo herege luterano retornar à unidade cristã até a Páscoa subsequente, além disso, as pregações de conteúdo luterano foram suspensas em todo o império. Como se não bastasse o rigor dessas medidas, ao fim desta Dieta orquestrou-se “entre os príncipes católicos a formação de uma liga de defesa do império, medida que deliberadamente impunha uma ameaça militar direta aos protestantes” (skinner, 2000, p. 473). Silvio Gabriel Serrano Nunes Doutor em Filosofia, Universidade de São Paulo, São Paulo, Brasil serrano.nunes@gmail.com Diante dessa realidade, que certamente evoluiria para um prog- nóstico desolador face aos ataques que sofria a causa protestante na Ale- manha, em outubro de 1530, Filipe de Hesse escreveu a Lutero e também a João da Saxônia e seu chanceler Gregory Brück, “exortando-os a acei- tar seu plano para uma aliança protestante e pedindo-lhes que aceitassem seu pressuposto de que na verdade era possível justificar a resistência armada ao imperador” (skinner, 2000, p. 473). João da Saxônia se sensibiliza com a crise enfrentada pela causa de sua nova crença e decide consultar seu chanceler Brück e os demais juristas de sua corte acerca da viabilidade e licitude ou não das ideias apresentadas pelo governante da região de Hesse. Apesar de Brück e os juristas inicialmente se inclinarem a acei- tar a “interpretação federalista da constituição imperial” manifestada por Hesse, optaram pelo fundamento privado do direito de resistência e, no final de outubro de 1530, entregaram ao eleitor da Saxônia uma súmula jurídica com a resposta requerida acerca da licitude ou não do exercício do direito de resistência. O documento intitulado Se é Justo Defender-se de um Juiz que Pro- ceda Injustamente, Iudici Procedenti Iniuste an Licitum Sit Resistire, apresenta o argumento de resistência ativa com lastro no direito privado, segundo o qual, em certos contextos, “o uso da violência não constitui necessa- riamente uma injúria”, recuperando teses já desenvolvidas por civilistas e canonistas na Idade Média nas quais se declara “ser justificável repelir com a força a força injusta” (skinner, 2000, p. 473-474). Silvio Gabriel Serrano Nunes p. 189 - 205 199 Revisitando canonistas como Panormitanus, cuja tese sustentava a plena licitude de resistir a um juiz injusto – que nessas circunstâncias passaria a ser considerado um indivíduo privado , válida por extensão ao imperador, posto “que o estatuto do imperador era com efeito o de um juiz”, Brück transpunha as lições daqueles (algo que dificilmente um católico poderia contestar) para o contexto da lide entre os protestantes e Carlos v. Cadernos Espinosanos São Paulo n.38 jan-jun 2018 Silvio Gabriel Serrano Nunes Doutor em Filosofia, Universidade de São Paulo, São Paulo, Brasil serrano.nunes@gmail.com Em sua súmula, Brück expõe três possíveis circunstâncias que va- lidam a resistência ativa lícita, inclusive por meio da violência, contra um juiz (agente político comparável ao imperador nesse documento) injusto quando ocorre uma apelação: “quando o juiz procede fora de sua jurisdição; [...] quando o dano é irreparável ou notório; [...] se o juiz age de acordo com sua jurisdição, porém, injustamente, e se o dano for irreparável” (heins, 1969, p. 64). Diante dessas premissas, a súmula menciona que “os príncipes e as cidades apelaram a César (imperador) e a um Concílio; então [estaria] suspensa a jurisdição” do imperador e seu caráter de exclusividade sobre a questão (heins, 1969, p. 64). Brück também alegava ser um completo despropósito a obedi- ência ao imperador nessas circunstâncias, uma vez que “obedecer ordens do imperador [em seus] éditos ou [em] seus mandatos contra a Palavra [de Deus] é um dano irreparável. É mais útil, nas causas da fé, obedecer a Deus e às verdades do Evangelho do que ao homem” (heins, 1969, p. 64). Em reforço ao dever de desobedecer ao imperador quando aten- tasse contra os ditames do Evangelho, é relembrada a tese luterana sobre a noção de tirania, na qual se conceitua o tirano como um governante que excede os limites de seu poder (ultra vires), ao disciplinar o conteúdo da fé verdadeira: “Além disso, o imperador, em questão de fé, não tem ne- nhuma jurisdição. Pode, com negligência, convocar um Concílio Papal, mas nada nele pode estatuir” (heins, 1969, p. 65). A título de conclusão, a súmula explicita as atitudes do imperador que afligiam os luteranos depois da Dieta de 1530, comparáveis apenas às Cadernos Espinosanos São Paulo n.38 jan-jun 2018 200 de um juiz que age injustamente, razão pela qual deveria ser considera- do uma pessoa privada, despojado de autoridade pública, passível de ser objeto do lícito exercício do direito de resistência eventualmente perpe- trado pelos protestantes (heins, 1969, p. 66). Para alguns comentadores (skinner, 2000; bowman, 1997), a re- sistência ao soberano considerado indivíduo privado e despojado da au- toridade pública deve ser exercida por um agente competente, ou seja, investido da vocação para tanto, o que exclui o povo em geral e autoriza os magistrados inferiores, conclusão oriunda da combinação lógica entre os dois argumentos de resistência de matriz luterana. Silvio Gabriel Serrano Nunes Doutor em Filosofia, Universidade de São Paulo, São Paulo, Brasil serrano.nunes@gmail.com Exemplo notório da combinação entre tais argumentos em um mesmo texto de matriz luterana acerca do problema da resistência é a Confissão de Magdeburgo (skinner, 2000, p. 493). Quanto ao dever de respeitar a vocação daquele que exerce o direito de resistência, salienta que, no âmbito do pensamento político lu- terano, haveria sempre um rechaço à participação popular simples e pura sem a colaboração e comando dos magistrados inferiores: Tanto a lei divina quanto a constitucional eram invocadas para apoiar o princípio de que a resistência era proibida para os cidadãos privados; desse modo se aquietavam os medos de que os súditos dos príncipes pudessem fazer o uso desses argumentos para justificar uma rebelião contra eles também. Deus não concedeu a espada para o indivíduo privado mas somente para os magistrados [...]. Indivíduos que não possuíssem um cargo público deviam obedecer às autoridades dentro de seus estados e juntarem-se na batalha contra o imperador somente por meio do comando de seus superiores locais (bowman, 1977, p. 75). O próprio Lutero sempre manteve postura e argumentação, em regra, refratárias ao direito de resistência ativa. O máximo que se apre- senta em sua biografia, em termos mais permissivos quanto ao direito de resistência ativa, consiste em uma capitulação autorizadora do direito de resistência no palácio de Torgau, nos fins de outubro de 1530, um escrito que foi redigido de próprio punho pelo reformador e assinado também Silvio Gabriel Serrano Nunes p. 189 - 205 201 por outras lideranças luteranas. Segundo Pierre Mesnard, as circunstân- cias vividas pelo movimento reformador alemão, nesse momento, teriam extorquido tal declaração de Lutero (mesnard, 1977, p. 228). Entre os dias 25 e 28 de outubro de 1527, os teólogos de Win- tenberg, liderados por Lutero, e os conselheiros e juristas do Eleitor da Saxônia, liderados por Brück, encontraram-se em Torgau para uma con- ferência. Os juristas apresentaram trechos de seus estudos sobre o direito de resistência, que levaram Lutero a capitular nos seguintes termos: Um pedaço de papel foi-nos apresentado no qual vemos o que os Doutores da Lei têm concluído sobre a questão: Em quais situações se pode resistir à autoridade governante? Silvio Gabriel Serrano Nunes Doutor em Filosofia, Universidade de São Paulo, São Paulo, Brasil serrano.nunes@gmail.com Se, então, [essa questão] tem sido colocada por esses Doutores da Lei ou especialistas nesse assunto, e [uma vez que] nós certamente estamos naquelas situações em que (como [os especialistas legais] demonstram) pode-se resistir à autori- dade governante, e [como] nós temos sempre ensinado que na medida em que o evangelho não contraria a lei secular e leva a lei secular a ser efetiva, válida e competente [naqueles problemas em que ela é apta a lidar], nós então somos incapazes de nos opor [a qualquer um com argumentos extraídos da] Escritura, se nessa instância é necessário lu- tar, mesmo se o próprio imperador [nos atacar], ou outro alguém que também o faça em seu nome. Além disso, agora em todo lugar há o perigo de que em algum dia outros incidentes possam ocorrer, como um resultado de que alguém tenha que se defender sozinho imediatamente, não apenas por causa da lei secular, mas também em razão do dever e da angústia da cons- ciência. Portanto, é também apropriado armar-se e estar pronto para opor-se à força que pode repentinamente se levantar, como pode fa- cilmente ocorrer, julgando a partir do padrão presente e do curso dos eventos (lutero, 1972, p. 431-432). Apesar da declaração acima, Lutero não perde por completo o tom de conservadorismo e fidelidade às instituições políticas de seu tempo e reafirma a coerência de seu pensamento político, uma vez que Cadernos Espinosanos São Paulo n.38 jan-jun 2018 202 a legitimidade dessa ação constitui uma transferência da sacralidade do dever de obediência ao qual os magistrados seculares são revestidos para a lei civil: De modo que, até agora, nós temos ensinado absolutamente a não re- sistir à autoridade governante; isto era devido ao fato de que nós não sabíamos que a própria lei da autoridade governante garante [o direito de resistência armada]; nós temos, claro, de forma diligente, ensinado que [essa lei] deve ser obedecida (lutero, 1972, p. 431-432). Compreendemos tais passagens mais como uma atitude polí- tica de Lutero do que propriamente uma elaboração teórica componen- te do conjunto de sua obra, o que permite afirmar ser quase nulo o grau de adesão ao direito de resistência ao longo do projeto reformador do teólogo de Wittenberg, que não envolvia apenas elucubrações teóricas, mas também atitudes políticas. Silvio Gabriel Serrano Nunes Doutor em Filosofia, Universidade de São Paulo, São Paulo, Brasil serrano.nunes@gmail.com Pelo exposto, podemos com clareza notar na filosofia política de John Locke traços do pensamento político de resistência de matriz lu- terana em suas duas vertentes: o argumento constitucional de resistência, que legitima a atuação do parlamento com prerrogativas de supremacia em relação ao monarca, e o argumento de direito privado, que autoriza considerar uma autoridade pública que se tornou um tirano como um particular e, portanto, passível de uma resposta pronta e justa à agressão que pratica. Silvio Gabriel Serrano Nunes p. 189 - 205 203 john locke and the theories of the right OF RESISTANCE OF LUTHERAN MATRIX abstract: This work aims to address how the Lutheran arguments – the constitutional argument of the “inferior magistracies” and the pri- vate law argument – about the right of resistance, developed in the late 1520s and early 1530s, were received in John Locke’s Second Treatise of Civil Government, written in the 17th century. The private law argument understands that every ruler who give up doing good actions in order to commit tyrannical acts, renounces his authority and, consequently, must be treated as a private person, therefore, subject to licit retaliation, which constitutes a kind of self-defence. The constitutional argument of the “inferior magistracies” asserts the possibility that intermediate organs (political agents such as legislative and judicial bodies) between the rul- er and the people are legitimated to resist tyranny. In John Locke, both arguments are aimed to solve the problem of tyranny in order to obtain political and religious freedom. keywords: Right of Resistance, Lutheran Theories, Constitutional Argu- ment, Private Law Argument, John Locke. Silvio Gabriel Serrano Nunes p. 189 - 205 205 referências bibliográficas: althusius, j. (2003). Política Metodicamente Apresentada e Ilustrada com Exemplos Sagrados e Profanos. Rio de Janeiro: Topbooks. althusius, j. (2003). Política Metodicamente Apresentada e Ilustrada com Exemplos Sagrados e Profanos. Rio de Janeiro: Topbooks. benert, r. r. (1967). Inferior Magistrates in Sixteenth-Century Political and Legal Thought. Minnesota. 388f. Thesis (PhD Philosophy). University of Minnesota. benert, r. r. (1967). Inferior Magistrates in Sixteenth-Century Political and Legal Thought. Minnesota. 388f. Thesis (PhD Philosophy). University of Minnesota. lia de jerusalém. (2006). São Paulo: Paulus. bíblia de jerusalém. (2006). São Paulo: Paulus. bowman, c. g. (1977). The development of the Lutheran Theory of Re- 04 Cadernos Espinosanos São Paulo n.38 jan-jun 2018 204 sistance: 1523-1530. The Sixteenth Century Journal, Kirksville, v. VIII, n. 1, p. 61-76, April/1977. heins, s. (1969). Das Widerstandrecht als Problem der deutschen Protestanten, 1523-1546. Gütersloh: Gütersloher Verlagshaus Gerd Mohn. kantorowicz, e. h. (1998). Os Dois Corpos do Rei. São Paulo: Companhia das Letras. locke, j. (2005). Dois Tratados Sobre o Governo. São Paulo: Martins Fontes. lutero. (1972). Luther’s Works - Letters II. v. 49. Philadelphia: Fortress Press. mesnard, p. (1977). L´essor de la Philosophie Politique au XVI Siècle. Paris: Libraire Philosophique J. Vrin. skinner, q. (2000). As Fundações do Pensamento Político Moderno. São Pau- lo: Companhia das Letras. Silvio Gabriel Serrano Nunes p. 189 - 205 205
https://openalex.org/W3047571307	https://discovery.dundee.ac.uk/ws/files/55039647/annurev_cellbio_100818_125512.pdf	English	null	Parkinson's: A Disease of Aberrant Vesicle Trafficking	Annual review of cell and developmental biology	2,020	cc-by	21,553	Parkinson’s Citation for published version (APA): Singh, P. K., & Muqit, M. M. K. (2020). Parkinson’s: A Disease of Aberrant Vesicle Trafficking. Annual Review of Cell and Developmental Biology, 36, 237-264. https://doi.org/10.1146/annurev-cellbio-100818-125512 Citation for published version (APA): Singh, P. K., & Muqit, M. M. K. (2020). Parkinson’s: A Disease of Aberrant Vesicle Trafficking. 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Published in: Annual Review of Cell and Developmental Biology DOI: 10.1146/annurev-cellbio-100818-125512 Publication date: 2020 Licence: CC BY Document Version Publisher's PDF, also known as Version of record Link to publication in Discovery Research Portal Citation for published version (APA): Singh, P. K., & Muqit, M. M. K. (2020). Parkinson’s: A Cell and Developmental Biology, 36, 237-264. https:// General rights i h d Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 24. Oct. 2024 Download date: 24. Oct. 2024 Download date: 24. Oct. 2024 Parkinson’s disease, α-synuclein, LRRK2, PINK1, Parkin, Rab, SNARE, exosome, vesicle trafficking, mitophagy Parkinson’s disease, α-synuclein, LRRK2, PINK1, Parkin, Rab, SNARE, exosome, vesicle trafficking, mitophagy The Annual Review of Cell and Developmental Biology is online at cellbio.annualreviews.org Annu. Rev. Cell Dev. Biol. 2020.36:237-264. Downloaded from www.annualreviews.org Access provided by 86.174.33.195 on 12/11/20. See copyright for approved use. Annu. Rev. Cell Dev. Biol. 2020. 36:237–64 Annu. Rev. Cell Dev. Biol. 2020. 36:237–64 Annu. Rev. Cell Dev. Biol. 2020. 36:237–64 First published as a Review in Advance on August 4, 2020 Keywords First published as a Review in Advance on August 4, 2020 Abstract https://doi.org/10.1146/annurev-cellbio-100818- 125512 Parkinson’s disease (PD) is a leading cause of neurodegeneration that is de- fined by the selective loss of dopaminergic neurons and the accumulation of protein aggregates called Lewy bodies (LBs). The unequivocal identification of Mendelian inherited mutations in 13 genes in PD has provided transform- ing insights into the pathogenesis of this disease. The mechanistic analysis of several PD genes, including α-synuclein (α-syn), leucine-rich repeat kinase 2 (LRRK2), PTEN-induced kinase 1 (PINK1), and Parkin, has revealed cen- tral roles for protein aggregation, mitochondrial damage, and defects in en- dolysosomal trafficking in PD neurodegeneration. In this review, we outline recent advances in our understanding of these gene pathways with a focus on the emergent role of Rab (Ras analog in brain) GTPases and vesicular trafficking as a common mechanism that underpins how mutations in PD genes lead to neuronal loss. These advances have led to previously distinct genes such as vacuolar protein–sorting-associated protein 35 (VPS35) and LRRK2 being implicated in a common signaling pathway. A greater under- standing of these common nodes of vesicular trafficking will be crucial for linking other PD genes and improving patient stratification in clinical trials underway against α-syn and LRRK2 targets. Copyright © 2020 Pawan Kishor Singh & Miratul M.K. Muqit. This work is licensed under a Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. See credit lines of images or other third-party material in this article for license information 237 Contents INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238 PARKINSON’S DISEASE GENETICS AND TRAFFICKING. . . . . . . . . . . . . . . . . . . . 239 The Genetic Landscape of Parkinson’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239 Rabs: RAB39B Mutations and PARK16 (RAB29) Risk Variants in Parkinson’s Disease . . . . Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241 VPS35 and VPS13C Mutations in Parkinson’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242 Synaptic Vesicle Trafficking: DNAJC6 and SYNJ1 Mutations in Parkinson’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243 PROTEIN AGGREGATION PATHWAYS AND VESICULAR TRAFFICKING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244 α-Synuclein and Rab GTPases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244 α-Synuclein and SNAREs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246 α-Synuclein and Exosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Abstract 247 LRRK2: AT THE NEXUS OF VESICULAR TRAFFICKING IN PARKINSON’S DISEASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248 The Landscape of Rab GTPases Regulated by LRRK2 . . . . . . . . . . . . . . . . . . . . . . . . . . . 248 Autophagy and LRRK2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250 Golgi Trafficking Mediated by LRRK2 and Rab29 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251 The Interplay Between Retromer Activity and LRRK2 Phosphorylation . . . . . . . . . . . 251 MITOCHONDRIAL QUALITY CONTROL AND VESICULAR TRAFFICKING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252 PINK1/Parkin Regulation of Mitophagy and Link to Rabs. . . . . . . . . . . . . . . . . . . . . . . . 252 PINK1/Parkin and Mitochondrial-Derived Vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254 PINK1 Regulation of the Rab SF3 Motif . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255 CONCLUDING REMARKS AND CLINICAL APPLICATION . . . . . . . . . . . . Abstract . . . . . . 256 INTRODUCTION Parkinson’s disease (PD) was first defined as a clinical entity just over two centuries ago, and it now represents the second most common neurodegenerative disorder, affecting from 2% to 3% of the population greater than 65 years age (Poewe et al. 2017). The hallmark motor symptoms of PD include resting tremor, postural instability, bradykinesia, and rigidity. However, PD also involves nonmotor symptoms that contribute to patients’ disability, including anosmia, cognitive dysfunc- tion, sleep–awake dysregulation, dysautonomia, depression, and constipation (Poewe et al. 2017). PD pathology is characterized by the loss of pigmented dopaminergic neurons in the substantia nigra pars compacta (SNpc) midbrain region, and PD motor symptoms are thought to occur with a 60–70% loss of dopaminergic neurons (Lang & Lozano 1998). In association with neuronal loss is the widespread accumulation of α-synuclein (α-syn)-containing aggregates in surviving neu- rons, termed Lewy bodies (LBs) (Spillantini & Goedert 2018). Although the common cause of PD, termed sporadic or idiopathic, is unknown, multiple risk factors including aging and environ- mental factors are believed to contribute to disease pathogenesis (Poewe et al. 2017). These risk factors are thought to interact with genetic susceptibility, and over the last decade, great advances have been made in uncovering the genetic architecture of PD (Bandres-Ciga et al. 2020). Our un- derstanding of mechanisms and pathways underlying PD has been transformed by the discovery of gene mutations in rare familial forms of PD that account for from 5% to 10% of all cases. The Singh • Muqit 238 mechanistic analysis of several genes,namely α-syn,PTEN-induced kinase 1 (PINK1),Parkin,and leucine-rich repeat kinase 2 (LRRK2), has broadly highlighted major pathways including protein aggregation, mitochondrial dysfunction, and defects in endolysosomal trafficking; this has thereby implicated Rab (Ras analog in brain) GTPases as playing a central role in the pathogenesis of PD.i Rab GTPases are the key regulators of intracellular trafficking in eukaryotic cells, with more than 60 members in mammals and 11 homologs in budding yeast (Muller & Goody 2018). Rabs exist within cells in two conformations: an active GTP-bound form, which associates with intra- cellular membranes, and an inactive GDP-bound conformation, which is present in the cytosol. Rabs are prenylated at their C-terminal cysteine residues with the help of geranylgeranyl trans- ferases (GGTs) and Rab escort proteins (REPs); this process is required for their association with the membrane. INTRODUCTION In their membrane-bound form, Rabs are activated by the exchange of GDP to GTP, which is catalyzed by guanine nucleotide exchange factors (GEFs). In their active state, Rabs interact with various effector proteins to regulate myriad functions including cargo selec- tion, vesicle budding, vesicle transport along the cytoskeletal track, and tethering and fusion of the vesicle with the target membrane compartments. There Rabs can hydrolyze their GTP into GDP with the aid of GTPase-activating proteins (GAPs) and thereby switch into an inactive state. In the GDP-bound inactive state, Rabs are retrieved and sequestered in the cytosol by GDP dissociation-inhibitor (GDI) proteins (Bonet-Ponce & Cookson 2019, Stenmark 2009). Whether the deregulation of Rab function and vesicular trafficking is a primary cause of PD neurodegen- eration, as well as the relevance of Rab biology to the more common general form of PD, is an outstanding question that is being intensively studied. PARKINSON’S DISEASE GENETICS AND TRAFFICKING The Genetic Landscape of Parkinson’s Disease The most recent meta-analysis of genome-wide association studies (GWAS) of sporadic PD has identified 90 independent gene risk variants that overall account for 16% to 36% of the heritable risk of PD (Nalls et al. 2019). The functional analysis of these genes has revealed links to lyso- somal biology, mitochondrial homeostasis, autophagy, and vesicular trafficking as well as protein aggregation (Nalls et al. 2019). However, whether the gene assignment of risk variants based on proximity is appropriate for all cases is unclear. Of the 23 PD genes and loci (designated PARK) that have been identified in rare families with monogenic forms of PD, clear-cut mutations have been confirmed in 13 genes (Lunati et al. 2018). These genes are classified into autosomal domi- nant genes, namely SNCA/PARK1/PARK4 (α-syn), LRRK2/PARK8 (LRRK2), and VPS35/PARK17 (vacuolar protein–sorting-associated protein 35; VPS35); X-linked gene mutations in RAB39B (Rab39B); autosomal recessive genes, including PRKN/PARK2 (Parkin), PINK1/PARK6 (PINK1), and PARK7 (DJ-1); and finally autosomal recessive genes associated with complex Parkinsonian phenotypes, including ATP13A2/PARK9 (ATPase 13A2; ATP13A2), PLA2G6/PARK14 (phospho- lipase A2 group VI; PLA2G6), FBX07/PARK15 (F-box only protein 7; FBX07), DNAJC6/PARK19 [DnaJ heat shock protein family member C6 (DNAJC6); auxilin], SYNJ1/PARK20 (Synaptojanin 1; SYNJ1),and VPS13C/PARK23 (vacuolar protein–sorting 13 homolog C; VPS13C) (Lunati et al. 2018). A further four loci/genes have been confirmed as major risk factors for late-onset PD in- cluding PARK10, MAPT (microtubule-associated protein tau), GBA (glucocerebrosidase; GBA) and PARK16, whose locus contains the gene RAB29 (Rab29) (Martin et al. 2011). The identi- fication of a PD-causing mutation in the α-syn gene and the subsequent discovery that α-syn (Figure 1a) is the major protein component of LBs have shown that protein misfolding and ag- gregation is a central pathway in PD pathogenesis. In parallel, the discovery of the physiological www.annualreviews.org • Aberrant Vesicle Trafficking in Parkinson’s 239 Amphipathic Acidic NAC 140 α-Syn UBL RING0 RING1 RING2 IBR 465 Parkin 581 MTS Kinase TM CTE PINK1 ARM ANK LRR ROC COR Kinase WD40 2,527 LRRK2 213 GTPase Rab39B 203 GTPase Rab7L1/Rab29 1,573 Sac1 5'-Phosphatase PRD SYNJ1 3,753 Chorein N rpt rpt C ATG SHR-BD rpt VPS13C α-Solenoid VSP29 binding VSP29 binding 796 VPS35 DNAJC6 PTEN CBD J 913 a b c d e f g h i j Figure 1 Domain architecture of PD proteins linked to membrane trafficking. Singh • Muqit PARKINSON’S DISEASE GENETICS AND TRAFFICKING The Genetic Landscape of Parkinson’s Disease Protein domains of human (a) α-syn (140 aa), (b) PINK1 (581 aa), (c) Parkin (465 aa), (d) LRRK2 (2,527 aa), (e) VPS35 (796 aa), ( f ) Rab39B (213 aa), (g) Rab7L1/Rab29 (203 aa), (h) DNAJC6/auxilin (913 aa), (i) SYNJ1/Synaptojanin (1,573 aa), and ( j) VPS13C (3,573 aa). Abbreviations: α-syn, α-synuclein; aa, amino acids; ANK, ankyrin; ARM, armadillo; ATG, autophagy-related protein; C, C-terminal domain; CBD, clathrin-binding domain; COR, C-terminal of ROC; CTE, C-terminal extension; DNAJC6, DnaJ heat shock protein family member C6; GTP, guanosine triphosphate; IBR, in-between RING; J, J-domain; LRRK2, leucine-rich repeat kinase 2; MTS, mitochondrial targeting sequence; N, N-terminal domain; NAC, nonamyloid-β component; PD, Parkinson’s disease; PINK1, PTEN-induced kinase 1; PRD, proline-rich domain; PTEN, phosphatase and tensin homolog; Rab, Ras analog in brain; RING, really interesting new gene; ROC, Ras of complex; rpt, repeat; SHR-BD, SHORT ROOT–binding domain; SYNJ1, Synaptojanin 1; TM, transmembrane; UBL, ubiquitin-like; VPS, vacuolar protein–sorting protein. Annu. Rev. Cell Dev. Biol. 2020.36:237-264. Downloaded from www.annualreviews.org Access provided by 86.174.33.195 on 12/11/20. See copyright for approved use. Amphipathic Acidic NAC 140 α-Syn UBL RING0 RING1 RING2 IBR 465 Parkin 581 MTS Kinase TM CTE PINK1 ARM ANK LRR ROC COR Kinase WD40 2,527 LRRK2 213 GTPase Rab39B 203 GTPase Rab7L1/Rab29 1,573 Sac1 5'-Phosphatase PRD SYNJ1 Chorein N rpt rpt C ATG SHR-BD rpt VPS13C α-Solenoid VSP29 binding VSP29 binding 796 VPS35 DNAJC6 PTEN CBD J 913 a b c d e f g h i j Figure 1 Domain architecture of PD proteins linked to membrane trafficking. Protein domains of human (a) α-syn (140 aa), (b) PINK1 (58 (c) Parkin (465 aa), (d) LRRK2 (2,527 aa), (e) VPS35 (796 aa), ( f ) Rab39B (213 aa), (g) Rab7L1/Rab29 (203 aa), (h) DNAJC6/auxil 2020.36:237-264. Downloaded from www.annualreviews.org 174.33.195 on 12/11/20. See copyright for approved use. Annu. Rev. Cell Dev. Biol. 2020.36:237-264. Downloaded from www.annualreviews.org Access provided by 86.174.33.195 on 12/11/20. See copyright for approved use. LRRK2 encodes a multidomain protein comprising core kinase and GTPase [Ras of complex (Roc)-C-terminal of Roc (COR)] domains (Figure 1d ) together with several protein-interaction domains; mutations in this gene represent the most common cause of PD (Taylor & Alessi 2020). To date nine missense mutations have been confirmed as pathogenic that are mainly lo- calized within the kinase or Roc-COR domains, including [Asn1437His], [Arg1441Gly/Cys/His], [Arg1628Pro], [Tyr1699Cys], [Gly2019Ser], [Ile2020Thr], and [Gly2385Arg] (Lunati et al. 2018). The [Gly2019Ser] mutation is the most frequent, varying from 1% to 5% of PD cases in Europe and up to 40% of cases in North Africa; however, the penetrance is incomplete and increases with every decade of patients’ lives to greater than 90% by the ninth decade (Lunati et al. 2018). The phenotype resembles typical PD with asymmetrical onset,nonmotor symptoms,and good L-Dopa response that is indistinguishable from typical PD (Lunati et al. 2018). However, the mean age of onset is lower at 58 years and the progression slower than that of typical PD cases (Lunati et al. 2018). Interestingly, there does not appear to be any difference in severity between heterozygous and homozygous [Gly2019Ser] patients (Lunati et al. 2018). Furthermore, the neuropathology of LRRK2 is variable, with LBs found in [Gly2019Ser] cases but few LBs and a predominance of tau inclusions found in the other mutations (Lunati et al. 2018). All pathogenic mutations lead to hyperactivation of the kinase activity of LRRK2, and Type I kinase inhibitors are currently undergoing clinical trials (Alessi & Sammler 2018). Annu. Rev. Cell Dev. Biol. 2020.36:237-264. Downloaded from www.annualreviews.org Access provided by 86.174.33.195 on 12/11/20. See copyright for approved use. Annu. Rev. Cell Dev. Biol. 2020.36:237-264. Downloaded from www.annualreviews.org Access provided by 86.174.33.195 on 12/11/20. See copyright for approved use. Domain architecture of PD proteins linked to membrane trafficking. Protein domains of human (a) α-syn (140 aa), (b) PINK1 (581 aa), (c) Parkin (465 aa), (d) LRRK2 (2,527 aa), (e) VPS35 (796 aa), ( f ) Rab39B (213 aa), (g) Rab7L1/Rab29 (203 aa), (h) DNAJC6/auxilin (913 aa), (i) SYNJ1/Synaptojanin (1,573 aa), and ( j) VPS13C (3,573 aa). Abbreviations: α-syn, α-synuclein; aa, amino acids; ANK, ankyrin; ARM, armadillo; ATG, autophagy-related protein; C, C-terminal domain; CBD, clathrin-binding domain; COR, C-terminal of ROC; CTE, C-terminal extension; DNAJC6, DnaJ heat shock protein family member C6; GTP, guanosine triphosphate; IBR, in-between RING; J, J-domain; LRRK2, leucine-rich repeat kinase 2; MTS, mitochondrial targeting sequence; N, N-terminal domain; NAC, nonamyloid-β component; PD, Parkinson’s disease; PINK1, PTEN-induced kinase 1; PRD, proline-rich domain; PTEN, phosphatase and tensin homolog; Rab, Ras analog in brain; RING, really interesting new gene; ROC, Ras of complex; rpt, repeat; SHR-BD, SHORT ROOT–binding domain; SYNJ1, Synaptojanin 1; TM, transmembrane; UBL, ubiquitin-like; VPS, vacuolar protein–sorting protein. nnu. Rev. Cell Dev. Biol. 2020.36 Access provided by 86.174.33 role that α-syn plays in SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex assembly and synaptic vesicle trafficking (see the section titled Protein Aggre- gation Pathways and Vesicular Trafficking) and how this is disrupted by α-syn mutations have also been of major interest. Mutations in α-syn are rare, accounting for only from 1% to 2% of familial PD. These comprise gene duplications, triplications, or missense mutations ([Ala30Pro], [Glu46Lys], [Gly51Asp], [Ala53Glu], and [Ala53Thr]). In general, the phenotype of these patients does not resemble that of patients with typical PD, instead having a high prevalence of dementia, neuropsychiatric signs, and a moderate response to L-Dopa. Pathologically there is a high burden of LBs, Lewy neurites (LNs), and neuronal loss in the SNpc (Lunati et al. 2018). The discovery of mutations in the PINK1 and Parkin genes has spearheaded understand- ing of the roles of these genes in mitochondrial quality control pathways linked to PD Singh • Muqit 240 neurodegeneration (Harper et al. 2018). Autosomal recessive mutations in Parkin were first dis- covered in 1998 and are the most common cause of early-onset PD, accounting for nearly 40% of all cases (Kitada et al. 1998). Annu. Rev. Cell Dev. Biol. 2020.36:237-264. Downloaded from www.annualreviews.org Access provided by 86.174.33.195 on 12/11/20. See copyright for approved use. Parkin encodes a multidomain ubiquitin (Ub) E3 ligase containing an N-terminal Ub-like (Ubl) domain and RING0, RING1, in-between RING (IBR), and RING2 domains (Figure 1c). The catalytic cysteine (Cys431) lies within the RING2 domain (Wenzel et al. 2011). Numerous PD-causing mutations have been identified including rearrangements that dras- tically destabilize the protein and missense mutations across the whole protein that disrupt critical regulatory amino acid residues, e.g., [Cys431Phe] within RING2 that disrupts the Ub thioester formation required for Ub transfer to substrates (Wenzel et al. 2011) and [Ser65Asn] that prevents phosphorylation and activation by PINK1 (McWilliams et al. 2018). Autosomal recessive muta- tions in PINK1 that encode a mitochondria-localized protein kinase represent the second leading cause of early-onset PD. Over 30 missense and nonsense mutations have been reported, mainly localized within the kinase domain (Figure 1b), that lead to loss of function through catalytic inactivation or truncations, respectively (Valente et al. 2004). Structural and biochemical studies have elaborated the mechanism by which PINK1 phosphorylates and activates Parkin, and cell biological studies have demonstrated that these enzymes function in a common signaling pathway to stimulate the removal of damaged mitochondria by mitophagy (see the section titled Mito- chondrial Quality Control and Vesicular Trafficking) (Harper et al. 2018, McWilliams & Muqit 2017). Consistent with this, patients harboring PINK1 or Parkin mutations exhibit identical phe- notypes with early-onset disease, an excellent response to L-Dopa, and a relatively high frequency of dystonia and dyskinesias (Lunati et al. 2018). Pathologically, PINK1 and Parkin patient brains exhibit relatively restricted neurodegeneration within the substantia nigra (SN) and a paucity of LBs, although variability in LB burden has been reported in select cases for each gene (Kunath et al. 2019). Rabs: RAB39B Mutations and PARK16 (RAB29) Risk Variants in Parkinson’s Disease The most compelling evidence for the deregulation of Rabs as a primary cause of PD has been the discovery of direct Rab mutations in familial PD cases. X-linked mutations in RAB39B were www.annualreviews.org • Aberrant Vesicle Trafficking in Parkinson’s 241 first reported in 2014, including a ∼45 kb loss-of-function deletion mutation in an Australian family with intellectual disability and early-onset PD and a missense [Thr168Lys] mutation in a Wisconsin family with a similar phenotype (Wilson et al. 2014). These mutations led to complete loss of protein expression, and patients exhibited a typical PD phenotype that evolved later in life (usually before age 45) and had a good response to L-Dopa. Women were reported to exhibit a milder phenotype with a later age of onset of PD and less intellectual disability (Wilson et al. 2014). Pathological analysis of a male patient revealed typical PD pathology with extensive loss of dopaminergic neurons from the SN and classic LBs and LNs (Wilson et al. 2014). A loss-of- function [Trp108Stop], null-truncating mutation was reported in a 39-year-old man with typical early-onset PD and mild intellectual disability (Lesage et al. 2015). A missense [Gly192Arg] muta- tion has been reported in families with L-Dopa-responsive, early-onset PD without any intellec- tual disability and affecting both males and females (albeit with reduced penetrance), suggesting that some mutations may be able to exert a dominant-negative effect (Mata et al. 2015). This mutation lies within the C-terminal hypervariable domain of Rab39B and is predicted to disrupt intracellular localization and interaction with effectors (Figure 1f ). Rab39B is highly expressed in the brain and in situ hybridization studies indicate this occurs exclusively in neurons (Giannandrea et al. 2010). The subcellular localization of Rab39B is predominantly within the Golgi apparatus, and RAB39B knockdown studies reveal effects on vesicle trafficking and a reduction in synapse formation of neurons in culture (Giannandrea et al. 2010). Genetic studies have identified and confirmed a locus, PARK16, as a susceptibility factor for sporadic PD (Nalls et al. 2014, Satake et al. 2009). Of 5 genes within this region, RAB29 (RAB7L1) has emerged as the leading candidate (Figure 1g), although the mechanism of how the risk variant leads to PD is unclear; in one study using protein–protein interaction arrays, analysis suggested that Rab29 is a binding partner of LRRK2 and promotes the removal of Golgi-derived vesicles through the autophagy-lysosome pathway (ALP) (Beilina et al. 2014). Rabs: RAB39B Mutations and PARK16 (RAB29) Risk Variants in Parkinson’s Disease Other genetic studies of PD patient cohorts have found common variants within the LRRK2 and RAB29 genes that function coordinately to increase PD risk nonadditively (Pihlstrøm et al. 2015). Perhaps the most com- pelling evidence for RAB29 has been functional studies showing that the Rab29 protein functions as a critical upstream regulator of LRRK2 leading to increased LRRK2 activity and phosphory- lation of Rabs (Liu et al. 2018, Purlyte et al. 2018). Rab29 is closely related to Rab32 and Rab38, and Rab32 has been found to interact with LRRK2 via its N-terminal armadillo (ARM) domain (Figure 1d ); similar to Rab29, however, there is no evidence for RAB32 mutations in familial PD cases (McGrath et al. 2019). VPS35 and VPS13C Mutations in Parkinson’s Disease Autosomal dominant mutations in VPS35 were identified in 2011 in patients with late-onset PD (Vilariño-Güell et al. 2011, Zimprich et al. 2011). VPS35 encodes the cargo-binding subunit of the trimeric retromer complex (also containing VPS29 and VPS26) whose function is to trans- port specific endosomal cargoes into vesicles and tubules,delivering these to either the trans-Golgi network or the plasma membrane (Williams et al. 2017). To date, the only definite pathogenic mu- tation identified is an [Asp620Asn] (D620N) mutation that structural analysis indicates is located at the surface of the convex face of an α-helical solenoid domain (Figure 1e), suggesting that the D620N mutation may impact interaction with an as-yet-unknown effector (Williams et al. 2017). The symptoms of these patients generally resemble those of patients with typical PD but with a mean age of onset of 50 years; however, several cases have been reported with earlier age of onset in the 30s and 40s (Lunati et al. 2018). Disease progression tends to be slow, cognitive decline rare, and response to L-Dopa good (Lunati et al. 2018). The discovery that the D620N mutation Singh • Muqit 242 significantly enhances LRRK2 activation, as assessed by autophosphorylation and Rab phospho- rylation, places VPS35 upstream of LRRK2 in a common pathway, although the mechanism by which it activates LRRK2 is unknown (Mir et al. 2018). Cell biological studies have shown that the D620N mutation also disrupts the association of VPS35 with the WASH complex via impaired binding to FAM21,which leads to trafficking defects (McGough et al.2014,Zavodszky et al.2014); further studies are required to assess whether this is linked to LRRK2 hyperactivation. Autosomal recessive mutations in VPS13C have been identified in patients with early-onset PD presenting between 25 and 45 years of age (Lesage et al. 2016). VPS13C encodes a large 3,753– amino acid protein that belongs to the VPS13 (A-D) family of proteins. VPS13C and VPS13A function as lipid transporters at organelle contact sites (Kumar et al. 2018). VPS13C is local- ized at sites bridging the endoplasmic reticulum (ER) with an endolysosomal system, specifi- cally vesicles containing the lysosomal marker LAMP1 and Rab7. VPS13C is anchored to the ER via its N-terminal domain (Figure 1j) through a conserved FFAT motif that binds both the ER vesicle-associated membrane protein (VAMP)-associated protein (VAP) and lipid droplets via its C-terminus (Kumar et al. 2018). VPS35 and VPS13C Mutations in Parkinson’s Disease Interestingly, the analysis of cells expressing PD-associated VPS13C mutants found mitochondrial defects suggesting that this may be an indirect consequence of defects in the endolysosomal system (Lesage et al. 2016). The clinical phenotype is atypical for PD, with a moderate response to L-Dopa and a rapid and severe disease progression with early cognitive decline. Pathological analysis of a single case has revealed severe pathological changes that resemble diffuse LB disease (Lesage et al. 2016). α-Synuclein and Rab GTPases α-Synuclein and Rab GTPases Multiple studies have established the role of Rab GTPases in α-syn aggregation–induced PD neu- rodegeneration in different model systems. An initial study reported the role of Rab5a, an early- endosomal Rab GTPase, in the endocytosis of α-syn leading to neuronal cell death, whereas the expression of GTPase-deficient Rab5a rescued the phenotype due to perturbed endocytosis (Sung et al. 2001). The role of Rab5b, another isoform of Rab5, was also linked to the α-syn-induced en- docytosis of the NMDA receptor (a glutamate and ion channel protein receptor) by upregulating its expression (Cheng et al. 2011). The role of endocytic Rabs contributing to α-syn trafficking was further established following the discovery of Rab7 in the autolysosomal clearance of α-syn aggregates (Figure 2b). When a pathogenic A53T mutant of α-syn was expressed in HEK293 Annu. Rev. Cell Dev. Biol. 2020.36:237-264. Downloaded from www.annualreviews.org Access provided by 86.174.33.195 on 12/11/20. See copyright for approved use. Annu. Rev. Cell Dev. Biol. 2020.36:237-264. Downloaded from www.annualreviews.org Access provided by 86.174.33.195 on 12/11/20. See copyright for approved use. gg g Microglia Neuron Exosomes Synapse a Synaptic cleft Synaptic cleft Postsynaptic Postsynaptic Presynaptic Presynaptic α-Syn α-Syn Annu. Rev. Cell Dev. Biol. 2020.36:237-264. Downloaded from www.annualreviews.org Access provided by 86.174.33.195 on 12/11/20. See copyright for approved use. Microglia Neuron Exosomes Exosomes ILV formation ESCRTs (I, II, and III) ESCRT 0 α-Syn α-Syn α-Syn α-Syn Rab7 Rab7 Rab5 Golgi TGN α-SNAP Munc18-1 Syntaxin-1 SNAP25 Synaptobrevin-2 (VAMP2) Synaptobrevin-2 (VAMP2) NSF SGT CSPα Hsc70 Synapse Presynaptic membrane Neurotransmitter Neurotransmitter Synaptic vesicle PM a b c MVBs MVBs MVBs MVBs Synaptic cleft Synaptic cleft Postsynaptic Postsynaptic Presynaptic Presynaptic α-Syn α-Syn Cytoplasm Synaptic cleft MHC II MHC I Tetraspanins Tetraspanins Hsps miRNA mRNA CPs α-Syn Exosome d Rab27b EE LY LY (Caption appears on following page) 244 Singh • Muqit Annu. Rev. Cell Dev. Biol. 2020.36:237-264. Downloaded from www.annualreviews. Access provided by 86.174.33.195 on 12/11/20. See copyright for approved use. Microglia Exosomes Exosomes ILV formation ESCRTs (I, II, and III) ESCRT 0 α-Syn α-Syn Rab7 Rab7 Rab5 Golgi TGN PM a b MVBs MVBs MVBs MVBs MHC II MHC I Hsps mRNA CPs d Rab27b EE LY LY y 86.174.33.195 on 12/11/20. See copyright for approved use b Annu. Rev. Cell Dev. Biol. Synaptic Vesicle Trafficking: DNAJC6 and SYNJ1 Mutations in Parkinson’s Disease Synaptic transmission at dopaminergic synapses is critically dependent on the recycling of dopamine receptors and other proteins via synaptic vesicle endocytosis followed by trafficking and sorting within the endosomal pathway. Interestingly, mutations in two genes encoding com- ponents of this process have been identified in autosomal recessive early-onset complex PD pa- tients. Mutations in DNAJC6 encoding auxilin (Figure 1h) cause juvenile-onset PD (Edvardson et al. 2012) and are loss-of-function with exonic deletions and splice-defective mutants. Auxilin is part of the conserved DNAJ/Hsp40 family that stimulates ATPase activity of heat shock cog- nate 70 (Hsc70) to mediate uncoating of clathrin-coated vesicles (CCVs) following endocytosis (Edvardson et al. 2012). Without the removal of clathrin, vesicles are unable to fuse and be taken up into the endosomal pathway for cargo sorting. Loss-of-function mutations in Synaptojanin-1 (SYNJ1), which encodes a lipid 5′ phosphatidylinositide phosphatase, lead to early-onset PD (Drouet & Lesage 2014, Fasano et al. 2018). SYNJ1 dephosphorylates phosphatidylinositide 4,5- bisphosphate to trigger the release of adaptor proteins from CCVs that enables auxilin-mediated clathrin uncoating (Drouet & Lesage 2014). Mutations including [Arg258Gln] and [Arg459Pro] have been identified within the Sac1 domain (Figure 1i) and have been shown to reduce phos- phatase activity (Hardies et al. 2016, Vanhauwaert et al. 2017). Patients harboring DNAJC6 or SYNJ1 mutations have a similar phenotype, with typical onset between 20 and 40 years of age and complex deficits including Parkinsonism with poor response to L-Dopa and epilepsy and cogni- tive decline (Lunati et al. 2018). Consistent with this observation, SYNJ1 or DNAJC6-knockout mice exhibit the same phenotype with neurological deficits and accumulate CCVs at the synaptic terminal (Cao et al. 2017). SYNJ1 and DNAJC6 mutations highlight the critical role of synaptic vesicle endocytosis and the endosomal pathway in the maintenance of dopaminergic striatal ter- minals to prevent PD; whether these factors interact with other PD gene components remains unknown. www.annualreviews.org • Aberrant Vesicle Trafficking in Parkinson’s 243 Annu. Rev. Cell Dev. Biol. 2020.36:237-264. Downloaded from www.annualreviews.org Access provided by 86.174.33.195 on 12/11/20. See copyright for approved use. cells and Drosophila melanogaster, Rab7 overexpression was found to lead to the clearance of α-syn aggregates, reduced cell death, and rescue of the locomotor deficit. These effects were specific to Rab7, as other Rab GTPases such as Rab5, Rab9, or Rab23 failed to rescue the pathogenic phe- notype (Dinter et al. 2016). Increased levels of GTP-bound Rab5 and Rab7 were also found upon α-syn overexpression that interacted with the motor protein dynein in a transgenic mouse model of PD (Fang et al. 2017). Rab11A, which regulates the trafficking of recycling endosomes, also regulates the secretion of extracellular α-syn through its interaction with Hsp90 (Liu et al. 2009). In Drosophila models of PD, Rab11 rescues the α-syn-induced increased synaptic vesicle size, the degeneration of dopaminergic neurons, and reduced life span in both larval and adult flies (Breda et al. 2015). Secretory pathway Rabs have also been shown to work in concert with endocytic Rabs to reg- ulate α-syn trafficking. Pull-down studies (Dalfó et al. 2004b) have identified the interaction of secretory pathway Rabs such as Rab3A and Rab8 as well as endocytic Rab5 with mutant A30P human α-syn in a transgenic mouse model. Immunoprecipitation studies (Dalfó et al. 2004a) have also showed an increased interaction of Rab3A with α-syn aggregates and a decreased affinity for its effector Rabphilin in LB disease (LBD) cases. Rab3A was also immunoprecipitated with α-syn from the SN but not cerebral cortex extracts from the postmortem brains of patients with PD (Dalfó & Ferrer 2005). In PC12 cells, soluble α-syn interacts with Rab3A to enhance exocytosis through vesicle priming and fusion by releasing calcium (Ca2+) from thapsigargin (an inhibitor of ER Ca2+ ATPase)-sensitive Ca2+ pools (Huang et al. 2018). In an RNA interference (RNAi)- based microscopic screen of α-syn aggregation in human neuroglial cells, Rab8B, Rab11A, and Rab13 knockdown were found to rescue α-syn-induced cytotoxicity, whereas Rab11A and Rab13 expression induced α-syn endocytic recycling and secretion (Gonçalves et al. 2016). α-Synuclein also blocks trafficking between the ER and Golgi compartments in yeast cells and increased Rab1 (yeast homolog Ypt1) expression protects against dopaminergic neuronal loss in PD animal models (Cooper et al. 2006). Similarly, in a neuronal model of PD, post–Golgi vesicle– associated Rab8A also inhibited α-syn toxicity (Gitler et al. 2008). α-Synuclein and Rab GTPases 2020.36:237- A id d b 86 174 33 195 Access provided by 86.174.33.195 Neuron α-Syn α-Syn α-SNAP Munc18-1 Syntaxin-1 SNAP25 Synaptobrevin-2 (VAMP2) Synaptobrevin-2 (VAMP2) NSF SGT CSPα Hsc70 Presynaptic membrane Neurotransmitter Neurotransmitter Synaptic vesicle c Postsynaptic Postsynaptic Cytoplasm Synaptic cleft Tetraspanins Tetraspanins miRNA α-Syn Exosome (Caption appears on following page) c Exosome Presynaptic Presynaptic Cytoplasm Synaptic cleft Synaptic cleft Synaptic cleft 244 Singh • Muqit Figure 2 (Figure appears on preceding page) Figure 2 (Figure appears on preceding page) www.annualreviews.org • Aberrant Vesicle Trafficking in Parkinson’s Figure 2 (Figure appears on preceding page) Vesicular trafficking mechanisms of α-syn aggregation–mediated neurotoxicity. (a) α-Syn aggregates can be transmitted to other neurons through endocytic mechanisms or exosomes, or they can block neurotransmitter release at synapses. (b) Under normal conditions, α-syn aggregates can enter the cell through an endocytic mechanism, first reaching Rab5-positive EEs, where they can be recycled back through a recycling mechanism (not shown) or sorted within the MVBs to form ILVs through the involvement of ESCRT machinery (complexes 0, I, II, and III). When MVBs fuse with the plasma membrane, exosomes are secreted into the extracellular milieu. Rab27b has been suggested to play a role in the exosome-mediated secretion of α-syn aggregates, or these aggregates can be degraded within lysosomes through Rab7-mediated endolysosomal fusion. The perturbation of these processes in PD causes the accumulation of pathogenic α-syn aggregates within the cell, leading to toxicity. (c) Under physiological conditions, α-syn functions as a SNARE chaperone to regulate the exocytic release of neurotransmitter at the presynaptic terminal. In PD, α-syn aggregates block neurotransmitter release by engaging VAMP2 and SNAP25, leading to neurodegeneration. Panel c is adapted from Rizo & Sudhof (2012). (d ) Exosomes containing α-syn aggregates may facilitate propagation to other cells. Other constituents of exosomes include proteins such as tetraspanins, MHCs I and II, CPs, Hsps, miRNA, and mRNA. Abbreviations: α-SNAP, α-soluble NSF-attachment protein; α-syn, α-synuclein; CP, cytoskeletal protein; CSPα, cysteine string protein α; EE, early endosome; ESCRT, endosomal sorting complex required for transport; Hsc70, heat shock cognate 70; Hsp, heat shock protein; ILV, intraluminal vesicle; LE, late endosome; LY, lysosome; MHC, major histocompatibility complex; miRNA, microRNA; mRNA, messenger RNA; Munc18-1, mammalian uncoordinated 18-1; MVB, multivesicular body; NSF, N-ethylmaleimide-sensitive factor; PM, plasma membrane; Rab, Ras analog in brain; SGT, small glutamine-rich tetratricopeptide repeat; SNAP25, synaptosomal-associated protein 25; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptors; TGN, trans-Golgi network; VAMP2, vesicle-associated membrane protein 2. α-Synuclein and SNAREs SNAREs are a family of proteins that associates with intracellular membranes and regulates the docking and fusion of donor intracellular vesicles with target compartment membranes. SNAREs can be functionally categorized into v-SNAREs, which associate with donor vesicles, and t-SNAREs, which are associated with target compartments ( Jahn & Scheller 2006). The specific interaction of v-SNAREs with t-SNAREs leads to the formation of a quaternary helix complex that facilitates membrane fusion. These complexes are further recycled with the help of two solu- ble proteins: NSF (N-ethylmaleimide-sensitive factor), an AAA (ATPases associated with diverse cellular activities)-type ATPase, and α-SNAP (α soluble NSF-attachment protein), which main- tains SNARE availability for subsequent fusion events (Chen & Scheller 2001). Similar to Rab GTPases, SNAREs also define a layer of specificity by localizing to specific membrane compart- ments,but their role is considered more promiscuous than that of Rabs.Synaptic vesicle fusion and neurotransmission are regulated by the neuronal SNAREs syntaxin-1, synaptobrevin/VAMP1, and synaptosomal-associated protein 25 (SNAP25) (Rizo & Sudhof 2012). Therefore, any pertur- bation in SNARE proteins per se or in regulatory proteins such as α-syn leads to the neurotrans- mission defects underpinning many neurological diseases including PD (Chua & Tang 2011). p g y g g g Our understanding of the role of SNARE proteins involved in PD came from an earlier metabolic labeling study in the late nineties ( Jensen et al. 1999), when SNAP25, a predominantly neuronal SNARE, was found to be colocalized with and involved in α-syn axonal transport in rat retinal ganglion cells. α-Syn was later discovered to cooperate with cysteine string protein α (CSPα) in mediating chaperone activity toward neuronal SNARE complexes (Figure 2c) and to prevent neurodegeneration in mice. This modulation was achieved through the phospholipid binding of wild-type α-syn but not of the A30P mutant, which failed to attach to the membrane and could not rescue the phenotype (Chandra et al. 2005). A subsequent study (Darios et al. 2010) suggested that α-syn modulated SNARE complex formation and exocytosis through sequestering arachidonic acid, a common component of phospholipids. A transgenic mouse model of PD expressing truncated human α-syn (1–120) showed an age-dependent reduction of dopamine release accompanied by a redistribution of the SNARE proteins SNAP25, syntaxin-1, and synaptobrevin-2 in the striatum (Garcia-Reitböck et al. 2010). However, another study (Anwar et al. 2011) suggested no difference in the distribution of synaptic SNAREs in the dorsal striatum of triple-synuclein knockout mice. Annu. Rev. Cell Dev. Biol. 2020.36:237-264. Downloaded from www.annualreviews.org Access provided by 86.174.33.195 on 12/11/20. See copyright for approved use. In a cellular model of PD, Rab1, Rab2, and Rab8 regulated Golgi fragmentation, leading to the aggregation of α-syn as well as the impairment of anterograde and retrograde transport between the ER and the Golgi apparatus www.annualreviews.org • Aberrant Vesicle Trafficking in Parkinson’s 245 (Rendón et al. 2013). Taken together, Rab GTPases can regulate α-syn aggregation and secretion as well as its clearance in the various model systems of PD. α-Synuclein and Exosomes Exosomes are a type of extracellular vesicle measuring 30–100 nm in diameter that are secreted into the extracellular medium by many cell types in both normal and pathological conditions. Exo- somes are composed of a lipid bilayer and are found to contain various proteins, lipids, RNAs, and other substances released by the secreting cell (Mathivanan et al. 2010). Exosomes are formed through inward budding in multivesicular bodies (MVBs) that generates intraluminal vesicles (ILVs) (Figure 2d ), which are then secreted extracellularly by the fusion of MVBs with the plasma membrane. ESCRT (endosomal sorting complex required for transport) machinery, which in- cludes ESCRT complexes 0, I, II, and III, are involved in cargo sorting, budding, and fission of exosomes within MVBs. Several families of proteins such as tetraspanins, Hsps, cytoskeletal proteins, and trafficking proteins as well as various lipids, miRNAs, mRNAs, immune regulatory molecules such as major histocompatibility complexes (MHCs) I and II, and pathological com- ponents including viral proteins are all found in exosomes (Gupta & Pulliam 2014) (Figure 2d ). Initial hints about the role of exosomes in α-syn trafficking came through its detection in human plasma and cerebrospinal fluid and reports of the secretion of its monomeric and oligomeric forms from primary cortical neurons as well as from human neuroblastoma cells (Borghi et al. 2000; El- Agnaf et al. 2003, 2006; Lee et al. 2005; Tokuda et al. 2010). Later it was reported that α-syn is secreted through exosomes in a Ca2+-dependent manner and affects the survival of neighbor- ing neurons (Emmanouilidou et al. 2010). Another study (Alvarez-Erviti et al. 2011) in SH-SY5Y cells reported that lysosomal dysfunction, which has been reported in PD, can lead to the increased exosome-mediated secretion of α-syn and to its transmission to neighboring cells. In transgenic mice expressing human phosphorylation mimic S129D α-syn, which aggregates, exosomes loaded with small interfering RNA (siRNA) against α-syn and expressing neuron-specific rabies viral gly- coprotein were found to be effective in reducing intraneuronal aggregates (Cooper et al. 2014). Exosomes have also been suggested to provide a catalytic environment for nucleation that pro- motes α-syn aggregation (Grey et al. 2015). Microglia have been suggested to play an important role in the transmission of α-syn- containing exosomes through the uptake of pathogenic exosomes (Figure 2a). www.annualreviews.org • Aberrant Vesicle Trafficking in Parkinson’s α-Synuclein and SNAREs The first evidence of the direct interaction of α-syn with SNAREs came through a study (Burré et al. 2010) suggesting its binding with synaptobrevin-2 (VAMP2) through its C-terminus to maintain the presynaptic SNARE complex for efficient neurotransmitter release. Remarkably, a follow-up study by the same group (Sharma et al. 2011) also discovered that CSPα, which forms complexes with Hsc70 and the small glutamine-rich tetratricopeptide-repeat protein (SGT), directly interacts with SNAP25 to promote SNARE- complex formation (Figure 2c) by preventing its aggregation and ubiquitylation-dependent proteasomal degradation. Analysis of the CSPα-knockout mouse that leads to neurodegeneration revealed that the neurodegenerative phenotype was mainly due to low levels of SNAP-25 leading to aberrant synaptic SNARE-complex assembly (Sharma et al. 2012). While soluble α-syn monomers promote the assembly of such complexes, how α-syn oligomers cause neurotoxicity has also been a major question. One report (Choi et al. 2013) suggested that α-syn oligomers bind to the N-terminal domain of VAMP2 and inhibit SNARE-mediated lipid mixing by pre- venting SNARE-complex formation. Interestingly, another study (DeWitt & Rhoades 2013) also Singh • Muqit 246 suggested that α-syn inhibits SNARE-dependent vesicle fusion by binding to the lipid membrane without direct interaction with SNAREs. α-Syn aggregates have also been shown to directly sequester SNARE proteins such as VAMP2 and SNAP25 (Figure 2c), and this interaction, which is resistant to SDS treatment, leads to neurotoxicity. Furthermore, this sequestration is partially blocked by a VAMP2 peptide, resulting in reduced neurotoxicity (Choi et al. 2018). y g y Proteins regulating SNARE functions have also been found to be involved in modulating α- syn activity. Munc18-1 (mammalian uncoordinated 18-1), which is important for regulating the SNARE-dependent fusion of synaptic vesicles and thereby neurotransmitter release, regulates α-syn aggregation by acting as a molecular chaperone (Chai et al. 2016). In a mouse model of lysosomal storage disorders, α-syn aggregation and proteasomal degradation of CSPα have been reported, resulting in inhibition of SNARE-complex assembly and reduced recycling of synaptic vesicles and leading to neurodegeneration (Sambri et al. 2017). In conclusion, α-syn interacts with SNAREs to regulate neurotransmitter release, but this process is disrupted by oligomeric α-syn; conversely, SNAREs can, in turn, regulate α-syn function. α-Synuclein and Exosomes This uptake re- sults in inhibition of the autophagy and scavenging activities of microglia, which leads to further enhanced secretion of pathogenic exosomes and transmission to other cells (Xia et al. 2019). The reduced phagocytosis of exosomes containing α-syn with increased tumor necrosis factor (TNF)- α secretion was shown in mice microglia as well as in human monocytes derived from aged donors. www.annualreviews.org • Aberrant Vesicle Trafficking in Parkinson’s 247 This might lead to an age-dependent susceptibility to misfolded proteins and infections in neu- rodegenerative diseases such as PD (Bliederhaeuser et al. 2016). Exosomes derived from the serum of PD patients have also been shown to be pathogenic to mice due to the presence of an increased abundance of α-syn and inflammatory factors such as interleukin (IL)-1β and TNF-α in compari- son to healthy controls. This leads to α-syn, Ub, and p62 aggregation, leading in turn to microglial activation, dopaminergic neuronal cell death, and motor defects in recipient cells (Han et al. 2019). Quantitative proteomic profiling using laser-capture microdissection has identified the enrich- ment of extracellular exosomal proteins in the SN of human brain samples (Griesser et al. 2020). A recent study also suggested the role of Rab27b, a GTPase involved in the endolysosomal pathway, in α-syn secretion through both exosomal (Figure 2b) and nonexosomal pathways as well as its clearance and toxicity (Underwood et al. 2020). Microglia secreted α-syn through exosomes when they were treated with human α-syn preformed fibrils, which help in the transmission of α-syn pathology and cause aggregation in neuronal cells (Guo et al. 2020). Collectively, α-syn secreted in exosomes may play an important role in PD progression and appears to be a promising biomarker and therapeutic target. However, future studies are required to uncover the fusion mechanism by which exosomes are taken up by recipient cells, and how the RNA or protein contents of exosomes escapes from its membrane coat following engulfment of exosomes to mediate cellular effects. This might lead to an age-dependent susceptibility to misfolded proteins and infections in neu- rodegenerative diseases such as PD (Bliederhaeuser et al. 2016). Exosomes derived from the serum of PD patients have also been shown to be pathogenic to mice due to the presence of an increased abundance of α-syn and inflammatory factors such as interleukin (IL)-1β and TNF-α in compari- son to healthy controls. α-Synuclein and Exosomes This leads to α-syn, Ub, and p62 aggregation, leading in turn to microglial activation, dopaminergic neuronal cell death, and motor defects in recipient cells (Han et al. 2019). Quantitative proteomic profiling using laser-capture microdissection has identified the enrich- ment of extracellular exosomal proteins in the SN of human brain samples (Griesser et al. 2020). A recent study also suggested the role of Rab27b, a GTPase involved in the endolysosomal pathway, in α-syn secretion through both exosomal (Figure 2b) and nonexosomal pathways as well as its clearance and toxicity (Underwood et al. 2020). Microglia secreted α-syn through exosomes when they were treated with human α-syn preformed fibrils, which help in the transmission of α-syn pathology and cause aggregation in neuronal cells (Guo et al. 2020). Collectively, α-syn secreted in exosomes may play an important role in PD progression and appears to be a promising biomarker and therapeutic target. However, future studies are required to uncover the fusion mechanism by which exosomes are taken up by recipient cells, and how the RNA or protein contents of exosomes escapes from its membrane coat following engulfment of exosomes to mediate cellular effects. Annu. Rev. Cell Dev. Biol. 2020.36:237-264. Downloaded from www.annualreviews.org Access provided by 86.174.33.195 on 12/11/20. See copyright for approved use. LRRK2: AT THE NEXUS OF VESICULAR TRAFFICKING IN PARKINSON’S DISEASE The Landscape of Rab GTPases Regulated by LRRK2 Early studies implicating LRRK2 with Rab GTPases came from localization of the Drosophila ho- molog of LRRK2 (Lrrk) to the membranes of late endosomes and lysosomes and found that it regulates endolysosomal transport and autophagy (Dodson et al. 2012, 2014). Further, LRRK2 was found to interact with Rab7 to inhibit the perinuclear positioning of lysosomes, whereas the G2019S disease mutant promoted perinuclear positioning through microtubules and dynein (Dodson et al. 2012). Mutant LRRK2 was further shown to delay EGFR receptor degradation by decreasing Rab7 activity, which led to the blocking of late endosomal budding (Gómez-Suaga et al. 2014) (Figure 3). Another study also suggested that LRRK2 directly interacts with Rab32 and Rab38 to regulate endolysosomal trafficking (Waschbüsch et al. 2014). The interaction of LRRK2 with Rab32 and Rab38 (a related Rab32 subfamily of GTPases) occurred via the LRRK2 N-terminal armadillo domain (Figure 1d ) with GTP-bound Rab proteins (McGrath et al. 2019). Interestingly, Rab2a was also identified as an LRRK2-interacting partner that regulated the traf- ficking of lysozyme-containing dense core vesicles to promote the symbiosis of commensal bac- teria in Paneth cells (Zhang et al. 2015). In human pluripotent stem cell–derived macrophages, LRRK2 was also found to be recruited onto LAMP1- and Rab9-positive maturing phagosomes, where it in turn recruits Rab8 and Rab10 and might regulate phagocytic trafficking (Lee et al. 2020). These latter studies suggested the role of LRRK2 in infection and inflammatory bowel disease that could also be linked to PD pathogenesis (Ahmadi Rastegar & Dzamko 2020, Herbst & Gutierrez 2019, Houser & Tansey 2017, Wallings & Tansey 2019). Another study in mice (Kuwahara et al.2016) suggested that LRRK2 and Rab29 work in a coordinated fashion to regulate axonal morphology and lysosome integrity, as knockout of either gene resulted in a similar pheno- type. Furthermore, altered LRRK2 kinase activity is associated with defects in neurite outgrowth, and PD pathogenic mutants of LRRK2, including R1441C, R1441G, Y1699C, and I2020T, have been shown by immunoelectron microscopy and electron tomography studies (Kett et al. 2012) to exhibit enhanced binding to microtubules appearing as filamentous structures in primary neuron cultures. www.annualreviews.org • Aberrant Vesicle Trafficking in Parkinson’s LRRK2: AT THE NEXUS OF VESICULAR TRAFFICKING IN PARKINSON’S DISEASE Cryo-electron tomography analysis has recently demonstrated that pathogenic LRRK2 Singh • Muqit 248 Rab29 ARM ANK LRR ROC COR α-Syn Damaged mitochondria Kinase WD40 LRRK2 Retromer complex VPS35 VPS26 VPS29 8A 10 43 3 12 5 1A/B 29 35 Golgi TGN CCV LE LE Rab7 Rab5 Rab7 EGFR PM Autolysosomes Phagophore Autophagosomes 8A 10 RILPL1/2 RILPL1/2 RILPL1/2 RILPL1/2 Ciliogenesis Thr73 Thr72 8A Thr72 Ser111 Parkinson’s disease ? PPM1H protein phosphatase ? Rab GTPases Phosphorylation at Thr72 or homologous residues at switch II– effector-binding region LY LY Rab10 Rab8A Rab8A PINK1 EE Figure 3 The regulation and downstream function of LRRK2. Pathogenic mutations of LRRK2 have been identified in PD that lead to various trafficking perturbations. Rab29 can activate LRRK2, which leads to its recruitment to the TGN through the ARM and ANK domains. VPS35, a core component of the retromer complex, is mutated in PD (D620N) and is associated with the hyperactivation of LRRK2 activity by an unknown mechanism. LRRK2 has been reported to disrupt endocytic pathways (e.g., EGFR trafficking) via the inhibition of Rab7 activity. LRRK2 phosphorylates Thr72 or homologous residues in the switch II–effector-binding region of a subset of Rab GTPases. PPM1H phosphatase reverses the LLRK2-mediated phosphorylation of Rab GTPases. The phosphorylation of Rab10 and Rab8A regulates ciliogenesis through binding to RILPL1 and RILPL2. Rab8A is phosphorylated in response to PINK1 activation at the Ser111 residue, which can modulate phosphorylation by LRRK2. Abbreviations: α-syn, α-synuclein; ANK, ankyrin; ARM, armadillo; CCV, clathrin-coated vesicle; COR, C-terminal of ROC; EE, early endosome; EGFR, epidermal growth factor receptor; LE, late endosome; LRRK2, leucine-rich repeat kinase 2; LY, lysosome; PD, Parkinson’s disease; PINK1, PTEN-induced kinase 1; Rab, Ras analog in brain; RILPL, Rab-interacting lysosomal protein-like; ROC, Ras of complex; TGN, trans-Golgi network; VPS, vacuolar protein–sorting. Rab29 ARM ANK LRR ROC COR α-Syn Damaged mitochondria Kinase WD40 LRRK2 Retromer complex VPS35 VPS26 VPS29 8A 10 43 3 12 5 1A/B 29 35 Golgi TGN CCV LE LE Rab7 Rab5 Rab7 EGFR PM Autolysosomes Phagophore Autophagosomes 8A 10 RILPL1/2 RILPL1/2 RILPL1/2 RILPL1/2 Ciliogenesis Thr73 Thr72 8A Thr72 Ser111 Parkinson’s disease ? PPM1H protein phosphatase ? Rab GTPases Phosphorylation at Thr72 or homologous residues at switch II– effector-binding region LY LY Rab10 Rab8A Rab8A PINK1 EE Figure 3 Annu. Rev. Cell Dev. Biol. 2020.36:237-264. Downloaded from www.annualreviews.org Access provided by 86.174.33.195 on 12/11/20. See copyright for approved use. Parkinson’s disease Figure 3 The regulation and downstream function of LRRK2. Pathogenic mutations of LRRK2 have been identified in PD that lead to various trafficking perturbations. Rab29 can activate LRRK2, which leads to its recruitment to the TGN through the ARM and ANK domains. VPS35, a core component of the retromer complex, is mutated in PD (D620N) and is associated with the hyperactivation of LRRK2 activity by an unknown mechanism. LRRK2 has been reported to disrupt endocytic pathways (e.g., EGFR trafficking) via the inhibition of Rab7 activity. LRRK2 phosphorylates Thr72 or homologous residues in the switch II–effector-binding region of a subset of Rab GTPases. PPM1H phosphatase reverses the LLRK2-mediated phosphorylation of Rab GTPases. The phosphorylation of Rab10 and Rab8A regulates ciliogenesis through binding to RILPL1 and RILPL2. Rab8A is phosphorylated in response to PINK1 activation at the Ser111 residue, which can modulate phosphorylation by LRRK2. Abbreviations: α-syn, α-synuclein; ANK, ankyrin; ARM, armadillo; CCV, clathrin-coated vesicle; COR, C-terminal of ROC; EE, early endosome; EGFR, epidermal growth factor receptor; LE, late endosome; LRRK2, leucine-rich repeat kinase 2; LY, lysosome; PD, Parkinson’s disease; PINK1, PTEN-induced kinase 1; Rab, Ras analog in brain; RILPL, Rab-interacting lysosomal protein-like; ROC, Ras of complex; TGN, trans-Golgi network; VPS, vacuolar protein–sorting. oligomerizes around microtubules (Watanabe et al. 2019), and a high-resolution cryo-electron mi- croscopy structure has been solved that shows the C-terminal half of the LRRK2 kinase domain bound to tubulin (Deniston et al. 2020). In future work it will be interesting to understand how LRRK2 regulation of Rab GTPase influences vesicle trafficking along microtubules (Horgan & McCaffrey 2011). However, the most compelling evidence for a link between LRRK2 and Rab biology was the discovery that a subset of Rab GTPases, including Rab1A/B, Rab3, Rab5, Rab8A, Rab10, Rab12, Rab29, Rab35, and Rab43, were substrates of LRRK2 via phosphorylation of a con- served Thr72 or homologous residue within their switch II–effector-binding regions (Figure 3). The phosphorylated Rab8A and Rab10 were suggested to regulate cilia formation through their antagonistic roles by interacting with RILPL1 and RILPL2 (Rab-interacting lysosomal protein-like 1/2), respectively (Dhekne et al. 2018; Steger et al. 2016, 2017) (Figure 3). In- terestingly, Rab phosphorylation by LRRK2 was found to be counteracted by the action of the Golgi-localized PPM1H phosphatase, which dephosphorylates Rab proteins (Figure 3) www.annualreviews.org • Aberrant Vesicle Trafficking in Parkinson’s 249 to regulate primary cilia formation (Berndsen et al. 2019). Figure 3 The LRRK2 activation and subse- quent phosphorylation of various Rab GTPases have been suggested to depend on the LRRK2 association at the membrane of different intracellular compartments (Gomez et al. 2019). Rab8A phosphorylation by LRRK2 was also suggested to promote lipid storage, which might be indirectly linked to PD pathogenesis (Yu et al. 2018). In iPSC (induced pluripotent stem cell)-derived dopaminergic neurons carrying the LRRK2 G2019S mutation, the use of the integrated transcriptomic and proteomic method led to the observation of endocytic dysreg- ulation, which might be due to changes in the expression of several Rab proteins, including Rab5B, Rab7A, and Rab10, along with other endocytic proteins (Connor-Robson et al. 2019). Interestingly, Rab29 was suggested to be an activator of LRRK2 that recruits it to the trans- Golgi network (TGN) to stimulate its kinase activity (Liu et al. 2018, Purlyte et al. 2018) (Figure 3). A major question has been which Rabs are relevant to neurodegeneration in PD. One study ( Jeong et al. 2018) using in vitro kinase assays and tandem mass spectrometry suggested that the dysregulation of Rab35 phosphorylation was associated with neurodegeneration. In a transgenic mouse model expressing mutant LRRK2, Rab35 protein expression was found to be increased in the SN and was suggested as a potential biomarker (Chiu et al. 2016). In an in vivo electroretinogram screen in selected Drosophila dopaminergic neurons, Rab10 was found to be the strongest interactor and Rab3 the weakest with the PD-associated LRRK2-G2019S mutant form (Petridi et al. 2020). Autophagy and LRRK2 A role for LRRK2 in regulating autophagy has been suggested by many studies using knockout or expressing wild-type or PD-associated mutants in many model systems. That said, whether LRRK2 positively or negatively regulates autophagy remains unclear (Figure 3). In differentiated SH-SY5Y cells, the expression of the LRRK2 PD-associated mutant G2019S led to an increase in autophagic vacuoles (AVs) marked by LC3 and neurite shortening (Plowey et al. 2008). This phenotype was similar to that seen in the deletion of Atg7 (an essential autophagy gene) in Purk- inje cells of conditional knockout mutant mice and also leads to the presynaptic accumulation of LRRK2 (Friedman et al. 2012, Komatsu et al. 2007). Moreover, the localization of LRRK2 R1441C to ILVs of MVBs as well as in caveolae neck and filopodia in the endosomal-autophagic pathway has been reported. Here also LRRK2 was reported to cause autophagic impairment leading to an increase in AVs with partly digested cargoes, the accumulation of MVBs, and increased levels of the p62 autophagy receptor (Alegre-Abarrategui et al. 2009). In Saccharomyces cerevisiae, the GTPase domain of LRRK2 (Figure 3) has been shown to be important for mod- ulating endocytic and autophagic activity (Xiong et al. 2010). In mice studies (Tong et al. 2010, 2012), knockout of LRRK2 showed age-dependent biphasic alterations in autophagy pathways in kidneys leading to altered LC3-II and p62 levels with an accumulation of lipofuscin granules. Additionally, transgenic mice expressing LRRK2 G2019S showed autophagic impairment in the aged brain (Ramonet et al. 2011). Interestingly, the ALP was found to be impaired in the brains of double-knockout mice of both LRRK2 and LRRK1 (a functional homolog of LRRK2, which is not mutated in PD) but not in the single knockout of either gene (Giaime et al. 2017). Using LRRK2 kinase inhibitors in SH-SY5Y cells, LRRK2 activates a protective autophagic process through the disruption of dynamin-related protein-1 (DRP-1)-dependent mitochondrial dynamics and mitochondrial-derived reactive oxygen species signaling (Saez-Atienzar et al. 2014). Overall, LRRK2 likely modulates autophagic activity, but in future work it will be important to address whether these are direct or indirect effects of LRRK2. With the knowledge that LRRK2 phosphorylates Rab GTPase, future studies investigating autophagy should be undertaken within the prism of potential Rab-dependent mechanisms. Singh • Muqit Singh • Muqit 250 Golgi Trafficking Mediated by LRRK2 and Rab29 Golgi Trafficking Mediated by LRRK2 and Rab29 The association of LRRK2 and Rab29 (also known as Rab7L1), a TGN-associated GTPase that regulates the retrograde trafficking of the mannose-6-phosphate receptor, came from both ge- netic and protein interaction studies, further suggesting the involvement of aberrant trafficking defects in PD (Feng et al. 2018, Wang et al. 2014). In primary rodent neurons as well as a fly model of PD, Rab29 was found to interact with LRRK2 and to rescue a pathogenic degeneration defect associated with the expression of familial PD–associated LRRK2 G2019S or R1441C mu- tants (MacLeod et al. 2013). Later, a large-scale GWAS also identified Rab29 as a risk locus for PD (Nalls et al. 2014). In an unbiased screen, Rab29 was found to be an interacting partner of LRRK2 in both in vitro and in vivo systems and promoted the clearance of Golgi-derived vesicles through the ALP (Beilina et al. 2014). This TGN-associated activity of LRRK2 might involve its constitutive phosphorylation by the CK1α kinase and its interaction with VPS52, a subunit of the Golgi-associated retrograde protein complex that regulates fusion at the TGN (Beilina et al. 2020, Chia et al. 2014). The finding that LRRK2 phosphorylates many members of the Rab GTPase family, including Rab29, opens a new avenue of research regarding trafficking perturba- tion in PD. LRRK2 was found to phosphorylate Rab29 at both the Thr71 and Ser72 residues, and the N-terminal domain of LRRK2 is important for optimal phosphorylation at Thr71 (Kalogeropulou et al. 2018, Steger et al. 2017). The Ser72 phosphorylation of Rab29 by LRRK2 has been suggested to alter the morphology and distribution of the TGN (Fujimoto et al. 2018). Furthermore, LRRK2 directly phosphorylates Rab29 in its GTP-bound, membrane-associated form, which in turn regulates LRRK2 activity and recruits it to the TGN (Liu et al. 2018, Purlyte et al. 2018). The Rab29-mediated recruitment of wild-type LRRK2 to the Golgi (Figure 3) leads to centrosomal cohesion deficits due to aberrant Rab8A phosphorylation (Madero-Pérez et al. 2018a, Madero-Pérez et al. 2018b). This centrosomal cohesion deficit resembles defects associ- ated with pathogenic PD-associated LRRK2 mutants that also showed ciliogenesis defects and depends upon both Rab8A and Rab10 phosphorylation as well as on RILP1 (Lara Ordónez et al. 2019) (Figure 3). PINK1/Parkin Regulation of Mitophagy and Link to Rabs PINK1/Parkin Regulation of Mitophagy and Link to Rabs PINK1 and Parkin function in a common mitochondrial signaling pathway to generate the Ub- dependent elimination of damaged mitochondria by autophagy (mitophagy) (Harper et al. 2018, McWilliams & Muqit 2017) (Figure 4). Under healthy cellular conditions, PINK1 is imported into mitochondria, where it undergoes proteolytic cleavage and then is released back into the cy- toplasm where it is degraded via the N-end rule pathway (Yamano & Youle 2013), and Parkin resides in the cytosol in an autoinhibited conformation mediated in part by its N-terminal Ubl domain (Figure 1c) and partly by an autoinhibitory interface between the RING2 and RING0 domains (Chaugule et al. 2011, Trempe et al. 2013, Wauer & Komander 2013). However, in re- sponse to mitochondrial damage (specifically mitochondrial depolarization that can be induced by mitochondrial uncouplers), PINK1 accumulates at the outer mitochondrial membrane (OMM), where it becomes activated and autophosphorylated (Kondapalli et al. 2012, Narendra et al. 2010) (Figure 4). The major role of active PINK1 is to phosphorylate and activate the Parkin E3 ligase; it achieves this via a three-step activation process whereby it phosphorylates Ser65 on Ub resident on protein substrates on the OMM to yield Ser65-phosphorylated Ub (phospho-Ub). Phospho-Ub binds with high affinity to Parkin via a phosphate-binding pocket that leads to release of the Ubl domain, thereby enabling PINK1 to phosphorylate Parkin at a conserved Ser65 site within the Ubl domain (equivalent to the Ub residue) (Kane et al. 2014, Kazlauskaite et al. 2014, Kondapalli et al. 2012, Koyano et al. 2014, Ordureau et al. 2014) (Figure 4). The formation of the cocomplex of Ser65-phosphorylated Parkin bound to phospho-Ub represents the active Parkin conforma- tion and leads to the ubiquitylation of multiple OMM substrates including mitofusins 1 and 2, voltage-dependent anion-selective channel 1–3 (VDAC1–3), and CDGSH iron–sulfur domain- containing protein 1 (CISD1) (Ordureau et al. 2018, 2020; Sarraf et al. 2013). The assembly of new Ub chains at the OMM of mitochondria leads to further PINK-dependent phosphorylation, Parkin recruitment, and activation, thereby creating a powerful feed-forward amplification step that drives mitophagy (Ordureau et al. 2014) (Figure 4). Furthermore, pSer65 poly-Ub chains are resistant to degradation by deubiquitinases, which promotes mitophagic signaling (Wauer et al. 2015). The Interplay Between Retromer Activity and LRRK2 Phosphorylation Our understanding of the dynamic interplay between retromer activity and LRRK2 activity has come from many studies. In a Drosophila model of PD, the overexpression of VPS35, a core com- ponent of the retromer complex (Figure 3), rescued the disease phenotype linked with the ex- pression of mutant LRRK2 associated with PD (Linhart et al. 2014). Loss of function of lrrk, the Drosophila homolog of LRRK2, causes aberrant autophagy and endolysosomal trafficking that was rescued by the expression of the constitutively active form of Rab9 (a small GTPase that regulates retromer-dependent recycling from late endosomes to the Golgi) (Dodson et al. 2014). Further- more, LRRK2 and VPS35, along with Rab5 and Rab11, work in cooperation to regulate synaptic vesicle recycling in Drosophila (Inoshita et al. 2017). In addition, in brain tissues, reduced LRRK2 expression in LRRK2 mutant carriers also showed a reduced retromer function with a decreased level of VPS35 (Zhao et al. 2018). Moreover, the retromer VPS35 PD-associated mutant D620N was found to enhance the kinase activity of LRRK2 as measured by Rab8A, Rab10, and Rab12 phosphorylation (Mir et al. 2018, Vilarino-Guell et al. 2011, Zimprich et al. 2011). The Rab32 interaction with sorting nexin 6 (SNX6) was also suggested to regulate LRRK2-dependent Golgi trafficking (Waschbüsch et al. 2019). This further strengthened the evidence of an interplay be- tween these two genes in trafficking perturbations associated with PD. Overall these studies have firmly placed VPS35 upstream of LRRK2, and the mechanism of how the D620N mutant of VPS35 activates LRRK2 remains to be solved in future work. www.annualreviews.org • Aberrant Vesicle Trafficking in Parkinson’s 251 PINK1/Parkin Regulation of Mitophagy and Link to Rabs PINK1 phosphorylates both Parkin and Ub at the Ser65 residue. Ser65- phosphorylated Ub binds to Parkin, leading to the activation of its E3 Ub ligase activity. Parkin ubiquitylates multiple OMM proteins. Damaged mitochondria also lead to the recruitment of several proteins, including Rab7, Rab7-GAPs, TBC1D15, and TBC1D17, as well as TBK1 kinase, which phosphorylates Rab7 at Ser72. In addition, this damage recruits autophagy receptors that bind ubiquitylated proteins and they in turn recruit autophagy machinery to degrade mitochondria through the selective autophagy known as mitophagy. In response to oxidative stress, mitochondria can also release MDVs that fuse with lysosomes through the recruitment of syntaxin-17 that forms a SNARE complex with SNAP29 and VAMP7. PINK1 activation also leads to the phosphorylation of several Rab GTPases such as Rab8A, Rab8B, and Rab13 at the Ser111 residue. The kinases or phosphatases involved in these phosphorylation events remain unknown. Abbreviations: CCV, clathrin-coated vesicle; EE, early endosome; ER, endoplasmic reticulum; GAP, GTPase-activating protein; LE, late endosome; LY, lysosome; MDV, mitochondria-derived vesicle; NSF, N-ethylmaleimide-sensitive factor; OMM, outer mitochondrial membrane; PARL, presenilins-associated rhomboidlike; PINK1, PTEN-induced kinase 1; PM, plasma membrane; Rab, Ras analog in brain; SNAP, soluble NSF-attachment protein; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptors; TBK1, TANK-binding kinase 1; TGN, trans-Golgi network; Ub, ubiquitin; VAMP, vesicle-associated membrane protein. Annu. Rev. Cell Dev. Biol. 2020.36:237-264. Downloaded from www.annualreviews.org Access provided by 86.174.33.195 on 12/11/20. See copyright for approved use. Annu. Rev. Cell Dev. Biol. 2020.36:237-264. Downloaded from www.annualreviews.org Access provided by 86.174.33.195 on 12/11/20. See copyright for approved use. Annu. Rev. Cell Dev. Biol. 2020.36:237-264. Downloaded from www.annualreviews.org Access provided by 86.174.33.195 on 12/11/20. See copyright for approved use. Annu. Rev. Cell Dev. Biol. 2020.36:237-264. Downloaded from www.annualreviews.org Access provided by 86.174.33.195 on 12/11/20. See copyright for approved use. The PINK1-Parkin signaling pathway. Under healthy conditions, PINK1 is degraded in the cytosol after its cleavage following precursor import into mitochondria by the intramembrane protease PARL, which leads to its release into the cytoplasm. Upon mitochondrial depolarization, PINK1 accumulates on the outer mitochondrial membrane through an unknown mechanism and is activated and autophosphorylated (small red spheres). PINK1 phosphorylates both Parkin and Ub at the Ser65 residue. Ser65- phosphorylated Ub binds to Parkin, leading to the activation of its E3 Ub ligase activity. Parkin ubiquitylates multiple OMM proteins. PINK1/Parkin Regulation of Mitophagy and Link to Rabs The mechanism of Parkin activation by phosphorylation has been comprehensively studied by structural analysis, revealing the sequential release of autoinhibitory interfaces culminating in the binding of the phospho-Ser65 Ubl domain to the RING0 domain to release its autoinhibition of the catalytic Cys431 within the RING2 domain. This release enables the transthiolation of the Cys431 by Ub from the charged E2 to allow substrate ubiquitylation (Gladkova et al. 2018, Sauvé et al. 2018). While early cell biology studies used human cell lines and Parkin overexpression, this model of Parkin activation by PINK1 has been validated in both primary mouse neurons and human- induced neurons (McWilliams et al. 2018; Ordureau et al. 2018, 2020). The model has also been demonstrated by showing that mutation of Parkin Ser65 to Ala or Asn inactivates Parkin activity in a mouse knock-in model in vivo and leads to early-onset PD in humans, respectively (McWilliams et al. 2018). The contribution of endogenous PINK1 to stress-evoked mitophagy has recently been demonstrated in human-induced neurons exposed to mitochondrial depolar- ization using the mitophagy reporter mito-Keima (Ordureau et al. 2020). This conclusion also follows from previous studies using PINK1 and Parkin knockout models that showed that, in response to mitochondrial stress in vivo (induced genetically in a mouse model of mitochondrial stress or acquired through exhaustive exercise regimes), PINK1 and Parkin are critical for Singh • Muqit 252 PM CCV Ser111 Nucleus Ub Ser72 Ser65 Ser65 TBK1 Damaged mitochondria Phosphatases? Rab8A Rab7 Rab7 Rab5 Rab13 TGN Golgi ER Rab8B Parkin Syntaxin-17 VAMP7 LY LE EE SNAP29 Kinases? Degradation PINK1 PINK1 Mitochondrial depolarization Mitochondrial depolarization Healthy mitochondria TBC1D15 TBC1D15 TBC1D17 TBC1D17 MDVs ? ? ? Autolysosomes Phagophore Autophagosomes Autophagy receptors Autophagy adapters OMM proteins Rab7A Rab7A PM CCV Ser111 Nucleus Ub Ser72 Ser65 Ser65 TBK1 Damaged mitochondria Phosphatases? Rab8A Rab7 Rab7 Rab5 Rab13 TGN Golgi ER Rab8B Parkin Syntaxin-17 VAMP7 LY LE EE SNAP29 Kinases? Degradation PINK1 PINK1 Mitochondrial depolarization Mitochondrial depolarization Healthy mitochondria TBC1D15 TBC1D15 TBC1D17 TBC1D17 MDVs ? ? ? Autolysosomes Phagophore Autophagosomes Autophagy receptors Autophagy adapters OMM proteins Rab7A Rab7A Figure 4 The PINK1-Parkin signaling pathway. Under healthy conditions, PINK1 is degraded in the cytosol after its cleavage following precursor import into mitochondria by the intramembrane protease PARL, which leads to its release into the cytoplasm. Upon mitochondrial depolarization, PINK1 accumulates on the outer mitochondrial membrane through an unknown mechanism and is activated and autophosphorylated (small red spheres). PINK1/Parkin Regulation of Mitophagy and Link to Rabs Damaged mitochondria also lead to the recruitment of several proteins, including Rab7, Rab7-GAPs, TBC1D15, and TBC1D17, as well as TBK1 kinase, which phosphorylates Rab7 at Ser72. In addition, this damage recruits autophagy receptors that bind ubiquitylated proteins and they in turn recruit autophagy machinery to degrade mitochondria through the selective autophagy known as mitophagy. In response to oxidative stress, mitochondria can also release MDVs that fuse with lysosomes through the recruitment of syntaxin-17 that forms a SNARE complex with SNAP29 and VAMP7. PINK1 activation also leads to the phosphorylation of several Rab GTPases such as Rab8A, Rab8B, and Rab13 at the Ser111 residue. The kinases or phosphatases involved in these phosphorylation events remain unknown. Abbreviations: CCV, clathrin-coated vesicle; EE, early endosome; ER, endoplasmic reticulum; GAP, GTPase-activating protein; LE, late endosome; LY, lysosome; MDV, mitochondria-derived vesicle; NSF, N-ethylmaleimide-sensitive factor; OMM, outer mitochondrial membrane; PARL, presenilins-associated rhomboidlike; PINK1, PTEN-induced kinase 1; PM, plasma membrane; Rab, Ras analog in brain; SNAP, soluble NSF-attachment protein; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptors; TBK1, TANK-binding kinase 1; TGN, trans-Golgi network; Ub, ubiquitin; VAMP, vesicle-associated membrane protein. www.annualreviews.org • Aberrant Vesicle Trafficking in Parkinson’s 253 maintaining dopaminergic neuronal survival and preventing the development of Parkinsonian features (Pickrell et al. 2015, Sliter et al. 2018). Vesicular trafficking machinery is required for multiple steps in autophagy including the delivery of membranes to the site of phagophore formation and autophagosome-lysosome fusion (Søreng et al. 2018). The initial evidence for a specific role of Rabs in mitophagy was the report that the Rab7 GTPase-activating protein (Rab7-GAP) TBC1D15 (Figure 4) regulates mito- autophagosome biogenesis, and that TBC1D15 knockout cells exhibit excessive LC3-positive autophagosome structures around damaged mitochondria (Yamano et al. 2014). A similar effect was seen with TBC1D17 (Figure 4) and was mitigated by knockdown of Rab7, suggesting that overactivation of Rab7 activity underlies the phenotype and highlighting the critical role of Rab7 in the regulation of mito-autophagosome formation (Yamano et al. 2014). Because Rab7 is found primarily on late endosomes and lysosomal membranes, the mechanism was initially not clear. However, Yamano and colleagues (2018) further demonstrated that Rab7 is recruited to damaged mitochondria together with the upstream regulator Rab5. The activation of Rab5 at mitochondria led to the recruitment of its effectors MON1/CCZ1, which in turn recruited GDP-bound Rab7 dependent on Parkin activation (Yamano et al. 2018). PINK1/Parkin Regulation of Mitophagy and Link to Rabs An alternative model for the role of Rab7 in mitophagy has been proposed through which Rab7 activity and localization to the mitochondria is regulated by the interaction between the retromer complex and TBC1D5 (retromer-associated Rab7-specific GAP) ( Jimenez-Orgaz et al. 2018). Further, mitophagy was suppressed in cells in which VPS29, VPS35, and TBC1D5 were knocked out, and Rab7 localization was shifted to late endosomal-lysosomal membranes where it was hyperactive ( Jimenez-Orgaz et al. 2018). Several Ub-binding autophagy receptors have been identified, including Optineurin (OPTN1), Tax-1 binding protein (TAX1BP1), p62/SQSTM1, and coiled-coil domain-containing protein 2 (CALCOCO2/NDP52), that are recruited to Ub chains assembled at the OMM and are required for mitophagy (Heo et al. 2015, Lazarou et al. 2015, Richter et al. 2016). Akin to other forms of selective autophagy such as xenophagy, PINK1/Parkin–induced recruitment of OPTN1 is associated with the activation of TANK-binding kinase 1 (TBK1), which phosphory- lates OPTN1 to increase its affinity for Ub chain binding (Heo et al. 2015, Richter et al. 2016). This promotes further OPTN1 recruitment to damaged mitochondria, in turn increasing fur- ther TBK1 activation (Figure 4) and creating a feed-forward loop that is critical for mitophagy (Heo et al. 2015, Richter et al. 2016). TBK1 has also been demonstrated to phosphorylate Rab7 at Ser72 (Figure 4) that lies within the switch II domain and is the equivalent site on other Rabs targeted by the LRRK2 kinase (Heo et al. 2018). The phosphorylation of Rab7 at Ser72 is critical for mitophagy because cells expressing the nonphosphorylatable Rab7 [Ser72Ala] mutant could no longer undergo mitophagy and were associated with a defect in the recruitment of autophago- some components to sites of damaged mitochondria (Heo et al. 2018). The contribution of Rab Ser72 phosphorylation to mitophagy has been established in HeLa cells overexpressing Parkin, and probing the contribution of Rab 7 phosphorylation in cells more relevant to PD pathogenesis will be interesting. Because TBK1 is also implicated in regulating the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway in innate immune signaling, it will also be intriguing to explore the role of Rab7 in the STING-mediated neuroinflammation that has been found in PINK1/Parkin mouse models in which STING inflammation is increased by the accumulation of mtDNA mutations in the cytosol following exhaustive exercise (Sliter et al. 2018). PINK1/Parkin Regulation of Mitophagy and Link to Rabs PINK1/Parkin and Mitochondrial-Derived Vesicles In Drosophila, while PINK1/Parkin–dependent mitophagy is evident, the half-lives of several mi- tochondrial proteins dependent on PINK1/Parkin were also found to be independent of ATG7, Singh • Muqit 254 suggesting that there are additional autophagy-independent mechanisms of mitochondrial pro- tein turnover (Vincow et al. 2013). This has been further extended in cultured mammalian cell lines in which mitochondria-derived vesicles (MDVs) have been detected that traffic specific mi- tochondrial protein cargoes to lysosomes for degradation in a strict PINK1/Parkin–dependent manner (McLelland et al. 2014, Soubannier et al. 2012). MDVs occur under basal conditions but are upregulated in response to oxidative stress, which promotes the generation of MDVs con- taining oxidized cargoes (McLelland et al. 2014). The composition and identity of MDVs are dependent on the source of oxidative stress, with intramitochondrial stressors such as antimycin A (complex III inhibitor) inducing double-membrane MDVs positive for inner membrane and ma- trix components (e.g., pyruvate dehydrogenase [PDH]) but negative for OMM proteins, whereas more general oxidative stress outside the mitochondria led to the formation of single-membrane MDVs that were positive for OMM components (e.g., Tom20 or VDAC1) but negative for PDH1 (McLelland et al. 2014, 2016). Interestingly, endogenous PINK1 and Parkin have not been de- tected in MDVs, although this may be due to limitations on detection with current antibody tools. While disease-associated Parkin mutants (e.g., C431F) affect the formation of MDVs under ox- idative stress, the Ub-dependent mechanisms of MDV formation remain to be fully elucidated— whether there are differential substrates, Ub linkages, and Ub receptors to those of mitophagy are unknown (McLelland et al. 2014). Furthermore, it remains to be established whether phospho-Ub is required for MDV generation. However, the regulation of PINK1/Parkin–dependent MDVs appears to be distinct from mitophagy, as the former can occur independently of Drp1, which promotes mitochondrial fission (Soubannier et al. 2012). Furthermore, the targeting of MDVs to the late endosome/lysosome compartment is dependent on the SNARE syntaxin-17 that forms a ternary SNARE complex with SNAP29 and VAMP7 to mediate MDV-endolysosome fusion (Figure 4), and this SNARE machinery is not required for mitophagy (McLelland et al. 2016). www.annualreviews.org • Aberrant Vesicle Trafficking in Parkinson’s PINK1/Parkin Regulation of Mitophagy and Link to Rabs In striking contrast to cultured cell lines, PINK1 and Parkin have been demonstrated to inhibit MDV formation in immune cells, which is required for mitochondrial antigen presentation (MitAP) crit- ical for the expression of MHC I peptides at the cell surface for T cell recognition and activation (Matheoud et al. 2016). The generation of MDVs and MitAP is dependent on the SNX9 required for clathrin-mediated endocytosis and the Rab9 required for late endosome fusion, and SNX9 levels have been observed to be regulated by Parkin (Matheoud et al. 2016). The relevance of this pathway to PD has been strikingly demonstrated in PINK1-knockout mice exposed to intestinal infection by gram-negative bacteria (Matheoud et al. 2019). This led to a MitAP response and the induction of cytotoxic mitochondrial CD8+ T cells in the periphery, which in turn triggered dopaminergic pathological deficits in the brain associated with L-Dopa-responsive motor deficits (Matheoud et al. 2019). This has opened a new area of biological research in which PINK1 and Parkin may have critical roles in adaptive immunity to repress mitochondrial antigens that may k th CNS i fl ti t ib ti t PD th i suggesting that there are additional autophagy-independent mechanisms of mitochondrial pro- tein turnover (Vincow et al. 2013). This has been further extended in cultured mammalian cell lines in which mitochondria-derived vesicles (MDVs) have been detected that traffic specific mi- tochondrial protein cargoes to lysosomes for degradation in a strict PINK1/Parkin–dependent manner (McLelland et al. 2014, Soubannier et al. 2012). MDVs occur under basal conditions but are upregulated in response to oxidative stress, which promotes the generation of MDVs con- taining oxidized cargoes (McLelland et al. 2014). The composition and identity of MDVs are dependent on the source of oxidative stress, with intramitochondrial stressors such as antimycin A (complex III inhibitor) inducing double-membrane MDVs positive for inner membrane and ma- trix components (e.g., pyruvate dehydrogenase [PDH]) but negative for OMM proteins, whereas more general oxidative stress outside the mitochondria led to the formation of single-membrane MDVs that were positive for OMM components (e.g., Tom20 or VDAC1) but negative for PDH1 (McLelland et al. 2014, 2016). Interestingly, endogenous PINK1 and Parkin have not been de- tected in MDVs, although this may be due to limitations on detection with current antibody tools. PINK1/Parkin Regulation of Mitophagy and Link to Rabs While disease-associated Parkin mutants (e.g., C431F) affect the formation of MDVs under ox- idative stress, the Ub-dependent mechanisms of MDV formation remain to be fully elucidated— whether there are differential substrates, Ub linkages, and Ub receptors to those of mitophagy are unknown (McLelland et al. 2014). Furthermore, it remains to be established whether phospho-Ub is required for MDV generation. However, the regulation of PINK1/Parkin–dependent MDVs appears to be distinct from mitophagy, as the former can occur independently of Drp1, which promotes mitochondrial fission (Soubannier et al. 2012). Furthermore, the targeting of MDVs to the late endosome/lysosome compartment is dependent on the SNARE syntaxin-17 that forms a ternary SNARE complex with SNAP29 and VAMP7 to mediate MDV-endolysosome fusion (Figure 4), and this SNARE machinery is not required for mitophagy (McLelland et al. 2016). In striking contrast to cultured cell lines, PINK1 and Parkin have been demonstrated to inhibit MDV formation in immune cells, which is required for mitochondrial antigen presentation (MitAP) crit- ical for the expression of MHC I peptides at the cell surface for T cell recognition and activation (Matheoud et al. 2016). The generation of MDVs and MitAP is dependent on the SNX9 required for clathrin-mediated endocytosis and the Rab9 required for late endosome fusion, and SNX9 levels have been observed to be regulated by Parkin (Matheoud et al. 2016). The relevance of this pathway to PD has been strikingly demonstrated in PINK1-knockout mice exposed to intestinal infection by gram-negative bacteria (Matheoud et al. 2019). This led to a MitAP response and the induction of cytotoxic mitochondrial CD8+ T cells in the periphery, which in turn triggered dopaminergic pathological deficits in the brain associated with L-Dopa-responsive motor deficits (Matheoud et al. 2019). This has opened a new area of biological research in which PINK1 and Parkin may have critical roles in adaptive immunity to repress mitochondrial antigens that may provoke the CNS neuroinflammation contributing to PD pathogenesis. PINK1 Regulation of the Rab SF3 Motif A subset of Rab GTPases, including Rab8A, Rab8B, Rab13, and Rab1A/1B, have been reported to be phosphorylated at a highly conserved Ser111 residue upon PINK1 activation (Lai et al. 2015) (Figure 4). The regulation of Rabs by PINK1 appears to be indirect, and the identity of the upstream Rab kinase remains unknown. The Ser111 residue lies within a C-terminal α3/β5 loop of Rab8A known as the SF3 motif and lies adjacent to the effector-binding switch II domain. Structural heterogeneity within the SF3 motif across diverse Rabs suggests that this region also determines effector binding. Previous studies (Ostermeier & Brunger 1999) have reported a role for the SF3 motif in the selective recruitment of effectors via direct effector-specific contact sites, www.annualreviews.org • Aberrant Vesicle Trafficking in Parkinson’s 255 e.g., the recruitment of Rabphilin by Rab3A via SF3 motif–binding sites. Clues to how phospho- rylation of the SF3 motif alters such effector interactions have recently been obtained by exploit- ing gene codon expansion technologies to purify preparative amounts of Ser111-phosphorylated Rab8A (Vieweg et al. 2020). Phosphorylation of Rab8A led to impaired interactions with its GEFs, Rabin8, and GAPs. Analysis of the phospho-Ser111 Rab8A crystal structure revealed an in- tramolecular H bond created by the presence of the phosphate at Ser111 with an Arg79 side chain (Vieweg et al. 2020). Molecular dynamics (MD) simulation studies of the Rab8A:Rabin8 complex structure demonstrate a salt-bridge interaction between residue Asp187 of Rabin8 with Arg79; this structure would be predicted to be weakened or disrupted by the addition of a negative charge by pSer111 that lies opposite the negative surface patch of Rabin8 containing Asp187 (Pourjafar- Dehkordi et al. 2019). The specific effectors for phospho-Ser111 Rab8A remain unknown, a gap that has hampered progress in understanding the downstream biology of phospho-Ser111 Rab8A; however, phospho-Ser111 Rab8A has been shown to impair the ability of the LRRK2 kinase to phosphorylate its Thr72 residue within the switch II domain (Vieweg et al. 2020) (Figure 3). Because hyperactive R1441G LRRK2-phosphorylated Rab Thr72 recruits RILPL1 and RILPL2 leading to inhibition of primary cilia in the brains of mice (Dhekne et al. 2018), understanding the contribution of PINK1-regulated Rab phosphorylation to this process in vivo would be of inter- est. CONCLUDING REMARKS AND CLINICAL APPLICATION The identification of monogenic forms of PD has led to the elaboration of vesicular trafficking pathways whose disruption are major causal mechanisms in PD. This knowledge has been crucial for the development of therapeutic strategies against PD. Currently, clinical trials are advancing for the evaluation of human monoclonal antibodies that preferentially bind oligomeric α-syn and prevent cell-to-cell transmission and progression, including the PASDENA Phase II trial being undertaken by Roche. Trials for LRRK2 inhibitors are also underway in several companies in- cluding Denali Therapeutics, whose LRRK2 inhibitor DNL201 was found to be safe for humans in Phase I studies. In addition, Biogen is currently evaluating an antisense oligonucleotide against LRRK2 in Phase I studies. A major question is whether these strategies will be beneficial for pa- tients beyond those monogenic individuals harboring causal mutations, and this will be dependent on uncovering common overlapping pathways between genes. An exemplar of this has been the discovery that VPS35 [D620N] mutations lead to increased LRRK2-mediated Rab phosphoryla- tion, thereby placing VPS35 upstream of LRRK2 and suggesting that VPS35 patients may also benefit from LRRK2 inhibitors and should be assessed in ongoing clinical trials (Mir et al. 2018). Our increased understanding of Rab biology and vesicular trafficking mechanisms between dis- tinct PD gene pathways now provides a tractable framework to uncover further knowledge that will expand our ability to stratify PD patients for future disease-modifying therapies. PINK1 Regulation of the Rab SF3 Motif The contribution of the Ser111 phosphorylation of Rabs to mitophagy and MDV generation is also unknown, although none of these Rab components were found to be essential in a recent CRISPR genome-wide screen for mitophagy regulators (Heo et al. 2019). Annu. Rev. Cell Dev. Biol. 2020.36:237-264. Downloaded from www.annualreviews.org Access provided by 86.174.33.195 on 12/11/20. See copyright for approved use. ACKNOWLEDGMENTS We apologize to our colleagues whose work we were unable to discuss or cite due to space limitations. DISCLOSURE STATEMENT M.M.K.M. is a member of the Scientific Advisory Board of Mitokinin Inc. and serves as a member of the Wellcome Trust Clinical Interview Committee. M.M.K.M. is funded by a Wellcome Trust Senior Research Fellowship in Clinical Science (210753/Z/18/Z), the Michael J. Fox Foundation for Parkinson’s Research, the Medical Research Council, the Rosetrees Trust, Astex Pharmaceuti- cals Ltd.,and an EMBO (European Molecular Biology Organization) Young Investigator Program Award. Singh • Muqit 256 Annu. Rev. Cell Dev. Biol. 2020.36:237-264. Downloaded from www.annualreviews.org Access provided by 86.174.33.195 on 12/11/20. See copyright for approved use. Anwar S, Peters O, Millership S, Ninkina N, Doig N, et al. 2011. Functional alterations to the nigrostriatal system in mice lacking all three members of the synuclein family. J. Neurosci. 31:7264–74 Bandres-Ciga S, Diez-Fairen M, Kim JJ, Singleton AB. 2020. Genetics of Parkinson’s disease: an introspection of its journey towards precision medicine. Neurobiol. Dis. 137:104782 Beilina A, Bonet-Ponce L, Kumaran R, Kordich JJ, Ishida M, et al. 2020. The Parkinson’s disease protein LRRK2 interacts with the GARP complex to promote retrograde transport to the trans-Golgi network. Cell Rep. 31:107614 Beilina A, Rudenko IN, Kaganovich A, Civiero L, Chau H, et al. 2014. Unbiased screen for interactors of leucine-rich repeat kinase 2 supports a common pathway for sporadic and familial Parkinson disease. PNAS 111:2626–31 Berndsen K, Lis P, Yeshaw WM, Wawro PS, Nirujogi RS, et al. 2019. PPM1H phosphatase counteracts LRRK2 signaling by selectively dephosphorylating Rab proteins. eLife 8:e50416 Bliederhaeuser C, Grozdanov V, Speidel A, Zondler L, Ruf WP, et al. 2016. Age-dependent defects of alpha- synuclein oligomer uptake in microglia and monocytes. Acta Neuropathol. 131:379–91 Bonet-Ponce L, Cookson MR. 2019. The role of Rab GTPases in the pathobiology of Parkinson’ disease. Curr. Opin. Cell Biol. 59:73–80 Borghi R, Marchese R, Negro A, Marinelli L, Forloni G, et al. 2000. Full length α-synuclein is present in cerebrospinal fluid from Parkinson’s disease and normal subjects. Neurosci. Lett. 287:65–67 Breda C, Nugent ML, Estranero JG, Kyriacou CP, Outeiro TF, et al. 2015. Rab11 modulates α-synuclein- mediated defects in synaptic transmission and behaviour. Hum. Mol. Genet. 24:1077–91 Burré J,Sharma M,Tsetsenis T,Buchman V,Etherton MR,Südhof TC.2010.α-Synuclein promotes SNARE- complex assembly in vivo and in vitro. Science 329:1663–67 Cao M, Wu Y, Ashrafi G, McCartney AJ, Wheeler H, et al. 2017. Parkinson Sac domain mutation in synap- tojanin 1 impairs clathrin uncoating at synapses and triggers dystrophic changes in dopaminergic axons. Neuron 93:882–96 Chai YJ, Sierecki E, Tomatis VM, Gormal RS, Giles N, et al. 2016. Munc18-1 is a molecular chaperone for α-synuclein, controlling its self-replicating aggregation. J. Cell Biol. 214:705–18 Chandra S, Gallardo G, Fernández-Chacón R, Schlüter OM, Südhof TC. 2005. α-Synuclein cooperates with CSPα in preventing neurodegeneration. Cell 123:383–96 Chaugule VK, Burchell L, Barber KR, Sidhu A, Leslie SJ, et al. 2011. Autoregulation of Parkin activity through its ubiquitin-like domain. EMBO J. 30:2853–67 Chen YA, Scheller RH. 2001. SNARE-mediated membrane fusion. Nat. Rev. Mol. Cell Biol. LITERATURE CITED Ahmadi Rastegar D, Dzamko N. 2020. Leucine rich repeat kinase 2 and innate immunity. Front. Neurosci. 14:193 Alegre-Abarrategui J, Christian H, Lufino MMP, Mutihac R, Venda LL, et al. 2009. LRRK2 regulates au- tophagic activity and localizes to specific membrane microdomains in a novel human genomic reporter cellular model. Hum. Mol. Genet. 18:4022–34 Alessi DR, Sammler E. 2018. LRRK2 kinase in Parkinson’s disease. Science 360:36–37 Alvarez-Erviti L, Seow Y, Schapira AH, Gardiner C, Sargent IL, et al. 2011. Lysosomal dysfunction increases di d l h l i l d i i N bi l Di 42 360 67 Alvarez-Erviti L, Seow Y, Schapira AH, Gardiner C, Sargent IL, et al. 2011. Lysosomal dysfunction increases exosome-mediated alpha-synuclein release and transmission. Neurobiol. Dis. 42:360–67 Annu. Rev. Cell Dev. Biol. 2020.36:237-264. Downloaded from www.annualreviews.org Access provided by 86.174.33.195 on 12/11/20. See copyright for approved use. Dis. 16:92–97 Dalfó E, Ferrer I. 2005. α-Synuclein binding to rab3a in multiple system a Dalfó E, Ferrer I. 2005. α-Synuclein binding to rab3a in multiple system atrophy. Neurosci. Lett. 380:170–75 Dalfó E, Gómez-Isla T, Rosa JL, Nieto Bodelón M, Cuadrado Tejedor M, et al. 2004b. Abnormal α-synuclein interactions with Rab proteins in α-synuclein A30P transgenic mice. J. Neuropathol. Exp. Neurol. 63:302– 13 Darios F, Ruipérez V, López I, Villanueva J, Gutierrez LM, Davletov B. 2010. α-Synuclein sequesters arachi- donic acid to modulate SNARE-mediated exocytosis. EMBO Rep. 11:528–33 Deniston CK, Salogiannis J, Mathea S, Snead DM, Lahiri I, et al. 2020. Parkinson’s disease-linked LRRK2 structure and model for microtubule interaction. bioRxiv 895367. https://doi.org/10.1101/2020.01. 06.895367 DeWitt DC, Rhoades E. 2013. α-Synuclein can inhibit SNARE-mediated vesicle fusion through direct inter- actions with lipid bilayers. Biochemistry 52:2385–87 Dhekne HS, Yanatori I, Gomez RC, Tonelli F, Diez F, et al. 2018. A pathway for Parkinson’s Disease LRRK2 kinase to block primary cilia and Sonic hedgehog signaling in the brain. eLife 7:e40202 Dinter E, Saridaki T, Nippold M, Plum S, Diederichs L, et al. 2016. Rab7 induces clearance of aggregates. J. Neurochem. 138:758–74 Dodson MW, Leung LK, Lone M, Lizzio MA, Guo M. 2014. Novel ethyl methanesulfonate (EMS)-induced null alleles of the Drosophila homolog of LRRK2 reveal a crucial role in endolysosomal functions and autophagy in vivo. Dis. Model. Mech. 7:1351–63 Dodson MW, Zhang T, Jiang C, Chen S, Guo M. 2012. Roles of the Drosophila LRRK2 homolog in Rab7- dependent lysosomal positioning. Hum. Mol. Genet. 21:1350–63 V, Lesage S. 2014. Synaptojanin 1 mutation in Parkinson’s disease brings further insight into th uropathological mechanisms Biomed Res Int 2014:289728 Drouet V, Lesage S. 2014. Synaptojanin 1 mutation in Parkinson’s disease brings further insight into the neuropathological mechanisms. Biomed. Res. Int. 2014:289728 Edvardson S, Cinnamon Y, Ta-Shma A, Shaag A, Yim YI, et al. 2012. A deleterious mutation in DNAJC6 en- coding the neuronal-specific clathrin-uncoating co-chaperone auxilin, is associated with juvenile parkin- sonism. PLOS ONE 7:e36458 El-Agnaf OMA, Salem SA, Paleologou KE, Cooper LJ, Fullwood NJ, et al. 2003. α-Synuclein implicated in Parkinson’s disease is present in extracellular biological fluids, including human plasma. FASEB J. 17:1945–47 El-Agnaf OMA, Salem SA, Paleologou KE, Curran MD, Gibson MJ, et al. 2006. Detection of oligomeric forms of α-synuclein protein in human plasma as a potential biomarker for Parkinson’s disease. FASEB J. Annu. Rev. Cell Dev. Biol. 2020.36:237-264. Downloaded from www.annualreviews.org Access provided by 86.174.33.195 on 12/11/20. See copyright for approved use. 2:98–106 Cheng F, Li X, Li Y, Wang C, Wang T, et al. 2011. α-Synuclein promotes clathrin-mediated NMDA receptor endocytosis and attenuates NMDA-induced dopaminergic cell death. J. Neurochem. 119:815–25 Chia R, Haddock S, Beilina A, Rudenko IN, Mamais A, et al. 2014. Phosphorylation of LRRK2 by casein kinase 1α regulates trans-Golgi clustering via differential interaction with ARHGEF7. Nat. Commun. 5:5827 www.annualreviews.org • Aberrant Vesicle Trafficking in Parkinson’s 257 Chiu CC, Yeh TH, Lai SC, Weng YH, Huang YC, et al. 2016. Increased Rab35 expression is a potential biomarker and implicated in the pathogenesis of Parkinson’s disease. Oncotarget 7:54215–27 Choi BK,Choi MG,Kim JY,Yang Y,Lai Y,et al.2013.Large α-synuclein oligomers inhibit neuronal SNARE- mediated vesicle docking. PNAS 110:4087–92 g Choi MG, Kim MJ, Kim DG, Yu R, Jang YN, Oh WJ. 2018. Sequestration of synaptic proteins by alpha- synuclein aggregates leading to neurotoxicity is inhibited by small peptide. PLOS ONE 13:e0195339 Chua CE, Tang BL. 2011. Rabs, SNAREs and α-synuclein–membrane trafficking defects in synucleinopathies. B i R R 67 268 81 Choi MG, Kim MJ, Kim DG, Yu R, Jang YN, Oh WJ. 2018. Sequestration of synaptic proteins by alpha- synuclein aggregates leading to neurotoxicity is inhibited by small peptide. PLOS ONE 13:e0195339 h b d l bi k d l h J J g J q y p p y p synuclein aggregates leading to neurotoxicity is inhibited by small peptide. PLOS ONE 13:e0195339 Chua CE, Tang BL. 2011. Rabs, SNAREs and α-synuclein–membrane trafficking defects in synucleinopathies. Brain Res. Rev. 67:268–81 Chua CE, Tang BL. 2011. Rabs, SNAREs and α-synuclein–membrane trafficking defects in synucleinopathies. Brain Res. Rev. 67:268–81 Connor-Robson N, Booth H, Martin JG, Gao B, Li K, et al. 2019. An integrated transcriptomics and pro- teomics analysis reveals functional endocytic dysregulation caused by mutations in LRRK2. Neurobiol. Dis. 127:512–26 Cooper AA, Gitler AD, Cashikar A, Haynes CM, Hill KJ, et al. 2006. α-Synuclein blocks ER-Golgi traffic and Rab1 rescues neuron loss in Parkinson’s models. Science 313:324–28 Cooper JM, Wiklander PB, Nordin JZ, Al-Shawi R, Wood MJ, et al. 2014. Systemic exosomal siRNA delivery reduced alpha-synuclein aggregates in brains of transgenic mice. Mov. Disord. 29:1476–85 Dalfó E, Barrachina M, Rosa JL, Ambrosio S, Ferrer I. 2004a. Abnormal α-synuclein interactions with rab3a and rabphilin in diffuse Lewy body disease. Neurobiol. Dis. 16:92–97 Dalfó E, Barrachina M, Rosa JL, Ambrosio S, Ferrer I. 2004a. Abnormal α and rabphilin in diffuse Lewy body disease. Neurobiol. Annu. Rev. Cell Dev. Biol. 2020.36:237-264. Downloaded from www.annualreviews.org Access provided by 86.174.33.195 on 12/11/20. See copyright for approved use. Giannandrea M, Bianchi V, Mignogna ML, Sirri A, Carrabino S, et al. 2010. Mutations in the small GTPase gene RAB39B are responsible for X-linked mental retardation associated with autism, epilepsy, and macrocephaly. Am. J. Hum. Genet. 86:185–95 Gitler AD, Bevis BJ, Shorter J, Strathearn KE, Hamamichi S, et al. 2008. The Parkinson’s disease protein α-synuclein disrupts cellular Rab homeostasis. PNAS 105:145–50 Gladkova C, Maslen SL, Skehel JM, Komander D. 2018. Mechanism of parkin activation by PINK1. Nature 559:410–14 Gomez RC, Wawro P, Lis P, Alessi DR, Pfeffer SR. 2019. Membrane association but not identity is required for LRRK2 activation and phosphorylation of Rab GTPases. J. Cell Biol. 218:4157–70 Gómez-Suaga P, Rivero-Ríos P, Fdez E, Blanca Ramírez M, Ferrer I, et al. 2014. LRRK2 delays degradative receptor trafficking by impeding late endosomal budding through decreasing Rab7 activity. Hum. Mol. Genet. 23:6779–96 Gonçalves SA, Macedo D, Raquel H, Simões PD, Giorgini F, et al. 2016. shRNA-based screen identifies endocytic recycling pathway components that act as genetic modifiers of alpha-synuclein aggregation, secretion and toxicity. PLOS Genet. 12:e1005995 Grey M, Dunning CJ, Gaspar R, Grey C, Brundin P, et al. 2015. Acceleration of α-synuclein aggregation by exosomes. J. Biol. Chem. 290:2969–82 Griesser E, Wyatt H, Ten Have S, Stierstorfer B, Lenter M, Lamond AI. 2020. Quantitative profiling of the human substantia nigra proteome from laser-capture microdissected FFPE tissue. Mol. Cell Proteom. 19:839–51 Guo M, Wang J, Zhao Y, Feng Y, Han S, et al. 2020. Microglial exosomes facilitate α-synuclein transmission in Parkinson’s disease. Brain 143:1476–97 Gupta A, Pulliam L. 2014. Exosomes as mediators of neuroinflammation. J. Neuroinflammation 11:68 Han C, Xiong N, Guo X, Huang J, Ma K, et al. 2019. Exosomes from patients with Parkinson’s disease are pathological in mice. J. Mol. Med. 97:1329–44 Hardies K, Cai Y, Jardel C, Jansen AC, Cao M, et al. 2016. Loss of SYNJ1 dual phosphatase activity leads to early onset refractory seizures and progressive neurological decline. Brain 139:2420–30 Harper JW, Ordureau A, Heo JM. 2018. Building and decoding ubiquitin chains for mitophagy. Nat. Rev. Mol. Cell Biol. 19:93–108 Heo JM, Harper NJ, Paulo JA, Li M, Xu Q, et al. 2019. Integrated proteogenetic analysis reveals the landscape of a mitochondrial-autophagosome synapse during PARK2-dependent mitophagy. Sci. Adv. 5:eaay4624 Heo JM, Ordureau A, Paulo JA, Rinehart J, Harper JW. 2015. The PINK1-PARKIN mitochondrial ubiq- uitylation pathway drives a program of OPTN/NDP52 recruitment and TBK1 activation to promote mitophagy. Annu. Rev. Cell Dev. Biol. 2020.36:237-264. Downloaded from www.annualreviews.org Access provided by 86.174.33.195 on 12/11/20. See copyright for approved use. 20:419–25 Emmanouilidou E, Melachroinou K, Roumeliotis T, Garbis SD, Ntzouni M, et al. 2010. Cell-produced α-synuclein is secreted in a calcium-dependent manner by exosomes and impacts neuronal survival. J. Neurosci. 30:6838–51 Fang F, Yang W, Florio JB, Rockenstein E, Spencer B, et al. 2017. Synuclein impairs trafficking and signaling of BDNF in a mouse model of Parkinson’s disease. Sci. Rep. 7:3868 Singh • Muqit 258 Fasano D, Parisi S, Pierantoni GM, De Rosa A, Picillo M, et al. 2018. Alteration of endosomal trafficking is associated with early-onset parkinsonism caused by SYNJ1 mutations. Cell Death Dis. 9:385 Feng M, Hu X, Li N, Hu F, Chang F, et al. 2018. Distinctive roles of Rac1 and Rab29 in LRRK2 mediated membrane trafficking and neurite outgrowth. J. Biomed. Res. 32:145–56 Friedman LG, Lachenmayer ML, Wang J, He L, Poulose SM, et al. 2012. Disrupted autophagy leads to dopaminergic axon and dendrite degeneration and promotes presynaptic accumulation of α-synuclein and LRRK2 in the brain. J. Neurosci. 32:7585–93 Fujimoto T, Kuwahara T, Eguchi T, Sakurai M, Komori T, Iwatsubo T. 2018. Parkinson’s disease-associated mutant LRRK2 phosphorylates Rab7L1 and modifies trans-Golgi morphology. Biochem. Biophys. Res. Commun. 495:1708–15 Garcia-Reitböck P, Anichtchik O, Bellucci A, Iovino M, Ballini C, et al. 2010. SNARE protein redistribution and synaptic failure in a transgenic mouse model of Parkinson’s disease. Brain 133:2032–44 Giaime E, Tong Y, Wagner LK, Yuan Y, Huang G, Shen J. 2017. Age-dependent dopaminergic neurodegen- eration and impairment of the autophagy-lysosomal pathway in LRRK-deficient mice. Neuron 96:796– 807.e6 Annu. Rev. Cell Dev. Biol. 2020.36:237-264. Downloaded from www.annualreviews.org Access provided by 86.174.33.195 on 12/11/20. See copyright for approved use. Mol. Cell 60:7–20 Heo JM, Ordureau A, Swarup S, Paulo JA, Shen K, et al. 2018. RAB7A phosphorylation by TBK1 promotes mitophagy via the PINK-PARKIN pathway. Sci. Adv. 4:eaav0443 Herbst S, Gutierrez MG. 2019. LRRK2 in infection: friend or foe? ACS Infect. Dis. 5:809–15 www.annualreviews.org • Aberrant Vesicle Trafficking in Parkinson’s 259 Horgan CP, McCaffrey MW. 2011. Rab GTPases and microtubule motors. Biochem. Soc. Trans. 39 Houser MC, Tansey MG. 2017. The gut-brain axis: is intestinal inflammation a silent driver of Parkinson’s disease pathogenesis? NPJ Park. Dis. 3:3 Huang C-C, Chiu T-Y, Lee T-Y, Hsieh H-J, Lin C-C, Kao L-S. 2018. Soluble α-synuclein facilitates prim- ing and fusion by releasing Ca2+ from the thapsigargin-sensitive Ca2+ pool in PC12 cells. J. Cell Sci. 131:jcs213017 Inoshita T, Arano T, Hosaka Y, Meng H, Umezaki Y, et al. 2017. Vps35 in cooperation with LRRK2 regulates synaptic vesicle endocytosis through the endosomal pathway in Drosophila. Hum. Mol. Genet. 26:2933–48 h h ll f b f Jahn R, Scheller RH. 2006. SNAREs—engines for membrane fusion. Nat. Rev. Mol. Cell Biol. 7:63 Jensen PH, Li JY, Dahlström A, Dotti CG. 1999. Axonal transport of synucleins is mediated by all rate com- ponents. Eur. J. Neurosci. 11:3369–76 Jeong GR, Jang E-H, Bae JR, Jun S, Kang HC, et al. 2018. Dysregulated phosphorylation of Rab GTPases by LRRK2 induces neurodegeneration. Mol. Neurodegener. 13:8 Jimenez-Orgaz A, Kvainickas A, Nägele H, Denner J, Eimer S, et al. 2018. Control of RAB7 activity and localization through the retromer-TBC1D5 complex enables RAB7-dependent mitophagy. EMBO J. 37:235–54 Kalogeropulou AF,Zhao J,Bolliger MF,Memou A,Narasimha S,et al.2018.P62/SQSTM1 is a novel leucine- rich repeat kinase 2 (LRRK2) substrate that enhances neuronal toxicity. Biochem. J. 475:1271–93 Kane LA, Lazarou M, Fogel AI, Li Y, Yamano K, et al. 2014. PINK1 phosphorylates ubiquitin to activate Parkin E3 ubiquitin ligase activity. J. Cell Biol. 205:143–53 Kazlauskaite A, Kondapalli C, Gourlay R, Campbell DG, Ritorto MS, et al. 2014. Parkin is activated by PINK1-dependent phosphorylation of ubiquitin at Ser65. Biochem. J. 460:127–39 Kett LR, Boassa D, Ho CC, Rideout HJ, Hu J, et al. 2012. LRRK2 Parkinson disease mutations enhance its microtubule association. Hum. Mol. Genet. 21:890–9 Kitada T, Asakawa S, Hattori N, Matsumine H, Yamamura Y, et al. 1998. Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism. Nature 392:605–8 Komatsu M, Wang QJ, Holstein GR, Friedrich VL Jr., Iwata J, et al. 2007. Annu. Rev. Cell Dev. Biol. 2020.36:237-264. Downloaded from www.annualreviews.org Access provided by 86.174.33.195 on 12/11/20. See copyright for approved use. Essential role for autophagy pro- tein Atg7 in the maintenance of axonal homeostasis and the prevention of axonal degeneration. PNAS 104:14489–94 Kondapalli C, Kazlauskaite A, Zhang N, Woodroof HI, Campbell DG, et al. 2012. PINK1 is activated by mitochondrial membrane potential depolarization and stimulates Parkin E3 ligase activity by phospho- rylating Serine 65. Open Biol. 2:120080 Koyano F, Okatsu K, Kosako H, Tamura Y, Go E, et al. 2014. Ubiquitin is phosphorylated by PINK1 to activate parkin. Nature 510:162–66 Kumar N, Leonzino M, Hancock-Cerutti W, Horenkamp FA, Li P, et al. 2018. VPS13A and VPS13C are lipid transport proteins differentially localized at ER contact sites. J. Cell Biol. 217:3625–39 Kunath T, Natalwala A, Chan C, Chen Y, Stecher B, et al. 2019. Are PARKIN patients ideal candidates for dopaminergic cell replacement therapies? Eur. J. Neurosci. 49:453–62 Kuwahara T, Inoue K, D’Agati VD, Fujimoto T, Eguchi T, et al. 2016. LRRK2 and RAB7L1 coordinately regulate axonal morphology and lysosome integrity in diverse cellular contexts. Sci. Rep. 6:29945 Lai YC, Kondapalli C, Lehneck R, Procter JB, Dill BD, et al. 2015. Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1. EMBO J. 34:2840–61 Lang AE, Lozano AM. 1998. Parkinson’s disease. First of two parts. N. Engl. J. Med. 339:1044–53 Lara Ordónez AJ, Fernández B, Fdez E, Romo-Lozano M, Madero-Pérez J, et al. 2019. RAB8, RAB10 and RILPL1 contribute to both LRRK2 kinase–mediated centrosomal cohesion and ciliogenesis deficits. Hum. Mol. Genet. 28:3552–68 Lazarou M, Sliter DA, Kane LA, Sarraf SA, Wang C, et al. 2015. The ubiquitin kinase PINK1 recruits au- tophagy receptors to induce mitophagy. Nature 524:309–14 Lee H, Flynn R, Sharma I, Haberman E, Carling PJ, et al. 2020. LRRK2 is recruited to phagosomes and co- recruits RAB8 and RAB10 in human pluripotent stem cell-derived macrophages.Stem Cell Rep.14:940–55 Lee H, Flynn R, Sharma I, Haberman E, Carling PJ, et al. 2020. LRRK2 is recruited to phagosomes and co- recruits RAB8 and RAB10 in human pluripotent stem cell-derived macrophages.Stem Cell Rep.14:940–55 Lee HJ, Patel S, Lee SJ. 2005. Intravesicular localization and exocytosis of α-synuclein and its aggregates. y g p g recruits RAB8 and RAB10 in human pluripotent stem cell-derived macrophages.Stem Cell Rep.14:940–55 Lee HJ, Patel S, Lee SJ. 2005. Intravesicular localization and exocytosis of α-synuclein and its aggregates. J. Neurosci. 25:6016–24 Lee HJ, Patel S, Lee SJ. 2005. Intravesicular localization and exocytosis of α-synuclein and its aggregates. J. Neurosci. Annu. Rev. Cell Dev. Biol. 2020.36:237-264. Downloaded from www.annualreviews.org Access provided by 86.174.33.195 on 12/11/20. See copyright for approved use. MacLeod DA, Rhinn H, Kuwahara T, Zolin A, Di Paolo G, et al. 2013. RAB7L1 interacts with LRRK2 to modify intraneuronal protein sorting and Parkinson’s disease risk. Neuron 77:425–39 Madero-Pérez J, Fdez E, Fernández B, Lara Ordónez AJ, Blanca Ramírez M, et al. 2018a. Parkinson disease- associated mutations in LRRK2 cause centrosomal defects via Rab8a phosphorylation. Mol. Neurodegener. 13:3 Madero-Pérez J, Fernández B, Lara Ordónez AJ, Fdez E, Lobbestael E, et al. 2018b. RAB7L1-mediated relo- calization of LRRK2 to the Golgi complex causes centrosomal deficits via RAB8A. Front. Mol. Neurosci. 11:417 Martin I, Dawson VL, Dawson TM. 2011. Recent advances in the genetics of Parkinson’s disease. Annu. Rev. Genom. Hum. Genet. 12:301–25 Mata IF, Jang Y, Kim CH, Hanna DS, Dorschner MO, et al. 2015. The RAB39B p.G192R mutation causes X-linked dominant Parkinson’s disease. Mol. Neurodegener. 10:50 Mata IF, Jang Y, Kim CH, Hanna DS, Dorschner MO, et al. 2015. The R X-linked dominant Parkinson’s disease. Mol. Neurodegener. 10:50 Matheoud D,Cannon T,Voisin A,Penttinen AM,Ramet L,et al.2019.Intestinal infection triggers Parkinson’s disease-like symptoms in Pink1−/−mice. Nature 571:565–69 Matheoud D, Sugiura A, Bellemare-Pelletier A, Laplante A, Rondeau C, et al. 2016. Parkinson’s disease-related proteins PINK1 and Parkin repress mitochondrial antigen presentation. Cell 166:314–27 Mathivanan S, Ji H, Simpson RJ. 2010. Exosomes: extracellular organelles important in intercellular commu- nication. J. Proteom. 73:1907–20 McGough IJ, Steinberg F, Jia D, Barbuti PA, McMillan KJ, et al. 2014. Retromer binding to FAM21 and the WASH complex is perturbed by the Parkinson disease-linked VPS35(D620N) mutation. Curr. Biol. 24:1670–76 McGrath E, Waschbüsch D, Baker BM, Khan AR. 2019. LRRK2 binds to the Rab32 subfamily in a GTP- dependent manner via its armadillo domain. Small GTPases. https://doi.org/10.1080/21541248.2019. 1666623 McLelland GL, Lee SA, McBride HM, Fon EA. 2016. Syntaxin-17 delivers PINK1/parkin-dependent mito- chondrial vesicles to the endolysosomal system. J. Cell Biol. 214:275–91 McLelland GL, Soubannier V, Chen CX, McBride HM, Fon EA. 2014. Parkin and PINK1 function in a vesicular trafficking pathway regulating mitochondrial quality control. EMBO J. 33:282–95 McWilliams TG, Barini E, Pohjolan-Pirhonen R, Brooks SP, Singh F, et al. 2018. Phosphorylation of Parkin at serine 65 is essential for its activation in vivo. Open Biol. 8. https://doi.org/10.1098/rsob.180108 McWilliams TG, Barini E, Pohjolan-Pirhonen R, Brooks SP, Singh F, et al. 2018. Phosphorylation of Parkin at serine 65 is essential for its activation in vivo. Open Biol. 8. https://doi.org/10.1098/rsob.180108 McWilliams TG, Muqit MMK. 2017. Annu. Rev. Cell Dev. Biol. 2020.36:237-264. Downloaded from www.annualreviews.org Access provided by 86.174.33.195 on 12/11/20. See copyright for approved use. 25:6016–24 Singh • Muqit 260 Lesage S, Bras J, Cormier-Dequaire F, Condroyer C, Nicolas A, et al. 2015. Loss-of-function mutations in RAB39B are associated with typical early-onset Parkinson disease. Neurol. Genet. 1:e9 Lesage S, Drouet V, Majounie E, Deramecourt V, Jacoupy M, et al. 2016. Loss of VPS13C function in autosomal-recessive Parkinsonism causes mitochondrial dysfunction and increases PINK1/Parkin- dependent mitophagy. Am. J. Hum. Genet. 98:500–13 Linhart R,Wong SA,Cao J,Tran M,Huynh A,et al.2014.Vacuolar protein sorting 35 (Vps35) rescues locomo- tor deficits and shortened lifespan in Drosophila expressing a Parkinson’s disease mutant of Leucine-Rich Repeat Kinase 2 (LRRK2). Mol. Neurodegener. 9:23 Liu J, Zhang JP, Shi M, Quinn T, Bradner J, et al. 2009. Rab11a and HSP90 regulate recycling of extracellular α-synuclein. J. Neurosci. 29:1480–85 Liu Z, Bryant N, Kumaran R, Beilina A, Abeliovich A, et al. 2018. LRRK2 phosphorylates membrane-bound Rabs and is activated by GTP-bound Rab7L1 to promote recruitment to the trans-Golgi network. Hum. Mol. Genet. 27:385–95 Lunati A, Lesage S, Brice A. 2018. The genetic landscape of Parkinson’s disease. Rev. Neurol. 174:628–43 Annu. Rev. Cell Dev. Biol. 2020.36:237-264. Downloaded from www.annualreviews.org Access provided by 86.174.33.195 on 12/11/20. See copyright for approved use. PINK1 and Parkin: emerging themes in mitochondrial homeostasis. at serine 65 is essential for its activation in vivo. Open Biol. 8. https://doi.org/10.1098/rsob.180108 McWilliams TG, Muqit MMK. 2017. PINK1 and Parkin: emerging themes in mitochondrial homeostasis. Curr. Opin. Cell Biol. 45:83–91 McWilliams TG, Muqit MMK. 2017. PINK1 and Parkin: emerging themes in mitochondrial homeostasis. Curr. Opin. Cell Biol. 45:83–91 Mir R, Tonelli F, Lis P, Macartney T, Polinski NK, et al. 2018. The Parkinson’s disease VPS35[D620N] mutation enhances LRRK2-mediated Rab protein phosphorylation in mouse and human. Biochem. J. 475:1861–83 Muller MP, Goody RS. 2018. Molecular control of Rab activity by GEFs, GAPs and GDI. Small GTPases 9:5–21 www.annualreviews.org • Aberrant Vesicle Trafficking in Parkinson’s 261 Nalls MA, Blauwendraat C, Vallerga CL, Heilbron K, Bandres-Ciga S, et al. 2019. Identification of novel risk loci,causal insights,and heritable risk for Parkinson’s disease: a meta-analysis of genome-wide association studies. Lancet Neurol. 18:1091–102 Nalls MA, Pankratz N, Lill CM, Do CB, Hernandez DG, et al. 2014. Large-scale meta-analysis of genome- wide association data identifies six new risk loci for Parkinson’s disease. Nat. Genet. 46:989–93 Narendra DP, Jin SM, Tanaka A, Suen DF, Gautier CA, et al. 2010. PINK1 is selectively stabilized on impaired mitochondria to activate Parkin. PLOS Biol. 8:e1000298 Ordureau A, Paulo JA, Zhang J, An H, Swatek KN, et al. 2020. Global landscape and dynamics of Parkin and USP30-dependent ubiquitylomes in iNeurons during mitophagic signaling. Mol. Cell 77:1124–42.e10 Ordureau A, Paulo JA, Zhang W, Ahfeldt T, Zhang J, et al. 2018. Dynamics of PARKIN-dependent mito- chondrial ubiquitylation in induced neurons and model systems revealed by digital snapshot proteomics. Mol. Cell 70:211–27.e8 Ordureau A, Sarraf SA, Duda DM, Heo JM, Jedrychowski MP, et al. 2014. Quantitative proteomics re- veal a feedforward mechanism for mitochondrial PARKIN translocation and ubiquitin chain synthesis. Mol. Cell 56:360–75 Ostermeier C, Brunger AT. 1999. Structural basis of Rab effector specificity: crystal structure of the small G protein Rab3A complexed with the effector domain of rabphilin-3A. Cell 96:363–74 Petridi S, Middleton CA, Ugbode C, Fellgett A, Covill L, Elliott CJH. 2020. In vivo visual screen for dopamin- ergic Rab ↔LRRK2-G2019S interactions in Drosophila discriminates Rab10 from Rab3. Genes Genomes Genet. 10:1903–14 Pickrell AM, Huang CH, Kennedy SR, Ordureau A, Sideris DP, et al. 2015. Endogenous Parkin preserves dopaminergic substantia nigral neurons following mitochondrial DNA mutagenic stress. Neuron 87:371– 81 Pihlstrøm L, Rengmark A, Bjørnarå KA, Dizdar N, Fardell C, et al. 2015. Annu. Rev. Cell Dev. Biol. 2020.36:237-264. Downloaded from www.annualreviews.org Access provided by 86.174.33.195 on 12/11/20. See copyright for approved use. Fine mapping and resequencing of the PARK16 locus in Parkinson’s disease. J. Hum. Genet. 60:357–62 Plowey ED, Cherra SJ III, Liu Y-J, Chu CT. 2008. Role of autophagy in G2019S-LRRK2-associated neurite shortening in differentiated SH-SY5Y cells. J. Neurochem. 105:1048–56 Poewe W, Seppi K, Tanner CM, Halliday GM, Brundin P, et al. 2017. Parkinson disease. Nat. Rev. Dis. Primers 3:17013 Pourjafar-Dehkordi D, Vieweg S, Itzen A, Zacharias M. 2019. Phosphorylation of Ser111 in Rab8a modulates Rabin8-dependent activation by perturbation of side chain interaction networks.Biochemistry 58:3546–54 Purlyte E, Dhekne HS, Sarhan AR, Gomez R, Lis P, et al. 2018. Rab29 activation of the Parkinson’s disease- associated LRRK2 kinase. EMBO J. 37:1–18. Corrigendum. 2019. EMBO J. 38:e101237 Ramonet D, Daher JP, Lin BM, Stafa K, Kim J, et al. 2011. Dopaminergic neuronal loss, reduced neurite complexity and autophagic abnormalities in transgenic mice expressing G2019S mutant LRRK2. PLOS ONE 6:e18568 Rendón WO, Martínez-Alonso E, Tomás M, Martínez-Martínez N, Martínez-Menárguez JA. 2013. Golgi fragmentation is Rab and SNARE dependent in cellular models of Parkinson’s disease. Histochem. Cell Biol. 139:671–84 Richter B, Sliter DA, Herhaus L, Stolz A, Wang C, et al. 2016. Phosphorylation of OPTN by TBK1 enhances its binding to Ub chains and promotes selective autophagy of damaged mitochondria. PNAS 113:4039– 44 Rizo J, Sudhof TC. 2012. The membrane fusion enigma: SNAREs, Sec1/Munc18 proteins, and their accomplices—guilty as charged? Annu. Rev. Cell Dev. Biol. 28:279–308 Saez-Atienzar S, Bonet-Ponce L, Blesa JR, Romero FJ, Murphy MP, et al. 2014. The LRRK2 inhibitor GSK2578215A induces protective autophagy in SH-SY5Y cells: involvement of Drp-1-mediated mi- tochondrial fission and mitochondrial-derived ROS signaling. Cell Death Dis. 5:e1368 Sambri I, D’Alessio R, Ezhova Y, Giuliano T, Sorrentino NC, et al. 2017. Lysosomal dysfunction disrupts presynaptic maintenance and restoration of presynaptic function prevents neurodegeneration in lysoso- mal storage diseases. EMBO Mol. Med. 9:112–32 g Sarraf SA, Raman M, Guarani-Pereira V, Sowa ME, Huttlin EL, et al. 2013. Landscape of the PARKIN- dependent ubiquitylome in response to mitochondrial depolarization. Nature 496:372–76 Singh • Muqit 262 Satake W, Nakabayashi Y, Mizuta I, Hirota Y, Ito C, et al. 2009. Genome-wide association study identifies common variants at four loci as genetic risk factors for Parkinson’s disease. Nat. Genet. 41:1303–7 Sauvé V, Sung G, Soya N, Kozlov G, Blaimschein N, et al. 2018. Mechanism of parkin activation by phospho- rylation. Nat. Struct. Mol. Biol. Annu. Rev. Cell Dev. Biol. 2020.36:237-264. Downloaded from www.annualreviews.org Access provided by 86.174.33.195 on 12/11/20. See copyright for approved use. Spillantini MG, Goedert M. 2018. Neurodegeneration and the ordered assembly of α-synuclein. Cell Tissue Res. 373:137–48 Steger M, Diez F, Dhekne HS, Lis P, Nirujogi RS, et al. 2017. Systematic proteomic analysis of LRRK2- mediated Rab GTPase phosphorylation establishes a connection to ciliogenesis. eLife 6:e31012 Steger M, Tonelli F, Ito G, Davies P, Trost M, et al. 2016. Phosphoproteomics reveals that Parkinson’s disease kinase LRRK2 regulates a subset of Rab GTPases. eLife 5:e12813 g f tenmark H. 2009. Rab GTPases as coordinators of vesicle traffic. Nat. Rev. Mol. Cell Biol. 10:513–2 Sung JY, Kim J, Paik SR, Park JH, Ahn YS, Chung KC. 2001. Induction of neuronal cell death by Rab5A- dependent endocytosis of α-synuclein. J. Biol. Chem. 276:27441–48 Taylor M, Alessi DR. 2020. Advances in elucidating the function of leucine-rich repeat protein kinase-2 in normal cells and Parkinson’s disease. Curr. Opin. Cell Biol. 63:102–13 Tokuda T, Qureshi MM, Ardah MT, Varghese S, Shehab SA, et al. 2010. Detection of elevated levels of α-synuclein oligomers in CSF from patients with Parkinson disease. Neurology 75:1766–72 Tong Y, Giaime E, Yamaguchi H, Ichimura T, Liu Y, et al. 2012. Loss of leucine-rich repeat kinase 2 causes age-dependent bi-phasic alterations of the autophagy pathway. Mol. Neurodegener. 7:2 Tong Y, Yamaguchi H, Giaime E, Boyle S, Kopan R, et al. 2010. Loss of leucine-rich repeat kinase 2 causes impairment of protein degradation pathways, accumulation of α-synuclein, and apoptotic cell death in aged mice. PNAS 107:9879–84 Trempe JF, Sauvé V, Grenier K, Seirafi M, Tang MY, et al. 2013. Structure of parkin reveals mechanisms for ubiquitin ligase activation. Science 340:1451–55 Underwood R, Wang B, Carico C, Whitaker RH, Placzek WJ, Yacoubian TA. 2020. The GTPase Rab27b regulates the release, autophagic clearance, and toxicity of alpha-synuclein. J. Biol. Chem. http://doi. org/10.1074/jbc.RA120.013337 Valente EM, Abou-Sleiman PM, Caputo V, Muqit MMK, Harvey K, et al. 2004. Hereditary early-onset Parkinson’s disease caused by mutations in PINK1. Science 304:1158–60 Vanhauwaert R, Kuenen S, Masius R, Bademosi A, Manetsberger J, et al. 2017. The SAC1 domain in synap- tojanin is required for autophagosome maturation at presynaptic terminals. EMBO J. 36:1392–411 Vieweg S, Mulholland K, Bräuning B, Kacharia N, Lai Y-C, et al. 2020. PINK1-dependent phosphorylation of Serine111 within the SF3 motif of Rab GTPases impairs effector interactions and LRRK2-mediated phosphorylation at Threonine72. Biochem. J. 477:1651–68 Vilariño-Güell C, Wider C, Ross OA, Dachsel JC, Kachergus JM, et al. 2011. Annu. Rev. Cell Dev. Biol. 2020.36:237-264. Downloaded from www.annualreviews.org Access provided by 86.174.33.195 on 12/11/20. See copyright for approved use. 25:623–30 Sharma M, Burré J, Bronk P, Zhang Y, Xu W, Südhof TC. 2012. CSPα knockout causes neurodegeneration by impairing SNAP-25 function. EMBO J. 31:829–41 Sharma M, Burré J, Südhof TC. 2011. CSPα promotes SNARE-complex assembly by chaperoning SNAP-25 during synaptic activity. Nat. Cell Biol. 13:30–39 Sliter DA, Martinez J, Hao L, Chen X, Sun N, et al. 2018. Parkin and PINK1 mitigate STING-induced inflammation. Nature 561:258–62 Søreng K, Neufeld TP, Simonsen A. 2018. Membrane trafficking in autophagy. Int. Rev. Cell Mol. Biol. 336:1– 92 Soubannier V, McLelland GL, Zunino R, Braschi E, Rippstein P, et al. 2012. A vesicular transport pathway shuttles cargo from mitochondria to lysosomes. Curr. Biol. 22:135–41 Annu. Rev. Cell Dev. Biol. 2020.36:237-264. Downloaded from www.annualreviews.org Access provided by 86.174.33.195 on 12/11/20. See copyright for approved use. VPS35 mutations in Parkinson disease. Am. J. Hum. Genet. 89:162–67 Vincow ES, Merrihew G, Thomas RE, Shulman NJ, Beyer RP, et al. 2013. The PINK1–Parkin pathway promotes both mitophagy and selective respiratory chain turnover in vivo. PNAS 110:6400–5 Wallings RL, Tansey MG. 2019. LRRK2 regulation of immune-pathways and inflammatory disease. Biochem. Soc. Trans. 47:1581–95 Wang S, Ma Z, Xu X, Wang Z, Sun L, et al. 2014. A role of Rab29 in the integrity of the trans-Golgi network and retrograde trafficking of mannose-6-phosphate receptor. PLOS ONE 9:e96242 www.annualreviews.org • Aberrant Vesicle Trafficking in Parkinson’s 263 Waschbüsch D, Hübel N, Ossendorf E, Lobbestael E, Baekelandt V, et al. 2019. Rab32 interacts with SNX6 and affects retromer-dependent Golgi trafficking. PLOS ONE 14:e0208889 Waschbüsch D, Michels H, Strassheim S, Ossendorf E, Kessler D, et al. 2014. LRRK2 transport is regulated by its novel interacting partner Rab32. PLOS ONE 9:e111632 Watanabe R, Buschauer R, Böhning J, Audagnotto M, Lasker K et al. 2019. The in situ structure of Parkinson’s disease-linked LRRK2. bioRxiv 837203. https://doi.org/10.1101/837203 Wauer T, Komander D. 2013. Structure of the human Parkin ligase domain in an autoinhibited state. EMBO J. 32:2099–112 Wauer T, Swatek KN, Wagstaff JL, Gladkova C, Pruneda JN, et al. 2015. Ubiquitin Ser65 phosphorylation affects ubiquitin structure, chain assembly and hydrolysis. EMBO J. 34:307–25 Wenzel DM, Lissounov A, Brzovic PS, Klevit RE. 2011. UBCH7 reactivity profile reveals parkin and HHARI to be RING/HECT hybrids. Nature 474:105–8 Williams ET, Chen X, Moore DJ. 2017. VPS35, the retromer complex and Parkinson’s disease. J. Park. Dis. 7:219–33 Wilson GR, Sim JCH, McLean C, Giannandrea M, Galea CA, et al. 2014. Mutations in RAB39B cause X-linked intellectual disability and early-onset Parkinson disease with α-synuclein pathology. Am. J. Hum. Genet. 95:729–35 Xia Y, Zhang G, Han C, Ma K, Guo X, et al. 2019. Microglia as modulators of exosomal alpha-synuclein transmission. Cell Death Dis. 10:174 Xiong Y, Coombes CE, Kilaru A, Li X, Gitler AD, et al. 2010. GTPase activity plays a key role in the patho- biology of LRRK2. PLOS Genet. 6:e1000902 gy Yamano K, Fogel AI, Wang C, van der Bliek AM, Youle RJ. 2014. Mitochondrial Rab GAPs govern autophago- some biogenesis during mitophagy. eLife 3:e01612 Yamano K, Fogel AI, Wang C, van der Bliek AM, Youle RJ. some biogenesis during mitophagy. eLife 3:e01612 Yamano K, Wang C, Sarraf SA, Münch C, Kikuchi R, et al. 2018. Contents Structural Biology of RNA Polymerase II Transcription: 20 Years On Sara Osman and Patrick Cramer p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1 F-Actin Cytoskeleton Network Self-Organization Through Competition and Cooperation Rachel S. Kadzik, Kaitlin E. Homa, and David R. Kovar p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p35 Integrating Chemistry and Mechanics: The Forces Driving Axon Growth Kristian Franze p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p61 Nuclear Membrane Rupture and Its Consequences John Maciejowski and Emily M. Hatch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p85 Protein Quality Control and Lipid Droplet Metabolism Melissa A. Roberts and James A. Contents Olzmann p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 115 Msp1/ATAD1 in Protein Quality Control and Regulation of Synaptic Activities Lan Wang and Peter Walter p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 141 Developing Therapies for Neurodegenerative Disorders: Insights from Protein Aggregation and Cellular Stress Responses Giovanna R. Mallucci, David Klenerman, and David C. Rubinsztein p p p p p p p p p p p p p p p p p p p 165 Cellular Mechanisms of NETosis Hawa Racine Thiam, Siu Ling Wong, Denisa D. Wagner, and Clare M. Waterman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 191 Scaling of Subcellular Structures Wallace F. Marshall p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 219 Parkinson’s: A Disease of Aberrant Vesicle Trafficking Pawan Kishor Singh and Miratul M.K. Annu. Rev. Cell Dev. Biol. 2020.36:237-264. Downloaded from www.annualreviews.org Access provided by 86.174.33.195 on 12/11/20. See copyright for approved use. Endosomal Rab cycles regulate Parkin- mediated mitophagy. eLife 7:e31326 Yamano K, Youle RJ. 2013. PINK1 is degraded through the N-end rule pathway. Autophagy 9:1758–69 Yu M, Arshad M, Wang W, Zhao D, Xu L, Zhou L. 2018. LRRK2 mediated Rab8a phosphorylation promotes lipid storage. Lipids Health Dis. 17:34 Zavodszky E, Seaman MNJ, Moreau K, Jimenez-Sanchez M, Breusegem SY, et al. 2014. Mutation in VPS35 associated with Parkinson’s disease impairs WASH complex association and inhibits autophagy. Nat. Commun. 5:3828 Zhang Q, Pan Y, Yan R, Zeng B, Wang H, et al. 2015. Commensal bacteria direct selective cargo sorting to promote symbiosis. Nat. Immunol. 16:918–26 Zhao Y, Perera G, Takahashi-Fujigasaki J, Mash DC, Vonsattel JPG, et al. 2018. Reduced LRRK2 in asso- ciation with retromer dysfunction in post-mortem brain tissue from LRRK2 mutation carriers. Brain 141:486–95 Zimprich A, Benet-Pages A, Struhal W, Graf E, Eck SH, et al. 2011. A mutation in VPS35, encoding a subunit of the retromer complex, causes late-onset Parkinson disease. Am. J. Hum. Genet. 89:168–75 264 Singh • Muqit Contents Muqit p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 237 Mitochondrial Quality Control and Restraining Innate Immunity Andrew T. Moehlman and Richard J. Youle p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 265 Annu. Rev. Cell Dev. Biol. 2020.36:237-264. Downloaded from www.annualreviews.org Access provided by 86.174.33.195 on 12/11/20. See copyright for approved use. vii Combinatorial Control of Plant Specialized Metabolism: Mechanisms, Functions, and Consequences Elia Lacchini and Alain Goossens p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 291 Cellular, Molecular, and Physiological Adaptations of Hibernation: The Solution to Environmental Challenges Sarah M. Mohr, Sviatoslav N. Bagriantsev, and Elena O. Gracheva p p p p p p p p p p p p p p p p p p p p p 315 Synthetic Developmental Biology: Understanding Through Reconstitution Gavin Schlissel and Pulin Li p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 339 Processing Temporal Growth Factor Patterns by an Epidermal Growth Factor Receptor Network Dynamically Established in Space Aneta Koseska and Philippe I.H. Bastiaens p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 359 Shaping Organs: Shared Structural Principles Across Kingdoms O. Hamant and T.E. Saunders p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 385 Human Embryogenesis: A Comparative Perspective Claudia Gerri, Sergio Menchero, Shantha K. Mahadevaiah, James M.A. Turner, and Kathy K. Annu. Rev. Cell Dev. Biol. 2020.36:237-264. Downloaded from www.annualreviews.org Access provided by 86.174.33.195 on 12/11/20. See copyright for approved use. Niakan p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 411 The Role of Immune Factors in Shaping Fetal Neurodevelopment Alice Lu-Culligan and Akiko Iwasaki p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 441 The Lineage Before Time: Circadian and Nonclassical Clock Influences on Development Amita Sehgal, Daniel Maxim Iascone, and Joseph Bedont p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 469 Pediatric Allergic Diseases, Food Allergy, and Oral Tolerance Kirsty Logan, George Du Toit, Mattia Giovannini, Victor Turcanu, and Gideon Lack p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 511 The Source and Dynamics of Adult Hematopoiesis: Insights from Lineage Tracing Joseph N. Pucella, Samik Upadhaya, and Boris Reizis p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 529 B Cell Immunosenescence Daniela Frasca, Alain Diaz, Maria Romero, Denisse Garcia, and Bonnie B. Annu. Rev. Cell Dev. Biol. 2020.36:237-264. Downloaded from www.annualreviews.org Access provided by 86.174.33.195 on 12/11/20. See copyright for approved use. Blomberg p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 551 Indexes Cumulative Index of Contributing Authors, Volumes 32–36 p p p p p p p p p p p p p p p p p p p p p p p p p p p 575 Indexes Cumulative Index of Contributing Authors, Volumes 32–36 p p p p p p p p p p p p p p p p p p p p p p p p p p p 575 Errata Errata An online log of corrections to Annual Review of Cell and Developmental Biology articles may be found at http://www.annualreviews.org/errata/cellbio Contents
https://openalex.org/W3105486817	https://www.biorxiv.org/content/biorxiv/early/2016/12/11/093021.full.pdf	English	null	Cox-nnet: an artificial neural network method for prognosis prediction on high-throughput omics data	bioRxiv (Cold Spring Harbor Laboratory)	2,016	cc-by	19,061	. CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 11, 2016. ; https://doi.org/10.1101/093021 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 11, 2016. ; https://doi.org/10.1101/093021 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made availab The copyright holder for this preprint (which w this version posted December 11, 2016. ; https://doi.org/10.1101/093021 doi: bioRxiv preprint Cox-nnet: an artificial neural network 1 method for prognosis prediction on high- 2 throughput omics data 3 Travers Ching,†, Xun Zhu,†, Lana X. Garmire,† 5 Molecular Biosciences and Bioengineering Graduate Program, University of Hawaii at Manoa, 6 Honolulu, HI 96822, USA 7 †Epidemiology Program, University of Hawaii Cancer Center, Honolulu, HI 96813, USA 8 1 1 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 11, 2016. ; https://doi.org/10.1101/093021 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 11, 2016. ; https://doi.org/10.1101/093021 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 11, 2016. ; https://doi.org/10.1101/093021 doi: bioRxiv preprint Running title 9 Artificial neural network for prognosis 10 Keywords 11 Survival analysis, RNA-Seq, cancer, machine learning, bioinformatics 12 Corresponding author 13 Lana Garmire 14 701 Ilalo Street, Honolulu HI 96813 15 (808) 441-8193 16 lgarmire@cc.hawaii.edu 17 18 2 2 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 11, 2016. ; https://doi.org/10.1101/093021 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 11, 2016. ; https://doi.org/10.1101/093021 doi: bioRxiv preprint Abstract 19 Artificial neural networks (ANN) are computing architectures with massively parallel interconnections of 20 simple neurons and has been applied to biomedical fields such as imaging analysis and diagnosis. We 21 have developed a new ANN framework called Cox-nnet to predict patient prognosis from high throughput 22 transcriptomics data. In over 10 TCGA RNA-Seq data sets, Cox-nnet achieves a statistically significant 23 increase in predictive accuracy, compared to the other three methods including Cox-proportional hazards 24 (Cox-PH), Random Forests Survival and CoxBoost. Cox-nnet also reveals richer biological information, 25 from both pathway and gene levels. The outputs from the hidden layer node can provide a new approach 26 for survival-sensitive dimension reduction. In summary, we have developed a new method for more 27 accurate and efficient prognosis prediction on high throughput data, with functional biological insights. 28 The source code is freely available at https://github.com/lanagarmire/cox-nnet. 29 Artificial neural networks (ANN) are computing architectures with massively parallel interconnections of 20 simple neurons and has been applied to biomedical fields such as imaging analysis and diagnosis. We 21 have developed a new ANN framework called Cox-nnet to predict patient prognosis from high throughput 22 transcriptomics data. In over 10 TCGA RNA-Seq data sets, Cox-nnet achieves a statistically significant 23 increase in predictive accuracy, compared to the other three methods including Cox-proportional hazards 24 (Cox-PH), Random Forests Survival and CoxBoost. Cox-nnet also reveals richer biological information, 25 from both pathway and gene levels. The outputs from the hidden layer node can provide a new approach 26 for survival-sensitive dimension reduction. In summary, we have developed a new method for more 27 accurate and efficient prognosis prediction on high throughput data, with functional biological insights. 28 The source code is freely available at https://github.com/lanagarmire/cox-nnet. 29 30 30 3 3 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 11, 2016. ; https://doi.org/10.1101/093021 doi: bioRxiv preprint Introduction 31 Artificial Neural networks (ANNs) were developed in 1943 in order to model the activity of neurons 1. In 32 recent years, ANNs have caught renewed attention, thanks to the increased parallel computing power and 33 the promise of deep learning 2. The original ANN extension of Cox Regression was not designed to 34 handle high throughput input data3. Some recent attempts using ANNs to high dimensional survival data 35 simplified the regression problem as either a binary classification or fitting discrete variables of survival 36 time through binning , leading to loss of accuracy 4,5. Another study used time as an additional input in 37 order to predict patient survival or censoring status6, with the potential to overfit if the survival time and 38 censoring time are correlated. To avoid all these issues, we herein leverage the neural network extension 39 of Cox regression by a high-performance and easy-to-use package, particularly fit for high dimensional 40 data. 41 Artificial Neural networks (ANNs) were developed in 1943 in order to model the activity of neurons 1. In 32 recent years, ANNs have caught renewed attention, thanks to the increased parallel computing power and 33 the promise of deep learning 2. The original ANN extension of Cox Regression was not designed to 34 handle high throughput input data3. Some recent attempts using ANNs to high dimensional survival data 35 simplified the regression problem as either a binary classification or fitting discrete variables of survival 36 time through binning , leading to loss of accuracy 4,5. Another study used time as an additional input in 37 order to predict patient survival or censoring status6, with the potential to overfit if the survival time and 38 censoring time are correlated. To avoid all these issues, we herein leverage the neural network extension 39 of Cox regression by a high-performance and easy-to-use package, particularly fit for high dimensional 40 data. 41 Besides Cox-nnet, some other modeling methods exist to predict patient survival. The standard method is 42 Cox proportional hazards (Cox-PH) regression, a semi-parametric and generalized linear model with an 43 exponential link function7. Another method, CoxBoost 8, is an iterative “boosting” method modified from 44 the Cox-PH model. In each boosting iteration, it refits the parameters by maximizing the penalized 45 likelihood function. Introduction 31 CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 11, 2016. ; https://doi.org/10.1101/093021 doi: bioRxiv preprint comparison with the other methods mentioned above (Cox-PH, RF-S and CoxBoost), Cox-nnet has better 56 overall predictive accuracy. It is also optimized on graphics processing unit (GPU) with at least an order 57 of computational speed-up over the central processing unit (CPU), making it a compelling new tool to 58 predict disease prognosis in the era of precision medicine. Second, Cox-nnet utilizes feature importance 59 scores based on the partial derivatives of gene features selected by the model, so that the relative 60 importance of the genes to prognosis outcome can be directly assessed. Thirdly, the hidden layer node 61 structure in ANN can be harnessed to reveal much richer information of featuring genes and biological 62 pathways, compared to the Cox-PH method. Overall, Cox-nnet is a desirable survival analysis method 63 with both excellent predictive power and usage to gain biological functions related to prognosis. 64 comparison with the other methods mentioned above (Cox-PH, RF-S and CoxBoost), Cox-nnet has better 56 overall predictive accuracy. It is also optimized on graphics processing unit (GPU) with at least an order 57 of computational speed-up over the central processing unit (CPU), making it a compelling new tool to 58 predict disease prognosis in the era of precision medicine. Second, Cox-nnet utilizes feature importance 59 scores based on the partial derivatives of gene features selected by the model, so that the relative 60 importance of the genes to prognosis outcome can be directly assessed. Thirdly, the hidden layer node 61 structure in ANN can be harnessed to reveal much richer information of featuring genes and biological 62 pathways, compared to the Cox-PH method. Overall, Cox-nnet is a desirable survival analysis method 63 with both excellent predictive power and usage to gain biological functions related to prognosis. 64 Introduction 31 Rather than using L2 ridge regression common in Cox-PH, the number of boosting 46 iterations is used as the complexity parameter in CoxBoost and optimized via cross-validation (CV) 8. 47 Random Forests Survival (RF-S) is another ensemble, non-linear method 9. It combines many 48 bootstrapped decision trees in order to reduce the variance in the model, and then calculates a weighted 49 average of all the decision trees. Unlike Cox-PH and CoxBoost, RF-S does not use the log likelihood 50 function to determine the fitness of the model. Instead, it predicts estimated survival times, and uses 51 Harrel’s C-Index, a score that measures the correct ranking of individuals 9. 52 Besides Cox-nnet, some other modeling methods exist to predict patient survival. The standard method is 42 Cox proportional hazards (Cox-PH) regression, a semi-parametric and generalized linear model with an 43 exponential link function7. Another method, CoxBoost 8, is an iterative “boosting” method modified from 44 the Cox-PH model. In each boosting iteration, it refits the parameters by maximizing the penalized 45 likelihood function. Rather than using L2 ridge regression common in Cox-PH, the number of boosting 46 iterations is used as the complexity parameter in CoxBoost and optimized via cross-validation (CV) 8. 47 Random Forests Survival (RF-S) is another ensemble, non-linear method 9. It combines many 48 bootstrapped decision trees in order to reduce the variance in the model, and then calculates a weighted 49 average of all the decision trees. Unlike Cox-PH and CoxBoost, RF-S does not use the log likelihood 50 function to determine the fitness of the model. Instead, it predicts estimated survival times, and uses 51 Harrel’s C-Index, a score that measures the correct ranking of individuals 9. 52 4 The new software package we have developed here, named Cox-nnet, advances the ANN extension of 53 Cox regression for survival prediction on high-throughput data. The caliber of this package is manifested 54 in several aspects. First, it has improved technical performance in terms of both accuracy and speed. In 55 4 4 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 11, 2016. ; https://doi.org/10.1101/093021 doi: bioRxiv preprint . The Cox model 66 The Cox-PH model is a log-linear model that estimates individual hazard, i.e., an instantaneous measure 67 g , , of the likelihood of an event, based on a set of features. The hazard is given by the equation: 68 of the likelihood of an event, based on a set of features. The hazard is given by the equation: 68 ( 1 ) ( 2 )    0 \| exp i h t h t   i X i T i X β ( 1 ) ( 2 )    0 \| exp i h t h t   i X i T i X β ( 1 )    0 \| exp i h t h t   i X ( 1 ) ( ) ( 2 )   0 exp i h t   i X i T i X β ( 2 ) i T i X β ( 2 ) i T i X β ( 2 ) Where is the log hazard ratio for patient . The partial likelihood is represented by the following 69 formula: 70 i i Where is the log hazard ratio for patient . The partial likelihood is represented by the following 69 formula: 70 i i ( 3 )  1 exp exp j i i C i j t t PL      β ( 3 ) Where is the censoring status of a patient, and if the patient was censored or 1 if the 71  C i  0 C i  Where is the censoring status of a patient, and if the patient was censored or 1 if the 71  C i  0 C i  patient died or had a recurrence event, etc. The partial log-likelihood is used as the cost function: 72 patient died or had a recurrence event, etc. The partial log-likelihood is used as the cost function: 72 5 5 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 11, 2016. ; https://doi.org/10.1101/093021 doi: bioRxiv preprint . The Cox model 66 In the k-th iteration of a K-fold CV, the 79 optimal coefficients are found by minimizing the cost function on the training sub-samples. If 80 , ˆ k  β The cross-validated performance metric may be Harrel’s concordance index (C-index) 10 or the “cross- 76 validated partial likelihood” 11. Since the contribution of each patient in the partial likelihood is 77 determined only in the context of all the other patients, the cross-validated partial likelihood is calculated 78 subtracting full partial likelihood from the training set in the CV. In the k-th iteration of a K-fold CV, the 79 optimal coefficients are found by minimizing the cost function on the training sub-samples. If 80 , ˆ k  β The cross-validated performance metric may be Harrel’s concordance index (C-index) 10 or the “cross- 76 validated partial likelihood” 11. Since the contribution of each patient in the partial likelihood is 77 determined only in the context of all the other patients the cross-validated partial likelihood is calculated 78 is the partial likelihood of the training sub-samples, and is the partial likelihood of 81   , ˆ k k pl  β   , ˆ k pl  β the full dataset, then the cross-validated partial likelihood is the sum of differences: 82 the full dataset, then the cross-validated partial likelihood is the sum of differences: 82 ( 6 ) ( 6 )    , , 1 ˆ ˆ ( ) K k k k k cvpl pl pl        β β ( 6 )    , , 1 ˆ ˆ ( ) K k k k k cvpl pl pl        β β ( 6 )    , , 1 ˆ ˆ ( ) K k k k k cvpl pl pl        β β    , , 1 ˆ ˆ ( ) K k k k k cvpl pl pl        β β ANN extension of Cox regression 83 The Cox model 66 CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 11, 2016. ; https://doi.org/10.1101/093021 doi: bioRxiv preprint ( 4 )  1 log exp j i i j C i t t pl                 β ( 4 )  1 log exp j i i j C i t t pl                 β ( 4 ) In a Cox model with L2 ridge regression, a penalty term is added which is proportional to the L2 norm of 73 the coefficients. The cost function is minimized to find the best coefficients for the model: 74 In a Cox model with L2 ridge regression, a penalty term is added which is proportional to the L2 norm of 73 the coefficients. The cost function is minimized to find the best coefficients for the model: 74 In a Cox model with L2 ridge regression, a penalty term is added which is proportional to the L2 norm of 73 the coefficients. The cost function is minimized to find the best coefficients for the model: 74 In a Cox model with L2 ridge regression, a penalty term is added which is proportional to the L2 norm of 73 the coefficients The cost function is minimized to find the best coefficients for the model: 74 ( 5 )   2 Cost pl    β β β ( 5 ) ( 5 )   2 Cost pl    β β β where the tuning parameter is determined by maximizing CV. 75  where the tuning parameter is determined by maximizing CV. 75  The cross-validated performance metric may be Harrel’s concordance index (C-index) 10 or the “cross- 76 validated partial likelihood” 11. Since the contribution of each patient in the partial likelihood is 77 determined only in the context of all the other patients, the cross-validated partial likelihood is calculated 78 subtracting full partial likelihood from the training set in the CV. ANN extension of Cox regression 83 The tanh function is: 91 In this manuscript, the tanh activation function is used, as it results in faster training time compared to the 90 sigmoid activation 12. The tanh function is: 91 ( 9 )        exp exp exp exp z z G z z z      ( 9 )        exp exp exp exp z z G z z z      ( 9 ) In addition to ridge regularization, we also employ dropout regularization13. In this approach, nodes are 92 removed during each training iteration with probability 1-p. During evaluation, output from the nodes are 93 multiplied by p. The optimal dropout parameter, p, is determined through cross-validation on the training 94 set. Dropout regularization has been shown to reduce overfitting and improve performance over other 95 regularization schemes13. 96 The source code of cox-nnet can be found at: https://github.com/lgarmire/cox-nnet, and can be installed 97 through the Python Package Index (PyPI). Documentation of package can be found at 98 http://lgarmire.github.io/cox-nnet/docs. 99 The source code of cox-nnet can be found at: https://github.com/lgarmire/cox-nnet, and can be installed 97 through the Python Package Index (PyPI). Documentation of package can be found at 98 http://lgarmire.github.io/cox-nnet/docs. 99 ANN extension of Cox regression 83 ANN extension of Cox regression 83 The ANN extension of Cox regression (Cox-nnet) is a neural network whose output layer is replaced by a 84 Cox model. In a Cox-nnet model with one input layer of input features and one hidden layer composed 85 of hidden nodes, the linear predictor is replaced by the outputs of the hidden layer: 86 J H The ANN extension of Cox regression (Cox-nnet) is a neural network whose output layer is replaced by a 84 The ANN extension of Cox regression (Cox-nnet) is a neural network whose output layer is replaced by a 84 Cox model. In a Cox-nnet model with one input layer of input features and one hidden layer composed 85 of hidden nodes, the linear predictor is replaced by the outputs of the hidden layer: 86 J H ( 7 )   i G   T T i W X b β ( 7 ) 6 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 11, 2016. ; https://doi.org/10.1101/093021 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 11, 2016. ; https://doi.org/10.1101/093021 doi: bioRxiv preprint Where is the coefficient weight matrix between the input and hidden layer with the size H x J, is 87 W b the bias term for each hidden node and is the activation function (applied element-wise on a vector). 88 G Subsequently, the ridge regression cost function is modified to: 89 y, the ridge regression cost function is modified to: ( 8 )       2 2 , , Cost pl     β W β W β W ( 8 ) In this manuscript, the tanh activation function is used, as it results in faster training time compared to the 90 sigmoid activation 12. The tanh function is: 91 In this manuscript, the tanh activation function is used, as it results in faster training time compared to the 90 sigmoid activation 12. Implementation in Python with Theano 100 We implement Cox-nnet using a feed forward, back propagation network with gradient descent. The 101 partial log likelihood is usually written as a double conditional sum (equation 4). To avoid the 102 computational inefficiency of calculating the partial log likelihood (equation 4) using two nested for 103 loops, we convert it into a formulation of matrix operations and basic sums. First we define an indicator 104 matrix with elements: 105 R We implement Cox-nnet using a feed forward, back propagation network with gradient descent. The 101 partial log likelihood is usually written as a double conditional sum (equation 4). To avoid the 102 computational inefficiency of calculating the partial log likelihood (equation 4) using two nested for 103 loops, we convert it into a formulation of matrix operations and basic sums. First we define an indicator 104 matrix with elements: 105 R 7 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 11, 2016. ; https://doi.org/10.1101/093021 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 11, 2016. ; https://doi.org/10.1101/093021 doi: bioRxiv preprint (10 ) 1, 0, i j ij i j t t R t t      (10 ) 1, 0, i j ij i j t t R t t      1, 0, i j ij i j t t R t t      (10 ) We also define an indicator vector with elements given by the censoring of each patient. An operation 106 using replaces the conditional sum over , and an operation using replaces the conditional 107 C R j i t t  C sum over in equation 4. In Theano, the partial log likelihood is: 108  1 C i  sum over in equation 4. In Theano, the partial log likelihood is: 108  1 C i  pl=T.sum((theta - T.log(T.sum(T.exp(theta) * R,axis=1))) * C) ( 11 ) pl=T.sum((theta - T.log(T.sum(T.exp(theta) * R,axis=1))) * C) ( 11 ) ( 11 ) Model evaluation 109 To evaluate the performance of all methods in comparison, we trained each model on 80% of the samples 110 for each dataset (chosen randomly) and evaluated the performance on the 20% holdout test set. The 111 output of Cox-PH, Cox-nnet and CoxBoost are the log hazard ratios (i.e., Prognosis Index, or PI) for each 112 patient. The hazard ratio describes the relative risk of a patient compared to a non-parametric baseline. 113 On the other hand, the output of RF-S is an estimation of the survival time for each patient. 114 We use C-index and log-ranked p-value based on dichotomization of the hold-out test data of the holdout 115 test data to measure the performance of each model. The C-index is a measure of how well the model 116 prediction corresponds to the ranking of the survival data 14. It is calculated for censored survival data, 117 which evaluates a value between 0 and 1, with 0.5 equivalent to a random process. The C-index can be 118 computed as a summation over all events in the dataset, whereby patients with a higher survival time and 119 lower log hazard ratios (and conversely patients with a lower survival time but higher log hazard ratios) 120 are considered concordant. The C-index is a measure of concordance of the data with the model 121 prediction. To calculate the log-ranked p-value, a PI cutoff threshold is used to dichotomize the patients 122 in the data set into higher and lower risk groups, similar to our earlier report 15,16. A log-ranked p-value is 123 then computed to differentiate the Kaplan-Meier survival curves between the higher vs. lower risk groups. 124 In this report, we used the median log hazard ratio as the cutoff threshold. 125 To evaluate the performance of all methods in comparison, we trained each model on 80% of the samples 110 for each dataset (chosen randomly) and evaluated the performance on the 20% holdout test set. The 111 output of Cox-PH, Cox-nnet and CoxBoost are the log hazard ratios (i.e., Prognosis Index, or PI) for each 112 patient. The hazard ratio describes the relative risk of a patient compared to a non-parametric baseline. 113 On the other hand, the output of RF-S is an estimation of the survival time for each patient. Model evaluation 109 CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 11, 2016. ; https://doi.org/10.1101/093021 doi: bioRxiv preprint Feature evaluation 126 For computing the importance of a feature in Cox-nnet, we use a method of partial derivatives (PaD) 17,18. 127 For each patient, we compute the partial derivatives of each input with respect to the linear output of the 128 model (e.g., the log hazard ratio). The average of the partial derivatives for each input across all patient 129 samples is calculated as the feature score. 130 For computing the importance of a feature in Cox-nnet, we use a method of partial derivatives (PaD) 17,18. 127 For each patient, we compute the partial derivatives of each input with respect to the linear output of the 128 model (e.g., the log hazard ratio). The average of the partial derivatives for each input across all patient 129 samples is calculated as the feature score. 130 Model evaluation 109 114 We use C-index and log-ranked p-value based on dichotomization of the hold-out test data of the holdout 115 test data to measure the performance of each model. The C-index is a measure of how well the model 116 prediction corresponds to the ranking of the survival data 14. It is calculated for censored survival data, 117 which evaluates a value between 0 and 1, with 0.5 equivalent to a random process. The C-index can be 118 computed as a summation over all events in the dataset, whereby patients with a higher survival time and 119 lower log hazard ratios (and conversely patients with a lower survival time but higher log hazard ratios) 120 are considered concordant. The C-index is a measure of concordance of the data with the model 121 prediction. To calculate the log-ranked p-value, a PI cutoff threshold is used to dichotomize the patients 122 in the data set into higher and lower risk groups, similar to our earlier report 15,16. A log-ranked p-value is 123 then computed to differentiate the Kaplan-Meier survival curves between the higher vs. lower risk groups. 124 In this report, we used the median log hazard ratio as the cutoff threshold. 125 We use C-index and log-ranked p-value based on dichotomization of the hold-out test data of the holdout 115 test data to measure the performance of each model. The C-index is a measure of how well the model 116 prediction corresponds to the ranking of the survival data 14. It is calculated for censored survival data, 117 which evaluates a value between 0 and 1, with 0.5 equivalent to a random process. The C-index can be 118 computed as a summation over all events in the dataset, whereby patients with a higher survival time and 119 lower log hazard ratios (and conversely patients with a lower survival time but higher log hazard ratios) 120 are considered concordant. The C-index is a measure of concordance of the data with the model 121 8 8 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 11, 2016. ; https://doi.org/10.1101/093021 doi: bioRxiv preprint . Datasets 131 148 As a proof of concept, the current ANN architecture is composed of three layers: one input layer, one 149 Datasets 131 In order to evaluate the performance of Cox-nnet, we analyzed 10 TCGA datasets which were combined 132 into a pan-cancer dataset. The TCGA datasets included the following cancer types: Bladder Urothelial 133 Carcinoma (BLCA), Breast invasive carcinoma (BRCA), Head and Neck squamous cell carcinoma 134 (HNSC), Kidney renal clear cell carcinoma (KIRC), Brain Lower Grade Glioma (LGG), Liver 135 hepatocellular carcinoma (LIHC), Lung adenocarcinoma (LUAD), Lung squamous cell carcinoma 136 (LUSC), Ovarian serous cystadenocarcinoma (OV) and Stomach adenocarcinoma (STAD). RNA-Seq 137 expression and clinical data were downloaded from the Broad Institute GDAC 19. Overall survival time 138 and censoring information were extracted from the clinical follow-up data. Raw count data were 139 normalized using the DESeq2 R package 20 and then log-transformed. Datasets were selected from 140 TCGA based on the following criteria: > 300 samples with both RNASeq and survival data and > 50 141 survival events. In total, 5031 patient samples were used (see Table S1 for a patient tabulation by 142 individual dataset). 143 In order to evaluate the performance of Cox-nnet, we analyzed 10 TCGA datasets which were combined 132 into a pan-cancer dataset. The TCGA datasets included the following cancer types: Bladder Urothelial 133 Carcinoma (BLCA), Breast invasive carcinoma (BRCA), Head and Neck squamous cell carcinoma 134 (HNSC), Kidney renal clear cell carcinoma (KIRC), Brain Lower Grade Glioma (LGG), Liver 135 hepatocellular carcinoma (LIHC), Lung adenocarcinoma (LUAD), Lung squamous cell carcinoma 136 (LUSC), Ovarian serous cystadenocarcinoma (OV) and Stomach adenocarcinoma (STAD). RNA-Seq 137 expression and clinical data were downloaded from the Broad Institute GDAC 19. Overall survival time 138 and censoring information were extracted from the clinical follow-up data. Raw count data were 139 normalized using the DESeq2 R package 20 and then log-transformed. Datasets were selected from 140 TCGA based on the following criteria: > 300 samples with both RNASeq and survival data and > 50 141 survival events. In total, 5031 patient samples were used (see Table S1 for a patient tabulation by 142 individual dataset). 143 Results 144 Cox-nnet structure and optimization 145 Cox-nnet is the neural network extension of the Cox-PH model. We created a package suitable for high 146 dimensional datasets using the Theano math library in Python. The neural network model used in this 147 paper is shown in Figure 1 and an overview of modules in the Cox-nnet package is shown in Figure S1. Cox-nnet structure and optimization 145 Moreover, this package can be run on multiple threads or a Graphics Processing 158 Unit (GPU), and it achieves slightly faster training time compared to Random Forest and CoxBoost 159 (Figure S2B). Thus, Cox-nnet is a modern software implementation that can achieve efficient 160 computational time. 161 fully connected hidden layer and an output “Cox regression” layer. The output layer of Cox-nnet replaces 150 the linear predictors in the standard Cox-PH model. Many other functions are implemented to improve 151 the usability of the package, including CVSearch, CVProfile, CrossValidation, and TrainCoxMlp. 152 CVSearch, CVProfile, CrossValidation are methods that perform CV to find the optimal regularization 153 parameter. TrainCoxMlp performs optimization of coefficients on the regularized partial likelihood 154 function. The optimization strategies include momentum gradient descent 21, Nesterov accelerated 155 gradient 22 and Ada Delta 23. A comparison of these descent methods is shown in Figure S2A, where 156 Nesterov accelerated gradient method achieved the best efficiency based on TCGA kidney renal clear cell 157 carcinoma (KIRC) data. Moreover, this package can be run on multiple threads or a Graphics Processing 158 Unit (GPU), and it achieves slightly faster training time compared to Random Forest and CoxBoost 159 (Figure S2B). Thus, Cox-nnet is a modern software implementation that can achieve efficient 160 computational time. 161 Cox-nnet structure and optimization 145 9 Cox-nnet is the neural network extension of the Cox-PH model. We created a package suitable for high 146 dimensional datasets using the Theano math library in Python. The neural network model used in this 147 paper is shown in Figure 1 and an overview of modules in the Cox-nnet package is shown in Figure S1. 148 As a proof of concept, the current ANN architecture is composed of three layers: one input layer, one 149 Cox-nnet is the neural network extension of the Cox-PH model. We created a package suitable for high 146 dimensional datasets using the Theano math library in Python. The neural network model used in this 147 paper is shown in Figure 1 and an overview of modules in the Cox-nnet package is shown in Figure S1. 148 As a proof of concept, the current ANN architecture is composed of three layers: one input layer, one 149 9 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 11, 2016. ; https://doi.org/10.1101/093021 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 11, 2016. ; https://doi.org/10.1101/093021 doi: bioRxiv preprint fully connected hidden layer and an output “Cox regression” layer. The output layer of Cox-nnet replaces 150 the linear predictors in the standard Cox-PH model. Many other functions are implemented to improve 151 the usability of the package, including CVSearch, CVProfile, CrossValidation, and TrainCoxMlp. 152 CVSearch, CVProfile, CrossValidation are methods that perform CV to find the optimal regularization 153 parameter. TrainCoxMlp performs optimization of coefficients on the regularized partial likelihood 154 function. The optimization strategies include momentum gradient descent 21, Nesterov accelerated 155 gradient 22 and Ada Delta 23. A comparison of these descent methods is shown in Figure S2A, where 156 Nesterov accelerated gradient method achieved the best efficiency based on TCGA kidney renal clear cell 157 carcinoma (KIRC) data. reports 15,16,24. A log-ranked p-value is then computed to differentiate the Kaplan-Meier survival curves 174 from these two groups. 175 The comparison of C-indices among the four methods over the 10 TCGA data is shown in Figure 2A. 176 Overall, Cox-nnet has higher predictive accuracy over the other three methods, regardless of the 177 regularization method. Cox-PH performs the second best, followed by CoxBoost and RF-S in descending 178 order (Figure 2B). The comparison of log-ranked p-values on the dichotomized survival risk groups is 179 shown in Figure S3. Generally, log-ranked p-values in the 10 TCGA datasets are more significant in Cox- 180 nnet, compared to other methods. However, the dichotomization of patients ignores the differences within 181 each dichotomized group, thus the resulting log-ranked p-values are less consistent than C-indices on the 182 same data. 183 The comparison of C-indices among the four methods over the 10 TCGA data is shown in Figure 2A. 176 Overall, Cox-nnet has higher predictive accuracy over the other three methods, regardless of the 177 regularization method. Cox-PH performs the second best, followed by CoxBoost and RF-S in descending 178 order (Figure 2B). The comparison of log-ranked p-values on the dichotomized survival risk groups is 179 shown in Figure S3. Generally, log-ranked p-values in the 10 TCGA datasets are more significant in Cox- 180 nnet, compared to other methods. However, the dichotomization of patients ignores the differences within 181 each dichotomized group, thus the resulting log-ranked p-values are less consistent than C-indices on the 182 same data. 183 Performance comparison of survival prediction methods 162 CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 11, 2016. ; https://doi.org/10.1101/093021 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 11, 2016. ; https://doi.org/10.1101/093021 doi: bioRxiv preprint reports 15,16,24. A log-ranked p-value is then computed to differentiate the Kaplan-Meier survival curves 174 from these two groups. 175 Performance comparison of survival prediction methods 162 We compared four methods, including Cox-nnet, Cox-PH, CoxBoost and RF-S, on 10 datasets from The 163 Cancer Genome Atlas (TCGA), which were selected based on having at least 50 death events (Table S1). 164 For each dataset, we trained the model on 80% of the randomly selected samples and determined the 165 regularization parameter using 5-fold CV on the training set. We used two types of regularizations, L2 166 ridge regularization (also known as weight decay) and dropout regularization. We evaluated the 167 performance on the remaining 20% holdout test set. Two metrics are used to evaluate the performance of 168 the model. The first one is Harrell’s concordance index (C-index) calculated for censored survival data 169 10,24. It evaluates the relative ordering of the samples and ranges between 0 and 1, with 0.5 equivalent to a 170 random process. The second metric is the log-ranked p-value from Kaplan-Meier survival curves of two 171 different survival risk groups. This is done by using the median threshold of Prognosis Index (PI), the 172 output of Cox-nnet, to dichotomize the patients into higher and lower risk groups, similar to our earlier 173 We compared four methods, including Cox-nnet, Cox-PH, CoxBoost and RF-S, on 10 datasets from The 163 Cancer Genome Atlas (TCGA), which were selected based on having at least 50 death events (Table S1). 164 For each dataset, we trained the model on 80% of the randomly selected samples and determined the 165 regularization parameter using 5-fold CV on the training set. We used two types of regularizations, L2 166 ridge regularization (also known as weight decay) and dropout regularization. We evaluated the 167 performance on the remaining 20% holdout test set. Two metrics are used to evaluate the performance of 168 the model. The first one is Harrell’s concordance index (C-index) calculated for censored survival data 169 10,24. It evaluates the relative ordering of the samples and ranges between 0 and 1, with 0.5 equivalent to a 170 random process. The second metric is the log-ranked p-value from Kaplan-Meier survival curves of two 171 different survival risk groups. This is done by using the median threshold of Prognosis Index (PI), the 172 output of Cox-nnet, to dichotomize the patients into higher and lower risk groups, similar to our earlier 173 10 . Biological relevance of hidden layer nodes of Cox-nnet 184 To explore the biological relevance of the hidden nodes of Cox-nnet, we used the TCGA KIRC dataset as 185 an example. We first extracted the contribution of each hidden node to the PI score for each patient 186 (Figure 3A). The contribution was calculated as the output value of each hidden node weighted by the 187 corresponding coefficient at the Cox regression output layer. As expected, the value of the hidden nodes 188 strongly correlated to the PI score. However, there is still significant heterogeneity among the nodes, 189 suggesting that individual nodes may reflect different biological processes. We hypothesize that the top 190 nodes may serve as surrogate features to discriminate patient survival. To explore this idea, we selected 191 the top 20 nodes with the highest variances, and presented the patients PI scores using t-SNE, a popular 192 method to enhance the separation among samples25. The nodes represent a dimension reduction of the 193 original data and clearly discriminate samples by their PI scores (Figure 3B). In contrast, the top 20 194 principle components obtained from principal component analysis (PCA) in combination with t-SNE fail 195 to separate the patient samples (Figure 3B). This drastic difference demonstrates that the nodes in Cox- 196 nnet effectively capture the survival information, and the top node PI scores can be used as features for 197 dimension reduction in survival analysis. 198 To explore the biological relevance of the hidden nodes of Cox-nnet, we used the TCGA KIRC dataset as 185 an example. We first extracted the contribution of each hidden node to the PI score for each patient 186 (Figure 3A). The contribution was calculated as the output value of each hidden node weighted by the 187 corresponding coefficient at the Cox regression output layer. As expected, the value of the hidden nodes 188 strongly correlated to the PI score. However, there is still significant heterogeneity among the nodes, 189 suggesting that individual nodes may reflect different biological processes. We hypothesize that the top 190 nodes may serve as surrogate features to discriminate patient survival. To explore this idea, we selected 191 the top 20 nodes with the highest variances, and presented the patients PI scores using t-SNE, a popular 192 method to enhance the separation among samples25. The nodes represent a dimension reduction of the 193 original data and clearly discriminate samples by their PI scores (Figure 3B). Biological relevance of hidden layer nodes of Cox-nnet 184 In contrast, the top 20 194 principle components obtained from principal component analysis (PCA) in combination with t-SNE fail 195 to separate the patient samples (Figure 3B). This drastic difference demonstrates that the nodes in Cox- 196 nnet effectively capture the survival information, and the top node PI scores can be used as features for 197 dimension reduction in survival analysis. 198 11 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 11, 2016. ; https://doi.org/10.1101/093021 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 11, 2016. ; https://doi.org/10.1101/093021 doi: ioRxiv preprint To further explore the biological relevance of the top 20 hidden nodes, we conducted Gene Set 199 Enrichment Analysis (GSEA)26 using KEGG pathways 27. We calculated significantly enriched pathways 200 using gene correlation to the output score of each node (Figure 3C and Table S2), and compared these 201 enriched pathways to those from GSEA of the Cox-PH model (Table S3). To calculate statistical 202 significance of the pathways, we performed 10,000 permutations, followed by multiple hypothesis testing 203 with Benjamini Hochberg adjustment. A total of 110 (out of 187) significantly enriched pathways (Table 204 S2) were identified in at least one node, including seven pathways enriched in all 20 nodes that were not 205 found by the Cox-PH method (Table 1). In contrast, Cox-PH only identified 30 significantly enriched 206 pathways using the same significance threshold. Among the seven pathways, the P53 signaling pathway 207 stands out as an important biologically relevant pathway (Figure 4 and Figure S4), since it was shown to 208 be highly prognostic of patient survival in kidney cancer28. 209 Next, we estimated the predicative accuracies of the leading edge genes (LEG) enriched in the KEGG 210 pathways from Cox-nnet vs. those enriched in Cox-PH model. We used the C-index of each LEG, 211 obtained from single-variable analysis (Figure 4). Biological relevance of hidden layer nodes of Cox-nnet 184 Collectively, LEGs from Cox-nnet have significantly 212 higher C-index scores (p = 5.79e-05) than those from Cox-PH, suggesting that Cox-nnet has selected 213 more informative features. In order to visualize these gene level and pathway level differences between 214 Cox-nnet and Cox-PH, we reconstructed a bipartite graph between LEGs for Cox-nnet or feature genes 215 (for Cox-PH) and their corresponding enriched pathways (Figure 5). Besides P53 pathway mentioned 216 earlier that is specific to Cox-nnet, several other pathways, such as insulin signaling pathway, endocytosis 217 and adherens junction, also have many more genes enriched in Cox-nnet. Among them, some have been 218 previously reported to relevant to renal carcinoma development and prognosis, such as CASP929, 219 TGFBR230, KDR (VEGFR)31. These results demonstrate that Cox-nnet model reveals richer biological 220 information than Cox-PH. 221 Next, we estimated the predicative accuracies of the leading edge genes (LEG) enriched in the KEGG 210 pathways from Cox-nnet vs. those enriched in Cox-PH model. We used the C-index of each LEG, 211 obtained from single-variable analysis (Figure 4). Collectively, LEGs from Cox-nnet have significantly 212 higher C-index scores (p = 5.79e-05) than those from Cox-PH, suggesting that Cox-nnet has selected 213 more informative features. In order to visualize these gene level and pathway level differences between 214 Cox-nnet and Cox-PH, we reconstructed a bipartite graph between LEGs for Cox-nnet or feature genes 215 (for Cox-PH) and their corresponding enriched pathways (Figure 5). Besides P53 pathway mentioned 216 earlier that is specific to Cox-nnet, several other pathways, such as insulin signaling pathway, endocytosis 217 and adherens junction, also have many more genes enriched in Cox-nnet. Among them, some have been 218 previously reported to relevant to renal carcinoma development and prognosis, such as CASP929, 219 TGFBR230, KDR (VEGFR)31. These results demonstrate that Cox-nnet model reveals richer biological 220 Next, we estimated the predicative accuracies of the leading edge genes (LEG) enriched in the KEGG 210 pathways from Cox-nnet vs. those enriched in Cox-PH model. Biological relevance of hidden layer nodes of Cox-nnet 184 We used the C-index of each LEG, 211 To further examine the importance of each gene relative to the survival outcome, we calculated the 222 averaged partial derivative (PaD) of each input gene feature over all patients, with respect to the linear 223 To further examine the importance of each gene relative to the survival outcome, we calculated the 222 averaged partial derivative (PaD) of each input gene feature over all patients, with respect to the linear 223 12 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 11, 2016. ; https://doi.org/10.1101/093021 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 11, 2016. ; https://doi.org/10.1101/093021 doi: bioRxiv preprint output of the model (e.g., the log hazard ratio). As demonstrated by the LEGs in seven common pathways 224 of all nodes in Cox-nnet, the feature importance scores produce stronger biological insight (Figure S4). 225 For example, the feature importance for the BAI1 gene in the P53 pathway is much higher in the Cox- 226 nnet model compared to the Cox-PH model. Corresponding to our finding, the BAI gene family was 227 found to be involved in several types of cancers including renal cancer32 33 34 35. BAI1 acts as an inhibitor 228 to angiogenesis and is transcriptionally regulated by P53 36. Its expression level was significantly 229 decreased in tumor vs. normal kidney tissue, and was even lower in advanced stage renal carcinoma35. 230 Mice kidney cancer models treated with BAI1 showed slower tumor growth and proliferation 37. 231 Additionally, the MAPK1 gene (also known as ERK2) has a much higher feature importance score in 232 Cox-nnet compared to Cox-PH, and is annotated in the Adherens Junction pathway as well as the Insulin 233 Signalling Pathway found by Cox-nnet. MAPK1 is one of the key kinases in intra-cellular transduction, 234 and was found constitutively activated in renal cell carcinoma 38. Drugs inhibiting the MAPK cascade 235 have been targeted for development39. Biological relevance of hidden layer nodes of Cox-nnet 184 236 output of the model (e.g., the log hazard ratio). As demonstrated by the LEGs in seven common pathways 224 of all nodes in Cox-nnet, the feature importance scores produce stronger biological insight (Figure S4). 225 For example, the feature importance for the BAI1 gene in the P53 pathway is much higher in the Cox- 226 nnet model compared to the Cox-PH model. Corresponding to our finding, the BAI gene family was 227 found to be involved in several types of cancers including renal cancer32 33 34 35. BAI1 acts as an inhibitor 228 to angiogenesis and is transcriptionally regulated by P53 36. Its expression level was significantly 229 decreased in tumor vs. normal kidney tissue, and was even lower in advanced stage renal carcinoma35. 230 Mice kidney cancer models treated with BAI1 showed slower tumor growth and proliferation 37. 231 Additionally, the MAPK1 gene (also known as ERK2) has a much higher feature importance score in 232 Cox-nnet compared to Cox-PH, and is annotated in the Adherens Junction pathway as well as the Insulin 233 Signalling Pathway found by Cox-nnet. MAPK1 is one of the key kinases in intra-cellular transduction, 234 and was found constitutively activated in renal cell carcinoma 38. Drugs inhibiting the MAPK cascade 235 have been targeted for development39. 236 Discussion 238 Additionally, it is possible to embed a priori 263 biological pathway information into the network architecture, e.g., by connecting genes in a pathway to a 264 common node in the next hidden layer of neurons. In the future, we plan to further analyze how different 265 neural network architectures affect the performance of Cox-nnet and compare the biological insights from 266 the various models. 267 Discussion 238 In this report, we have implemented Cox-nnet, a new non-linear ANN method, to predict patient survival 239 from high throughput omics data. Cox-nnet is an improved, modern alternative to the standard Cox-PH 240 regression, as demonstrated by increased performance for survival prediction and the capabilities to 241 explore more deeply the biological information. 242 In this report, we have implemented Cox-nnet, a new non-linear ANN method, to predict patient survival 239 from high throughput omics data. Cox-nnet is an improved, modern alternative to the standard Cox-PH 240 regression, as demonstrated by increased performance for survival prediction and the capabilities to 241 explore more deeply the biological information. 242 In this report, we have implemented Cox-nnet, a new non-linear ANN method, to predict patient survival 239 from high throughput omics data. Cox-nnet is an improved, modern alternative to the standard Cox-PH 240 regression, as demonstrated by increased performance for survival prediction and the capabilities to 241 explore more deeply the biological information. 242 First, through in-depth comparison of 10 TCGA RNA-Seq, Cox-nnet achieves overall statistically 243 significant improvements over Cox-PH on its predictive accuracy, as measured by C-indices. 244 Interestingly, the ensemble-based method RF-S consistently ranks worse than Cox-nnet and Cox-PH. 245 Because RF-S bootstraps both samples and features for individual trees, many uninformative features in 246 each tree may be chosen for node splitting in particularly high dimensional datasets, leading to a decrease 247 Interestingly, the ensemble-based method RF-S consistently ranks worse than Cox-nnet and Cox-PH. 245 Because RF-S bootstraps both samples and features for individual trees, many uninformative features in 246 each tree may be chosen for node splitting in particularly high dimensional datasets, leading to a decrease 247 13 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 11, 2016. ; https://doi.org/10.1101/093021 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 11, 2016. ; https://doi.org/10.1101/093021 doi: bioRxiv preprint in overall accuracy 40. Discussion 238 In contrast, the dropout and L2-regularization approach used by both Cox-nnet and 248 Cox-PH can prune out uninformative features. 249 in overall accuracy 40. In contrast, the dropout and L2-regularization approach used by both Cox-nnet and 248 Cox-PH can prune out uninformative features. 249 Second, Cox-nnet can reveal a lot richer biological information than Cox-PH. This is manifested both at 250 the pathway and gene levels. The hidden nodes in the Cox-nnet model have distinct expression patterns, 251 and can serve as surrogate features for survival-sensitive dimension reduction. Many more significant 252 KEGG pathways are enriched which correlate with top nodes in Cox-nnet, as compared to those from the 253 Cox-PH model. A critical pathway for renal cancer development, P53 pathway, is only enriched by Cox- 254 nnet but not Cox-PH model in TCGA KIRC. Other pathways, including insulin signaling pathway, 255 endocytosis and adherens junction, have many more genes enriched by Cox-mmet. Moreover, leading 256 edge genes (LEGs) obtained from these KEGG pathways enriched by Cox-nnet (which are a fraction of 257 the gene features considered by the model) have collectively higher associations with survival. 258 As a promising new predictive method for prognosis, the current Cox-nnet implementation has some 259 limitations. Its architecture includes 3-layer ANN, and it is possible to incorporate other more 260 sophisticated architecture into the model, such as including more layers of neurons. A convolutional 261 neural network approach using convolutional and pooling layers could also be used, as those reported in 262 processing imaging or other types of positional data 41. Additionally, it is possible to embed a priori 263 biological pathway information into the network architecture, e.g., by connecting genes in a pathway to a 264 common node in the next hidden layer of neurons. In the future, we plan to further analyze how different 265 neural network architectures affect the performance of Cox-nnet and compare the biological insights from 266 the various models. 267 As a promising new predictive method for prognosis, the current Cox-nnet implementation has some 259 limitations. Its architecture includes 3-layer ANN, and it is possible to incorporate other more 260 sophisticated architecture into the model, such as including more layers of neurons. A convolutional 261 neural network approach using convolutional and pooling layers could also be used, as those reported in 262 processing imaging or other types of positional data 41. Competing financial interests 272 The author(s) declare no competing financial interests. 273 The author(s) declare no competing financial interests. 273 The author(s) declare no competing financial interests. 273 Acknowledgements 274 This research was supported by grants K01ES025434 awarded by NIEHS thro 275 trans-NIH Big Data to Knowledge (BD2K) initiative (www.bd2k.nih.gov), P2 276 awarded by NIH/NIGMS, R01 LM012373 awarded by NLM, R01 HD084633 277 Hawaii Community Foundation Medical Research Grant 14ADVC-64566 to L 278 References 279 1 McCulloch, W. S. & Pitts, W. A logical calculus of the ideas immanent 280 bulletin of mathematical biophysics 5, 115-133 (1943). 281 2 Jones, N. (Nature Publishing Group MACMILLAN BUILDING, 4 CRINA 282 ENGLAND, 2014). 283 3 Faraggi, D. & Simon, R. A neural network model for survival data. Stat 284 (1995). 285 4 Petalidis, L. P. et al. Improved grading and survival prediction of huma 286 by artificial neural network analysis of gene expression microarray da 287 therapeutics 7, 1013-1024 (2008). 288 5 Chi, C.-L., Street, W. N. & Wolberg, W. H. in AMIA Annual Symposium 289 (American Medical Informatics Association). 290 6 Joshi, R. & Reeves, C. in Proceedings of the eighteenth international c 291 engineering. 179-184. 292 7 Therneau, T. M. & Grambsch, P. M. Modeling survival data: extending 293 Science & Business Media, 2000). 294 8 Binder, H. CoxBoost: Cox models by likelihood based boosting for a si 295 competing risks. R package version 1 (2013). 296 9 Ishwaran, H., Kogalur, U. B., Blackstone, E. H. & Lauer, M. S. Random 297 of Applied Statistics, 841-860 (2008). 298 10 Koziol, J. A. & Jia, Z. The concordance index C and the Mann–Whitney 299 randomly censored data. Biometrical Journal 51, 467-474 (2009). 300 11 van Houwelingen, H. C., Bruinsma, T., Hart, A. A., van't Veer, L. J. & W 301 Cox regression on microarray gene expression data. Statistics in medi 302 12 Haykin, S. & Network, N. A comprehensive foundation. Neural Netwo 303 13 Srivastava, N., Hinton, G. E., Krizhevsky, A., Sutskever, I. & Salakhutdin 304 way to prevent neural networks from overfitting. Journal of Machine 305 1929 1958 (2014) 306 Acknowledgements 274 This research was supported by grants K01ES025434 awarded by NIEHS through funds provided by the 275 trans-NIH Big Data to Knowledge (BD2K) initiative (www.bd2k.nih.gov), P20 COBRE GM103457 276 awarded by NIH/NIGMS, R01 LM012373 awarded by NLM, R01 HD084633 awarded by NICHD, and 277 Hawaii Community Foundation Medical Research Grant 14ADVC-64566 to L.X. Competing financial interests 272 Garmire. 278 References 279 1 McCulloch, W. S. & Pitts, W. A logical calculus of the ideas immanent in nervous activity. The 280 bulletin of mathematical biophysics 5, 115-133 (1943). 281 2 Jones, N. (Nature Publishing Group MACMILLAN BUILDING, 4 CRINAN ST, LONDON N1 9XW, 282 ENGLAND, 2014). 283 3 Faraggi, D. & Simon, R. A neural network model for survival data. Statistics in medicine 14, 73-82 284 (1995). 285 4 Petalidis, L. P. et al. Improved grading and survival prediction of human astrocytic brain tumors 286 by artificial neural network analysis of gene expression microarray data. Molecular cancer 287 therapeutics 7, 1013-1024 (2008). 288 5 Chi, C.-L., Street, W. N. & Wolberg, W. H. in AMIA Annual Symposium Proceedings. 130 289 (American Medical Informatics Association). 290 6 Joshi, R. & Reeves, C. in Proceedings of the eighteenth international conference on systems 291 engineering. 179-184. 292 7 Therneau, T. M. & Grambsch, P. M. Modeling survival data: extending the Cox model. (Springer 293 Science & Business Media, 2000). 294 8 Binder, H. CoxBoost: Cox models by likelihood based boosting for a single survival endpoint or 295 competing risks. R package version 1 (2013). 296 9 Ishwaran, H., Kogalur, U. B., Blackstone, E. H. & Lauer, M. S. Random survival forests. The Annals 297 of Applied Statistics, 841-860 (2008). 298 10 Koziol, J. A. & Jia, Z. The concordance index C and the Mann–Whitney parameter Pr (X> Y) with 299 randomly censored data. Biometrical Journal 51, 467-474 (2009). 300 11 van Houwelingen, H. C., Bruinsma, T., Hart, A. A., van't Veer, L. J. & Wessels, L. F. Cross‐validated 301 Cox regression on microarray gene expression data. Statistics in medicine 25, 3201-3216 (2006). 302 12 Haykin, S. & Network, N. A comprehensive foundation. Neural Networks 2 (2004). 303 13 Srivastava, N., Hinton, G. E., Krizhevsky, A., Sutskever, I. & Salakhutdinov, R. Dropout: a simple 304 way to prevent neural networks from overfitting. Journal of Machine Learning Research 15, 305 1929-1958 (2014). 306 Author contributions 268 LXG and TC envisioned the project and designed the work. TC coded the project and conducted the 269 analysis. XZ provided insight and discussion on neural networks. All authors have read, revised and 270 approved the final manuscript. 271 LXG and TC envisioned the project and designed the work. TC coded the project and conducted the 269 analysis. XZ provided insight and discussion on neural networks. All authors have read, revised and 270 approved the final manuscript. 271 14 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 11, 2016. ; https://doi.org/10.1101/093021 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 11, 2016. ; https://doi.org/10.1101/093021 doi: bioRxiv preprint Acknowledgements 274 This research was supported by grants K01ES025434 awarded by NIEHS through funds provided by the 275 trans-NIH Big Data to Knowledge (BD2K) initiative (www.bd2k.nih.gov), P20 COBRE GM103457 276 awarded by NIH/NIGMS, R01 LM012373 awarded by NLM, R01 HD084633 awarded by NICHD, and 277 Hawaii Community Foundation Medical Research Grant 14ADVC-64566 to L.X. Garmire. 278 This research was supported by grants K01ES025434 awarded by NIEHS through funds provided by the 275 trans-NIH Big Data to Knowledge (BD2K) initiative (www.bd2k.nih.gov), P20 COBRE GM103457 276 awarded by NIH/NIGMS, R01 LM012373 awarded by NLM, R01 HD084633 awarded by NICHD, and 277 Hawaii Community Foundation Medical Research Grant 14ADVC-64566 to L.X. Garmire. 278 1 McCulloch, W. S. & Pitts, W. A logical calculus of the ideas immanent in nervous activity. The 280 bulletin of mathematical biophysics 5, 115-133 (1943). 281 2 Jones, N. (Nature Publishing Group MACMILLAN BUILDING, 4 CRINAN ST, LONDON N1 9XW, 282 ENGLAND, 2014). 283 3 Faraggi, D. & Simon, R. A neural network model for survival data. Statistics in medicine 14, 73-82 284 (1995). 285 4 Petalidis, L. P. et al. Improved grading and survival prediction of human astrocytic brain tumors 286 by artificial neural network analysis of gene expression microarray data. Molecular cancer 287 therapeutics 7, 1013-1024 (2008). 288 5 Chi, C.-L., Street, W. N. & Wolberg, W. H. in AMIA Annual Symposium Proceedings. 130 289 (American Medical Informatics Association). 290 6 Joshi, R. & Reeves, C. in Proceedings of the eighteenth international conference on systems 291 engineering. 179-184. 292 7 Therneau, T. M. & Grambsch, P. M. Modeling survival data: extending the Cox model. (Springer 293 Science & Business Media, 2000). 294 8 Binder, H. CoxBoost: Cox models by likelihood based boosting for a single survival endpoint or 295 competing risks. R package version 1 (2013). 296 9 Ishwaran, H., Kogalur, U. B., Blackstone, E. H. & Lauer, M. S. Random survival forests. The Annals 297 of Applied Statistics, 841-860 (2008). 298 10 Koziol, J. A. & Jia, Z. The concordance index C and the Mann–Whitney parameter Pr (X> Y) with 299 randomly censored data. Biometrical Journal 51, 467-474 (2009). 300 11 van Houwelingen, H. C., Bruinsma, T., Hart, A. A., van't Veer, L. J. & Wessels, L. F. Cross‐validated 301 Cox regression on microarray gene expression data. Statistics in medicine 25, 3201-3216 (2006). 302 12 Haykin, S. & Network, N. A comprehensive foundation. Neural Networks 2 (2004). Acknowledgements 274 Brain-specific angiogenesis inhibitor 1 expression is inversely co 352 vascularity and distant metastasis of colorectal cancer. International journal of onco 353 970 (1998). 354 14 Harrell, F. E., Lee, K. L. & Mark, D. B. Tutorial in biostatistics multivariable prognostic models: 307 issues in developing models, evaluating assumptions and adequacy, and measuring and reducing 308 errors. Statistics in medicine 15, 361-387 (1996). 309 14 Harrell, F. E., Lee, K. L. & Mark, D. B. Tutorial in biostatistics multivariable prognostic models: 307 issues in developing models, evaluating assumptions and adequacy, and measuring and reducing 308 errors. Statistics in medicine 15, 361-387 (1996). 309 15 Huang, S., Yee, C., Ching, T., Yu, H. & Garmire, L. X. A Novel Model to Combine Clinical and 310 Pathway-Based Transcriptomic Information for the Prognosis Prediction of Breast Cancer. PLoS 311 computational biology 10, e1003851 (2014). 312 16 Huang, S. et al. Novel personalized pathway-based metabolomics models reveal key metabolic 313 pathways for breast cancer diagnosis. Genome medicine 8, 1 (2016). 314 17 Gevrey, M., Dimopoulos, I. & Lek, S. Review and comparison of methods to study the 315 contribution of variables in artificial neural network models. Ecological modelling 160, 249-264 316 (2003). 317 18 Olden, J. D., Joy, M. K. & Death, R. G. An accurate comparison of methods for quantifying 318 variable importance in artificial neural networks using simulated data. Ecological Modelling 178, 319 389-397 (2004). 320 19 Broad. Broad Institute TCGA Genome Data Analysis Center (2014): Analysis Overview for 15 July 321 2014. Broad Institute of MIT and Harvard, doi:10.7908/C1DN43V9 (2014). 322 20 Love, M., Anders, S. & Huber, W. Differential analysis of RNA-Seq data at the gene level using 323 the DESeq2 package. (2013). 324 21 Qian, N. On the momentum term in gradient descent learning algorithms. Neural networks 12, 325 145-151 (1999). 326 22 Bengio, Y., Boulanger-Lewandowski, N. & Pascanu, R. in Acoustics, Speech and Signal Processing 327 (ICASSP), 2013 IEEE International Conference on. 8624-8628 (IEEE). 328 23 Battiti, R. Accelerated backpropagation learning: Two optimization methods. Complex systems 3, 329 331-342 (1989). 330 24 Wei, R. et al. Meta-dimensional data integration identifies critical pathways for susceptibility, 331 tumorigenesis and progression of endometrial cancer. Oncotarget (2016). 332 25 Maaten, L. v. d. & Hinton, G. Visualizing data using t-SNE. Journal of Machine Learning Research 333 9, 2579-2605 (2008). 334 26 Subramanian, A. et al. Acknowledgements 274 Gene set enrichment analysis: a knowledge-based approach for 335 interpreting genome-wide expression profiles. Proceedings of the National Academy of Sciences 336 102, 15545-15550 (2005). 337 , ( ) 27 Kanehisa, M. & Goto, S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic acids research 338 28, 27-30 (2000). 339 27 Kanehisa, M. & Goto, S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic acids research 338 28, 27-30 (2000). 339 Girgin, C. et al. P53 mutations and other prognostic factors of renal cell carcinoma. Urol internationalis 66, 78-83 (2001). 28 Girgin, C. et al. P53 mutations and other prognostic factors of renal cell carcinoma. Urologia 340 internationalis 66, 78-83 (2001). 341 28 Girgin, C. et al. P53 mutations and other prognostic factors of renal cell carcinoma. Urologia 340 internationalis 66, 78-83 (2001). 341 29 Marques, I. et al. Influence of survivin (BIRC5) and caspase-9 (CASP9) functional polymorphisms 342 in renal cell carcinoma development: a study in a southern European population. Molecular 343 biology reports 40, 4819-4826 (2013). 344 29 Marques, I. et al. Influence of survivin (BIRC5) and caspase-9 (CASP9) functional polymorphisms 342 in renal cell carcinoma development: a study in a southern European population. Molecular 343 biology reports 40, 4819-4826 (2013). 344 29 Marques, I. et al. Influence of survivin (BIRC5) and caspase-9 (CASP9) functional polymorphisms 342 in renal cell carcinoma development: a study in a southern European population. Molecular 343 biology reports 40, 4819-4826 (2013). 344 30 Akhurst, R. J. & Derynck, R. TGF-β signaling in cancer–a double-edged sword. Trends in cell 345 biology 11, S44-S51 (2001). 346 30 Akhurst, R. J. & Derynck, R. TGF-β signaling in cancer–a double-edged sword. Trends in cell 345 biology 11, S44-S51 (2001). 346 30 Akhurst, R. J. & Derynck, R. TGF-β signaling in cancer–a double-edged sword. Trends in cell 345 biology 11, S44-S51 (2001). 346 31 Choueiri, T. K. et al. Phase II and biomarker study of the dual MET/VEGFR2 inhibitor foretinib in 347 patients with papillary renal cell carcinoma. Journal of Clinical Oncology 31, 181-186 (2013). 348 31 Choueiri, T. K. et al. Phase II and biomarker study of the dual MET/VEGFR2 inhibitor foretinib in 347 patients with papillary renal cell carcinoma. Journal of Clinical Oncology 31, 181-186 (2013). 348 31 Choueiri, T. K. et al. Phase II and biomarker study of the dual MET/VEGFR2 inhibitor foretinib in 347 patients with papillary renal cell carcinoma. Acknowledgements 274 303 13 Srivastava, N., Hinton, G. E., Krizhevsky, A., Sutskever, I. & Salakhutdinov, R. Dropout: a simple 304 way to prevent neural networks from overfitting. Journal of Machine Learning Research 15, 305 1929-1958 (2014). 306 8 Binder, H. CoxBoost: Cox models by likelihood based boosting for a single survival endpoint or 295 competing risks. R package version 1 (2013). 296 9 Ishwaran, H., Kogalur, U. B., Blackstone, E. H. & Lauer, M. S. Random survival forests. The Annals 297 of Applied Statistics, 841-860 (2008). 298 10 Koziol, J. A. & Jia, Z. The concordance index C and the Mann–Whitney parameter Pr (X> Y) with 299 randomly censored data. Biometrical Journal 51, 467-474 (2009). 300 13 Srivastava, N., Hinton, G. E., Krizhevsky, A., Sutskever, I. & Salakhutdinov, R. Dropout: a simple 304 way to prevent neural networks from overfitting. Journal of Machine Learning Research 15, 305 1929-1958 (2014). 306 13 Srivastava, N., Hinton, G. E., Krizhevsky, A., Sutskever, I. & Salakhutdinov, R. Dropout: a simple 304 way to prevent neural networks from overfitting. Journal of Machine Learning Research 15, 305 1929-1958 (2014). 306 15 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 11, 2016. ; https://doi.org/10.1101/093021 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 11, 2016. ; https://doi.org/10.1101/093021 doi: bioRxiv preprint 14 Harrell, F. E., Lee, K. L. & Mark, D. B. Tutorial in biostatistics multivariable prognosti 307 issues in developing models, evaluating assumptions and adequacy, and measuring 308 errors. Statistics in medicine 15, 361-387 (1996). 309 15 Huang, S., Yee, C., Ching, T., Yu, H. & Garmire, L. X. A Novel Model to Combine Clinic 310 Pathway-Based Transcriptomic Information for the Prognosis Prediction of Breast C 311 computational biology 10, e1003851 (2014). 312 16 Huang, S. et al. Novel personalized pathway-based metabolomics models reveal key 313 pathways for breast cancer diagnosis. Genome medicine 8, 1 (2016). 314 17 Gevrey, M., Dimopoulos, I. & Lek, S. Acknowledgements 274 Review and comparison of methods to study th 315 contribution of variables in artificial neural network models. Ecological modelling 16 316 (2003). 317 18 Olden, J. D., Joy, M. K. & Death, R. G. An accurate comparison of methods for quant 318 variable importance in artificial neural networks using simulated data. Ecological M 319 389-397 (2004). 320 19 Broad. Broad Institute TCGA Genome Data Analysis Center (2014): Analysis Overvie 321 2014. Broad Institute of MIT and Harvard, doi:10.7908/C1DN43V9 (2014). 322 20 Love, M., Anders, S. & Huber, W. Differential analysis of RNA-Seq data at the gene l 323 the DESeq2 package. (2013). 324 21 Qian, N. On the momentum term in gradient descent learning algorithms. Neural ne 325 145-151 (1999). 326 22 Bengio, Y., Boulanger-Lewandowski, N. & Pascanu, R. in Acoustics, Speech and Signa 327 (ICASSP), 2013 IEEE International Conference on. 8624-8628 (IEEE). 328 23 Battiti, R. Accelerated backpropagation learning: Two optimization methods. Compl 329 331-342 (1989). 330 24 Wei, R. et al. Meta-dimensional data integration identifies critical pathways for susc 331 tumorigenesis and progression of endometrial cancer. Oncotarget (2016). 332 25 Maaten, L. v. d. & Hinton, G. Visualizing data using t-SNE. Journal of Machine Learni 333 9, 2579-2605 (2008). 334 26 Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach 335 interpreting genome-wide expression profiles. Proceedings of the National Academ 336 102, 15545-15550 (2005). 337 27 Kanehisa, M. & Goto, S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic a 338 28, 27-30 (2000). 339 28 Girgin, C. et al. P53 mutations and other prognostic factors of renal cell carcinoma. 340 internationalis 66, 78-83 (2001). 341 29 Marques, I. et al. Influence of survivin (BIRC5) and caspase-9 (CASP9) functional pol 342 in renal cell carcinoma development: a study in a southern European population. M 343 biology reports 40, 4819-4826 (2013). 344 30 Akhurst, R. J. & Derynck, R. TGF-β signaling in cancer–a double-edged sword. Trends 345 biology 11, S44-S51 (2001). 346 31 Choueiri, T. K. et al. Phase II and biomarker study of the dual MET/VEGFR2 inhibitor 347 patients with papillary renal cell carcinoma. Journal of Clinical Oncology 31, 181-186 348 32 Cork, S. M. & Van Meir, E. G. Emerging roles for the BAI1 protein family in the regul 349 phagocytosis, synaptogenesis, neurovasculature, and tumor development. Journal o 350 medicine 89, 743-752 (2011). 351 33 Fukushima, Y. et al. Acknowledgements 274 Journal of Clinical Oncology 31, 181-186 (2013). 348 p p p y f gy , ( ) 32 Cork, S. M. & Van Meir, E. G. Emerging roles for the BAI1 protein family in the regulation of 349 phagocytosis, synaptogenesis, neurovasculature, and tumor development. Journal of molecular 350 medicine 89, 743-752 (2011). 351 32 Cork, S. M. & Van Meir, E. G. Emerging roles for the BAI1 protein family in the regulation of 349 phagocytosis, synaptogenesis, neurovasculature, and tumor development. Journal of molecular 350 medicine 89, 743-752 (2011). 351 33 Fukushima, Y. et al. Brain-specific angiogenesis inhibitor 1 expression is inversely correlated with 352 vascularity and distant metastasis of colorectal cancer. International journal of oncology 13, 967- 353 970 (1998). 354 16 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 11, 2016. ; https://doi.org/10.1101/093021 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 11, 2016. ; https://doi.org/10.1101/093021 doi: bioRxiv preprint 34 Lee, J. et al. Comparative study of angiostatic and anti-invasive gene expressions as prognostic 355 factors in gastric cancer. International journal of oncology 18, 355-362 (2001). 356 35 Izutsu, T., Konda, R., Sugimura, J., Iwasaki, K. & Fujioka, T. Brain-specific angiogenesis inhibitor 1 357 is a putative factor for inhibition of neovascular formation in renal cell carcinoma. The Journal of 358 urology 185, 2353-2358 (2011). 359 36 Nishimori, H. et al. A novel brain-specific p53-target gene, BAI1, containing thrombospondin 360 type 1 repeats inhibits experimental angiogenesis. Oncogene 15, 2145-2150 (1997). 361 37 Kudo, S. et al. Inhibition of tumor growth through suppression of angiogenesis by brain-specific 362 angiogenesis inhibitor 1 gene transfer in murine renal cell carcinoma. Oncology reports 18, 785- 363 792 (2007). 364 38 Oka, H. et al. Constitutive activation of mitogen-activated protein (MAP) kinases in human renal 365 cell carcinoma. Cancer research 55, 4182-4187 (1995). 366 39 Friday, B. B. & Adjei, A. A. Advances in targeting the Ras/Raf/MEK/Erk mitogen-activated protein 367 kinase cascade with MEK inhibitors for cancer therapy. Acknowledgements 274 Clinical Cancer Research 14, 342-346 368 (2008). 369 40 Nguyen, T.-T., Huang, J. Z. & Nguyen, T. T. Unbiased Feature Selection in Learning Random 370 Forests for High-Dimensional Data. The Scientific World Journal 2015 (2015). 371 41 LeCun, Y. & Bengio, Y. Convolutional networks for images, speech, and time series. The 372 handbook of brain theory and neural networks 3361, 1995 (1995). 373 374 375 376 Figures 377 Figure 1. An overview of the neural network architecture used in this study. 378 35 Izutsu, T., Konda, R., Sugimura, J., Iwasaki, K. & Fujioka, T. Brain-specific angiogenesis inhibitor 1 357 is a putative factor for inhibition of neovascular formation in renal cell carcinoma. The Journal of 358 urology 185, 2353-2358 (2011). 359 36 Nishimori, H. et al. A novel brain-specific p53-target gene, BAI1, containing thrombospondin 360 type 1 repeats inhibits experimental angiogenesis Oncogene 15 2145-2150 (1997) 361 7 Kudo, S. et al. Inhibition of tumor growth through suppression of angiogenesis by brain-specific angiogenesis inhibitor 1 gene transfer in murine renal cell carcinoma. Oncology reports 18, 785- 792 (2007). 38 Oka, H. et al. Constitutive activation of mitogen-activated protein (MAP) kinases in human renal 365 cell carcinoma. Cancer research 55, 4182-4187 (1995). 366 39 Friday, B. B. & Adjei, A. A. Advances in targeting the Ras/Raf/MEK/Erk mitogen-activated protein 367 kinase cascade with MEK inhibitors for cancer therapy. Clinical Cancer Research 14, 342-346 368 (2008). 369 40 Nguyen, T.-T., Huang, J. Z. & Nguyen, T. T. Unbiased Feature Selection in Learning Random 370 Forests for High-Dimensional Data. The Scientific World Journal 2015 (2015). 371 40 Nguyen, T.-T., Huang, J. Z. & Nguyen, T. T. Unbiased Feature Selection in Learning Random 370 Forests for High-Dimensional Data. The Scientific World Journal 2015 (2015). 371 41 LeCun, Y. & Bengio, Y. Convolutional networks for images, speech, and time series. The 372 handbook of brain theory and neural networks 3361, 1995 (1995). 373 Figures 377 CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 11, 2016. ; https://doi.org/10.1101/093021 doi: bioRxiv preprint Figure 5. Enriched pathway-gene bipartite network from the leading edge genes and significantly 387 enriched pathways. Significantly enriched pathways common to all 20 hidden nodes are labeled in green. 388 Leading edge genes found uniquely in Cox-nnet are labeled in orange, and genes found in both Cox-nnet 389 and Cox-PH are labeled in blue. 390 enriched pathways. Significantly enriched pathways common to all 20 hidden nodes are labeled in green. 388 Leading edge genes found uniquely in Cox-nnet are labeled in orange, and genes found in both Cox-nnet 389 and Cox-PH are labeled in blue. 390 Table 1. Cox-nnet node-associated pathways. Significantly enriched pathways from common to all 20 391 hidden nodes that are not found in the Cox-PH Gene Set Enrichment Analysis (Adjusted p < 0.05). 392 Pathway P.value P.adjusted Nodes KEGG adherens junction 0.000 0.001 1-20 KEGG endocytosis 0.000 0.001 1-20 KEGG insulin signaling pathway 0.000 0.001 1-20 KEGG lysine degradation 0.000 0.003 1-20 KEGG p53 signaling pathway 0.000 0.003 1-20 KEGG pyruvate metabolism 0.000 0.001 1-20 KEGG sphingolipid metabolism 0.001 0.005 1-20 393 Supplemental Figures 394 Figure S1. An overview of the structure, methods and classes in Cox-nnet package. 395 Figure S2. A: comparison of descent methods on the TCGA KIRC dataset. The change in cost function 396 is evaluated over 100,000 iterations for three methods: gradient descent, momentum gradient descent and 397 the Nesterov accelerated gradient. B: Training time comparing CPU training time vs. GPU training time 398 on the same dataset. 399 Figure S3. Log-rank p-value bar plot of the 10 TCGA datasets. 400 Table 1. Cox-nnet node-associated pathways. Significantly enriched pathways from common to all 20 391 hidden nodes that are not found in the Cox-PH Gene Set Enrichment Analysis (Adjusted p < 0.05). 392 Pathway P.value P.adjusted Nodes KEGG adherens junction 0.000 0.001 1-20 KEGG endocytosis 0.000 0.001 1-20 KEGG insulin signaling pathway 0.000 0.001 1-20 KEGG lysine degradation 0.000 0.003 1-20 KEGG p53 signaling pathway 0.000 0.003 1-20 KEGG pyruvate metabolism 0.000 0.001 1-20 KEGG sphingolipid metabolism 0.001 0.005 1-20 393 Figure S3. Log-rank p-value bar plot of the 10 TCGA datasets. Figures 377 Figures 377 Figure 1. An overview of the neural network architecture used in this study. 378 Figure 2. A. Barplot of the C-index of the 10 TCGA datasets using four prognosis-predicting methods 379 (Cox-nnet, CoxBoost, Cox-PH and RF-S). Each dataset was randomly split into 80% training and 20% 380 testing sets. B. Heatmap of the performance rank of each dataset. 381 Figure 3. A. Hidden node output of the TCGA KIRC dataset. B. t-SNE plot of the top 20 hidden nodes 382 and the top 20 principal components in PCA analysis. C. Gene Set Enrichment Analysis: significantly 383 enriched KEGG pathways of the top 20 hidden nodes (adjusted p-value < 0.05). 384 Figure 4. Single variable C-index scores of the leading edge genes from Cox-nnet and Cox-PH. Cox-nn 385 has significantly higher C-index scores (p = 5.79e-5). 386 Figure 1. An overview of the neural network architecture used in this study. 378 Figure 2. A. Barplot of the C-index of the 10 TCGA datasets using four prognosis-predicting methods 379 (Cox-nnet, CoxBoost, Cox-PH and RF-S). Each dataset was randomly split into 80% training and 20% 380 testing sets. B. Heatmap of the performance rank of each dataset. 381 Figure 3. A. Hidden node output of the TCGA KIRC dataset. B. t-SNE plot of the top 20 hidden nodes 382 and the top 20 principal components in PCA analysis. C. Gene Set Enrichment Analysis: significantly 383 enriched KEGG pathways of the top 20 hidden nodes (adjusted p-value < 0.05). 384 Figure 3. A. Hidden node output of the TCGA KIRC dataset. B. t-SNE plot of the top 20 hidden nodes 382 and the top 20 principal components in PCA analysis. C. Gene Set Enrichment Analysis: significantly 383 enriched KEGG pathways of the top 20 hidden nodes (adjusted p-value < 0.05). 384 Figure 4. Single variable C-index scores of the leading edge genes from Cox-nnet and Cox-PH. Cox-nnet 385 has significantly higher C-index scores (p = 5.79e-5). 386 17 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 11, 2016. ; https://doi.org/10.1101/093021 doi: bioRxiv preprint . Figure S3. Log-rank p-value bar plot of the 10 TCGA datasets. 400 Figures 377 400 18 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 11, 2016. ; https://doi.org/10.1101/093021 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 11, 2016. ; https://doi.org/10.1101/093021 doi: bioRxiv preprint Figure S4. Variable importance of the common leading edge genes of enriched KEGG pathways. 401 402 Table S1. Tabulation of TCGA patients by individual dataset. 403 Table S2. Significantly enriched pathways from the Cox-PH method (p < 0.05). 404 Table S3. Significantly enriched pathways from the Cox-nnet method (p < 0.05). 405 406 407 Table S1. Tabulation of TCGA patients by individual dataset. 403 Table S2. Significantly enriched pathways from the Cox-PH method (p < 0.05). 404 Table S3. Significantly enriched pathways from the Cox-nnet method (p < 0.05). 405 407 19 ( ) T i H G W X b = + ( ) T T i H G W X b β = + ( ) 1 ( ) ( log exp ) i j i j C i t t pl β θ θ = ≥ = − ∑ ∑ * * ** * * ** * * ** 0.5 0.6 0.7 0.8 0.9 BLCA BRCA HNSC KIRC LGG LIHC LUAD LUSC OV STAD Dataset c−index Mtehod Cox−nnet ( Cox−nnet ( Cox−PH CoxBoost Random Fo Performance across TCGA datasets Cox-nnet s better * Performance Rank A B . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 11, 2016. Figures 377 CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 11, 2016. ; https://doi.org/10.1101/093021 doi: bioRxiv preprint Prognostic Index of patients Node value −2 0 2 Node output value . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 11, 2016. Figures 377 ; https://doi.org/10.1101/093021 doi: bioRxiv preprint node_19 node_9 node_16 node_14 node_4 node_15 node_6 node_13 node_5 node_20 node_11 node_2 node_7 node_8 node_17 node_10 node_1 node_3 node_18 node_12 MELANOGENE CALCIUM_SIG LONG_TERM_ CHRONIC_MY MELANOMA THYROID_CAN UBIQUITIN_ME GLIOMA SMALL_CELL_ ACUTE_MYEL NON_SMALL_ ERBB_SIGNAL PANCREATIC_ MISMATCH_R ARRHYTHMOG SPLICEOSOM PRION_DISEA PURINE_META GLYCOSAMIN ALZHEIMERS_ HUNTINGTON O_GLYCAN_B LONG_TERM_ GAP_JUNCTIO LYSOSOME ARGININE_AN TASTE_TRANS LEUKOCYTE_ PARKINSONS_ OXIDATIVE_PH PRIMARY_BIL AMYOTROPHI VIRAL_MYOCA ASTHMA FC_GAMMA_R T_CELL_RECE STEROID_BIO CELL_ADHES AUTOIMMUNE JAK_STAT_SIG CHEMOKINE_ ANTIGEN_PRO ALLOGRAFT_ GRAFT_VERS LEISHMANIA_ NATURAL_KIL TYPE_I_DIABE TOLL_LIKE_RE HEMATOPOIET FC_EPSILON_ BASAL_CELL_ DRUG_METAB ASCORBATE_ GLYCINE_SER PYRIMIDINE_M PATHOGENIC_ GLYCOSAMIN HISTIDINE_ME DNA_REPLICA NOD_LIKE_RE COMPLEMENT GLYCOLYSIS_ REGULATION_ BASE_EXCISI CYTOSOLIC_D RIBOSOME INTESTINAL_I VASOPRESSIN TERPENOID_B PRIMARY_IMM WNT_SIGNAL TGF_BETA_SI PATHWAYS_IN RENAL_CELL_ ALDOSTERON MTOR_SIGNA NEUROTROPH COLORECTAL FOCAL_ADHE HOMOLOGOU ADIPOCYTOK NITROGEN_M INOSITOL_PH PHOSPHATIDY BETA_ALANIN SYSTEMIC_LU PROSTATE_CA ENDOMETRIA PROXIMAL_TU INSULIN_SIGN RENIN_ANGIO TIGHT_JUNCT ADHERENS_J VASCULAR_S PEROXISOME ENDOCYTOSI P53_SIGNALIN CELL_CYCLE CYTOKINE_CY PPAR_SIGNAL PROTEASOME BUTANOATE_M PROPANOATE PYRUVATE_M SPHINGOLIPID TRYPTOPHAN LYSINE_DEGR VALINE_LEUC CITRATE_CYC FATTY_ACID_M Prognostic Index of patients Node value −2 0 2 Node output value t−SNE plot of top 20 Cox−nnet nodes B ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● −20 −10 0 10 20 −20 −10 0 10 20 30 t−SNE_1 t−SNE_2 Top 20 Cox−nnet nodes ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● −20 0 20 −25 0 25 t−SNE_1 t−SNE_2 −4 0 4 PI Top 20 principal components . Figures 377 ; https://doi.org/10.1101/093021 doi: bioRxiv preprint * * * ** * * ** BRCA HNSC KIRC LGG LIHC LUAD LUSC OV STAD Dataset Mtehod Cox−nnet (Dropout) Cox−nnet (Ridge) Cox−PH CoxBoost Random Forest Performance across TCGA datasets Cox-nnet significantly better * (Dropout) et (Ridge) Cox−PH CoxBoost RF BLCA BRCA HNSC KIRC LGG LIHC LUAD LUSC OV STAD Dataset Rank 1 2 3 4 5 Performance Rank . CC-BY 4.0 International license under a y peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 11, 2016. ; https://doi.org/10.1101/093021 doi: t * * * ** * * ** BRCA HNSC KIRC LGG LIHC LUAD LUSC OV STAD Dataset Mtehod Cox−nnet (Dropout) Cox−nnet (Ridge) Cox−PH CoxBoost Random Forest Performance across TCGA datasets Cox-nnet significantly better * . CC-BY 4.0 International license under a y peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 11, 2016. ; https://doi.org/10.1101/093021 doi: t A A Performance across TCGA datasets . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 11, 2016. ; https://doi.org/10.1101/093021 doi: bioRxiv preprint Performance across TCGA datasets . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 11, 2016. ; https://doi.org/10.1101/093021 doi: bioRxiv preprint Cox−nnet (Dropout) Cox−nnet (Ridge) Cox−PH CoxBoost RF BLCA BRCA HNSC KIRC LGG LIHC LUAD LUSC OV STAD Dataset Method Rank 1 2 3 4 5 Performance Rank B B Performance Rank Method C A Node clustering by pathway Hidden layer node outputs of KIRC dataset Node clustering by pathway y p Prognostic Index of patients Node value −2 0 2 Node output value . Figures 377 CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 11, 2016. Figures 377 ; https://doi.org/10.1101/093021 doi: bioRxiv preprint Prognostic Index of patients Enriched Pathway Not significant 2 B t−SNE plot of top 20 Cox−nnet nodes B ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● −20 −10 0 10 20 −20 −10 0 10 20 30 t−SNE_1 t−SNE_2 Top 20 Cox−nnet nodes ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● −20 0 20 −25 0 25 t−SNE_1 t−SNE_2 −4 0 4 PI Top 20 principal components e_19 de_9 e_16 e_14 de_4 e 15 t−SNE plot of top 20 Cox−nnet nodes B ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● −20 −10 0 10 20 −20 −10 0 10 20 30 t−SNE_1 t−SNE_2 Top 20 Cox−nnet nodes ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● −20 0 20 −25 0 25 t−SNE_1 t−SNE_2 −4 0 4 PI Top 20 principal components t−SNE plot of top 20 Cox−nnet nodes Cox nnet nodes ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● −20 0 20 −25 0 25 t−SNE_1 t SNE_2 −4 0 4 PI Top 20 principal components p p ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● −20 −10 0 10 20 −20 −10 0 10 20 30 t−SNE_1 t−SNE_2 Top 20 Cox−nnet nodes Top 20 principal components Top 20 Cox−nnet nodes t−SNE_2 t−SNE_2 node_19 node_9 node_16 node_14 node_4 node_15 node_6 node_13 node_5 node_20 node_11 node_2 node_7 node_8 node_17 node_10 node_1 node_3 node_18 node_12 0 50 0 55 0 60 Single variable c−index of leading edge genes Method Cox−nnet Cox−PH Cox-nnet specific leading edge genes Leading edge genes found by both Cox-nnet and Cox-PH models Enriched Pathway Single variable c−index of leading edge genes Single variable c−index of leading edge genes 20 density 0 0.50 0.55 0.60 c_index Enriched pathway-gene bipartite network ADHERENS_JUNCTION BUTANOATE_METABOLISM CITRATE_CYCLE_TCA_CYCLE ENDOCYTOSIS ENDOMETRIAL_CANCER FATTY_ACID_METABOLISM INSULIN_SIGNALING_PATHWAY LYSINE_DEGRADATION PEROXISOME PPAR_SIGNALING_PATHWAY PROPANOATE_METABOLISM PROSTATE_CANCER PROXIMAL_TUBULE_BICARBONATE_RECLAMATION INSR CREBBP EP300 MAPK1 TJP1 SMAD4 ERBB2 WASL FER TCF7L2 CTNNA1 TGFBR2 PVRL3 LMO7 CTNNB1 RHOA IGF1R PTPRB ACSM3 ALDH2 ALDH7A1 OXCT1 HADHA BDH2 ABAT HMGCS2 HMGCS1 EHHADH ACAT1 L2HGDH ALDH3A2 HADH PCK2 SDHAIDH3A SUCLG2 ACO1 SDHD OGDH SDHB OGDHL DLD PCK1 DLST ACO2 DLAT SUCLA2 FLT1 C3 3 IQSEC1 NEDD4L KIT ASAP2RABEP1 ZFYVE20 DCD6IP EHD3 STAM AB11FIP5 DNAJC6 CHMP4C RAB11FIP2 EHD4 ITCH STAM2 PIP5K1B 5C AP2B1 AGAP1 KDR ADRB2 USP8 MP5 VPS4B VPS37D VPS24 TCF7L1 CCND1 FOXO3 ARAF PIK3CA GSK3B AKT3 PTEN APC BRAF PIK3R3 PDPK1 SOS2 CYP4A22 CYP4A11 ACADL CPT1A HADHB ACSL1 ACOX1 PECI ACAA2 ADH5 CPT2 ACADM ACADSB PRKAA1 MAPK10 RAPGEF1 LIPE FBP1 PRKCZ PRKAR1A FOXO1 MAPK8 G6PC CRK PPP1CB PRKAR2A PHKB SOCS2 CALM1 PPARGC1A PRKAA2 SUV420H1 SETDB2 ASH1L AASDH BBOX1 TMLHE NUDT12 DDO PXMP2 HSD17B4 PEX11A HACL1 NOS2 PEX7 ACOX2 HAO2 PECR ABCD3 PEX26 CAT PHYH PEX1 CROT PEX12 PEX19 SCP2 EPHX2 CRAT SLC27A2 MPV17L AQP7 PPARG PPARA SCD5 ACSS3 MUT HIBCH PCCA ALDH6A1 PDGFB BCL2 CHUK PDGFD AR ATP1B1 SLC9A3 FXYD2 SLC4A4 CA2 GLS2 AQP1 GLUD2 CA4 ATP1A1 CYTOKINE_CYTOKINE RECEPTOR_INTERACTION PROTEASOME AMH IL20RB RELT TNFSF14 TNFRSF25 IL15RA INHBE LIF IL6 CXCL2 IL23A IFNB1 IFNE TNFRSF6B CSF2 OSM TNFRSF18 CXCL5 IFNGR2 CXCL3 CXCL13 IFNG TNFRSF8 CCR10 CCL7 TNFSF4 CXCL1 IL4R CSF3R CCL8 LTA CCL5 CCL25 FLT3LG CXCR5 XCL1 TNFRSF1A TNFSF9 XCL2 CSF1 LTBR FASLG IL8 TNFRSF12A IL2RG IL21R TGFB1 LTB CCL11 IL2RB IL10RB IL1B IL1A GDF5 TNFSF11 CCL26 TNFSF13B IL1R2 IL20RA CCL19 CCR8 PSMD13 PSMA4 PSMB4 PSME2 PSMB10 PSMD4 PSMC5 PSMB3 SHFM1 PSMD7 PSMB2 PSMA5 PSMB6 PSMA1 PSMC3 PSMD14 PSMC4 CELL_CYCLE P53_SIGNALING PATHWAY PLK1 CDC25B CHEK2 CCNA2 ANAPC7 PTTG1 ORC6L CDC20 CDC7 CCNB2 E2F3 PKMYT1 ANAPC5 E2F4 CDC25C BUB1B CCNE1 DBF4 TTK CDC45 CDK1 BUB1 SFN E2F2 CDK2 CDC6 CCNB1 CDC25A CCNA1 CDKN2C BID GTSE1 BAI1 STEAP3 RRM2 PMAIP1 SHISA5 CASP9 CASP3 et specific leading edge genes g edge genes found by both et and Cox-PH models ched Pathway Enriched pathway-gene bipartite network ADHERENS_JUNCTION ENDOCYTOSIS ENDOMETRIAL_CANCER INSULIN_SIGNALING_PATHWAY PROSTATE_CANCER PROXIMAL_TUBULE_BICAR INSR CREBBP EP300 MAPK1 TJP1 SMAD4 ERBB2 WASL FER TCF7L2 CTNND1 CTNNA1 TGFBR2 PVRL3 LMO7 CTNNB1 RHOA IGF1R PTPRB PCK1 FLT1 IQSEC3 ERBB3 IQSEC1 CHMP2B NEDD4L KIT ASAP2RABEP1 ZFYVE20 PDCD6IP EHD3 STAM RAB11FIP5 DNAJC6 CHMP4C HSPA1L RAB11FIP2 EHD4 ITCH STAM2 PIP5K1B RAB5C AP2B1 AGAP1 KDR ADRB2 USP8 CHMP5 VPS4B VPS37D TRAF6 VPS24 TCF7L1 CCND1 FOXO3 ARAF PIK3CA GSK3B AKT3 PTEN APC BRAF PIK3R3 PDPK1 SOS2 PRKAA1 MAPK10 RAPGEF1 LIPE FBP1 PRKCZ PRKAR1A FOXO1 MAPK8 G6PC CRK PPP1CB PRKAR2A PHKB SOCS2 CALM1 PPARGC1A PRKAA2 PDGFB BCL2 CHUK PDGFD AR CELL_CYCLE P53_SIGNALING PATHWAY PLK1 CDC25B CHEK2 CCNA2 ANAPC7 PTTG1 ORC6L CDC20 CDC7 CCNB2 E2F3 PKMYT1 ANAPC5 E2F4 CDC25C BUB1B CCNE1 DBF4 TTK CDC45 CDK1 BUB1 SFN E2F2 CDK2 CDC6 CCNB1 CDC25A CCNA1 CDKN2C BID GTSE1 BAI1 STEAP3 RRM2 PMAIP1 SHISA5 CASP9 CASP3
W2301744751.txt	https://ejournal.undip.ac.id/index.php/jitaa/article/download/10589/8427	en		SYNCHRONIZATION OF CARBOHYDRATE AND PROTEIN SUPPLY IN THE SUGARCANE BAGASSE BASED RATION ON IN SITU NUTRIENT DEGRADABILITY	Journal of The Indonesian Tropical Animal Agriculture/Journal of the Indonesian tropical animal agriculture	2,016	cc-by-sa	4,229	Journal of the Indonesian Tropical Animal Agriculture (J. Indonesian Trop. Anim. Agric.) pISSN 2087-8273 eISSN 2460-6278 41(1):28-36, March 2016 DOI: 10.14710/jitaa.41.1.28-36 SYNCHRONIZATION OF CARBOHYDRATE AND PROTEIN SUPPLY IN THE SUGARCANE BAGASSE BASED RATION ON IN SITU NUTRIENT DEGRADABILITY F. F. Sani, L. K. Nuswantara, E. Pangestu, F. Wahyono and J. Achmadi1 Faculty of Animal and Agricultural Sciences, Diponegoro University, Tembalang Campus, Semarang 50275 - Indonesia 1Corresponding E-mail:jachmadi@gmail.com Received August 17, 2015; Accepted November 02, 2015 ABSTRAK Dua ekor domba jantan dewasa digunakan pada dua percobaan untuk mengkaji pengaruh sinkronisasi suplai karbohidrat dan protein dalam pakan komplit berbasis bagase tebu terhadap kecernaan nutrien secara in situ. Percobaan pertama ditujukan untuk menyusun tiga pakan komplit berbasis bagase dengan tiga indeks sinkroni yang berbeda. Semua bahan pakan penyusun dievaluasi untuk mendapatkan gambaran kinetika degradasi bahan organik (BO) dan nitrogen (N) secara in situ. Berdasarkan atas kinetika degradasi BO dan N, tiga pakan komplit masing-masing diformulasikan dengan indeks sinkroni 0,37; 0,50; dan 0,63. Ketiga pakan komplit mengandung protein kasar (PK), energi dan neutral detergent fiber (NDF) yang sama. Percobaan kedua merupakan evaluasi terhadap tiga pakan komplit berbasis bagase tebu dengan indeks sinkroni yang berbeda secara in situ. Degradasi BO pada pakan komplit secara in situ menurun (P<0,05) dengan peningkatan indeks sinkroni. Semakin meningkat indeks sinkroni semakin meningkatkan (P<0,05) degradasi PK pada pakan komplit. Perubahan indeks sinkroni pada pakan komplit menurunkan (P<0,05) degradasi NDF. Semakin meningkat indeks sinkroni pada pakan komplit semakin menurunkan degradasi sulfur, dan ini tidak mendukung tujuan pengaruh sinkronisasi suplai karbohidrat dan protein dalam pakan komplit berbasis bagase. Kata kunci: degradasi nutrien, bagase tebu, sinkronisasi, in situ ABSTRACT Two adult male sheeps fitted with rumen cannula were used in two experiments to study the effects of synchronization of carbohydrate and nitrogen supply in sugarcane bagasse based total mixed ration (TMR) on in situ nutrient degradation. The first experiment was aimed to create three TMR with different synchronization index. Ingredient feedstuffs of TMR were evaluated for its in situ organic matter (OM) and nitrogen (N) degradation kinetics. On the basis of the OM and N degradation kinetics of feedstuffs, three sugarcane bagasse based TMR were formulated with synchronization indexes of 0.37; 0.50; and 0.63; respectively. The TMR had similar levels of crude protein (CP), total digestible nutrients, and neutral detergent fiber (NDF). In the second experiment, the three TMR with different synchronization index were evaluated for in situ degradability characteristics of OM, CP, NDF, and sulfur. The in situ degradation of OM in TMR were decreased (P<0.05) with the increasing of synchronization index. The higher synchronization index in TMR increased (P<0.05) CP degradation of CP. The NDF degradation decreased slightly by the alteration of synchronization index in TMR. The higher synchronization index in TMR reduced (P<0.05) in situ sulfur degradation, and this may not support to effects of synchronization of carbohydrate and nitrogen supply. Key words: in situ, nutrient degradation, sugarcane bagasse, synchronization 28 J.Indonesian Trop.Anim.Agric. 41(1):28-36, March 2016 INTRODUCTION The synchronization of dietary carbohydrate and protein degradability in rumen is attempted to increase microbial protein synthesis, thus improving the post-ruminal flow of protein. The synchronization of energy and nitrogen supplies in the rumen enhance microbial protein synthesis, improving rumen fermentation efficiency, nurient utilization and animal performance (Seo et al., 2010; Chumpawadee et al., 2006; Sinclair et al., 1993). By contrast, Piao et al. (2012), Rotger et al. (2006) and Chanjula et al. (2004) reported that there is no significant synchronization effect of ruminal nutrient degradability characteristics on rumen fermentation and microbial growth. Sulfur is one of the most important element for synthesis of sulfur amino acid such as methionine, cystine and cysteine. Methionine is an essential amino acid which has an important role in protein synthesis, as a start codon in the formation of amino acids. Sulfur intake restriction may lower the synthesis of methionine by bacteria, thus microbial protein synthesis may be adjusted to the availability of ruminal sulfur (Karto, 1999). Likewise, dietary sulfur concentration has been found to effect on rumen microbial growth (Suttle, 2010; Pathak, 2008; Stevani and Durand, 1989). The synchronization of supply from ruminal product of carbohydrate and nitrogen degradations is expected to increase microbial protein synthesis. Therefore, ruminal sulfur availability may be an important factor in contributing the improved microbial protein synthesis. The objectives of this study were to evaluate the effects of synchronization of carbohydrate and protein in sugarcane bagasse based total mixed ration on the ruminal degradation of sulfur and other organic nutrients. This was accomplished using the in situ technique. MATERIALS AND METHODS Experiment 1. In situ Degradation Characteristics of Feedstuffs This experiment was aimed to determine the characteristics of ruminal nutrient degradation of feedstuffs. Two ruminally cannulated male sheeps with the average body weight of 20 kg were housed individually in metabolic cages. They were fed on a diet (12% CP; 62% TDN; 55% NDF) daily at a maintenance level, and drinking water was available ad libitum. After two weeks of adaptation period to the diet and environment, each sheep was subjected to the experiment of in situ degradation characteristics of feedstuffs. Samples of each feedstuffs were ground through a 2-mm screen and material less than 45 μm was removed by hand sieving. Approximately 5.0 g of DM of each test feedstuff was accurately weighed into nylon bags with a pore size of 46 μm. The bags were then placed into the rumen 30 min after morning feeding and removed after intervals of 0, 2, 4, 6, 12, 24, 48, 72 h. For each incubation time, each sample of feedstuff was tested in triplicates in each sheep, randomly. Following incubation, the bags and contents were washed using aquademineral through the cold rinse cycle of a washing machine. Zero-hour bags were not incubated in the rumen but were washed in the same manner as incubated bags. After washing, bags and contents were dried in an oven at 60°C for 48 h and reweighed. Residue in the bags from the incubation period were analyzed to know the percentage of disappearance organic matter (OM) and crude protein (CP). The degradability data obtained for N and OM for feedstuffs were fitted to the model of Ǿrskov dan McDonald (1979) p = a + b (1 – exp (-ct)), where p is the cumulative amount degraded at time t, a is the rapidly soluble fraction, b is the potentially degraded fraction and c is the rate of degradation of fraction b. The synchronization index of each feedstuff was calculated based on degradation values of N to OM as described by Sinclair et al. (1993): Synchronization Index = 25 − ∑ 1 − 24 (25 − N kg) 2 OM 24 25 where 25 is 25 g of N/kg of digested OM in the rumen (Czerkawski, 1986), a synchronization index of 1.0 represent perfect synchronization between N and OM supply throughout the day and values less than 1.0 indicate the degree of ansynchronization. Three formulas of experimental diets were created on the basis of synchronization index value of each ingredient. The experimental diets were formulated to have synchronization indexes of 0.37; 0.50; and 0.63; respectively. The diets were in the form of total mixed ration (TMR) and were designed to have similar CP, TDN and NDF levels. Urea and molasses were used in formulating the diets. This was assumed that 95% of urea N was degraded in the first hour after In situ Nutrient Degradability of TMR with Different Synchronization Index (F. F. Sani et al.) 29 feeding, with the remaining 5% of urea N degraded at a rate of 0.5/h (Sinclair et al., 1995). Molasses was assumed at 100% of N and OM was degraded in the first hour post feeding (Chumpawadee et al., 2006). Experiment 2. Evaluation of Bagasse Based TMR with Different Synchronization Index The Experiment 2 was aimed to study the effect of the synchronization index alteration in sugarcane bagasse based TMR on the in situ degradation of sulfur, OM, CP and NDF. Three formulas of sugarcane bagasse based TMR resulted from Experiment 1 were used in this evaluation (Table 2). Animal, environment, and procedures of the in situ technique were similar to the Experiment 1. During the in situ technique, sheep were fed on TMR according to the test of TMR. Animals were fed TMR daily at maintenance level in two equal meals at 06.00 h and 18.00 h. Drinking water was available at all times throughout experimental period. Feed and residue in nylon bags were analyzed for sulfur, OM, CP, and NDF. Sulfur, OM, and CP contents of feed and residue were analyzed according to the procedure of AOAC (2000). A crossover design (two animals; three synchronization index treatments) was used to allocate the treatment of synchronization index alteration with six replicates of each treatment, and one way analyse of variance was used to test the data. RESULTS Experiment 1 Nutrient composition and synchronization index of each feedstuff ingredient are presented in Table 1. The feedstuff ingredients varied widely in their chemical compositions. The CP content of cassava waste was the lowest, and the CP content of soybean mill was the highest. The NDF and ADF contents of soybean m was the lowest, and the NDF and ADF contents of sugarcane bagasse was the highest. The ash content of rice bran was the highest and the ash content of maize grain was the lowest. The synchronization index was calculated on the basis of N and OM ruminal degradation characteristics for each feedstuff ingredients. The feedstuff ingredients had variation in their synchronization indexes. The synchronization Table 1. Chemical Compositions of Feedstuffs for TMR Formulation Feedstuffs OM TDN CP Ash NDF ADF Sulfur SI ---------------------------------- % DM basis ------------------------------- Sugarcane bagasse 96.66 49.49 1.75 3.34 89.07 53.98 0.63 0.088 Rice bran 87.20 66.64 6.01 12.80 61.21 28.39 0.10 0.668 Molasses 92.91 80.64 1.15 7.09 - - 0.84 1.000 Copra mill 93.32 55.21 14.98 6.68 62.35 35.51 0.25 0.847 - - 287.5 - - - - 0.950 Palm frond mill 96.94 61.78 15.07 3.06 75.19 45.33 0.06 0.627 Coffe seed shell 90.13 47.68 7.37 9.87 72.58 51.69 0.15 0.558 Cassava waste 95.55 64.99 1.49 4.45 35.28 21.76 0.03 0.112 Wheat pollard 95.14 75.74 12.83 4.86 51.10 20.69 0.05 0.891 Groundnuts shell 91.47 34.38 5.59 8.53 69.68 56.89 0.12 0.286 Corn 98.79 80.06 8.84 1.21 51.07 17.56 0.16 0.528 Soybean mill 93.34 82.23 47.96 6.66 11.66 8.87 0.45 0.960 - - - - - - - - Urea Salt OM = Organic matter, TDN = Total digestible nutrients, CP = Crude protein, NDF = Neutral detergent fiber, ADF = Acid detergent fiber, SI = Synchronization Index 30 J.Indonesian Trop.Anim.Agric. 41(1):28-36, March 2016 index of soybean mill was the highest and the synchronization index of sugarcane bagasse was the lowest. Table 2 shows the experimental TMR which were formulated with synchronization indexes of 0.37; 0.50; and 0.63; respectively. These diets had similar contents of CP, TDN, and NDF. Experiment 2 Figure 1 shows the percentage loss of OM, CP, NDF, and sulfur of three TMR with respective synchronization indexes throughout the incubation times. There were rapid increases in nutrient disappearances during the first 24 h of incubation time, then the rates of nutrient disappearances were plateau throughout the second 24 h of incubation time. Table 3 shows the in situ nutrient degradation characteristics of TMR with different synchronization index, those were calculated on the basis of data from Figure 1. The higher synchronization index of TMR lowered (P<0.05) fraction a+b of OM (Table 3). In contrast, higher synchronization index of TMR Table 2. Ingredients and Chemical Composition of TMR with Different Synchronization Index Synchronization Index 0.37 Ingredient Sugarcane bagasse Rice bran Molasses Copra mill Urea Palm frond mill Coffe seed shell Cassava waste Wheat pollard Groundnuts shell Corn Soybean mill Salt Total Analyzed chemical composition (%) Organic matter Crude protein Total digestible nutrients1 Neutral detergent fiber Acid detergent fiber Cell contents Hemicellulose Sulfur contents N : S ratio 1 0.50 0.63 25.00 2.50 2.00 2.50 0.70 16.50 2.30 30.20 1.10 1.60 5.80 11.30 0.50 100.00 25.00 4.00 4.00 6.00 0.50 9.00 2.00 15.50 15.50 3.50 4.50 10.00 0.50 100.00 25.00 5.60 7.00 16.30 0.20 1.00 3.80 2.20 23.00 3.60 0.50 11.30 0.50 100.00 95.64 12.06 62.05 54.80 32.85 45.20 21.95 0.26 7.4 : 1 95.03 12.17 62.93 55.97 32.12 44.03 23.85 0.29 6.7 : 1 94.13 12.95 62.96 55.98 31.67 44.02 24.31 0.34 6.1 : 1 Calculated according to Harris et al. (1972) In situ Nutrient Degradability of TMR with Different Synchronization Index (F. F. Sani et al.) 31 Table 3. In situ Degradability of Bagasse Based TMR with Different Synchronization Index Parameters Synchronization Index 0.37 0.50 0.63 a (%) 34.88±0.60a 28.21±0.78c 31.11±0.70b b (%) 23.32 ±0.73c 29.12±1.18a 25.09±1.14b c (% / h) 5.97±0.46a 5.44±0.30b 4.14±0.32c a+b (%) 58.12±0.27a 57.33±0.49b 56.12±0.71c a (%) 38.82±0.84a 21.52±0.94b 17.65±1.80c b (%) 25.08±0.96c 47.11±1.14b 52.59±2.21a c (% / h) 6.82±0.50a 6.28±0.21b 6.98±0.42a a+b (%) 63.90 ±0.23c 68.63±0.36b 70.34±0.48a a (%) 10.45±0.94 8.51±1.40 9.66±1.40 b (%) 20.25±1.04 19.35±2.05 20.21±2.30 c (% / h ) 5.66±0.72 5.78±0.98 5.25±1.03 a+b (%) 30.71±0.45a 27.86±0.85b 29.89±1.41ab a (%) 53.47±2.44a 40.99±0.61b 27.09 ±1.19c b (%) 24.93±2.54a 22.85±0.23b 21.64 ±0.87c c (% / h) 1.48±0.29b 0.98 ±0.26b 4.15 ±1.30a a+b (%) 78.40±0.14a 63.84 ±0.42b 48.73 ±0.83c OM degradability CP degradability NDF degradability Sulfur degradability Values are means ±SE (n=6) a, b, c P<0.05 increased (P<0.05) the degradable fraction a+b of CP. The fraction a+b degradability of NDF decreased slightly with the increasing value of synchronization index of TMR. The higher synchronization index of TMR lowered (P<0.05) the fraction a+b of sulfur. DISCUSSIONS Sugarcane Bagasse Based TMR with Different Synchronization Index The sugarcane based TMR could be an alternative to secure feed availability for ruminants throughout the year, though the existence of most fiber components in sugarcane bagasse are tightly bound to lignin as 32 lignohemicelluloses and lignocelluloses. This study evaluated the in situ sulfur and other nutrient degradability of sugarcane bagasse based TMR with different synchronization index. The TMR were created using similar ingredient of feedstuffs, but the ruminal supply of energy and nitrogen of all ingredients were synchronized to favor the improvement of microbial protein synthesis. The levels of CP, TDN, NDF, and sulfur were similar among the TMR to avoid any effect of different chemical composition in the tested TMR (Table 2). The levels of CP and TDN in TMR were designed as the recommendation of Kearl (1982). Moreover, sugarcane bagasse portions were also similar in the TMR, because sugarcane bagasse had the lowest CP and J.Indonesian Trop.Anim.Agric. 41(1):28-36, March 2016 ratio of N to sulfur at 9.09 may maximize the in vitro growth of rumen microbes. The ratio of N to sulfur at 10 in diet results maximum nitrogen retention in Naeimi sheep (Al-Dobeeb, 2004). Figure 1. Dissapearance of sulfur (S), neutral detergent fiber (NDF), crude protein (CP) and organic matter (OM) with synchrony indexes of 0.37 ( ), 0.50 ( ) and 0.63 ( ), respectivelly. synchronization index and the highest NDF compared to other ingredients (Table 1). The ratios of N to sulfur in the TMR were relatively low to contribute an optimum rumen microbial growth. Suwandyastuti (2013) reported that the In situ Organic Nutrient Degradability of TMR Generally, ruminant diet is characterized with relatively more rapid ruminal degradation of dietary nitrogen than those of dietary carbohydrate. The synchronization of protein and carbohydrate supply means providing both rumen degradable protein (rumen degradable protein; non protein N and rumen degradable true protein) and energy (ruminally fermentable carbohydrates) to the rumen, so that ruminal microorganisms use both simultaneously (Yang et al., 2010; Hall and Huntington, 2008). The synchronization of the ruminal degradation rate of carbohydrates and protein has been proposed as a method to increase ruminal microbial protein synthesis, improve efficiency of N usage and animal performance, and decrease urinary N excretion. Using the in situ method, Orskov and McDonald (1979) explain the nutrient degradation characteristic of tested feed on the basis of the rapidly soluble fraction of nutrients, the potentially degradable fraction of nutrient, and degradation rate of the potentially degradable fraction of nutrient, respectively which were delineated as values of a, b, and c. Kim et al. (2014) suggested that the DM of tested feeds are classified into water-soluble and 45-mm filterable, insoluble degradable, and non-degradable fraction. The digestible NDF would be completely degraded in the rumen within 72 h. The portion of NDF that remained after 72 h of incubation was considered the indigestible fraction. The CP of tested feeds are classified into water-soluble and 45-mm filterable, insoluble degradable, and nondegradable fractions. The portion of CP that remained after 48 h of incubation was considered the non-degradable fraction. The higher synchronization index in TMR may prevent acids accumulation that cause an abrupt reduce in ruminal pH. Kim et al. (2014) concluded that higher portion of soluble carbohydrates in a diet increases ruminal concentration of lactic acid concomitantly with reduces ruminal pH. The decrease in ruminal pH do not favor to the growth of rumen microbes. Table 3 shows that the rapidly degradable OM fraction decreased (P<0.05) and the potentially degradable portion of OM fraction increased (P<0.05) by increasing the synchronization index. In situ Nutrient Degradability of TMR with Different Synchronization Index (F. F. Sani et al.) 33 Although the impact of synchronization index alteration in the lower digestive tract may have diluted the influence on rumen fermentation, there were some inconsistency results of in vivo studies. The in vivo OM digestibility are unchanged with the increasing synchronization index in the diets (Piao et al., 2012; Rotger et al., 2006; Chanjula et al., 2004). In contrast, the higher synchronization index of diets slightly increases in vivo OM digestibility of diets (Seo et al., 2010; Chumpawadee et al., 2006). Table 3 shows that the higher synchronization index decreased (P<0.05) the rapid degradable fractions of dietary CP and increased (P<0.05) the potentially degradable fractions of dietary CP. These results were in line with degradability characteristics of OM fraction. Therefore, the synchronization of ruminal dietary OM and CP releases could be used simultaneously for the growth of rumen microbes (Yang et al., 2010; Hall and Huntington, 2008). Moreover, the higher synchronization index increased (P<0.05) the sum of the rapid degradable and the potentially degradable fractions of dietary CP (Table 3). Although the impact of synchronization index alteration in the lower digestive tract may have diluted the influence on rumen fermentation, the in vivo CP digestibility increased according to the increasing of synchronization index in diets (Chumpawadee et al., 2006). The sum of rapid degradable and potentially degradable dietary NDF decreased (P<0.05) by the alteration of synchronization index, but both fractions were unaffected by the alteration of synchronization index in the TMR (Table 3). The discrepancy between degradation characteristic of dietary NDF with other organic nutrients may be caused by the degradability dietary lignin, although the in situ degradation characteristic of lignin was not determined in this study. Dietary NDF consists of hemicellulose, cellulose, lignin and other nutrients bound to lignin. The portion of sugarcane bagasse in TMR was the highest among ingredient feedstuffs (Table 2). Most fiber components of sugarcane bagasse are tightly bound to lignin as lignohemicelluloses and lignocelluloses (Ariyani et al., 2014; Ramli et al., 2005). In situ Sulfur Degradability of TMR The synchronization of ruminal carbohydrate and nitrogen supply may create an environment that favor optimum microbial synthesis. Optimum microbial protein synthesis and sulfur capture 34 occur when fermentable energy, degradable sulfur, nitrogen and phosphorus are supplied at rates that match the capacity of optimum microbial protein synthesis (Suttle, 2010). When a large amount of urea to substitute natural protein in a ruminant diet, limited sulfur intake and may restrict microbial protein synthesis (Pathak, 2008). The sulfur capture for synthesis of microbial methionine and cystine requires more degradable source of feed carbohydrate (Stevani and Durand, 1989). However, it is not clear why all in situ degradation characteristics of sulfur decreased (P<0.05) with increasing the synchronization index in TMR (Table 3). The reduced ruminal NDF degradation may attribute to the decreased sulfur degradation. Rumen microbes are well known to have the capability in degrading lignocellulolitic bonds, therefore rumen is the most important site for mineral availability to animal host and rumen microbes. Some parts of feed mineral are associated with the cell wall in cellulolitic diet, and ruminal release of mineral depends on degradability of feed NDF (Ceresnakova et al., 2007; Ibrahim and Zemmelink, 1999). CONCLUSION The synchronization of carbohydrate and nitrogen supply in TMR reduced ruminal OM digestibility and increased CP digestibility. It is suggested that the decreased ruminal OM degradation may match with rate of CP degradation. However, degradability of NDF and sulfur did not favor to microbial protein synthesis. Future study needs to consider some aspects concerning with micro flora fauna environment of rumen. REFERENCES Al-Dobeeb, S.N. 2004. Evaluation of digestibility, nitrogen and sulfur balances and rumen fermentation of diets supplemented with urea and/or potassium sulfate in Naeimi sheep. Pak. J. Biol. Sci. 7(12):2216-2221. AOAC. 2000. Official methods of analysis, 17 th Ed. Association of Official Analytical Chemists. Washington DC. Ariyani, S. A., L.K. Nuswantara, E. Pangestu, F. Wahyono and J. Achmadi. 2014. Parameters of potein metabolism in goats fed diets with different portion of sugarcane bagasse. J. Indonesia Trop. Anim. Agric. 39(2): 111- J.Indonesian Trop.Anim.Agric. 41(1):28-36, March 2016 116. Ceresnakova, Z., P. Flak, M. Polacikova and M. Chrenkova. 2007. In sacco macromineral release from selected foreges. Czech. J. Anim. Sci. 52(6):175-182. Chanjula, P., M. Wanapat, C. Wachirapaorn and P. Rowlinson. 2004. Effects of synchronizing starch sources and protein (NPN) in the rumen on feed intake, rumen microbial fermentation, nutrient utilization and performance of lactating dairy cows. AsianAust. J. Anim. Sci. 17:1400-1410. Chumpawadee, S., K. Sommart, T. Vongpralub and V. Pattarajinda. 2006. Effect of synchronizing the rate of dietary energy and nitrogen release on ruminal fermentation, microbal protein synthesis, blood urea nitrogen and nutrient digestibility in beef cattle. Asian-Aust. J. Anim. Sci. 19(2):181188. Czerkawski, J. W. 1986. An Introduction to Ruminal Studies. Oxford: Pergamon Press (Ed. H. J. Finalyson) 1986. The effect of pH on the growth and metabolism of Streptococcus bovis in continous culture. J. Appl. Bacteriol. 61:201-208. Hall, M. B. and G. B. Huntington. 2008. Nutrient synchrony: Sound in theory, elusive in practice. J. Anim. Sci. 86(E.Suppl.): E287E292. Harris, L. E., L. C. Kearl and P. V. Fonnesbeck. 1972. Use of regression equations in predicting availability of energy and protein. J. Animal Sci. 35 (3):658-680. Ibrahim, M. N. M. and G. Zemmelink. 1999. Effect of plant fibre on the solubility of mineral elements. Asian-Aust. J. Anim. Sci. 12(8):1277-1284. Karto, A. A. 1999. Peran dan kebutuhan sulfur pada ternak ruminansia. Wartazoa 8(2):3843. Kearl, L. C. 1982. Nutrient Requirements of Ruminants in Developing Countries. International Feed Stuffs Institute. Utah Agriculture Experimental Station. Utah State University, Logon, Utah, USA. pp. 45-58. Kim, Y.I., Y.K. Oh. K.K. Park, and W.S. Kwak. 2014. Ensiling characteristics and the in situ degradability of a by-product feed based silage. Asian-Aust. J. Anim. Sci. 27(2):201208. Ǿrskov, E. R. and I. McDonald. 1979. The estimation of protein degradability in the rumen from incubation measurements weight according to rate of passage. J. Agric. Sci., Cambridge. 92:499-503. Pathak, A. K. 2008. Various factors affecting microbial protein synthesis in rumen. Veterinary World. 1(6):186-189. Piao, M.Y., H.J. Kim, J.K. Seo, T.S. Park, J.S. Yoon, K.H. Kim, and J.K. Ha. 2012. Effects os synchronization of carbohydrate and protein supply in total mixed ration with Korean wine residue on ruminal fermentation, nitrogen metabolism and microbial protein synthesis in Holstein steers. Asian-Aust. J. Anim. Sci. 25(11):1568-1574. Ramli, M.N., Y. Imura, K. Takayama, and Y. Nakanishi. 2005. Bioconversion of sugarcane bagasse with Japanese koji by solid-state fermentation and its effects on nutritive value and preference in goats. Asian-Aust. J. Anim. Sci. 18(9):1279-1284. Rotger, A., A. Ferret, S. Calsamiglia and X. Manteca. 2006. Effects of nonstructural carbohydrate and protein sources on intake, apparent total tract digestibility, and ruminal metabolism in vivo and in vitro with highconcentrate beef cattle diets. J. Anim. Sci. 84:1188-1196. Seo, J. K., J. Yang, H. J. Kim, S. D. Upadhaya, W. M. Cho and J. K. Ha. 2010. Effects of synchronization of carbohydrate and protein supply on ruminal fermentation, nitrogen metabolism and microbial protein synthesis in holstein steers. Asian-Aust. J. Anim. Sci. 23(11):1455-1461. Sinclair, L. A., P. C. Garnsworthy, J. R. Newbold and P. J. Buttery. 1993. Effects of synchronizing the rate dietary energy and nitrogen release on rumen fermentation and microbal protein synthesis in sheep. J. Agric. Sci. 120:251-263. Sinclair, L. A., P. C. Garnsworthy, J. R. Newbold and P. J. Buttery. 1995. Effects of synchronizing the rate dietary energy and nitrogen release in diets with a similiar carbohydrate composition on rumen fermentation and microbial protein synthesis in sheep. J. Agric. Sci. 124:463-472. Stevani, J. and M. Durand. 1989. Effect of sulphur deficiency on fermentation of different carbohydrate sources in an artificial rumen (RUSITEC). Asian-Aust. J. Anim. Sci. 2(3):399-389. Suttle, N. F. 2010. Mineral Nutrition of Livestock. 4th Ed. CAB International, Cambridge. Pp. In situ Nutrient Degradability of TMR with Different Synchronization Index (F. F. Sani et al.) 35 206-222. Suwandyastuti, S. N. O. 2013. Pengaruh nisbah energi-protein, nitrogen-sulfur dan kalsiumfosfor terhadap produk metabolisme rumen dan kecernaan substrat. Agripet. 13(2): 1-6. 36 Yang, J. Y., J. Seo, H. J. Kim, S. Seo and J. K. Ha. 2010. Nutrient synchrony: Is it a suitable strategy to improve nitrogen utilization and animal performace?. Asian-Aust. J. Anim. Sci. 23: 972-979 J.Indonesian Trop.Anim.Agric. 41(1):28-36, March 2016
https://openalex.org/W4395090203	https://eajournals.org/gjahss/wp-content/uploads/sites/33/2024/04/Firm-Characteristics.pdf	English	null	Firm Characteristics and Corporate Social Responsibility of Listed Consumer Goods Sector Firms in Nigeria	Global journal of arts, humanities and social sciences	2,024	cc-by	7,079	doi: https://doi.org/10.37745/gjahss.2013/vol12n36376 Published April 24, 2024 Citation: Colma E. and Etale L.M. (2024) Firm Characteristics and Corporate Social Responsibility of Listed Consumer Goods Sector Firms in Nigeria, Global Journal of Arts, Humanities and Social Sciences, Vol.12, No.3, pp.63-76 ABSTRACT: This study assessed the impact of firm characteristics on corporate social responsibility (CSR) of listed consumer goods firms in Nigeria for the period of eleven years covering 2013 to 2023. The study adopted firm size and firm age as proxies for firm characteristics with the addition of firm growth (explanatory variables), while corporate social responsibility served as the response variable. Based on the ex post facto study design, secondary data collected from published financial statements of sampled five companies listed on the Nigerian Exchange Group were evaluated using Pearson correlation coefficient and multiple regression analysis based on OLS technique assisted by E-Views statistical software. The findings revealed that that firm size and firm growth had positive but insignificant impact on CSR, while firm age had negative insignificant effect on CSR practices of listed consumer goods firms in Nigeria. The study recommended that larger firms should dedicate specific departments or teams to CSR, while smaller firms can designate responsible individuals to CSR or outsource CSR functions if needed before implementing any CSR initiatives. Also, firms should conduct a thorough assessment of their competitive advantages and competencies in order to develop a comprehensive and sustainable CSR strategy that aligns with their business objectives, enhances their reputation, to satisfy the interest of all stakeholders in order to enjoy stable operations to enhance firm growth and sustainability. KEYWORDS: firm age, firm growth, firm size, legitimacy, stakeholder value integrati Firm Characteristics and Corporate Social Responsibility of Listed Consumer Goods Sector Firms in Nigeria Ebiye Colma and Lyndon M. Etale Department of Accounting, Faculty of Management Sciences, Niger Delta University, Wilberforce Island, Bayelsa State, Nigeria Ebiye Colma and Lyndon M. Etale Department of Accounting, Faculty of Management Sciences, Niger Delta University, Wilberforce Island, Bayelsa State, Nigeria Global Journal of Arts, Humanities and Social Sciences Vol.12, No.3, pp.63-76, 2024 Print ISSN: 2052-6350(Print) Online ISSN: 2052-6369(Online) Website: https://www.eajournals.org/ Publication of the European Centre for Research Training and Development -UK Firm Characteristics and Corporate Social Responsibility of Listed Consumer Goods Sector Firms in Nigeria Ebiye Colma and Lyndon M. Etale Department of Accounting, Faculty of Management Sciences, Niger Delta University, Wilberforce Island, Bayelsa State, Nigeria Firm Characteristics and Corporate Social Responsibility of Listed Consumer Goods Sector Firms in Nigeria Ebiye Colma and Lyndon M. Etale Department of Accounting, Faculty of Management Sciences, Niger Delta University, Wilberforce Island, Bayelsa State, Nigeria Publication of the European Centre for Research Training and Development -UK According to CBN (2018), consumer goods firms in Nigeria contributed over 16% of country’s total Gross Domestic Product in the 2018 fiscal year. Firms are expected to contribute positively to the environment where they carry out their operation and to the growth of the economy at large. However, most of these firms production process, waste or disposal pattern sometimes does more harm to the environment they operate than good especially the manufacturing companies. As a result of this, corporate social responsibility (CSR) has been employed as a crucial element of business strategy to curtail the environmental and social brunt, the activities of firms does to the environment where they carry out production and other activities (Hosam, et al., 2019). The generally acceptable CSR given by Rasche et al. (2017) is that CSR is the integration of an organization’s social, ethical, environmental, and philanthropic responsibilities towards society into its processes, operations, and core business strategy in cooperation with relevant stakeholders. Nwude and Comfort (2021) further said that, CSR is an act where corporate firms include in their social values a strategic arrangement that will enable it to behave responsibly towards society. The extent to which a firm will meet up with its planned CSR as agreed by the board in a financial year largely depends on the board and firms characteristics such as the board diversity, board independence, board size, firm size, firm age etc. Nwude and Comfort (2021), Onipe and James (2021) and Adamu, et al. (2020), asserted that there is a connection between the board of directors’ characteristics, firms characteristics and corporate social responsibility. Firm characteristics can be seen as the wide varieties of information disclosed in the financial statement of business entities that serve as the predictors of the firm’s quality of accounting information and performance. It is made up of firm structural characteristics and firm performance characteristics. The firm structural characteristics include firm age, firm size, capital expenditure, firm leverage, and management efficiency while firm performance characteristics include profitability and firm growth (Shehu, 2009, Shuaibu et al., 2019). The financial statement of an organization is expected to show with accuracy the information on CSR for the stakeholders to know since it is a disclosure required by the standard. INTRODUCTION Corporate entities do not exist in isolation but rather exist in an environment. They exist as an entity that can sue and be sued and has impact on the social, economic, legal, cultural, ecological, technological and political environment of an economy so as to achieve the stated goals and objectives of the entity in the long run. Due to this, firms are being seen to be more concerned about what benefits them more than the environment they are situated. Ibrahim and Hamid (2019), posited that in the past firms have been seen traditionally to be self-centered by only aiming to achieve organizational profit, thereby assuming that the betterment of the standard of living of people in the society is solely on the government. 63 Global Journal of Arts, Humanities and Social Sciences Vol.12, No.3, pp.63-76, 2024 Print ISSN: 2052-6350(Print) Online ISSN: 2052-6369(Online) Website: https://www.eajournals.org/ Global Journal of Arts, Humanities and Social Sciences Vol.12, No.3, pp.63-76, 2024 Print ISSN: 2052-6350(Print) Online ISSN: 2052-6369(Online) Website: https://www.eajournals.org/ Global Journal of Arts, Humanities and Social Sciences Publication of the European Centre for Research Training and Development -UK Publication of the European Centre for Research Training and Development -UK The reminder of this paper dealt with the review of relevant literature, the methodology used in carrying out the study, the results of data analysis and discussion of findings, and finally, the conclusion and recommendations. Firm Age Firm age is a concept which examines the years the business has been in existence in the corporate world. Yameen, et.al (2019) asserted that firm age connotes the age of a firm at the time span of assessment. It has always been thought that years of operation by a firm can influence the degree of her CSR. Mutende et al (2017) argued that firm age has an influence on its performance. The older companies disclose more social responsibility information than newer companies, therefore. Firm Characteristics Firm characteristics also known as firm attributes simply refer to some specific features that differentiate a firm from other firms. Some of these features are: firm age, firm size, firm liquidity, firm financial performance, ownership structure and industry classification (Bassiouny, et al. 2016). Mohammed (2017) opined that firm characteristics are the factors that exist internally in the firm which influences share price of firms. According to Abdullahi (2016), firm characteristics are firm size, dividend, corporate governance, firm age, profitability, operating expenses, cash flow, and leverage. Firm Size Firm size connotes the magnitude of firm’s business. Astute (2015), opined that the size of a firm do have significant effect on the corporate social responsibility disclosure in her financial statement. Sritharan (2015), further stated that out of the elements of the characteristics of firm like liquidity, capital structure, performance, dividend policy and firm size, firm size gives the most competitive advantage to firms in the form of, reduced costs and economies of scale. Bujaki and Richardson (2017), concluded in their study that the expenses to be incurred with a higher disclosure level is at a minimal in big companies due to a better organized and robust internal reporting system. Masika and Simiyu (2018) then suggested that the size of a firm is strongly and positively related to financial performance of firms. Publication of the European Centre for Research Training and Development -UK Despite the fact that CSR is planned and budgeted for by entities, it is pertinent that the effect of such act on profit maximization and shareholders is at a minimal because prospective investors tend to look at firms age, firm size, asset composition, sales volume, profit distribution and dividend of a firm before making vital investment decision. Onipe and James (2021) agreed that CSR is desirable and also valued by all the stakeholders of a firm but even with the desire, most shareholders will prefer to earn a high dividend and capital gains on their investments. It is against the aforementioned backdrop that this study sought to examine the effect of firm characteristics (proxy by firm age, firm growth and firm size) on corporate social responsibility practices of listed consumer goods firms in Nigeria bearing in mind that these companies have resultant waste from operation that can be harmful to the hosting communities. More specifically, the purpose of the study was to determine the influence of firm age, firm growth and firm size on corporate social responsibility practices of listed consumer goods firms in Nigeria. In line these goals, three hypotheses were formulated and tested. 64 Global Journal of Arts, Humanities and Social Sciences Vol.12, No.3, pp.63-76, 2024 Print ISSN: 2052-6350(Print) Online ISSN: 2052-6369(Online) Website: https://www.eajournals.org/ Global Journal of Arts, Humanities and Social Sciences Vol.12, No.3, pp.63-76, 2024 Print ISSN: 2052-6350(Print) Online ISSN: 2052-6369(Online) Website: https://www.eajournals.org/ Legitimacy Theory The Legitimacy theory provided the theoretical foundation for this study to examine the effect of firm characteristics on corporate social responsibility practices of consumer goods firms in Nigeria. Legitimacy theory posits that organizations always make an effort to make sure that their operations adhere to social norms and boundaries (Deegan et al, 2002). The company's relationships with society are the main emphasis of this legitimacy theory. According to this view, an organization should adhere to the social norms that are applicable in the community since it is a part of it. According to legitimacy theory, a company's reporting practices and business practices are shaped by the social values of the community and society in which it operates (Ahmad et al, 2003). Therefore, businesses will make an effort to obtain legitimacy if they are aware of the changes in public opinion and the need for them to be socially responsible. In order to accomplish its goals, assure its existence, and reap other benefits, the organization has to carry out a variety of socially expected acts; society undoubtedly plays a role in determining the efficacy and legality of any particular corporation's activities. Society is putting more pressure on companies to use a portion of their profits to address pressing environmental issues, worker welfare, consumer safety, and involvement in the community. This is a significant departure from the long-held belief that an organization's ability to make money is a reliable measure of its legitimacy (Muwazir, 2011). Ogunode (2022), postulated that the legitimacy theory has to do with a social contract between communities and corporate entities where the corporate entities carryout operation. The social contract is of great importance for the purpose of peaceful co-existence between them and for also mutual benefits to both parties. The theory is of the opinion that entities should naturally agree to the existing rules, regulations, social and environmental norms of the community where they carry out operation maintaining existing peace and at the same time promoting growth. Suddaby et al. (2017), posit that legitimacy theory entails such assurance of meeting the conditions and expectations of the society, while the entity is carrying on its legitimate business. Some authors that have adopted this theory includes: Bissadu et al. (2017), Cho et al. (2015) and Akhalumeh & Ohiokha (2018). Publication of the European Centre for Research Training and Development -UK Publication of the European Centre for Research Training and Development -UK concerning the various positive effect the company has on the growth of its various stakeholders through community contribution, product involvement, employee welfare, and philanthropic act. The carrying out of CSR activities can build a positive image for a company than that of its competitors in the same industry. Starineca (2016), is of the opinion that communities that have enjoyed CSR programs have a better and higher standard of living than those who have not. Corporate Social Responsibility CSR is basically incorporation of the aspirations of the communities in the business plan of companies operating in their domain at a given time. The needs of the communities could be economic, legal, ethical or philanthropic. CSR activities to employees, communities, suppliers, creditors, shareholders and other stakeholders are expected to be disclosed by entities in order to keep the public abreast of the contribution the company is making. Egbunike and Tarilaye (2017), opined that CSR disclosure adequately provide all parties in the community with the information 65 Global Journal of Arts, Humanities and Social Sciences Vol.12, No.3, pp.63-76, 2024 Print ISSN: 2052-6350(Print) Online ISSN: 2052-6369(Online) Website: https://www.eajournals.org/ Global Journal of Arts, Humanities and Social Sciences Vol.12, No.3, pp.63-76, 2024 Print ISSN: 2052-6350(Print) Online ISSN: 2052-6369(Online) Empirical Review p Velinov et al (2022) examined the perception of employees of brewery companies in Czech. Emphasis on this study was placed on the implementation of CSR practices in breweries across Czech in order to motivate employees. The study findings revealed that workers in Czech breweries were the least motivated because of the fact that CSR practices are not mandatory, therefore, employees’ perception of CSR practices are not positive. Cincalova and Novakova (2021) examined CSR practices of a selected Czech transport company. Primary data for the study was generated through the administration of a questionnaire on employees of the company. The findings revealed that the sampled transport company had very good CSR strategies, and the company’s CSR activities were adequately disclosed in line with requirements in Czech. Malik and Okere (2020) examined corporate social responsibility, environmental investments and financial performance with evidence from manufacturing companies. The study explored a total of 64 manufacturing companies as population from the period 2011 to 2016 and used descriptive and panel regression analysis to analyze the information obtained. Donations, staff training cost and employee benefits as variables while monetary output as reliant factor. The findings of the study revealed that environmental investments had positive significant impact on financial performance. Oladele and Mokuolu (2020) evaluated the relationship between corporate social responsibility expenditure and financial performance of quoted manufacturing firms in Nigeria between 1999 and 2015. The study used profit after tax of the firms as proxy for financial performance while total asset, working capital and leverage ratio were used as proxy for corporate social responsibility expenditure. The study results showed that CSR expenditure had positive impact on financial performance of the companies studied even though not significant. Tarus (2020) explore the effect of board size on environmental disclosure in 27 listed firms in Kenya between the period of 2008 and 2017. The random effect regression was used to analyze the data and content analysis was used as a measurement for environmental accounting disclosure. The regression results showed that board size and environmental disclosure were significant negatively related. Cincalova and Hedija (2020) investigated the relationship between firm characteristics and corporate social responsibility among Czech transportation and storage industry firms. Data collected from the Albertina database was evaluated using Pearson and Spearman correlation coefficients, and regression analysis. Legitimacy Theory This theory is relevant to this study because it explain the relationship that is expected to exist between a company, the community in which it operates and its stakeholders. It emphasis on the notion that no entity is assured continuity without putting the interests of one of its major stakeholders: the community into consideration. 66 Global Journal of Arts, Humanities and Social Sciences Vol.12, No.3, pp.63-76, 2024 Print ISSN: 2052-6350(Print) Online ISSN: 2052-6369(Online) Website: https://www.eajournals.org/ C t f R h T i i d D l t UK Global Journal of Arts, Humanities and Social Sciences Vol.12, No.3, pp.63-76, 2024 Print ISSN: 2052-6350(Print) Online ISSN: 2052-6369(Online) Website: https://www.eajournals.org/ blication of the European Centre for Research Training and Development -UK Publication of the European Centre for Research Training and Development -UK Publication of the European Centre for Research Training and Development -UK Publication of the European Centre for Research Training and Development -UK regression trend analysis and E-view statistical package. The study concluded that the extent of compliance by Nigerian manufacturing firms to CSR is far higher than the rate of non-compliance. Mugambi and Fatoki (2019) analyzed the between corporate social responsibility disclosure and financial performance of quoted manufacturing firms on Nairobi Securities Exchange (NSE). Return on asset was used to measure financial performance, while STATA 12 software was employed to analyze the data using descriptive and inferential method. The study reached a conclusion that community disclosure and environmental disclosure of listed manufacturing companies in Kenya have a positive but insignificant effect on financial performance. Kajola et al (2019) examined board features and corporate social responsibility practices in Oil and Gas firms in Nigeria. Ten Nigerian oil and gas firms were used as sample with the aim of investigating the effect of three board attributes on CSR between the period 2011 and 2020 and data were then obtained from their audited financial statement. The data were analyzed using panel data analysis. The findings revealed that corporate boards should have more numbers with most of them being non-executive directors with diverse skills, experience and expertise. Osemene and Fagbemi (2019) examined the association between corporate governance attributes and environmental reporting in Nigerian consumer goods firms using 20 listed consumer goods firms. The study covered period from 2008 – 2018 while data collected were analyzed with fixed effects estimation technique. The result of the study findings shows that board independence has a positive significant effect on environment reporting. Inua and Emeni (2019) executed a study on the influence of corporate governance attributes on social sustainability reporting using chief executive officer tenure, executive compensation, board gender diversity and board size to measure corporate governance. The studied used 35 Nigerian financial and non-financial companies as sample for the study obtaining data between 2010 and 2016. The analytical tool employed for data analysis was panel regression and the study revealed that only board gender diversity was the only driving variable has impact on social sustainability reporting. Ofoegbu et al (2018) investigated the impact of corporate board characteristics on environmental disclosure of listed firms in South Africa and Nigeria. The study collected data from 90 Nigeria companies and 213 South Africa companies for 2015 financial statement. Empirical Review The study found significant relationship between firm size, firm financial performance and CSR, whereas, firm age and board gender diversity had no effect on CSR of Czech transportation and storage industry companies. Igbekoyi et al (2019) explored corporate social responsibility compliance among manufacturing firms in Nigeria. A study employed a sample size of 25 and the information were obtained from the firm’s financial records between 2002 and 2016 and did analysis using graph, table, cross-sectional 67 Global Journal of Arts, Humanities and Social Sciences Vol.12, No.3, pp.63-76, 2024 Print ISSN: 2052-6350(Print) Online ISSN: 2052-6369(Online) Website: https://www.eajournals.org/ Global Journal of Arts, Humanities and Social Sciences Vol.12, No.3, pp.63-76, 2024 Print ISSN: 2052-6350(Print) Online ISSN: 2052-6369(Online) Website: https://www.eajournals.org/ Global Journal of Arts, Humanities and Social Sciences Publication of the European Centre for Research Training and Development -UK Ashafoke and Ilaboya (2017) assessed the effect of board features on environmental disclosure in Nigerian listed banks between 2012 and 2014. The sample used was 10 while board features was measured with gender diversity, foreign director, board size and board independence and CSR disclosure was measured using environmental disclosure index. The OLS regression was employed to analyze the data and these showed that gender diversity has a negative and significant effect on CSR disclosure. Study Population and Sample Size The study was based on consumer goods companies in Nigeria with focus on firm characteristics (using firm size and firm age as proxy) and the independent variable, with firm growth and control variable, while corporate social responsibility was used as the dependent variable for the period of 2013 to 2023. The population of the study comprised of twenty-six (26) consumer goods manufacturing companies listed on the Nigerian Exchange Group as at 2023. Through the application of purposive sampling technique five (5) companies were selected for inclusion in the study sample. These five companies were selected because their published financial statements were readily available online and the statements provided data required for the study. Research Design This study made use of an ex-post facto research design in order to achieve the objective of the study. Ex-post facto design was adopted due to the nature of the variables involved in this study whereby data was collected from past historical observations (secondary source). An after-the-fact research design was most suited for this study, because the data used was already in existence and the researcher lacked the capacity to manipulate the data. Publication of the European Centre for Research Training and Development -UK The ordinary least square was adopted to analyze the data collected and they concluded that both in Nigeria and South Africa, board size and environmental disclosure were positively related. Ali and Isa (2018) investigated the impact of board attributes on corporate social responsibility performance in Nigerian cement companies. The variables used for board attributes were board size, board composition and managerial ownership. The study used 3 cement companies and obtained data from 2004-2014. Pooled ordinary least square and generalized least square regression were used to analyze the data collected. The study discovered that board composition and CSR performance have no positive significant relationship. 68 Global Journal of Arts, Humanities and Social Sciences Vol.12, No.3, pp.63-76, 2024 Print ISSN: 2052-6350(Print) Online ISSN: 2052-6369(Online) Website: https://www.eajournals.org/ Global Journal of Arts, Humanities and Social Sciences Publication of the European Centre for Research Training and Development -UK Publication of the European Centre for Research Training and Development -UK Publication of the European Centre for Research Training and Development -UK Results This study employed descriptive statistics, Pearson correlation coefficient and multiple regression techniques for the analysis of data in assessing the effect of firm characteristics measured by firm size and firm age (with firm growth as a control variable) on corporate social responsibility of listed consumer goods firms in Nigeria. The results of the analysis and the discussions to follow are presented in this section. Estimation Model The study employed a multiple regression model adapted from Etale and Levi-Owonaro (2023) as stated below: CSR = ƒ (FSZ, FGE, FGR) The above regression model was further translated into an econometric equation to facilitate the analysis. CSR = βₒ + β1FSZ + β2FGE + β3FGR + µ Equation 1 Where; CSR = Dependent variable FSZ = Firm size FGE = Firm age FGR = Firm growth β0 = Constant β1, β2 & β3 = Coefficients of the independent variables to be determined µ = Stochastic error term CSR = ƒ (FSZ, FGE, FGR) The above regression model was further translated into an econometric equation to facilitate the analysis. CSR = ƒ (FSZ, FGE, FGR) The above regression model was further translated into an econometric equation to facilitate the analysis. Equation 1 69 Global Journal of Arts, Humanities and Social Sciences Vol.12, No.3, pp.63-76, 2024 Print ISSN: 2052-6350(Print) Online ISSN: 2052-6369(Online) Website: https://www.eajournals.org/ Vol.12, No.3, pp.63-76, 2024 Print ISSN: 2052-6350(Print) Online ISSN: 2052-6369(Online) Website: https://www.eajournals.org/ Descriptive Statistics In a bid to carry out this study, the descriptive statistics of the data used for the study were initially examined. Table 1 reports the descriptive statistics of data employed in this study from 2013 to 2023. Table 1: Descriptive Statistics CSR FSZ FGE FGR Mean 5.055396 7.923232 48.40000 0.781636 Maximum 6.896526 8.793314 57.00000 38.15000 Minimum 2.863323 6.960838 38.00000 -0.960000 Std. Dev. 1.031583 0.566203 5.475873 5.138131 Observations 55 55 55 55 Source: E-views Out put 2024 Table 1 presents the descriptive statistics for both the dependent and explanatory variables of the study for 55 panel observations. Corporate social responsibility (CSR) which is represented by firm donation and gift to the community reflect a mean value of 5.0554 and standard deviation of 1.0316, while 2.8633 and 6.8965 are the minimum and maximum values respectively. The standard deviation reveals that the data of CSR are far spread across the mean. This simply implies that the CSR of consumer goods firms in Nigeria are sparsely spread apart. This is further confirmed by the disparity between the maximum and minimum. Thus, the CSR of consumer goods firms varies a great deal from one company to the other. Furthermore, the firm size (FSZ) had a mean value of 7.9232 with a fluctuation of 0.5662. This indicates that on the average, the FSZ of listed consumer goods firms stood at 7.9232 during the period under investigation. The standard deviation of FSZ in Table 1 indicates that there is a low dispersion from the mean value of FSZ recorded within the period of study. The highest FSZ recorded within the study period is 8.7933 with a minimum value of 6.9608. Firm age (FGE) of the sampled companies, indicates that on the average, FGE of listed consumer goods firms stood at 48.400 mean value during the period under investigation. Also, as shown in Table 1, its standard deviation indicates that there is a high dispersion from the mean value of FGE recorded within the period of study. The maximum and minimum FGE recorded within the study period were 57.000 and 38.150 respectively. Publication of the European Centre for Research Training and Development -UK Pearson Correlation Coefficients The correlation results computer based on Pearson Moment Correlation Coeeficients are reported in Table 2. It shows that the coefficients are 0.1110 for FSZ, -0.4262 for FGE, and 0.1340 for FGR respectively. The Pearson correlation coefficients results have been relied upon for assessing the multicollinearity of the study variables. The values of Pearson correlation coefficients between the independent and dependent variables have been found to be lower than the standard value of 0.7000. Therefore, the results reveal that there is no problem of multicollinearity. Table 2: Pearson Correlation Matrix of the Variables Table 2: Pearson Correlation Matrix of the Variables Observations 55 CSR FSZ FGE FGR CSR 1.0000 -0.1110 -0.4262 0.1340 FSZ 0.1110 1.0000 0.1888 0.1426 FGE -0.4262 0.1888 1.0000 0.1880 FGR 0.1340 0.1426 0.1880 1.0000 Source: E-views Out put 2024 Regression Results Descriptive Statistics 70 Global Journal of Arts, Humanities and Social Sciences Vol.12, No.3, pp.63-76, 2024 Print ISSN: 2052-6350(Print) Online ISSN: 2052-6369(Online) Website: https://www.eajournals.org/ ublication of the European Centre for Research Training and Development -UK Global Journal of Arts, Humanities and Social Sciences Vol.12, No.3, pp.63-76, 2024 Print ISSN: 2052-6350(Print) Online ISSN: 2052-6369(Online) Website: https://www.eajournals.org/ pean Centre for Research Training and Development -UK Vol.12, No.3, pp.63-76, 2024 Print ISSN: 2052-6350(Print) Online ISSN: 2052-6369(Online) Website: https://www.eajournals.org/ Publication of the European Centre for Research Training and Development -UK Hypothesis Three wth (FGR) has no significant influence on corporate social responsibility (CSR). Similar, the figures in Table 3 revealed that the coefficient of FGR and its p-value are 0.1340 and 0.3970 respectively. Again the p-value of FGR is 0.3970 > 0.0500. Therefore, the study revealed that FGR has a positive but non-significant influence on CSR of listed consumer goods firms in Nigeria. Here also, the null hypothesis is accepted. Publication of the European Centre for Research Training and Development -UK Publication of the European Centre for Research Training and Development -UK The regression results as presented in Table 3 revealed that when all the predictor variables assume zero values, the CSR of listed Consumer Goods firms would be estimated at 9.1342 (the constant value in the equation). This simply implies that when all the other variables are not considered, there will be an insignificant improvement in the CSR of listed Consumer Goods firms to the tune of 9.1342. Regression Results g Regression Estimates of the Model Table 3: Regression Results Dependent variable: CSR Variable Coefficient Std. Error t-Statistic Prob. C 9.1342 5.3887 1.7000 0.0970 FSZ 0.1110 0.5797 0.0100 0.9910 FGE -0.4262 3.1784 -0.7800 0.4420 FGR 0.1340 0.0176 0.8500 0.3970 R-squared 0.8724 Mean dependent var 5.0554 Adjusted R-squared 0.8144 S.D. dependent var 1.0316 S.E. of regression 8.9234 Durbin-Watson stat 1.9987 F-statistic 0.4245 Prob (F-statistic) 0.0003 Source: E-views Out put 2024 Regression Estimates of the Model Table 3: Regression Results Dependent variable: CSR Table 3 presents the results of the regression. The result in Table 3 revealed an overall R2 value of 0.8724. The R2, which represents the coefficient of determination implies that 87 per cent of the variations in the dependent variable (CSR) of listed consumer goods firms in Nigeria is jointly explained by the combined changes in the predictor variables (firm size (FSZ), firm age (FGE)and firm growth (FGR). The remaining 13 per cent of the variation could be explained by other factors not captured in the study model but could help to enhance firm success. Table 3 further explained the nature and extent of relationship between the dependent variable and each of the independent variables of the study in terms of coefficients and the probability values. 71 Global Journal of Arts, Humanities and Social Sciences Vol.12, No.3, pp.63-76, 2024 Print ISSN: 2052-6350(Print) Online ISSN: 2052-6369(Online) Website: https://www.eajournals.org/ Global Journal of Arts, Humanities and Social Sciences Vol.12, No.3, pp.63-76, 2024 Print ISSN: 2052-6350(Print) Online ISSN: 2052-6369(Online) Website: https://www.eajournals.org/ Global Journal of Arts, Humanities and Social Sciences yp Hypothesis One e (FSZ) has no significant influence on corporate social responsibility (CSR). Firm size (FSZ) has no significant influence on corporate social responsibility (CSR). From Table 3, the coefficient of FSZ is 0.1110 and its p-value is 0.9910. The p-value of FSZ 0.9910 > 0.0500, the rule for accepting or rejecting the null hypothesis in all cases is if the calculated p- value is greater than the 5 per cent level of significance. Therefore at 5% level of significance, the null hypothesis one is accepted. It means that FSZ has a positive influence on CSR, giving a coefficient of 0.1110 and p-value of 0.9910 but the influence is not significant. Hypothesis Two GE) has no significant influence on corporate social responsibility (CSR). Firm age (FGE) has no significant influence on corporate social responsibility (CSR). Again from Table 3, the coefficient of FGE and the p-value are -0.4262 and 0.4420 respectively. The p-value of FSZ as shown in Table 3 is 0.4420 > 0.0500. This means that FGE has a negative but insignificant influence on CSR. So, the null hypothesis is accepted. Firm age has no significant effect on CSR. Test of Hypotheses yp Hypothesis One Publication of the European Centre for Research Training and Development -UK Publication of the European Centre for Research Training and Development -UK Finally, this found that firm growth which was introduced as a control variable has no significant influence on CSR of consumer goods firms in Nigeria. In specific terms, the relationship between FGR and CSR though positive, given the values of the coefficient and p-value as 0.1340 and 0.3970 respectively, FGR has positive but insignificant relationship with CSR. In the overall, the findings of the study are in part consistent with the findings of Tarus (2020), Osemene and Fagbemi (2019), Ofoegbu et al (2018) and Ashafoke and Ilaboya (2017). Conclusion This study assessed the impact of firm characteristics on corporate social responsibility (CSR) of listed consumer goods firms in Nigeria for the period of eleven years covering 2013 to 2023. The study adopted firm size and firm age as proxies for firm characteristics with the addition of firm growth (explanatory variables), while corporate social responsibility served as the response variable. Based on the ex post facto study design, secondary data collected from published financial statements of sampled five companies listed on the Nigerian Exchange Group were evaluated using Pearson correlation coefficient and multiple regression analysis based on OLS technique assisted by E-Views statistical software. The findings revealed that that firm size and firm growth had positive but insignificant impact on CSR, while firm age had negative insignificant effect on CSR practices of listed consumer goods firms in Nigeria. Recommendations The following recommendations are advanced: e following recommendations are advanced: 1. The management of larger firms to should dedicate specific departments or teams to CSR, while smaller firms can designate responsible individuals or outsource CSR functions (where necessary) before implementing any CSR initiatives. 2. Firms should conduct a thorough assessment of their competitive advantages and competencies in order to develop a comprehensive and sustainable CSR strategy that aligns with their business objectives, enhances their reputation, and generates positive social and environmental impact. 3. Firms should ensure they satisfy the interest of all stakeholders to enjoy stable operations to enhance firm growth and sustainability. 3. Firms should ensure they satisfy the interest of all stakeholders to enjoy stable operations to enhance firm growth and sustainability. DISCUSSION This study revealed a positive relationship between firm size (FSZ) and CSR with coefficient of correlation value of 0.1110. This means a unit change in FSZ will lead to an increase in CSR by 0.1110 units. This relationship is however insignificant (given a p-value of 0.9910). This finding is consistent with the findings of Činčalová and Hedija (2020) who in their study investigated firm characteristics and corporate social responsibility of Czech transportation and storage industry and found that firm size has a statistically insignificant relationship between firm size and CSR practice. Also, this study revealed a negative relationship between firm age (FGE) and CSR with coefficient of correlation value of -0.4262. This means a unit change in FGE will lead to a decrease in CSR by 0.4262 units. Again this relationship is not significant (given a p-value of 0.4420). This finding also supports the findings of Činčalová and Hedija (2020), whose study investigated the relationship between firm characteristics and corporate social responsibility of Czech transportation and storage industry and submitted that firm age is not a factor affecting the CSR practice. 72 Global Journal of Arts, Humanities and Social Sciences Vol.12, No.3, pp.63-76, 2024 Print ISSN: 2052-6350(Print) Online ISSN: 2052-6369(Online) Website: https://www.eajournals.org/ Global Journal of Arts, Humanities and Social Sciences Vol.12, No.3, pp.63-76, 2024 Print ISSN: 2052-6350(Print) Online ISSN: 2052-6369(Online) Website: https://www.eajournals.org/ Global Journal of Arts, Humanities and Social Sciences Website: https://www.eajournals.org/ Publication of the European Centre for Research Training and Development -U Ali, S.M., & Isa,M.A. (2018). Board attributes and corporate social responsibility performance of listed cement companies in Nigeria. Research Journal of Finance and Accounting, 9(7), 109-121. Akhalumeh, P., & Ohiokha, F.I. (2018). Firm characteristics and corporate social donations among Nigerian firms. CEDSAF Journal of Business & Economy, 6(1), 60-72 Ashafoke, T.O., & Ilaboya, J. (2017). Board characteristics and environmental disclosure in Nigerian banking sector. ICAN Journal of Accounting & Finance, 6(1), 87-102. Astute, R.P. (2015). The influence firm characteristics of manufacture Industries to corporate social responsibility (CSR) Disclosure and the impact on investors reaction. Economic and Business Faculty Accounting Department Muhammadiyah University of Surakarta y g p y y Bassiouny, S.W., Soliman, M.M. & Ragab, A (2016). The impact of firm characteristics on earnings management: An empirical study of listed firms in Egypt. The Business and Management Review, 7(2), 91-101. Bissadu, K. D, Koglo, Y.S, Johnson, D. B. & Akpoti, K. (2017). Coarse scale remote sensing and GIS evaluation of rainfall and anthropogenic land use changes on soil erosion in Nasarawa state, Nigeria. Journal of Geoscience and Geography, 5(6), 259–266. Bujaki, M.L., & Richardson, A.J. (2017). A citation trail review of the uses of firm size in accounting research. Journal of Accounting Literature, 16, 1–27. CBN (2018). Central Bank of Nigeria Annual Report 2018 Central Bank of Nigeria Annual Report 201 Cho, C., Laine, M, Roberts, R.W., & Rodrigue, M. (2015). Organized hypocrisy, organizational facades, and sustainability reporting. Accounting, Organizations and Society, 40(6), 78-94. y p g g g y Cincalova, S. & Hedija, V. (2020). Firm characteristics and corporate social responsibility: The case of Czech transportation and storage industry. Sustainability (MDPI), 1-15 Cincalova, S. & Vovakova, Z. (2021). Corporate social responsibility of the selected transport company operated on the Czech market. Problems of Management in the 21st Century, 16(2), 65-73 Egbunike, A.R., & Tarilaye, N. (2017). Firm’s specific attributes and voluntary environmental disclosure in Nigeria: Evidence from listed manufacturing companies. Academy of Accounting and Financial Studies Journal, 21(3), 1-9. Etale, L.M. & Levi-Owonaro, S. P. (2023). Environmental financial reporting and corporate performance of listed pharmaceutical companies in Nigeria. European Journal of Accounting, Auditing and Finance Research, 11(8), 1-14 Hosam, A.R., Eko, G.S., & Salsabila, S.A. (2019). The impact of corporate social responsibility disclosure and board characteristics on corporate performance. Journal of Cogent Business and Management, 6(1), 1 – 19. Ibrahim, M. & Hamid, K. T. (2019). REFERENCES Abdullahi, M. (2016). Firms characteristics governance and financial performance of listed building materials firms in Nigeria. (Unpublished M.Sc Dissertation submitted to the Department of Accounting, Faculty of administration, Ahmadu Bello University, Zaria, Nigeria). Adamu, I.A., Nafisat, M.A., & Oyindamola,E. (2020). The relationship between corporate social disclosures and board characteristics: Evidence from Nigeria. Asian People Journal, 3(2), 96 – 105. Adamu, I.A., Nafisat, M.A., & Oyindamola,E. (2020). The relationship between corporate social disclosures and board characteristics: Evidence from Nigeria. Asian People Journal, 3(2), 96 – 105. 73 Global Journal of Arts, Humanities and Social Sciences Vol.12, No.3, pp.63-76, 2024 Print ISSN: 2052-6350(Print) Online ISSN: 2052-6369(Online) Website: https://www.eajournals.org/ Website: https://www.eajournals.org/ Publication of the European Centre for Research Training and Development -UK Publication of the European Centre for Research Training and Development -U Corporate social responsibility and financial performance of listed Non-Financial Services companies in Nigeria. American Journal of Business and Society, 4(2), 56-71 y ( ) Igbekoyi, O. S., Alade, M. E., & Oladele, R. (2019). Corporate social responsibility compliance among manufacturing firms in Nigeria. Accounting and Taxation Review, 3(1), 27–41. Inua, O. I., & Emeni, A. A. (2019). Corporate governance attributes and social sustainability reporting. International Journal of Accounting & Finance, 8(1), 21-37 74 Global Journal of Arts, Humanities and Social Sciences Vol.12, No.3, pp.63-76, 2024 Print ISSN: 2052-6350(Print) Online ISSN: 2052-6369(Online) Website: https://www.eajournals.org/ Global Journal of Arts, Humanities and Social Sciences Vol.12, No.3, pp.63-76, 2024 Print ISSN: 2052-6350(Print) Online ISSN: 2052-6369(Online) Website: https://www.eajournals.org/ Publication of the European Centre for Research Training and Development -UK Kajola, S.O., Sanyaolu, W.A., Alao, A., & Ojurongbe, J. (2019). Liquidity and profitability dynamics: Evidence from the Nigerian banking sector. Accounting & Taxation Review, 3(2), 1-12. Malik, S.S., & Okere, W. (2020). The relationship between corporate social responsibility, environmental investments and financial performance: Evidence from manufacturing companies. Environmental Science and Pollution Research, 27, 39946–39957. Masika, D. & Simiyu, E. (2018). Effect of firm characteristics on financial performance of deposit taking SACCOS licensed by Sasra in Nairobi Kenya. African Journal of Emerging Issues. Mohammed, G., T, (2017). Firm Characteristics and Firm Value of Listed Healthcare Companies in Nigeria. (unpubished published) M.Sc Dissertation Submitted to the Department of accounting, Faculty of Administration, Ahmadu Bello University, Zaria Nigeria. g y y g Mugambi, C.M., & Fatoki, O.I. (2019). Effect of corporate social responsibility disclosure on financial performance of manufacturing firms quoted on Nairobi Securities Exchange. Canadian Social Science, 15(4), 15–25. Mutende, A. E., Mwangi, M., Njihia, M. J. & Ochieng, E. D. (2017). The moderating role of firm characteristics on the relationship between free cash flows and financial performance of firms listed at the Nairobi Securities Exchange. Journal of Finance and Investment Analysis, 6(4), 55-74 Nwude, C.E., & Comfort, A. N. (2021). Board structure and corporate social responsibility: Evidence from developing economy. SAGE Open Journal, 3(1), 1 – 12. Ofoegbu, G. N., Odoemelom, N., & Okafor, R. G. (2018). Corporate board characteristics and environmental disclosure quantity: Evidence from South Africa (integrated reporting) and Nigeria (traditional reporting). Cogent Business & Management, 5, 1-27. g p g g g Ogunode, O. A. (2022). Legitimacy Theory and Environmental Accounting Reporting and Practice: A Review. South Asian Journal of Social Studies and Economics. Yameen, M., Farhan, N. H. S. & Tabash, M. I. (2019). The impact of liquidity on firms’ performance: Empirical investigation from India pharmaceutical companies. Academic Journal of Interdisciplinary Studies, 8(3), 212-220 Publication of the European Centre for Research Training and Development -U 13(1): 17-28 Oladele, P. O., & Mokuolu, J. O. (2020). Corporate Social Responsibility Expenditure and the Financial Performance of Quoted Firms in Nigeria. International Journal of Research and Innovation in Social Science (IJRISS), 4(4), 4–1. Onipe, A.Y., & James, A. (2021). Board of directors and corporate social responsibility reporting of quoted companies in Nigeria. Journal of Accounting, Finance and Auditing Studies, 7(2), 38 – 52. Osemene, O. F., & Fagbemi, T. O. (2019). A study of the relationship between corporate governance and environmental reporting in Nigeria’s listed consumer goods companies. Fountain University Osogbo Journal of Management, 4(3), 1-22 Rasche, A., Morsing, M., & Moon, J. (2017). Corporate social responsibility: Strategy, communication, governance. Cambridge University Press: Cambridge, UK. Shehu, M.M. (2009). The impact of firm characteristics on market value of quoted manufacturing firms in Nigeria. (unpubished published) M.Sc Dissertation submitted to the Department of accounting, Faculty of Administration, Ahmadu Bello University, Zaria Nigeria. Shuaibu, K., Ali, I., & Moh’d Amin, I. (2019). Company attributes and firm value of listed consumer goods companies in Nigeria. Journal of Research in Humanities and Social Science Volume 7(5), 40-49. 75 Global Journal of Arts, Humanities and Social Sciences Vol.12, No.3, pp.63-76, 2024 Print ISSN: 2052-6350(Print) Online ISSN: 2052-6369(Online) Website: https://www.eajournals.org/ Publication of the European Centre for Research Training and Development -UK Publication of the European Centre for Research Training and Development -UK Sritharan, V, (2015). Does firm size influence on firm’s profitability? Evidence from firms of Sri- Lankan hotels and travel sector. Research Journal of Finance and Accounting, 6(6), 201- 207. Starineca, O. (2016). Human resource selection approaches and socially responsible strategy. Economics and Business, 28(1), 106–114. Suddaby, R., Bitektine, A. & Haack, P. (2017). Legitimacy. Academy of Management Annals, 11(1), 451-478 Tarus, J. K. (2020). Do board size and firm size affect environmental accounting disclosure? Evidence from selected listed firms in Kenya. Journal of Business Management and Economic Research, 4(1), 21-37 Velinov, E., Cincalova, S. & Toporova, V. (2022). Corporate social responsibility activities through the employees’ lenses: Evidence from the Czech brewery sector. Hradiec Economic Days, 1-8 1-8 Yameen, M., Farhan, N. H. S. & Tabash, M. I. (2019). The impact of liquidity on firms’ performance: Empirical investigation from India pharmaceutical companies. Academic Journal of Interdisciplinary Studies, 8(3), 212-220 76
https://openalex.org/W3214053507	https://www.e3s-conferences.org/10.1051/e3sconf/202132103008/pdf	English	null	Application of passengers’ thermoregulation integral model used in the evaluation of thermal comfort in vehicles compartment equipped with internal curtains	E3S web of conferences	2,021	cc-by	3,856	* Corresponding author: econcei@ualg.pt Application of passengers’ thermoregulation integral model used in the evaluation of thermal comfort in vehicles compartment equipped with internal curtains 1FCT - Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal 2CINTAL, Campus de Gambelas, 8005-139 Faro, Portugal 3Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal 4School of Built Environment, University of Reading, Reading, RG6 6AW, United Kingdom 1FCT - Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal 2CINTAL, Campus de Gambelas, 8005-139 Faro, Portugal 3Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal 4School of Built Environment, University of Reading, Reading, RG6 6AW, United Kingdom Abstract. This work evaluates the passengers thermal comfort level inside a vehicles compartment. The numerical study, made in winter conditions, consider a bus indoor environment equipped with internal curtains, internal seats, lateral panels, ceiling, floor and occupied by 52 passengers. The numerical model considers the passengers and vehicle grid generation, passengers body and clothing thermal response and passengers thermal comfort level. The grid generation is used to evaluate the view factors and Mean Radiant Temperature that the passengers are subjected. In this calculus the passengers and the vehicles surfaces shading devices are considered. The thermal response numerical models consider the energy and mass balance integral equations. The thermal comfort evaluation considers the heat produced inside the body and the heat exchange between the body and the environment. The human body numerical model considers also the thermoregulatory system to control the temperature. The numerical model is used to evaluate the thermal comfort level that seated passengers are subjected in a bus equipped with asymmetrical warm curtains. Three Cases studies were developed: the first one considers the temperature curtains equal to the indoor bus surfaces, while the other two consider higher temperatures values. All Cases are thermally comfortable according to the standards. © 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/). DP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 ses/by/4 0/) https://doi.org/10.1051/e3sconf/202132103008 https://doi.org/10.1051/e3sconf/202132103008 E3S Web of Conferences 321, 03008 (2021) ICCHMT 2021 d by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution Licens org/licenses/by/4.0/). 3 Vehicle mathematical model The numerical model that simulates the Vehicle Thermal Behaviour and work in transient conditions is based on energy and mass balance integral equations. The equations are based on conduction, convection and radiation phenomena and are solved by the Runge- Kutta-Felberg method with error control. The Human Thermal Response numerical model is used to evaluate the passengers thermal comfort level. This numerical model, based on a human multi-node comfort model, considers energy and mass balance integral equations and the Thermoregulatory system based in the work of Stolwijk and Hardy [18]. More details can be seen in studies of Conceição et al. [19, 20]. The equations are developed through the Computer Aided Design techniques. This methodology, that considers a vehicle with complex topology, can be used to simulate more realistic vehicles designs. The radiative heat exchanges numerical model between the vehicle and the passengers, based on Mean Radiant Temperature method, is developed and applied in the works of Conceição et al. [6,21]. The numerical model that simulates the vehicles thermal behavior calculates not only the solar radiation incident in the internal and external surfaces, but also the radiative heat exchange between internal, in each space, and external surfaces, between vehicle and surrounding bodies. In the radiative calculus are considered, in the internal and external calculus, the shading effect. The radiative heat exchange applications can be seen in Conceição and Lúcio [15]. All coefficients used in these equations are evaluated by sub-models that calculate the glass radiative properties, occupation cycle, ventilation strategy and heat and mass transfer coefficients by convection using empirical expressions. The Vehicle Thermal Behavior considers the convective (natural, forced and mixed), conductive (in the main bodies divided in several layers), radiative (shortwave and long wave radiation outside and inside the vehicle) and mass transfer (inside the compartments) phenomena. The present work is a continuation of the works developed in Conceição et al. [22,23]. Others studies with the same topic can be analysed in Shek and Chan [24] and Velt and Daanen [25]. The vehicle, in general, is divided into several compartments (passengers, luggage and motor), main bodies (panels, doors, ceiling and floor), interior bodies (benches and motors) and glasses (windows). In the present numerical study is analysed, in detail, the thermal comfort of the passengers seated inside a bus, on both sides with windows, without and with internal curtains on the left window subjected to solar radiation. 1 Introduction Percentage of Dissatisfied (PPD) index. Applications using the thermal comfort level, adaptive thermal comfort levels and empirical models can be analysed in, the works of Conceição et al. [7,8]. The people spend part of the time inside vehicles, as bus, trains, and others vehicles. This kind of vehicles can provide acceptable thermal conditions, using Heating, Ventilating and Air-Conditioning system. This system can promote similar thermal comfort conditions to the occupants, however external environmental conditions, as the solar radiation, can provide asymmetrical conditions to the occupants. In general, internal curtains are used to reduce this uncomfortable conditions. The curtains subjected to solar radiation reduce the inlet solar radiation and can be used as radiative surfaces that, in winter conditions, can improve the thermal comfort conditions that the occupants are subjected. ç [ , ] The software applied in this work simulates the Vehicle Thermal Response and the Human Thermal Response numerical models, that includes the human thermoregulatory system. The first one evaluates the vehicle design, using Computer Aid Design, and calculates the distribution of temperature and solar radiation inside vehicle spaces, in particular, the passenger compartment. In the vehicle thermal behavior numerical models, have been given special attention to the passenger compartment (main and interior bodies and windows) and thermal phenomena (radiation, convection, human generation, glass radiative properties, air renovation and others). To evaluate the thermal discomfort and thermal comfort levels, different models were developing [1]. These models, inclusively, are published in international standards [2,3]. The local thermal discomfort, as asymmetrical thermal conditions, can promote uncomfortable thermal conditions, as Draught Risks and others. The works of Conceição et al. [4] and Conceição [5] analyzed in detail these uncomfortable problems using experimental and numerical methodologies. The thermal comfort level, developed by Fanger [6], and showed in the standards [2,3], is evaluated using Predicted Mean Vote (PMV) index and Predicted Some works related with the Vehicle Thermal Response and the evaluation of thermal comfort were developed in the last years. Some examples about the thermal comfort evaluation can be analyzed in Walgama et al. [9] and Rugh et al. [10], while the numerical simulation of bus and passengers can be seen in Riachi [11] and Mezrhab and Bouzidi [12]. Vehicle Thermal Response numerical model is based on the Building Thermal Response. 1 Introduction The Building Thermal Response, developed in the last years, E3S Web of Conferences 321, 03008 (2021) ICCHMT 2021 https://doi.org/10.1051/e3sconf/202132103008 12 cylindrical or spherical layers and could be still protected of the external environment through several layers of clothing. considers energy and mass balance integral equations. Some applications can be seen in Conceição et al. [13], for an internal energy analysis, in Conceição and Lúcio [14], for an application of shading devices, in Conceição and Lúcio [15], for a passive analysis, in Conceição and Lúcio [16], for both passive and active analysis, and for a set of validations in Conceição and Lúcio [17]. 4 Heat exchanges by radiation The heat exchange by radiation numerical model, that uses the view factors calculated between the human body and the vehicles surrounding surfaces, is based on the Mean Radiant Temperature method. In the methodology used in view factors determination, each vehicle surface is sub-divided into several small areas and each human body element, with inclinations and dimensions equal to the respective body, are divided into infinitesimal areas. It is considered the shading effect caused by the surrounding surfaces, namely the vehicle surfaces and the occupant’s bodies. 3 Vehicle mathematical model The view factors and the radiative heat exchanges by radiation between the different human body sections and the surroundings surfaces, namely, the seats, lateral panels, ceiling, floor, curtains, and others, are considered. 5 Simplified bus model In the passenger compartment of the bus studied is considered two ventilation ducts over the seats, 99 main bodies, 18 glazed window, 9 curtains and 105 interior bodies (as can be seen in Fig. 1 and Fig. 2). The bus is occupied by 52 passengers. Each passenger has 1.7 m of height, 70 kg of weight, 1 met of activity level and 1 clo of clothing level. Each of these surfaces, vehicle and passengers, in order to evaluate the view factors, is divided into several infinitesimal areas. In general, all vehicle surfaces are subdivided in a minimum of 100 elementary areas. Regarding the passengers: Fig. 1. Scheme of a bus and the 52 occupants: detailed visualization of the external vehicle surfaces. Fig. 2. Scheme of a bus and the 52 occupants: detailed visualization of the occupants.  The spherical head is divided in 64 elementary areas (a grid in the azimuthal angle direction of 8 divisions and a grid in the polar angle direction of 8 divisions);  The cylindrical elements are also divided in 64 elements (a grid in the longitudinal direction of 8 divisions and a grid in the radial direction of 8 divisions). In the geometry definition, all vehicles surfaces (main bodies and transparent bodies) are in contact. However, in the interior bodies only the seats, defined by a vertical and an inclined surface, are defined. y All elements of the passengers are arranged in similar way to the seat human body posture. The neck, the arms and the legs, both divided in four sections, are connected to the trunk with dimensions and angles similar to the human body sections. The head is connected to the neck, the hands are connected to the arms and the feet are connected to the legs. In the neck, shoulders, elbows, hips, knees and ankles, they are placed rotation elements. The dimensions and inclination of each element are obtained using a numerical model based on the human body weight and height. All passengers are not in contact between them and all passengers are not in contact with the seat and with all vehicle surfaces. Fig. 2. Scheme of a bus and the 52 occupants: detailed visualization of the occupants. Fig. 2. Scheme of a bus and the 52 occupants: detailed visualization of the occupants. 2 Occupant mathematical model The numerical model, that simulates the Human Thermal Response, that works in transient conditions, is based on energy and mass balance integral equations. The equations are based on conduction, convection and radiation phenomena and are solved by the Runge- Kutta-Felberg method with error control. The human body considers:  Heat exchanges between the human body sections and the surrounding surfaces (main and interiors bodies and glass); The human body geometry is generated using the empirical equations and the equation system is generated in accordance with the inputs, namely, in accordance with the number of elements and layers considered in the human body and in the clothing level defined in the numerical simulation.  Heat exchanges between the human bodies sections of each passenger and between the human bodies sections of different passengers.  The vehicle body considers: All coefficients used in these equations are evaluated by sub-models that calculate the view factors, radiative heat exchanges, clothing level, human thermo- physiology and heat and mass transfer coefficients by convection using empirical expressions.  Heat exchanges between the vehicle surfaces (main and interiors bodies and glass) itself;  Heat exchanges between the vehicle surfaces (main and interiors bodies and glass) and the passengers; The occupants are divided in 25 cylindrical or spherical elements and each element is sub-divided in 2 2 E3S Web of Conferences 321, 03008 (2021) ICCHMT 2021 https://doi.org/10.1051/e3sconf/202132103008  Heat exchanges between the vehicle surfaces (main and interiors bodies and glass) and the external surfaces (floor, environment, and fire front). while Fig. 2 shows the details of the visualization of the external passenger surfaces. In Fig. 3, the location of the occupants in the bus and the identification of symbols in Fig. 4 to Fig. 7 are presented. The Mean Radiant Temperature method, with correction, calculates, step by step, the Mean Radiant Temperature, using the human body and vehicles bodies, calculated in each iteration, and the pre- calculated view factors. The Mean Radiant Temperature method, with correction, calculates, step by step, the Mean Radiant Temperature, using the human body and vehicles bodies, calculated in each iteration, and the pre- calculated view factors. Fig. 1. Scheme of a bus and the 52 occupants: detailed visualization of the external vehicle surfaces. Fig. 3. Location of the occupants in the bus and identification of symbols in the Fig. 4 to Fig. 7. 6 Results and discussion In Fig. 4, it is presented the Mean Radiant Temperature (MRT) that each occupant is subjected for Case A (point a), Case B (point b), and Case C (point c). a) b) c) Fig. 4. Mean Radiant Temperature (MRT) that each occupant is subjected for: a) Case A; b) Case B; c) Case C. 15 20 25 30 35 40 45 50 55 60 MRT (ºC) Body sections 15 20 25 30 35 40 45 50 55 60 MRT (ºC) Body sections 15 20 25 30 35 40 45 50 55 60 MRT (ºC) Body sections a) b) 28 30 32 34 36 38 Tskin (ºC) Body sections a) 15 Body sections 60 a) b) Body sections 15 20 25 30 35 40 45 50 55 60 MRT (ºC) Body sections b) y c) 28 30 32 34 36 38 Tskin (ºC) Body sections ) c) 15 20 25 30 35 40 45 50 55 60 MRT (ºC) Body sections Fig. 5. Skin temperature (Tskin) that each occupant is subjected for: a) Case A; b) Case B; c) Case C. -0.7 -0.6 -0.5 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 Case A Case B Case C PMV Occupant Number c) Body sections Fig. 4. Mean Radiant Temperature (MRT) that each occupant is subjected for: a) Case A; b) Case B; c) Case C. Regarding to the results obtained is possible to verify that:  When the curtain surface temperature increases the Mean Radiant Temperature increases; Fig. 6. PMV values that the occupants are subjected. Fig. 6. PMV values that the occupants are subjected.  The Mean Radiant Temperature is highest for the passenger located near the left side of the vehicle; Fig. 7. PPD values that the occupants are subjected. 5 Simplified bus model In these calculations, the air temperature and relative humidity are, respectively, 20ºC and 50%, while the internal vehicle surfaces temperatures are 20ºC. This work is divided in three parts: This work is divided in three parts:  The temperature of the internal left curtains is 20ºC (Case A);  The temperature of the internal left curtains is 40ºC (Case B);  The temperature of the internal left curtains is 60ºC (Case C). The scheme of a bus and the 52 occupants is presented in Fig. 1 and Fig. 2. Fig. 1 shows the details of the visualization of the external vehicle surfaces, 3 https://doi.org/10.1051/e3sconf/202132103008 E3S Web of Conferences 321, 03008 (2021) ICCHMT 2021 a) b) c) Fig. 5. Skin temperature (Tskin) that each occupant is subjected for: a) Case A; b) Case B; c) Case C. Fig. 6. PMV values that the occupants are subjected. 28 30 32 34 36 38 Tskin (ºC) Body sections 28 30 32 34 36 38 Tskin (ºC) Body sections 28 30 32 34 36 38 Tskin (ºC) Body sections -0.7 -0.6 -0.5 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 Case A Case B Case C PMV Occupant Number 5 10 15 Case A Case B Case C PPD (%) a) b) c) Fig. 5. Skin temperature (Tskin) that each occupant is subjected for: a) Case A; b) Case B; c) Case C. 28 30 32 34 36 38 Tskin (ºC) Body sections 28 30 32 34 36 38 Tskin (ºC) Body sections 28 30 32 34 36 38 Tskin (ºC) Body sections a) 28 30 32 34 36 38 Tskin (ºC) Body sections 28 30 32 34 36 38 Tskin (ºC) Body sections a) 28 30 32 34 36 38 Tskin (ºC) Body sections 38 6 Results and discussion 0 5 10 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 Case A Case B Case C PPD (%) Occupant Number  The Mean Radiant Temperature is higher for the left side passenger sections than for the right side passenger sections;  The Mean Radiant Temperature is higher in upper passenger sections than in lower passenger sections. In Fig. 5, it is presented the skin temperature (Tskin) that each occupant is subjected for Case A (point a), Case B (point b), and Case C (point c). The PMV and the PPV values that each occupant is subjected are presented, respectively, in Fig. 6 and Fig. 7. Fig. 7. PPD values that the occupants are subjected. The thermal comfort level for the Case A is acceptable, regarding to the category C [3], for all occupants, by negative PMV values. The skin temperature, that each occupant section is subjected, increases when the Mean Radiant Temperature increases. The highest values are verified in sections not protected by the clothing as the head, neck and hands. When the curtain surface temperature increases, Case B, the thermal comfort level of the occupants improves and the occupant seated near the warm curtain surface is comfortable according to the Categories A and 4 https://doi.org/10.1051/e3sconf/202132103008 E3S Web of Conferences 321, 03008 (2021) ICCHMT 2021 system, in Proceedings of 7th International Conference on Air Distribution in Room, ROOMVENT 2000, 9-12 July 2000, Reading, UK (2000) B [3], by negative PMV values. However, the other occupants are comfortable according to the Category C [3], also by negative PMV values. When the curtains surface temperature increases to 60ºC, Case C, the thermal comfort of the occupant seated near the warm curtain is in accordance with the Category C [3], but by positive PMV values. However, the occupant seated near the right window is comfortable in accordance with the Category C [3], by negative PMV values, and the other occupants are comfortable according to the Categories A and B [3], by negative or positive PMV values. 6. P. Fanger, Thermal comfort (Danish Technical Press, Copenhagen, 1970) 7. E. Conceição, J. Gomes, A. Ruano. 7 Conclusions 13. E. Conceição, M. Lúcio, M. Lopes. WSEAS Trans. Environ. Dev. 4, 644-654 (2008) In this study the application of passengers’ thermoregulation integral model used in the evaluation of thermal comfort in vehicles compartment equipped with internal curtains was made. The study was made for winter conditions and considering three Cases study. 14. E. Conceição, M. Lúcio. Indoor Built Environ. 19, 657-667 (2010) 15. E. Conceição, M. Lúcio. Indoor Built Environ. 18, 41-51 (2009) The numerical model was used to evaluate the thermal comfort level that seated passengers are subjected in a bus equipped with asymmetrical warm curtains. 16. E. Conceição, M. Lúcio. Build. Simul. 3, 245-261 (2010) 17. E. Conceição, M. Lúcio, Numerical study of thermal response of school buildings in summer conditions, in Proceedings of the 8th International Conference and Exhibition on Healthy Buildings (HB 2006), 4-8 June 2006, Lisbon, Portugal (2006) The warm curtains, subjected to solar radiation, can be used to improve the thermal comfort conditions of the occupants in winter conditions. However, if the increase in the curtains temperature is high, then the thermal comfort conditions can be thermal uncomfortable for the occupants. 18. J. Stolwijk, J. Hardy. Pflugers Arch. Gesamte Physiol. Menschen Tiere 291, 129-162 (1966) The authors would like to acknowledge to the project (SAICT- ALG/39586/2018) from Algarve Regional Operational Program (CRESC Algarve 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) and the National Science and Technology Foundation (FCT). 19. E. Conceição, M. Lúcio, H. Awbi. Build. Simul. 6, 51-67 (2013) 20. E. Conceição, S. Rosa, A. Custódio, R. Andrade, M. Meira, M. Lúcio. HVACR Res. 16, 401-412 (2010) 21. F. Incropera, D. De Witt, T. Bergman, A. Lavine. Principles of heat and mass transfer (Wiley, 2013) 6 Results and discussion IFAC PapersOnLine 51, 20–25 (2018) 8. E. Conceição, M. Lúcio, A. Ruano, E. Crispim. Build. Environ. 44, 871-877 (2009) 9. C. Walgama, S. Fackrell, M. Karimi, A. Fartaj, G. Rankin. P. I. Mech. Eng. D-J. Aut. 5, 543-562 (2006) Thus, regarding to results obtained, the warm curtains, subjected to solar radiation, can be used to improve the thermal comfort conditions of the occupants in winter conditions. 10. J. Raugh. R. Farrington, D. Bharathan, A. Vlahinos, R. Burke, C. Huinzenga, H. Zhang. Eur. J. Appl. Physiol. 92, 721-727 (2004) In future works, the influence of solar radiation that each passenger is subjected, also in winter conditions, will be analyzed. 11. Y. Riachi. Int. J. Environ. Prot. Policy 2, 1-8 (2014) 12. A. Mezrhab, M. Bouzidi. Appl. Therm. Eng. 26, 1697-1704 (2006) References . Awbi, Ventilation of Buildings (Routledge, 2004) 22. E. Conceição, M. Silva, J. André, D. Viegas. Int. J. Veh. Des. 24, 372-387 (2000) 2. ANSI/ASHRAE-55 Standard, Thermal environmental conditions for human occupancy (ASHRAE, Atlanta, 2017) 23. E. Conceição, M. Silva, D. Viegas. HVACR Res. 3, 311-323 (1997) 3. ISO 7730, Ergonomics of the thermal environment Analytical determination and interpretation of thermal comfort using calculation of the PMV and PPD indices and local thermal comfort criteria (International Organisation for Standardisation, Geneva, 2005) 24. K. Shek, W. Chan. Sci. Total Environ. 389, 277-82 (2008) 25. K. Velt, H. Daanen. Appl. Ergon. 62, 72-76 (2017) 4. E. Conceição, M. Lúcio, V. Vicente, V. Rosão. Int. J. Vent. 7, 267-277 (2008) 4. E. Conceição, M. Lúcio, V. Vicente, V. Rosão. Int. J. Vent. 7, 267-277 (2008) 5. E. Conceição, Evaluation of thermal comfort and local discomfort conditions using the numerical modelling of the human and clothing thermal 5
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https://openalex.org/W3143693011	https://www.degruyter.com/document/doi/10.1515/9783110731958-017/pdf	German	null	Weil STANDARD verständlich ist und DIALEKT authentisch macht	De Gruyter eBooks	2,021	cc-by	10,960	Vergeiner, Philip C.: Universität Salzburg, FB Germanistik, Erzabt-Klotz.Straße 1, A–5020 Salzburg, philip.vergeiner@sbg.ac.at Buchner, Elisabeth: Universität Salzburg, FB Germanistik, Erzabt-Klotz.Straße 1, A–5020 Salzburg, elisabeth.buchner@sbg.ac.at Fuchs, Eva: Pädagogische Hochschule Salzburg „Stefan Zweig“, Akademiestraße 23–25, A– 5020 Salzburg, eva.fuchs@phsalzburg.at Elspaß, Stephan: Universität Salzburg, FB Germanistik, Erzabt-Klotz.Straße 1, A–5020 Salzburg, stephan. elspass@sbg.ac.at Philip C. Vergeiner, Elisabeth Buchner, Eva Fuchs, Stephan Elspaß Weil STANDARD verständlich ist und DIALEKT authentisch macht Varietätenkonzeptionen im sekundären und tertiären Bildungsbereich in Österreich Zusammenfassung: Der vorliegende Beitrag zielt darauf ab, Varietätenkonzep- tionen im sekundären und tertiären Bildungsbereich in Österreich zu analysie- ren. Dabei wird gefragt, (a) was Sprecher/innen an Schule und Universität unter Varietäten wie DIALEKT und STANDARD verstehen, (b) wie sie sie jeweils bewerten und (c) welche normativen Erwartungen sie zu ihrer Verwendung in Bildungs- kontexten haben. Um diese Fragen zu beantworten, wurden halbstrukurierte Interviews einer inhaltsanalytischen Untersuchung zugeführt. Die Ergebnisse zeigen, dass es sowohl an Schule als auch Universität zum Teil ähnliche Varie- tätenkonzepte gibt. Zugleich werden jedoch auch Ambiguitäten und Widersprü- che zwischen beiden Bereichen ebenso wie innerhalb dieser offengelegt. Dies spiegelt die komplexe DIAGLOSSISCHE, z. T. auch DIGLOSSISCHE Situation in Öster- reich, wo DIALEKT und STANDARD im Alltag gleichermaßen präsent sind und ein- zelne Varietäten regionen-, situations- und wohl auch individuenspezifisch unterschiedlich bewertet und verwendet werden. Abstract: The aim of this paper is to analyse concepts of language varieties in secondary and tertiary education in Austria. The questions put forward are (a) what speakers’ notions of varieties such as dialect and standard are at schools and universities, (b) how they evaluate them and (c) what normative expecta- tions they have for their use in educational contexts. To answer these questions, semi-structured interviews were subjected to a content analysis. The results Vergeiner, Philip C.: Universität Salzburg, FB Germanistik, Erzabt-Klotz.Straße 1, A–5020 Salzburg, philip.vergeiner@sbg.ac.at Buchner, Elisabeth: Universität Salzburg, FB Germanistik, Erzabt-Klotz.Straße 1, A–5020 Salzburg, elisabeth.buchner@sbg.ac.at Fuchs, Eva: Pädagogische Hochschule Salzburg „Stefan Zweig“, Akademiestraße 23–25, A– 5020 Salzburg, eva.fuchs@phsalzburg.at Elspaß, Stephan: Universität Salzburg, FB Germanistik, Erzabt-Klotz.Straße 1, A–5020 Salzburg, stephan. elspass@sbg.ac.at Vergeiner, Philip C.: Universität Salzburg, FB Germanistik, Erzabt-Klotz.Straße 1, A–5020 Salzburg, philip.vergeiner@sbg.ac.at Buchner, Elisabeth: Universität Salzburg, FB Germanistik, Erzabt-Klotz.Straße 1, A–5020 Salzburg, elisabeth.buchner@sbg.ac.at Fuchs, Eva: Pädagogische Hochschule Salzburg „Stefan Zweig“, Akademiestraße 23–25, A– 5020 Salzburg, eva.fuchs@phsalzburg.at Elspaß, Stephan: Universität Salzburg, FB Germanistik, Erzabt-Klotz.Straße 1, A–5020 Salzburg, stephan. elspass@sbg.ac.at Open Access. © 2021 Philip C. Vergeiner, Elisabeth Buchner, Eva Fuchs, Stephan Elspaß, publiziert von De Gruyter. Dieses Werk ist lizenziert unter der Creative Commons Attribution 4.0 International Lizenz. https://doi.org/10.1515/9783110731958-017 https://doi.org/10.1515/9783110731958-017 418 \| Philip C. Vergeiner, Elisabeth Buchner, Eva Fuchs, Stephan Elspaß 418 show that there are some similar variety concepts at school and university. At the same time, however, ambiguities and contradictions between the two areas as well as within them are disclosed. Philip C. Vergeiner, Elisabeth Buchner, Eva Fuchs, Stephan Elspaß Weil STANDARD verständlich ist und DIALEKT authentisch macht Varietätenkonzeptionen im sekundären und tertiären Bildungsbereich in Österreich This reflects the complex diaglossic and partly also diglossic situation in Austria, where dialect and standard are equally present in everyday life and individual varieties are evaluated and used differ- ently depending on the region, situation and probably also on an individual basis. Schlüsselwörter: Standard, Dialekt, Varietätenkonzeptionen, Sprachwahrnehmungen, Spracheinstellungen, Normen Schlüsselwörter: Standard, Dialekt, Varietätenkonzeptionen, Sprachwahrnehmungen, Spracheinstellungen, Normen \|\| 1 Erste Ergebnisse zur Schule wurden im Rahmen des Projekts Österreichisches Deutsch als Unterrichts- und Bildungssprache erbracht, das sich allerdings stark auf den STANDARDNAHEN Bereich und verschiedene STANDARD-Varietäten des Deutschen fokussierte; vgl. bspw. de Cillia \|\| (2018). Zu Varietätenverhältnissen an der Universität kann vor allem auf Publikationen im Kontext des VAMUS-Projekts verwiesen werden, bspw. Dannerer (2018) oder Vergeiner (2021). 2 Das von Monika Dannerer und Peter Mauser geleitete Projekt wurde vom Jubiläumsfonds der Österreichischen Nationalbank finanziert (ÖNB-Projektnummer 15.827). 3 Das von Stephan Elspaß geleitete Projekt – Fonds zur Förderung der wissenschaftlichen Forschung (FWF), Projektnummer F 6010 G23 – wird als Teil des SFBs F 60 „Deutsch in Öster- reich“ vom FWF gefördert. 1 Problemaufriss Bildungsinstitutionen galten lange als die Domäne der STANDARDSPRACHE – dies wurde für den tertiären Bildungsbereich als ebenso selbstverständlich ange- nommen (vgl. zusammenfassend Dannerer 2018: 169, 174–175) wie für den se- kundären (vgl. bspw. Mattheier 1991). Der Gebrauch von NONSTANDARD- Varietäten wurde daher auch – ganz im Sinne der Sprachbarrieren-Hypothese (vgl. Ammon 1972) – als etwas Defizitäres und von allen Beteiligten Uner- wünschtes dargestellt. Mehrere Untersuchungen konnten seither jedoch nach- weisen, dass in Bildungseinrichtungen NONSTANDARD-Gebrauch sehr wohl häu- fig vorkommt – zumindest im Süden des deutschsprachigen Raums (vgl. bspw. Knöbl 2012; Steiner 2008). Anzunehmen ist, dass dies der Fall ist, weil die Spre- cher/innen an diesen Institutionen keine so negative Sicht auf den NONSTANDARD haben, wie ihnen früher bisweilen unterstellt wurde. Festzustellen ist jedoch, dass sowohl die tatsächliche Varietätenverwendung als auch die Konzepte von STANDARD und NONSTANDARD bei den Sprecher/inne/n im Bildungsbereich bis heute nur ausschnittweise untersucht wurden (vgl. für die Varietätenkonzepte von bundesdeutschen Deutschlehrer/inne/n bspw. Hochholzer 2004). Gerade in Österreich liegen zwar zur allgemeinen Sicht auf Varietäten viele Befunde vor (vgl. z. B. Bellamy 2012; Moosmüller 1991; Soukup 2009), in Hinblick auf Schule und Universität ist darüber aber nur wenig bekannt.1 Hier besteht ein klares Forschungsdesiderat. 419 Weil Standard verständlich ist und Dialekt authentisch macht \| Ziel des vorliegenden Beitrages ist es, sich dieses Desiderats anzunehmen. Gefragt werden soll, welche Varietätenkonzeptionen verschiedene Spre- cher/innen in sekundären und tertiären Bildungsinstitutionen zu STANDARD- Varietäten einerseits und NONSTANDARD-Varietäten andererseits haben. Dabei sollen auch Gemeinsamkeiten und Unterschiede zwischen den Institutionen herausgearbeitet werden. Dazu werden Interviewdaten inhaltsanalytisch unter- sucht, die in zwei Projekten entstanden sind: zum einen im Projekt VAMUS (= Verknüpfte Analyse von Mehrsprachigkeiten am Beispiel der Universität Salz- burg),2 zum anderen im Teilprojekt PP10 Wahrnehmung von und Einstellung zu Varietäten und Sprachen an österreichischen Schulen des Spezialforschungsbe- reichs Deutsch in Österreich (DiÖ).3 In der Folge wird zunächst geklärt, was wir im vorliegenden Aufsatz unter Laienkonzeptionen verstehen (Abschnitt 2). In Abschnitt 3 werden wir auf das Datenmaterial der Untersuchung eingehen, bevor wir in Abschnitt 4 die Unter- suchungsergebnisse präsentieren. Abschließend erfolgt eine Diskussion mit Fazit (Abschnitt 5). 3 Das von Stephan Elspaß geleitete Projekt – Fonds zur Förderung der wissenschaftlichen Forschung (FWF), Projektnummer F 6010 G23 – wird als Teil des SFBs F 60 „Deutsch in Öster reich“ vom FWF gefördert. 2 Konzeptionelle Grundlage Die Sichtweise linguistischer Laien auf Sprachen und Varietäten wird bereits seit längerem ausgiebig beforscht. Dies geschieht unter Bezug auf verschiedene Konzepte – zu nennen sind etwa EINSTELLUNGEN, IDEOLOGIEN, PERZEPTIONEN oder NORMEN. Nicht immer ist klar, ob und inwiefern diese Begriffe klar abgrenzbar sind. In der Folge sollen die einzelnen Begriffe kurz diskutiert werden, um letzt- lich für einen holistischen Blickwinkel auf diese Phänomene in Form von ‚Vari- etätenkonzeptionen‘ zu plädieren. Eines der ersten Paradigmen, welches die Laiensicht auf Sprache systema- tisch untersuchte, war die Einstellungsforschung. Ausgehend von sozialpsycho- logischen Zugängen werden EINSTELLUNGEN dabei bis heute häufig mit der klas- sischen Definition von Allport (1935: 810) als sozial erlernte Dispositionen 420 \| Philip C. Vergeiner, Elisabeth Buchner, Eva Fuchs, Stephan Elspaß 420 verstanden, die beeinflussen, wie auf gewisse Phänomene (z. B. Sprachen oder Varietäten) reagiert wird – dies im Denken (kognitiv), Werten (affektiv) und Handeln (konativ) (vgl. z. B. Ajzen 1989; aus linguistischer Sicht Lasagabaster 2004). Untersucht wurden Spracheinstellungen häufig indirekt mittels der „matched-guise“-Methode (Lambert et al. 1960), womit äußerst konsistente Befunde erbracht werden konnten, wie etwa, dass STANDARD-Sprecher/inne/n vor allem status-, NONSTANDARD-Sprecher/inne/n hingegen vor allem solidari- tätsbezogene Eigenschaften zugewiesen werden (vgl. für Österreich z. B. Soukup 2009; zu Erklärungsansätzen Schoel & Stahlberg 2012). In den letzten Jahren ist jedoch vermehrte Kritik an der (vermeintlichen) Kontextlosigkeit und Stabilität beim gängigen Einstellungskonzept aufgekom- men. Das führte zu einer Modifikation des Einstellungsbegriffs im Rahmen einer „sozial-konstruktivistische[n] Perspektive“ (Cuonz 2014: 32). Hierbei werden Einstellungen als soziale Konstrukte aufgefasst, die in Interaktionen laufend entstehen, modifiziert oder stabilisiert werden – die Folge war eine Öffnung der Einstellungsforschung hin zu qualitativen Methoden wie der Gesprächsanalyse (vgl. Liebscher & Dailey-O’Cain 2009). Zugleich näherte sich der Einstellungs- begriff dadurch (noch) stärker dem Ideologiebegriff an (vgl. bspw. Soukup 2009: 88–89; zur Ähnlichkeit beider Konzepte etwa Garrett, Coupland & Willi- ams 2003). Der Begriff IDEOLOGIE, der vorrangig in sprachanthropologischen Paradigmen verwendet wird, bezeichnet dabei diskursiv erzeugte „conceptual schemes“ (Irvine & Gal 2000: 35). Ideologien liefern „Just So Stories“, „charac- teristically by the way that they constitute rationalizing, systematizing, indeed most importantly naturalizing schemata that ‚explain‘ the indexical value of signs“ (Silverstein 1992: 316). Wie Einstellungen sind Ideologien sozial erlernt – dabei prägen sie das Verhalten der Sprecher/innen (vgl. Errington 1999: 115; Kroskrity 2009: 505) ebenso wie auch ihre Bewertungen (vgl. Cavanaugh 2013) bzw. das Denken und den Diskurs über Sprache (vgl. Coupland & Jaworski 2004: 36). 2 Konzeptionelle Grundlage Die Spannweite von Ideologien ist groß und reicht „from seemingly neutral ‚cultural conceptions‘ to strategies for maintaining social power“ (Woolard 1992: 239). Sprachideologien sind dabei häufig mit außersprachlichen Kategorien und Institutionen verwoben (wie Nation, Rasse, Geschlecht; vgl. Woolard & Schieffelin 1994: 55–56). In diesem Sinne verknüpft bspw. die Ideo- logie des „homogeneism“ (Elspaß & Niehaus 2014: 48) eine homogene STAN- DARDSPRACHE mit einer homogenen Gesellschaft. Sowohl Einstellungen als auch Ideologien sind vielfach normativ – sie ge- hen mit Handlungsbewertungen und -erwartungen einher. Damit überlappen sie sich mit dem Normkonzept. Charakteristisch für NORMEN ist jedoch ihr klarer Handlungsbezug. Normen beruhen auf sozial geteilten Erwartungen zum er- Weil Standard verständlich ist und Dialekt authentisch macht \| 421 laubten, verbotenen oder präferierten Handeln. Werden sie nicht befolgt, so ruft dies oft wertende Reaktionen bzw. Sanktionen hervor (vgl. zum Normbegriff ausführlich Bartsch 1987; Gloy 1975, 2004; Vergeiner 2021). Voraussetzung für die normbezogenen Erwartungen und Wertungen ist, dass Sprecher/innen Normvorstellungen haben, für die ein Geltungsanspruch kennzeichnend ist (der jedoch von faktischer Geltung zu unterscheiden ist, vgl. Hundt 2009: 118). Zur Herstellung und Stützung dieser Geltung werden Normen häufig im Rahmen von normativen Systemen mit anderen normativen Entitäten (z. B. auch Einstel- lungen, Ideologien) verbunden (vgl. Berger & Luckmann 2016). Eine sehr wich- tige Rolle spielen dabei auch Werte und Prinzipien – ähnlich wie Normen die- nen sie zur Handlungsorientierung in sozialen Gruppen, schreiben dabei aber keine konkreten Handlungen, sondern vielmehr sein-sollende Zustände vor (vgl. Gloy 1975: 33). Was sprachbezogene Einstellungen, Ideologien, Normen, Werte und Prinzi- pien verbindet, ist, dass sie letztlich (auch) auf die Laiensicht auf sprachliche Phänomene abzielen – wenn auch mit unterschiedlichen Schwerpunktsetzun- gen und wissenschaftsgeschichtlichem Hintergrund. Die Laiensicht auf Varietä- ten wird heutzutage zunehmend im Rahmen der Wahrnehmungsdialektologie zusammengefasst. Entstanden als folk linguistics beschäftigte sie sich anfangs vor allem mit der PERZEPTION von Laien auf DIALEKT-Grenzen und die (Un-)Ähnlichkeit von DIALEKTEN (vgl. bspw. Preston 2009). Gegenwärtig wird als Aufgabe der Wahrnehmungsdialektologie indes vielfach die allgemeine „Be- schreibung kognitiver Strukturen des Alltagswissens“ gesehen (Anders 2010: 3; vgl. auch Hundt, Palliwoda & Schröder 2015: 385–386 sowie kritisch bspw. Kre- feld & Pustka 2010: 13–14). Dies schließt Untersuchungen zur Laiensicht auf die Strukturierung und Verbreitung sprachlicher Varietäten ebenso ein wie varietä- tenbezogene Attribuierungen, Assoziationen und Einstellungen (vgl. bspw. Hundt, Palliwoda & Schröder 2015: 369; Purschke & Stoeckle 2019). Als Überbe- griff für all diese zusammenhängenden Phänomene bietet sich der Ausdruck „Sprachkonzeption“ an (vgl. z. B. 2. wie die GP die jeweiligen Varietäten und ihre Verwendung im Rahmen ei- nes Bildungskontextes jeweils bewerten und 3. wann die Varietäten in diesem Bereich den GP folgend Verwendung finden sollen. 3. wann die Varietäten in diesem Bereich den GP folgend Verwendung finden sollen. Zu beachten ist, dass wir den Laienbegriff in der vorliegenden Untersuchung sehr weit definieren – wir zählen als Laienäußerungen alle varietätenbezogenen Aussagen, die aus einer nicht-wissenschaftlichen Perspektive geäußert werden, auch wenn sie von GP stammen, die sich beruflich mit Sprache beschäftigen und/oder eine entsprechende Ausbildung aufweisen (z. B. Deutschlehrer/innen in der Schule oder Philolog/inn/en an der Universität). Wir tun dies, weil auch diese GP eine Alltagssicht auf Variation haben, die zwar beeinflusst von ihrem ‚Expertenwissen‘ sein mag, aber nichtsdestoweniger nicht damit zusammenfal- len dürfte. Auf welchen Daten und Methoden die Untersuchung beruht, wird im nächsten Abschnitt ausführlicher thematisiert. \|\| 4 Die Datenkorpora von DiÖ wie VAMUS beinhalten jeweils auch Fragebogendaten, die auf- grund des Fokus auf qualitative Daten hier ausgespart bleiben (vgl. zu diesen Daten auch Vergeiner et al. 2019). Ausgeklammert werden weiters die Sprachgebrauchsaufnahmen beider Projekte, die nur indirekt Aufschluss über Varietätenkonzepte geben. Da der Untersuchungs- schwerpunkt auf Salzburg und Tirol liegt, werden außerdem die Daten des DiÖ-Teilprojekts aus den Bundesländern Vorarlberg und Wien ausgespart. 2 Konzeptionelle Grundlage Hundt, Palliwoda & Schröder 2015: 369). Im Rahmen des vorliegenden Aufsatzes sollen solche Varietätenkonzeptionen fo- kussiert werden, ohne den Forschungsgegenstand näher auf die weiter oben diskutierten Einzelphänomene festlegen zu wollen. Dies geschieht, weil, wie gezeigt, die Begriffe oftmals nicht abgrenzbar sind, sondern vielmehr zusam- menhängen. Im Folgenden sollen laienlinguistische Sprachkonzeptionen in Hinblick auf drei Aspekte genauer betrachtet werden: 1. was die untersuchten Gewährspersonen (= GP) unter den jeweiligen Varie- tätenbegriffen verstehen, 1. was die untersuchten Gewährspersonen (= GP) unter den jeweiligen Varie- tätenbegriffen verstehen, 422 \| Philip C. Vergeiner, Elisabeth Buchner, Eva Fuchs, Stephan Elspaß 2. wie die GP die jeweiligen Varietäten und ihre Verwendung im Rahmen ei- nes Bildungskontextes jeweils bewerten und 2. wie die GP die jeweiligen Varietäten und ihre Verwendung im Rahmen ei- nes Bildungskontextes jeweils bewerten und 5 Die Interviewleitfäden enthielten Fragen zu den Sprachbiographien der GP, zum (wahrge- nommenen) Sprachgebrauch in den Institutionen sowie zu Spracheinstellungen. 6 Bei der Überführung in den STANDARD wurden auch Merkmale der GESPROCHENEN SPRACHE wie Rückversicherungs- und Häsitationssignale ausgespart. 3 Daten und Methoden In der vorliegenden Studie wird ein Vergleich zwischen Varietätenkonzeptionen an sekundären und tertiären Bildungsinstitutionen in Österreich unternommen. Die analysierten und gegenübergestellten Befragungsdaten stammen zum einen aus dem Teilprojekt Wahrnehmungen von und Einstellungen zu Varietäten und Sprachen an österreichischen Schulen des SFB Deutsch in Österreich (= DiÖ) und zum anderen aus dem Projekt Verknüpfte Analyse von Mehrsprachigkeiten am Beispiel der Universität Salzburg (= VAMUS) (s. o. Anm. 2 und 3). Beide Projekte verbindet das Ziel, Perzeptionen und Attitüden zur Varietätenverwendung in Bildungskontexten zu untersuchen, wobei der Erhebungsschwerpunkt jeweils in Westösterreich liegt. Tabelle 1 gibt einen Überblick über das Datenmaterial beider Projekte, soweit es für die vorliegende Studie herangezogen wurde.4 Die Weil Standard verständlich ist und Dialekt authentisch macht \| 423 aus halbstrukturierten Interviews5 gewonnenen Daten wurden standardortho- grafisch transkribiert6 und inhaltsanalytisch ausgewertet. Tab. 1: Untersuchtes Datenmaterial. Projektkorpora DiÖ VAMUS Erhebungszeitraum 2017/2018 2014/2015 Untersuchungsort(e) vier berufsbildende mittlere und höhere Schulen in den Bundes- ländern Salzburg und Tirol eine mittelgroße öffentliche Universität in Grenznähe zu Deutschland (Universität Salz- burg) GP Lehrer/innen aller Fächer und Schüler/innen der 10. Schulstu- fe aus 4 Klassen (50 % städ- tisch; 50 % maturaführend); großteils aus der unmittelbaren Region stammend; bei Schü- ler/inne/n etwas mehr als die Hälfte Sprecher/innen anderer Erstsprachen (vor allem Tür- kisch und die BKS-Sprachen) Lehrende und Studierende an allen Fakultäten; jeweils bedeu- tender Anteil von GP aus Deutschland sowie Spre- cher/innen mit anderer Erstspra- che (vor allem DaF-, zum Teil aber auch DaZ-Sprecher/innen) GP-Zahl in den Interviews Lehrer/innen: 22 Schüler/innen: 140 Lehrende: 36 Studierende: 66 GP Wie Tabelle 1 zeigt, lässt sich das Datenmaterial von DiÖ und VAMUS nicht nur wegen der Befragungsmethode, sondern auch wegen ähnlicher Erhebungsräu- me, -zeitpunkte und ähnlich großer GP-Zahlen (> 100) gut vergleichen. Es be- stehen allerdings gewisse Unterschiede bei den befragten Gruppen: Während im DiÖ-Korpus mehrere Untersuchungsorte berücksichtigt werden (je eine städ- tische und eine ländliche Schule in Salzburg und Tirol), beschränkt sich VAMUS auf nur einen Standort. Gleichwohl ist bei der Gruppe der universitären GP – entsprechend dem größeren universitären Einzugsgebiet – eine größere Hetero- genität gegeben: Anders als im Schulkontext kommen vermehrt Sprecher/innen vor, die nicht in Österreich bzw. im deutschsprachigen Raum aufgewachsen sind. Ein weiterer Unterschied besteht in der Altersverteilung, die sich bei den 424 \| Philip C. Vergeiner, Elisabeth Buchner, Eva Fuchs, Stephan Elspaß 424 Schüler/inne/n im Gegensatz zu den anderen GP-Gruppen als weitestgehend homogen (16–17 Jahre) beschreiben lässt. 4 Ergebnisse In diesem Abschnitt werden die Ergebnisse der Untersuchung präsentiert. Dabei wird zunächst auf die Befunde zur Universität, anschließend jene zur Schule eingegangen. Jeweils erfolgt zunächst eine Analyse der Konzeptionen von STAN- DARD, anschließend der von NONSTANDARD. Aus Darstellungsgründen werden in der Folge nur exemplarisch Intervie- wäußerungen wiedergegeben. Bei der Zitation verweisen die Zahlen auf die Zeitfenster in den Aufnahmen. Zur Wahrung ihrer Anonymität werden die GP mit Siglen belegt. Sowohl bei den GP aus der Schule als auch bei denen aus der Universität steht der erste Buchstabe dabei für die GP-Gruppe (S = Studieren- de(r) bzw. Schüler/in); L = Lehrende(r)). Bei den GP aus der Universität stellen die Folgebuchstaben pseudonymisierte Initialen dar (z. B. „SIH“). Bei den GP aus den Schulen setzen sich die Folgezeichen zum einen aus Buchstaben, die den Schulstandort kennzeichnen, zum anderen aus Zahlen, die den einzelnen Proband/inn/en zugewiesen wurden, zusammen (z. B. „SA3“). 3 Daten und Methoden Schüler/inne/n im Gegensatz zu den anderen GP-Gruppen als weitestgehend homogen (16–17 Jahre) beschreiben lässt. 4.1.1 Konzeptionen von STANDARD Universitäten gelten häufig als reine Domänen des STANDARDS; in ihnen werde, so die verbreitete Ansicht, nur die STANDARDSPRACHE gebraucht (vgl. Dannerer 2018: 169, 174–175). Im VAMUS-Korpus zeigt sich tatsächlich, dass in vielen universitären Situationen die Verwendung der STANDARDSPRACHE erwartet wird. Dies gilt vor allem für Prüfungen, den Sprachgebrauch von und mit Lehrenden sowie studentische Präsentationen. Diese Normvorstellungen wie auch die Rechtfertigungen dafür deuten auf eine Konzeption der STANDARDSPRACHE als formelle, distanzsprachliche Varietät hin. So meint etwa LIH, wenn „[du] offiziell […] als Lehrender vorne stehst, hast natürlich eine gewisse Rolle […] und das ist schon verbunden mit der Standardsprache“ (LIH 26:28–26:42). Auf die Universi- tät allgemein bezogen betont auch LUJ, er sei ein „Verfechter, dass man die Hochsprache sprechen sollte, gerade wenn es um offizielle Dinge geht, auch im Studium“ (01:36–02:01). Mit seiner distanzsprachlichen Funktion geht einher, Weil Standard verständlich ist und Dialekt authentisch macht \| 425 dass der Gebrauch des STANDARDS mit Höflichkeit und Respekt assoziiert wird. Vor allem in der Kommunikation mit Lehrenden halten Studierende den STAN- DARD daher für angemessen. So ist etwa SIC überzeugt, es gebe ein „Niveau an Sprache, Standardsprache“, das für einen „respektvolle[n] Umgang“ mit Leh- renden unabdingbar sei; man rede mit ihnen „halt natürlich nicht so wie mit […] Freunden“ (32:44–33:14). Neben der Konzeption, dass die STANDARDSPRACHE diejenige Varietät ist, mit der man dem Gegenüber in besonderer Weise Höflichkeit und Respekt zum Ausdruck bringt, wird sie auch häufig mit Korrektheit assoziiert. Dahinter mag die Ideologie stehen, dass (nur) die STANDARDSPRACHE korrekt sei. Für etliche GP, wie etwa SUI, ist die STANDARDSPRACHE das „korrekte Deutsch, das auch in ge- schriebener Sprache existiert und bestimmte grammatikalische Regeln hat und Normen“ (SUI 19:12–19:32). Gerade die Verbindung zwischen SCHRIFTLICHKEIT – NORMIERTHEIT – STANDARDSPRACHLICHKEIT ist an der Universität weit verbreitet. Der STANDARD sei „halt dann wirklich korrekt“, insofern man spricht, wie man schreibt, urteilt etwa SIH (27:21–27:28). Diese Gleichsetzungen sind wohl ur- sächlich dafür, dass STANDARD-Sprecher/inne/n auch eine höhere Kompetenz und Professionalität unterstellt wird. LOB bspw. ist „nicht dafür, dass wir […] im Lehrbetrieb Umgangssprache sprechen […]. Die Professionalität hängt auch ein bisschen daran, dass man gehobeneres Deutsch spricht“. Nicht nur auf sie wir- ken bspw. Sprecher/innen aus dem Norden Deutschlands allein wegen ihrer STANDARDNAHEN Sprechweise „unglaublich kompetent“ (16:18–17:37). 4.1.1 Konzeptionen von STANDARD In Verbin- dung mit solchen Einstellungen steht, dass die STANDARDSPRACHE als bildungs- sprachliche Varietät konzipiert wird: „Ich glaube schon […], dass es erwünscht ist, wenn man halt schöner spricht […], es klingt irgendwie gebildeter“, denkt etwa SUG (24:45–25:15). Auch SOE bekundet, in Referaten sei STANDARD zu ver- wenden, weil es „einfach seriöser klingt“ – „wissenschaftliche Sachen, die sind halt doch irgendwie immer eher bisschen steif und das ist für mich auch die Standardsprache“ (21:34–22:42). Eine besonders häufig vorgebrachte Zuschreibung der STANDARDSPRACHE ist ihre hohe Verständlichkeit. Im Allgemeinen wird die STANDARDSPRACHE als ver- ständlicher und damit als angemessener für universitären Sprachgebrauch angesehen als NONSTANDARD. So ist etwa SIC dafür, „dass die Professoren selber darauf schauen bisschen, dass alle Hochdeutsch sprechen“ – NONSTANDARD würden nämlich „nicht alle verstehen“ (05:31–05:37). Als besonders unver- ständlich werden vom STANDARD abweichende Varietäten für Sprecher/innen mit anderer L1 als Deutsch angesehen. So erwartet etwa LOE, dass man wegen der „Internationalisierung unserer Universitäten“ und der „zahlreichen Inco- mings“ generell STANDARD verwenden sollte. „Dass sie unsere Sprache verste- 426 \| Philip C. Vergeiner, Elisabeth Buchner, Eva Fuchs, Stephan Elspaß 426 hen“, sei ein „Argument der Höflichkeit“ (09:18–10:52). Bisweilen wird der Ge- brauch der STANDARDSPRACHE auch in Gegenwart von Sprecher/inne/n aus Mit- tel- und Norddeutschland befürwortet, da auch diesen unterstellt wird, keinen bairisch-österreichischen NONSTANDARD zu verstehen: „wenn ich mit […] jeman- dem aus Norddeutschland spreche, dann versteht er mich nicht, so wie wir zwei jetzt reden, oder hat seine Mühe nur, da muss ich mich anpassen und Hoch- deutsch verwenden“, wertet etwa die Österreicherin SUH (19:39–19:51).7 Bun- desdeutschen Sprecher/inne/n (vor allem aus dem Norden und der Mitte Deutschlands) wird an der Universität Salzburg allerdings nicht nur unterstellt, keinen regionalen NONSTANDARD zu verstehen; ihnen wird auch zugeschrieben, die ,echten‘ bzw. ,besten‘ STANDARD-Sprecher/innen zu sein: „sie sprechen na- türlich schöner, akkurater, und nicht so schlampig wie wir Österreicher“, urteilt z. B. die Österreicherin VIA (01:52–02:01). Das ist nicht nur die Fremdwahrneh- mung österreichischer GP, auch viele bundesdeutsche GP sind davon über- zeugt. LUJ vertritt etwa die Meinung, dass er in Österreich „eigentlich noch nie auf einen gestoßen [ist], der Hochdeutsch spricht“ (17:10–17:14), höchstens auf Sprecher/innen, die „Pseudohochdeutsch“ verwenden. Was er als Norddeut- scher spreche, „komme dem Hochdeutschen wahrscheinlich relativ nahe“, die STANDARDORIENTIERTE Sprechweise in Österreich sei hingegen „vom Hochdeut- schen relativ weit entfernt und hat noch immer ganz deutliche dialektische [sic] Einfärbungen“ (03:30–03:50). \|\| 7 Auffällig ist, dass die Sprechweise im Ausschnitt nicht sonderlich DIALEKTAL ist, vgl. die Übertragung der Transkription in GAT2: „we_wenn i mit am (-) m_mit am (.) mit jemanden aus NORDdeutschland sprech? \| dann verSTEHT er mi ned, \| so wie WIR zwa jetzt reden;=JA? \| °h äh oder (.) hot seine MÜhe nur;=JO?= \| =do muss i mi ONpassn.“ Zu erwähnen ist, dass nicht alle GP aus der Ansicht, dass Bundesdeutsche NONSTANDARD nicht verstehen, folgern, es sollte STANDARD mit ihnen gesprochen werden – vielmehr sind sie der Ansicht, Deutsche sollten NONSTANDARD verstehen lernen (vgl. dazu Vergeiner 2020). 9 Um Missverständnisse zu vermeiden, sei erwähnt, dass die Sprecherin hier natürlich eine völlig korrekte Ausspracheform vorbringt, diese aber für defizient hält. 8 Dies führt etwa zu der paradoxen Situation, dass Sprecher/innen, die ein vermeintlich ,von regionalen Merkmalen freies Deutsch‘ sprechen, in einer bestimmten Region verortet werden, z. B. „in der mitte von deutschland“ (vgl. Beuge 2019: 161; auch 188, 227, 274f.). \|\| 7 Auffällig ist, dass die Sprechweise im Ausschnitt nicht sonderlich DIALEKTAL ist, vgl. die Übertragung der Transkription in GAT2: „we_wenn i mit am (-) m_mit am (.) mit jemanden aus NORDdeutschland sprech? \| dann verSTEHT er mi ned, \| so wie WIR zwa jetzt reden;=JA? \| °h äh oder (.) hot seine MÜhe nur;=JO?= \| =do muss i mi ONpassn.“ Zu erwähnen ist, dass nicht alle GP aus der Ansicht, dass Bundesdeutsche NONSTANDARD nicht verstehen, folgern, es sollte STANDARD mit ihnen gesprochen werden – vielmehr sind sie der Ansicht, Deutsche sollten NONSTANDARD verstehen lernen (vgl. dazu Vergeiner 2020). 8 Dies führt etwa zu der paradoxen Situation, dass Sprecher/innen, die ein vermeintlich ,von regionalen Merkmalen freies Deutsch‘ sprechen, in einer bestimmten Region verortet werden, z. B. „in der mitte von deutschland“ (vgl. Beuge 2019: 161; auch 188, 227, 274f.). 9 Um Missverständnisse zu vermeiden, sei erwähnt, dass die Sprecherin hier natürlich eine völlig korrekte Ausspracheform vorbringt, diese aber für defizient hält. 10 Der durchaus umstrittene Begriff wird hier verwendet, da es sich bei vielen GP um eine Teilnehmerkategorie handelt (s. u.). Wir nutzen ihn als solchen, ohne damit eine diskrete Sprachschicht zwischen DIALEKT und STANDARD postulieren zu wollen (vgl. zur Diskussion bspw. Vergeiner 2019). 4.1.1 Konzeptionen von STANDARD STANDARDSPRACHLICHKEIT wird oft damit gleichgesetzt, dass eine Sprechweise frei von regionalen Merkmalen sei.8 Dabei werden Merkmale als STANDARDkon- stitutiv genannt, die auf ein sehr rigides, monozentrisches Konzept von STANDARD hinweisen – die Bayerin SOE bspw. ist davon überzeugt, sie könne „einfach nicht richtig Hochdeutsch sprechen […] ich sage zum Beispiel [ʃpɔɐ̯t]9 und nicht [ʃpɔʁt], oder im Bayrischen gibt es kein summendes [z] […], oder das [ɒ] ist ganz Weil Standard verständlich ist und Dialekt authentisch macht \| 427 anders“ (23:23–23:41). Diese starre Auffassung des STANDARDS hat zur Folge, dass nicht nur Österreicher/inne/n, sondern bisweilen überhaupt den meisten oder allen Deutschsprachigen abgesprochen wird, eine (mündliche) STANDARD- KOMPETENZ zu besitzen. LAF hält den GESPROCHENEN STANDARD etwa für eine „reine Kunstsprache“ – es gebe nur „Umgangssprache“ und „Österreichisch ist genau- so […] wenig Hochdeutsch wie jetzt irgendeine Umgangssprache in Deutsch- land“ (72:05–73:08). Andere GP sind indessen bereit, mehr Variation zu akzep- tieren und vertreten Positionen, die eher einem GEBRAUCHSSTANDARD (vgl. Elspaß & Kleiner 2019: 159–160) entsprechen – so charakterisiert etwa LED sein Deutsch zwar als „ein bisschen österreichisch gefärbt, aber grundsätzlich ist es die Hochsprache“, auch wenn er Strukturen gebraucht, „die man natürlich in Norddeutschland nicht verwenden würde“ (16:52–17:11). Insgesamt sind solche Konzeptionen aber eher selten – auch der tatsächliche Sprachgebrauch ist eher von einem am Monozentrismus und der Explizitlautung orientierten Konzept des STANDARDS geprägt (vgl. ausführlich Vergeiner 2021). 4.1.2 Konzeptionen von NONSTANDARD Auch wenn an der Universität vielfach der Gebrauch von STANDARD erwartet wird und die Konzeptionen von STANDARD generell eher positiv ausfallen, wird an der Universität nicht ausschließlich STANDARDSPRACHLICHKEIT propagiert. Etliche GP halten die Verwendung von DIALEKT und UMGANGSSPRACHE10 als infor- melle, eher nähesprachlichere Varietäten an der Universität für legitim, zumin- dest in gewissen Situationen wie spontanen Wortmeldungen oder Sprechstun- dengesprächen. Dafür scheinen nicht zuletzt gewisse Einstellungen verantwortlich, die ins- besondere DIALEKT mit Attributen wie Authentizität, Identität oder Spontanität verbinden. Dass DIALEKT an der Universität vorkomme, hält etwa SUK für „be- rechtigt“, denn „es macht das Ganze ein bisschen ungezwungen und lebendig, authentisch“ (20:48–20:58). Auch der Lehrende LAE bemerkt, dass durch die Verwendung von NONSTANDARD-Varietäten der Sprachgebrauch an der Universi- tät „aufgelockert“ werde – dadurch komme man „wesentlich authentischer rüber“, sei „pointierter“ und „vom alltäglichen Umgang her“ sei die Kommuni- 428 \| Philip C. Vergeiner, Elisabeth Buchner, Eva Fuchs, Stephan Elspaß 428 kation dadurch „einfach […] viel einfacher“ (26:20–26:50). Äußerungen wie diese verweisen einerseits auf eine Ambivalenz der Konzeptionen von STAN- DARD, insofern Zuschreibungen wie Formalität und Distanziertheit auch negativ wahrgenommen werden können. Sie belegen andererseits aber auch die hohe Loyalität, die etliche GP an der Universität gegenüber NONSTANDARD-sprach- lichen Varietäten aufweisen – wie etwa SOE, die von sich behauptet, „immer Dialekt“ zu reden, komme sie sich doch beim Sprechen des STANDARDS „immer so gekünstelt vor“, es sei dann ihre „Stimme ganz anders und das bin dann einfach nicht mehr ich“. Sie wertet es daher positiv, dass sie an der Universität auch ihren DIALEKT sprechen dürfe (17:33–18:42). Doch auch GP, die selbst keine DIALEKT-Sprecher/innen sind, verbinden mit dem DIALEKT vielfach positive Eigenschaften. Auffallend oft werden dabei ästhe- tische Urteile gefällt: „Mir […] gefallen Dialekte“, betont etwa SOF, der weiter ausführt, die Verwendung von DIALEKT bringe „ein bisschen Pepp“ und „Span- nung“ in Lehrveranstaltungen, während es ohne Sprachvariation „langweilig“ sei (02:33–03:02). Besonders häufig wird auch darauf verwiesen, dass es „gut“ sei, wenn man „hört, wo wer herkommt“ (SUA 15:31–15:48). Und während eini- ge GP durch den Gebrauch von DIALEKT zumindest „keine Nachteile“ sehen, „solange alle Anwesenden zurechtkommen“ – schließlich sei „Sprache“ nur „ein Mittel der Verständigung“ (LOH 03:37–04:49) – tragen DIALEKTE nach An- sicht anderer GP zur sprachlichen Vielfalt an der Universität bei. Das wird als positiver Wert gesehen: Z. B. 4.1.2 Konzeptionen von NONSTANDARD sollte SOL zufolge „nicht jeder sprachlich so zu- recht gedengelt werden […] dass alles uniform klingt“ (33:48–33:56) – „Diversi- tät“ sei schließlich „immer etwas Gutes“ (38:47–38:56). Ein Topos, der in die- sem Zusammenhang immer wieder angesprochen wird, ist der (vermeintliche) Schwund des DIALEKTS: SUK bspw. befürchtet, dass manche Personen DIALEKTE „ausrotten“ und „aussortieren“ wollen; das sei „schade“, habe der DIALEKT doch einen „gewissen Wert“, gerade im „Alpenraum“, der DIALEKTAL „so extrem viel- schichtig ist“ und wo DIALEKTE auch für die kulturelle Identität „prägend sind“; um den DIALEKT-Abbau zu verhindern, sollten DIALEKTE auch an der Universität Verwendung finden (31:42–33:38). Allerdings treten an der Universität auch negative Einstellungen zu und Wer- tungen von NONSTANDARD-Varietäten zutage. Im Vergleich zur STANDARDSPRACHE wird insbesondere der DIALEKT als potentielles Verständnishindernis dargestellt (s. o.), außerdem sei es so, wie etwa LOC bemerkt, dass viele „Intelligenz nicht mit Dialekt“ verbinden (12:41–12:59). Damit zusammenhängend wird NONSTAN- DARD oftmals als inkorrekte und falsche Form der Sprachverwendung begriffen. So charakterisiert etwa SIH den Begriff des DIALEKTS wie folgt: „Dialekt ist mehr so, ja, ich weiß eigentlich eh, wie es richtig ist, aber es fällt mir unheimlich Weil Standard verständlich ist und Dialekt authentisch macht \| 429 schwer, das im Sprachgebrauch anzuwenden“ (32:24–32:33). Andere GP vertre- ten zwar sehr wohl die Ansicht, dass auch der DIALEKT Regeln und Normen ha- be, obgleich diese vom STANDARD abweichen. So ist etwa nach SIC im STANDARD „Vieles nicht richtig“, was „im Dialekt einfach richtig ist […], er [= der DIALEKT] hat einfach eigene Regeln und eine eigene Grammatik“ (37:28–37:34). Nichts- destoweniger halten viele GP den DIALEKT zumindest in Bezug auf die wissen- schaftliche Sprachverwendung jedoch für nicht ausreichend ausgebaut. „Einen wissenschaftlichen Vortrag“, ist etwa LEA überzeugt, „komplett im Dialekt, das geht ja kaum […], Dialekt hat ja einfach nicht diesen funktionalen Geltungsbe- reich“ (05:40–05:50). Auch SUH äußert z. B. die Ansicht, im DIALEKT könne man nicht „kurz und prägnant und klar formulieren“, weshalb man in Prüfungen STANDARD nutzen solle (19:51–20:25). Häufiger vorgebracht wird auch der (Irr-)Glaube, der DIALEKT sei ein Resul- tat des Sprachverfalls oder eine, wie etwa SAB äußert, „verzerrte Form […] von der Standardsprache“ (29:41–29:49). 4.2 Schule Auch an der Schule existieren unterschiedliche Varietätenkonzeptionen, die z. T. mit denen an der Universität übereinstimmen, sich z. T. auch unterschei- den. In der Folge werden zunächst die STANDARD-, dann die NONSTANDARD- Konzepte in der Schule besprochen und mit der Universität verglichen. 4.1.2 Konzeptionen von NONSTANDARD Eine besonders drastische Ansicht ist, vermehrter Gebrauch des DIALEKTS sei schädlich, wie sie etwa SEG vertritt; sie berichtet in diesem Zusammenhang von einer Lehrveranstaltung in „Stimmbil- dung“, wo von einer externen Lehrenden (einer „Opernsängerin aus München“) vermittelt worden sei, der Gebrauch von DIALEKT schädige die Stimmbänder und wenn jemand „ständig Dialekt spricht“, könne der-/diejenige die „Stimme ver- lieren“ (36:42–38:00). Negative Konzeptionen finden sich häufig auch von der UMGANGSSPRACHE. Von manchen GP wird sie sogar als noch weniger angebracht für den Gebrauch an der Universität eingestuft als etwa der DIALEKT; so meint etwa LAC, man habe „weniger ein Problem“, dass das „Sprachniveau zu niedrig wird […], wenn man Dialekt spricht […] als wenn man Umgangssprache spricht“ (07:11–07:22). Sol- che Konzeptionen legen nahe, dass die UMGANGSSPRACHE nicht von allen GP als Sprechweise ,zwischen‘ DIALEKT und STANDARD begriffen wird – auch wenn viele GP dies tun –, sondern als SOZIOLEKT: „Umgangssprache ist halt, immer so viel Englisch drinnen […], das ist halt Jugendsprache“ definiert den Begriff etwa SIH (27:28–27:43). Andere GP bekunden, das Konzept überhaupt nicht zu kennen oder sie rücken es in die Nähe der STANDARDSPRACHE. So etwa LEG, wenn er meint, er habe eigentlich „immer in Dialekt und Schriftsprache gedacht“ – durch die Fragestellungen bei der VAMUS-Erhebung sei er jedoch darauf ge- kommen, dass es „dreigeteilt“ ist, sei doch ein „große[r] Unterschied zwischen wirklich Schriftdeutsch“ und dem, „was ein österreichischer Fernsehsprecher redet“ (06:08–06:31). Dass nach LEGs Ansicht selbst professionelle Spre- cher/innen NONSTANDARD in Form einer UMGANGSSPRACHE verwenden, belegt 430 \| Philip C. Vergeiner, Elisabeth Buchner, Eva Fuchs, Stephan Elspaß 430 nicht zuletzt die eher monozentrische Auffassung des STANDARDS vieler GP, von welcher oben die Rede war. nicht zuletzt die eher monozentrische Auffassung des STANDARDS vieler GP, von welcher oben die Rede war. 4.2.1 Konzeptionen von STANDARD Betrachtet man die Konzepte von STANDARD an der Schule, fällt zunächst ein Unterschied zwischen Stadt und Land auf: Der STANDARD wird von den meisten GP aus dem städtischen Raum als Sprache des täglichen Umgangs definiert. So ist es Schülerin SA47 zufolge eine „komplett normale Sprache“, die in der „All- tagskommunikation“ gesprochen wird (12:14–13:21). SA10 bestätigt das dahin- gehend, als diese Varietät etwas sei, „das alle reden“ und „das am Ende des Tages von allen verstanden wird“ (19:35–20:21). SA21 geht noch weiter, indem sie meint, dass die STANDARDSPRACHE diejenige Varietät sei, die „in Österreich allgemein gesprochen“ werde und „im täglichen Austausch“ die wichtigste Kommunikationsform darstelle – sei es nun „im Arbeitsleben“ oder „in der Freizeit“ (15:50–19:22). Auch Schüler SA23 ist der Meinung, dass der STANDARD „eine vorgegebene Sprache für alles“ sei, die auch allenthalben genutzt werden sollte. Ihm zufolge habe es „nur Vorteile“, dass in Salzburg-Stadt keine DIALEKTE gesprochen würden. Es gebe keine Verständnisprobleme und dadurch sei die Kommunikation „viel unkomplizierter“ (20:21–21:15). Interessanterweise wird der STANDARD von den Schüler/inne/n in Salzburg-Stadt viel deutlicher als „All- tagssprache“ definiert als von ihren Lehrer/inne/n, die dabei wesentlich vager bleiben. Trotzdem ist der STANDARD auch für etliche städtische Lehrpersonen eine Art GEBRAUCHSSTANDARD, der in den meisten Interaktionssituationen ange- messen ist und den Lehrenden zufolge dabei auch Potential für Variation auf- weist. Nicht überall gilt der STANDARD jedoch als „Sprache des täglichen Umgangs“ – vor allem in stark DIALEKTAL geprägten, ländlichen Regionen wird er im Gegen- teil als „eine Art Fremdsprache“ verstanden, wie es etwa LC4 ausdrückt, wobei er als solche zum Teil „nur wenig Ähnlichkeiten“ mit dem lokalen DIALEKT auf- weise. Dennoch sei es laut LC4 vor allem in Hinblick auf die (mündlichen) Ma- Weil Standard verständlich ist und Dialekt authentisch macht \| 431 turaprüfungen notwendig, dass die Schüler/innen auch mit dem HOCHDEUT- SCHEN konfrontiert werden (19:18–21:08). Auch andere GP konzipieren den STANDARD als Art Fremdsprache – wie beim Fremdspracherwerb erfolge laut LC2 etwa der Übergang vom DIALEKT in die STANDARDSPRACHE „prozesshaft“. Nach und nach könne die STANDARD-Kompetenz aufgebaut und dieser in verschiede- nen Situationen eingesetzt werden (17:33–19:09). Auch wenn der STANDARD zumindest in ländlichen Regionen als Art Fremd- sprache wahrgenommen wird, wird er nichtsdestoweniger an allen Schulstan- dardorten oft – wie an der Universität (vgl. Abschnitt 4.1.1) – als korrektes Deutsch vorgestellt. SC22 vertritt bspw. 4.2.1 Konzeptionen von STANDARD die Ansicht, dass das HOCHDEUTSCHE teilweise einen „ganz anderen Wortschatz“ als der DIALEKT umfasse, wobei HOCHDEUTSCH im Gegensatz zum DIALEKT auch „grammatikalisch korrekt“ sei. Das mache eine Verständigung außerhalb der eigenen Region schwierig. Schon allein deshalb sei „das Erlernen der Standardsprache notwendig“ (15:22–15:47). Auch an der Schule wird von den GP der STANDARD gemeinhin als „klarer“, „deutlicher“ und auch „gebildeter“ angesehen, wie etwa SA45 meint. Ihr zufol- ge versuche man mit HOCHDEUTSCH „etwas darzustellen“, „sich gut zu positio- nieren“ und dadurch auch „ernster genommen zu werden“ (17:03–17:55). Ähn- lich argumentiert SA52. Für sie zeugt HOCHDEUTSCH von „Respekt, von Hochachtung“. Indem man STANDARD spricht, zeige man, dass man das Gegen- über „akzeptiert“ und „schätzt“. Insofern SA52 den STANDARD mit der Sprache Goethes vergleicht, wird auch deutlich, dass sie HOCHDEUTSCH als Sprache der Gebildeten und der Bildung ansieht (15:22–16:03). Für SA2 ist die STANDARDSPRA- CHE dabei auch eine Sprechweise, die „so vorgegeben ist“ und „bestimmten Regeln entspricht, an die man sich halten muss“ (13:28–14:50). SA6 zufolge ist es wichtig, „die Wörter so auszusprechen, wie sie sind“. Erst dann sei es „richti- ges Hochdeutsch“. Obwohl es für diese GP „nicht schlimm“ ist, wenn dazwi- schen ein paar Wörter im DIALEKT vorkommen, zeuge das trotzdem für „weniger Intelligenz“ und sei auch „nicht so schön“ (13:58–14:32). Dass STANDARD mit Bildung, Respekt und Höflichkeit verbunden wird, entspricht der Sicht, die an der Universität vorherrscht (vgl. Abschnitt 4.1.1). Auch in Regionen, in denen der DIALEKT die unumstrittene Alltagsvarietät ist, stellt die STANDARDSPRACHE die Varietät mit dem höchsten Prestige dar, die mit der Assoziation hoher Bildung einhergeht. So bezeichnet SC5 den STANDARD als „höher, gebildeter“ (12:05–12:35). SC25 unterstreicht diese Meinung, indem er die STANDARDSPRACHE als „eine gehobene Weise von Dialekt“ bezeichnet, die „gepflogener und schöner“ ist. Für ihn steht sie in unmittelbarem Zusammen- hang mit „Vornehmheit“. STANDARD sei eine „saubere Sprache, ohne Fehler, in dem man alles klar ausdrücken“ könne. Vor allem „die Gebildeten“ würden 432 \| Philip C. Vergeiner, Elisabeth Buchner, Eva Fuchs, Stephan Elspaß 432 HOCHDEUTSCH sprechen, während in der Familie oder mit Freunden doch eher der DIALEKT im Vordergrund stehe. Vor Leuten, die die STANDARDSPRACHE im täglichen Leben benutzen, habe er „großen Respekt“ (11:05–12:01). Aus einem ähnlichen Grund hält es auch SA20 für unangebracht, in der Schule DIALEKT zu sprechen. HOCHDEUTSCH sei die „Höflichkeitsform“, die „förmlicher“ als der DIALEKT sei und „die man außerhalb der Familie oder des Freundeskreises“ immer sprechen sollte (07:52–09:19). 4.2.1 Konzeptionen von STANDARD Weil der STANDARD als förmlichere Sprechweise konzipiert wird, ist der aus- schließliche Gebrauch des STANDARDS sowohl Lehrer/inne/n als auch Schü- ler/inne/n zufolge in bestimmten schulischen Situationen anzustreben. Dies betrifft – ähnlich wie an der Universität (vgl. Abschnitt 4.1.1) – vor allem die fachlichen Teile bei Referaten (bei Lehrer/inne/n teilweise auch Fachvorträgen) oder Prüfungssituationen. Schüler SA1 betont in diesem Sinne, dass es sich „einfach nicht gehört, bei Referaten Dialekt zu sprechen“ (06:58–07:03). Eine ähnliche Ansicht vertritt der Lehrer LC3, der beteuert, dass „dialektal gefärbte“ Sprache bei einer Abschlussprüfung „natürlich nicht geht“ (04:28–04:55). Als oberstes Ziel wird dabei oft die mündliche Abschlussprüfung (d. h. Matura- bzw. Fachprüfung) genannt. Auch in Hinblick auf ein Universitätsstudium sei, so sind die schulischen GP nicht zu Unrecht (vgl. Abschnitt 4.1.1) überzeugt, eine gewisse Versiertheit in der STANDARDSPRACHE notwendig. Das ist dann auch der Grund für LA3, ihre Schüler/innen auf eine solche Versiertheit vorzubereiten; schließlich sei sie selbst, im Gegensatz zu „deutschen oder ostösterreichischen“ Kommiliton/inn/en, die sich die adäquate Sprache „nicht zuerst überlegen mussten“, an der Universität oft in einen „sprachlichen Notstand“ geraten, weil die „Grammatik und Lexik“, die sie aus dem DIALEKT gewohnt war, für formelle Situationen nicht mehr passend schien. Um den Lernenden solche Schwierig- keiten zu ersparen, versuche sie, ihnen „anhand eigener Erfahrungen Empfeh- lungen zu geben“ und in Vorbereitung auf das Studium mit ihnen die STAN- DARDVARIETÄT zu trainieren (09:37–10:18). Dass die Schule den Erwerb von STANDARD auch in Hinblick auf das spätere Berufsleben forcieren müsse, kommt häufiger vor. Dabei scheint das Erlernen von HOCHDEUTSCH in der Schule als überregionale Varietät von einem Großteil der GP als Vorteil für das spätere Berufsleben eingeschätzt zu werden. Verwie- sen wird im Zuge dessen insbesondere auf Wirtschaftsberufe oder Berufe, die ein Studium voraussetzen, wie etwa der Arzt- oder Lehrberuf. SC25 zufolge wer- de man in solchen Berufen „besser wahrgenommen“ und sei dadurch „erfolg- reicher“, wenn man sich „gut auf Hochdeutsch artikulieren“ könne (21:14– 21:57). Ein ähnliches Argument bemüht SC41, die der Meinung ist, dass die STANDARDSPRACHE „das Um und Auf im Geschäftsleben“ sei (19:18–21:03). Auch 433 Weil Standard verständlich ist und Dialekt authentisch macht \| Bewerbungsgespräche werden in diesem Zusammenhang öfter genannt. In ihnen spricht etwa SC4 „eher Hochdeutsch“, weil sie sich „gut ausdrücken“ will, außerdem ist der STANDARD für sie ein Mittel, um vom Gegenüber gut ver- standen zu werden (03:30–04:05). 4.2.1 Konzeptionen von STANDARD Für LA2 ist es gleichsam in einer „globalisier- ten Welt“ Voraussetzung, dass man „in einer Sprache spricht, wo man einen in Norddeutschland auch versteht.“ Gerade für ihren Schultyp sei das umso wich- tiger, wo doch ein Großteil der Absolvent/inn/en zukünftig in wirtschaftlichen Betätigungsfeldern arbeiten werde (09:07–09:28). Dass STANDARD verwendet werden soll, um den Erwerb des STANDARDS zu forcieren, ist ein Aspekt, der an der Universität zwar auch belegbar ist, aber nicht so oft, da STANDARD- Kompetenzen dort vielfach erwartet werden (vgl. ausführlicher etwa Vergeiner et al. 2020). Gerade in Hinblick auf das spätere Berufsleben zeigt sich, dass die GP an der Schule dem STANDARD nicht nur ein höheres Prestige, sondern auch eine bessere Verständlichkeit zuschreiben. Dieser Wert stellt für die meisten ein star- kes Argument für die Orientierung am STANDARD dar. Zumindest für manche GP spricht dies auch für einen verstärkten Gebrauch von STANDARD in der Schule. Für die Lehrperson LG1 ist es z. B. explizit „erwünscht“, dass „Schriftsprache“ gesprochen werde. Ihr zufolge hätten vorwiegend Schüler/innen aus stark dia- lektal geprägten Regionen zwar anfangs oft Schwierigkeiten, sich „sprachlich umzustellen“; „erstrebenswert“ sei das aber allemal, zumal Lernende aus DIA- LEKTFERNEREN Gebieten oft Schwierigkeiten hätten, den starken DIALEKT zu ver- stehen (05:22–06:18). Auch die große Zahl an Schüler/inne/n mit Deutsch als Zweitsprache mache es SG5 zufolge notwendig, sich an die STANDARDSPRACHE anzunähern (15:18–15:56). Auffällig an den schulischen Interviews im Vergleich zur Universität ist, dass manche GP zwar unterschiedslos von HOCHSPRACHE und STANDARDSPRACHE sprechen, dass andere GP jedoch zwischen beiden Begriffen sehr wohl differen- zieren. Sie unterscheiden dabei zwischen STANDARD, der in Österreich als „höchste Varietät“ möglich ist, und HOCHDEUTSCH. Letzteres sei eine Sprachform, die „rein in Deutschland“ gesprochen wird und für Österreicher/innen „gar nicht realisierbar“ sei. SA33 zufolge sei das aber auch nicht notwendig. Damit einher geht auch die Auffassung von einem eigenen ÖSTERREICHISCHEN STANDARD. Für viele GP ist dieser ÖSTERREICHISCHE STANDARD dem DEUTSCHEN STANDARD indes untergeordnet im Sinne einer asymmetrischen Plurizentrik (vgl. Scharloth 2005 für eine ähnliche Situation in der Deutschschweiz). Trotz der Überzeu- gung, dass verschiedene Zentren existierten und ein Einheitsdeutsch nie er- reicht werden könne, stellt ein solches den GP zufolge ein Ideal dar, das über allen anderen Varietäten steht. Das österreichische Deutsch, das innerhalb 434 \| Philip C. Vergeiner, Elisabeth Buchner, Eva Fuchs, Stephan Elspaß 434 Österreichs die „höchste Sprachlage“ darstelle, wird dabei als inferior betrach- tet. 4.2.1 Konzeptionen von STANDARD So ist nach Ansicht von LG2 das „deutsche Deutsch lupenreiner“ im Ver- gleich zum „holprigen“ österreichischen Deutsch, Deutsche wirkten „kompe- tenter“, „rhetorisch besser“ und seien viel eher in der Lage, etwas auf den Punkt zu bringen (07:22–08:23). Laut SA40 gibt es einen großen Unterscheid zwischen dem DEUTSCHEN und dem ÖSTERREICHISCHEN STANDARD. Bewusst sei ihr das erst geworden, als ihre vierjährige deutsche Cousine sie fragte, warum sie nicht „richtig Deutsch“ sprechen könne. Sie würde sie „nicht gut verstehen“, weil sie „so komisch“ rede (20:16–20:49). SA52 zufolge würden in Österreich „die gleichen Wörter benutzt“ wie in Deutschland, diese würden aber „schlampiger ausgesprochen“. Im Gegensatz dazu klinge das HOCHDEUTSCHE „viel hochgestochener“ und sei „viel näher an der Rechtschreibung und an der Norm“ (15:33–16:25). Dass die Schreibung als Maßstab für STANDARDSPRACHLICHKEIT gesehen wird, zeigt sich generell auch bei anderen GP – STANDARDSPRACHLICH ist nach Meinung von SC37 „das Schriftli- che“, was für die Alltagskommunikation „nicht relevant“ sei. Insofern gilt der STANDARD als „normierte und kodifizierte Schriftsprache“, wie sie in den Lehr- büchern verwendet wird (20:18–21:03). Laut SA12 ist HOCHDEUTSCH genau so, „wie man schreibt und formuliert“. Es sei in Bezug auf die Grammatik „genau so, wie man es in der Schule lernt“ (15:14–15:46). Auch für SC12 ist die STAN- DARDSPRACHE ebendas, was „im Wörterbuch“ zu finden sei. Es sei ein „sehr sach- liches und grammatikalisch korrektes Deutsch“, das in Österreich „die Amts- sprache“ darstelle und deshalb auch in der Schule gelernt werden müsse (23:30–24:45). Letztlich ist die Gleichsetzung von SCHRIFTLICHKEIT – NORMIERTHEIT – STANDARDSPRACHLICHKEIT also an der Schule genauso wie an der Universität verbreitet. Dasselbe gilt – wenn auch etwas weniger stark – für die areale Veror- tung des STANDARDS in Deutschland. 4.2.2 Konzeptionen von NONSTANDARD Obwohl die STANDARDVARIETÄT eng mit dem schulischen Alltag verwoben zu sein scheint und gerade den Lehrpersonen zufolge unabdingbar in formelleren Situ- ationen ist, wird oft für den Gebrauch von NONSTANDARD-Varietäten im Schulall- tag argumentiert: Analog zur Universität (vgl. Abschnitt 4.1.2) wird NONSTANDARD dabei vor- wiegend mit Nähe, Heimat und Verwurzelung in Zusammenhang gebracht. Von SC6 als „emotionale Herzenssprache“ mit kleinräumiger Verbreitung definiert, zeichne er sich durch „lokale Sprachnuancen“ aus und unterscheide sich in Weil Standard verständlich ist und Dialekt authentisch macht \| 435 „wesentlichen Elementen“ von der STANDARDSPRACHE. Insofern sei der DIALEKT stark mit „Heimat“ in Beziehung zu bringen und als solcher ein „wichtiger Be- standteil“ ihres Lebens (09:46–10:01). Für LC2 zeigt sich die regionale Verwur- zelung in der Verwendung von DIALEKT, der für sie „die Wurzel“, die „wirkliche Muttersprache“, in der man „fast alles“, insbesondere aber „Emotionen und Gefühle“, viel besser ausdrücken könne (12:06–13:10). LC4 teilt diese Sichtwei- se, wobei sie den Stellenwert des DIALEKTS für die Jugendlichen hervorstreicht. Die regionale Varietät würde ihnen ein „gewisses Selbstbewusstsein“ geben, sie hätten das Gefühl, „irgendwo dazuzugehören“. Der DIALEKT habe demgemäß viel mit „Identität, mit Persönlichkeit“ zu tun. Durch die Sprache würde man sich „mit einem Gebiet identifizieren“. In ihrem Unterricht sei es ihr daher wich- tig, dass die Schüler/innen „authentisch“ blieben und sich nicht „verstellten“ (15:14–15:23). Immer wieder wird, wie an der Universität (vgl. Abschnitt 4.1.2), dieser Zusammenhang von Identität und DIALEKT betont. LF5 zufolge betreffe er selbst Schüler/innen mit Migrationshintergrund: Sie wüssten oft nicht, welcher Nationalität sie sich zugehörig fühlen. Allein die regionale Varietät und die dadurch geschaffene Solidarität helfe ihnen, sich zu integrieren (07:18–08:52). Das Zugeständnis an den als authentisch empfundenen NONSTANDARD wird an der Schule – und hier unterschieden sich Schule und Universität – auch öfter auf die pädagogische Beziehung zwischen Lehrenden und Lernenden bezogen, zumindest wenn es sich nicht um offizielle Situationen handelt. Für LA2 ist es das Kennzeichen einer „guten Beziehung“, wenn die Schüler/innen mit ihr im DIALEKT sprechen. Die Lernenden würden sich dadurch „wohl“ und „angenom- men“ fühlen, was für eine „gute Vertrauensbasis“ unabdingbar sei. In Prü- fungssituationen müssten sie natürlich „ordentlich“ sprechen (05:10–06:04). An der Universität scheint die nähesprachliche Funktion von NONSTANDARD bei Lehrenden-Studierenden-Interaktionen weit weniger bedeutsam zu sein. Das gilt auch für den didaktischen Wert, der dem DIALEKT an der Schule zu- gemessen wird, etwa beim Sprachenlernen: Für LF2 z. B. 4.2.2 Konzeptionen von NONSTANDARD bildet der DIALEKT als „Muttersprache“ die Basis für den weiteren Varietäten- und Spracherwerb. In der regionalen Sprache seien „zusätzliche Informationen“ enthalten. „Über Jahrzehnte bzw. sogar Jahrhunderte entwickelt“, habe diese Varietät „eine ge- wisse Tradition“. Verstehe man das, „woher man kommt“, könne man auch „das große Ganze“ besser begreifen. Egal, ob man dann „gehobene Umgangs- sprache“, „Standard“ oder sogar eine „zweite oder dritte Fremdsprache“ lerne, mit dem DIALEKT als Basis habe man weit weniger Schwierigkeiten damit (14:22– 15:35). Im Unterschied zur Universität stellen NONSTANDARD-Varietäten an der Schu- le für manche Lehrpersonen auch eine Möglichkeit dar, ein Bewusstsein für eine 436 \| Philip C. Vergeiner, Elisabeth Buchner, Eva Fuchs, Stephan Elspaß 436 situationsangemessene Varietätenverwendung bei den Lernenden zu schaffen. LC4 zufolge ist es wichtig, die „sprachlichen Register für formelle und informel- le Kontexte“ bedienen zu können. Das schließe auch den DIALEKT mit ein. Die Lernenden sollten auf verschiedene sprachliche Anforderungen vorbereitet werden und dafür sensibilisiert werden, sich in verschiedenen sozialen Kontex- ten sprachlich angemessen zu verhalten. Nach Ansicht von LC4 trägt genau diese sprachliche Diversität dazu bei, dass man für andere Varietäten und Spra- chen sensibilisiert und im Zuge dessen „toleranter“ anderen Sprachformen gegenüber werde (15:41–16:12). Zudem würde die Auseinandersetzung mit an- deren Varietäten auch zu einer Veränderung des persönlichen Sprachgebrauchs und in weiterer Folge zu einem Ausbau STANDARDSPRACHLICHER Kompetenzen beigetragen. Das sei die Voraussetzung für eine umfassende Sprachkompetenz. Die Schule sollte als „Übungsplatz“ dafür dienen. Generell wird an der Schule – im Unterschied zur Universität – davon aus- gegangen, dass Kompetenzen im STANDARD bei den Lernenden nicht von Anfang an gegeben sind. Auch wenn STANDARD für LG4 in der Schule eine „wichtige Varietät“ sei, die auch die „größte kommunikative Reichweite“ aufweise, sei er für viele Schüler/innen de facto aber „mündlich nur schwer realisierbar“. Aus diesem Grund würden STANDARDABWEICHENDE Sprechweisen akzeptiert, um die Angst vor Überforderung angesichts zu hoher STANDARD-Erwartungen zu mini- mieren. So finde die Alltagskommunikation der meisten Schüler/innen im länd- lichen Raum ausschließlich im DIALEKT statt (15:20–16-11). LC2 zufolge sei der „Wechsel“ zum STANDARD „sehr schwierig“. Der DIALEKT sei somit eine Art „Hilfsvarietät“, die „natürlich mit Grenzen“ akzeptiert werde, um den Schü- ler/inne/n entgegenzukommen (06:15–07:22). Stellt der NONSTANDARD vorwiegend für Deutschlehrer/innen eine Hilfestel- lung dar, wird er in anderen Fächern großteils als die eigentliche Unterrichts- sprache definiert. So fällt es LC1 zufolge in anderen Gegenständen „nur zum Teil“ in den Aufgabenbereich einer Lehrperson, auf die Sprache zu achten. 4.2.2 Konzeptionen von NONSTANDARD Solange eine gegenseitige Verständigung gewährleistet sei, sei der DIALEKT dort durchaus adäquat (05:21–06:15). Auch SA1 argumentiert dahingehend, dass die Sprache in anderen Fächern lediglich „als Medium“ diene, explizit aber „kaum etwas mit dem Lernstoff zu tun“ habe. Insofern könne man durchaus gute Leis- tungen beispielsweise in Mathematik erbringen, ohne STANDARDSPRACHE im Unterricht zu sprechen (07:39–08:25). Ähnlich pragmatisch wird auch bisweilen an der Universität argumentiert (vgl. Abschnitt 4.1.2). 437 Weil Standard verständlich ist und Dialekt authentisch macht \| 437 11 Zu ausgewählten quantitativen (neben qualitativen) Ergebnissen vgl. Vergeiner et al. (2019). 5 Diskussion und Fazit Im vorliegenden Beitrag präsentierten wir Ergebnisse einer qualitativen Unter- suchung zu Varietätenkonzeptionen an Schulen in Salzburg und Tirol und an der Universität Salzburg. In Anlehnung an neuere qualitative Ansätze der Perzeptionslinguistik wurden für den vorliegenden Beitrag keine quantitativen Daten,11 sondern ausschließlich Gesprächsdaten ausgewertet, die im Rahmen halbstrukturierter Interviews mit insgesamt 264 GP erhoben wurden. Begründen lässt sich diese Vorgehensweise damit, dass die inhaltsanalytische Auswertung von Interviewdaten eine Tiefe der Rekonstruktion abstrakter Konzepte wie den Varietätenkonzeptionen gestattet, die rein quantitative Ansätze nicht zu leisten vermögen. Die Interviewdaten wurden daraufhin ausgewertet, (1) welche Varie- tätenkonzeptionen sich aus den Äußerungen erschließen lassen, (2) wie die Verwendung dieser Varietäten innerhalb und außerhalb des Schul- bzw. Uni- versitätsalltags bewertet wird sowie (3) wann welche Varietäten aus der Sicht der GP verwendet werden sollten (und wann nicht). Generell lässt sich festhalten, dass zwischen den Varietätenkonzeptionen der GP an der Universität und den Schulen viele Gemeinsamkeiten auftreten. Was beiden Institutionstypen gemeinsam ist, sind die heterogenen Sprachver- hältnisse in Bezug auf innere wie äußere Mehrsprachigkeit, wobei die Hetero- genität an der Universität aufgrund des höheren Anteils von Personen, die nicht in Österreich bzw. überhaupt im deutschsprachigen Raum aufgewachsen sind, ausgeprägter ist als an den Schulen (vgl. Abschnitt 3). Mit Blick auf die drei genannten Fragen lässt sich aus den Analysen Folgendes rekonstruieren: 1. Alle GP haben ein klares Bewusstsein für die Existenz von mindestens zwei Varietäten. Das STANDARDDEUTSCHE wird meist als HOCHDEUTSCH bezeichnet, der NONSTANDARD meist als DIALEKT, gelegentlich wird zusätzlich auch von einer „UMGANGSSPRACHE“ oder einfach von einem „DAZWISCHEN“ (von Leh- rer/inne/n an den Schulen) gesprochen. Bei jenen GP, deren Varietätenkon- zeption neben HOCHDEUTSCH und DIALEKT keine(n) Zwischenbereich(e) vor- sieht, muss man annehmen, dass der DIALEKT-Begriff sehr weit ist und neben den Basisdialekten auch den mittleren Bereich umfasst. Von GP an Schulen wie an der Universität werden DIALEKTE hauptsächlich als informelle, nähesprachliche Varietäten begriffen; der STANDARD/das HOCHDEUTSCHE wird entsprechend als formelle, distanzsprachliche Varietät Von GP an Schulen wie an der Universität werden DIALEKTE hauptsächlich als informelle, nähesprachliche Varietäten begriffen; der STANDARD/das HOCHDEUTSCHE wird entsprechend als formelle, distanzsprachliche Varietät 438 \| Philip C. Vergeiner, Elisabeth Buchner, Eva Fuchs, Stephan Elspaß 438 konzeptualisiert. Auffällig ist an beiden Institutionen, dass STAN- DARDSPRACHLICHKEIT dabei stark mit Schriftlichkeit assoziiert wird. 5 Diskussion und Fazit Insbeson- dere an den Universitäten lassen viele GP ein starres, monozentrisches, auf die Existenz einer homogenen Entität gerichtetes Verständnis von STANDARD erkennen, einige andere eher ein an einen GEBRAUCHSSTANDARD erinnerndes Konzept, das etwa auch österreichische Eigenheiten zulässt. Die Vorstel- lung eines eigenen ÖSTERREICHISCHEN HOCHDEUTSCH klingt nur in einigen In- terviews mit Schüler/inne/n an – allerdings meist mit dem Hinweis einer gewissen Höherwertigkeit des DEUTSCHEN HOCHDEUTSCH. Wenn es in den Köp- fen dieser GP also tatsächlich so etwas wie ein Konzept der Plurizentrizität gibt, dann ist es klar von einer Asymmetrie zugunsten des BUNDESDEUTSCHEN STANDARDS geprägt – so, wie es etwa für das Verhältnis zwischen DEUTSCH- SCHWEIZERISCHEM und BUNDESDEUTSCHEM STANDARD beschrieben wird (vgl. Scharloth 2005). 2. Die Bewertungen der Varietätenverwendungen lassen sich zum Teil explizi- ten Urteilen entnehmen, zum Teil müssen sie aus Äußerungen, die ihnen bestimmte Attribute zuschreiben, erschlossen werden. Explizit sind etwa den DIALEKT stigmatisierende Bewertungen, die an Vorstellungen von einem RESTRINGIERTEN CODE erinnern. Gemeinsam ist Schule und Universität dabei etwa, dass der NONSTANDARD vielfach als falsche, inkorrekte Sprechweise at- tribuiert wird. Zu den indirekten Bewertungen gehören die an beiden Insti- tutionstypen anzutreffenden Äußerungen, wonach dem STANDARD im Ver- gleich zu NONSTANDARD-Varietäten ein hoher Grad an Verständlichkeit attestiert wird – an der Universität nicht nur mit Blick auf Nichtmutter- sprachler/innen des Deutschen, sondern durchaus auch auf bundesdeut- sche Studierende und Lehrende (vgl. ausführlicher dazu Vergeiner 2020). Aus der These der besseren Verständlichkeit des STANDARDs lässt sich die Auffassung ableiten, dass sich für bestimmte universitäre Verwendungssi- tuationen, z. B. Lehrveranstaltungen, per se nur STANDARD-Varietäten eigne- ten (siehe Punkt 3). 2. Die Bewertungen der Varietätenverwendungen lassen sich zum Teil explizi- ten Urteilen entnehmen, zum Teil müssen sie aus Äußerungen, die ihnen bestimmte Attribute zuschreiben, erschlossen werden. Explizit sind etwa den DIALEKT stigmatisierende Bewertungen, die an Vorstellungen von einem RESTRINGIERTEN CODE erinnern. Gemeinsam ist Schule und Universität dabei etwa, dass der NONSTANDARD vielfach als falsche, inkorrekte Sprechweise at- tribuiert wird. Zu den indirekten Bewertungen gehören die an beiden Insti- tutionstypen anzutreffenden Äußerungen, wonach dem STANDARD im Ver- gleich zu NONSTANDARD-Varietäten ein hoher Grad an Verständlichkeit attestiert wird – an der Universität nicht nur mit Blick auf Nichtmutter- sprachler/innen des Deutschen, sondern durchaus auch auf bundesdeut- sche Studierende und Lehrende (vgl. ausführlicher dazu Vergeiner 2020). Aus der These der besseren Verständlichkeit des STANDARDs lässt sich die Auffassung ableiten, dass sich für bestimmte universitäre Verwendungssi- tuationen, z. B. 5 Diskussion und Fazit Wichtig sei mit Blick auf münd- liche Abschlussprüfungen und das spätere Berufsleben eine Sensibilisierung für situationsangemessene Varietätenverwendung, die eben den Gebrauch der STANDARD- wie der NONSTANDARD-Varietäten im Un- terricht umfasse. ) 4. Aus verschiedenen Attribuierungen und Bewertungen ergeben sich schließ- lich auch Erwartungen an Varietätenverwendungen. Wenn etwa auf der ei- nen Seite Universitätslehrer/innen den Gebrauch des STANDARDS mit Kom- petenz und Professionalität oder Studierende mit wissenschaftlicher Seriosität assoziieren, so ergibt sich daraus fast zwangsläufig, dass solche Akteure und Akteurinnen für universitäre Lehrveranstaltungen und Prü- fungsgespräche die Verwendung der STANDARDSPRACHE einfordern – wie auch immer sich diese am Ende wirklich anhört. Wird auf der anderen Seite – von Universitätsangehörigen wie auch von Lehrenden und Schü- ler/inne/n an den Schulen gleichermaßen – hervorgehoben, dass regionale NONSTANDARD-Varietäten wichtige identitätsstiftende Funktionen hätten und die Sprecher/innen ,authentischer‘ wirken ließen, so wird damit ihre Verwendung in Unterrichtssituationen legitimiert. Eine ganz eigene Recht- fertigung des NONSTANDARD-Gebrauchs im Unterricht findet sich in Äuße- rungen von Lehrer/inne/n vor allem ländlicher Schulen: Da DIALEKTE so stark in der Alltagskommunikation vieler Schüler/innen verankert und auch durchaus positiv konnotiert seien, sei die Verwendung von NONSTAN- DARD-Varietäten im Unterricht nicht nur üblich, sondern in einem bestimm- ten Rahmen auch angemessen und legitim. Wichtig sei mit Blick auf münd- liche Abschlussprüfungen und das spätere Berufsleben eine Sensibilisierung für situationsangemessene Varietätenverwendung, die eben den Gebrauch der STANDARD- wie der NONSTANDARD-Varietäten im Un- terricht umfasse. 4. Aus verschiedenen Attribuierungen und Bewertungen ergeben sich schließ- lich auch Erwartungen an Varietätenverwendungen. Wenn etwa auf der ei- nen Seite Universitätslehrer/innen den Gebrauch des STANDARDS mit Kom- petenz und Professionalität oder Studierende mit wissenschaftlicher Seriosität assoziieren, so ergibt sich daraus fast zwangsläufig, dass solche Akteure und Akteurinnen für universitäre Lehrveranstaltungen und Prü- fungsgespräche die Verwendung der STANDARDSPRACHE einfordern – wie auch immer sich diese am Ende wirklich anhört. Wird auf der anderen Seite 4. In inhaltlicher Perspektive fördern die Ergebnisse unserer Untersuchungen sowohl heterogene als auch sehr reflektierte Varietätenkonzeptionen von Ak- teur/inn/en im sekundären und im tertiären Bildungsbereich in Österreich zu- tage. Dabei dürften sie, wie überhaupt die Salzburger Sprachverhältnisse – aufgrund ähnlich diaglossischer Situationen –, mit denen in anderen Regionen in Österreich sowie im Süden Deutschlands vergleichbar sein. Wie gezeigt, be- steht auch eine gewisse Vergleichbarkeit der Varietätenkonzeptionen mit denen in der Schweiz, trotz der grundlegend anderen, Situation dort. 5 Diskussion und Fazit Lehrveranstaltungen, per se nur STANDARD-Varietäten eigne- ten (siehe Punkt 3). 2. 3. Was den schulischen Bereich betrifft, zeigen sich bei den indirekten Bewer- tungen von Varietätenverwendungen klare Unterschiede zwischen Schü- ler/inne/n, die städtische und solchen, die ländliche Schulen besuchen. Während Schüler/innen an Schulen im städtischen Umfeld den STANDARD als „Sprache des täglichen Umgangs“ charakterisieren, kommt dieses Attri- but am Land den DIALEKTEN zu. Schüler/innen im ländlichen Umfeld etiket- tieren das HOCHDEUTSCHE vielfach als so etwas wie ihre erste Fremdsprache, als Sprache der Gebildeten bzw. der Bildung oder als Sprache der Schrift – auf jeden Fall als eine exogene Varietät. Auch hier bestehen durchaus Pa- 3. Was den schulischen Bereich betrifft, zeigen sich bei den indirekten Bewer- tungen von Varietätenverwendungen klare Unterschiede zwischen Schü- ler/inne/n, die städtische und solchen, die ländliche Schulen besuchen. Während Schüler/innen an Schulen im städtischen Umfeld den STANDARD als „Sprache des täglichen Umgangs“ charakterisieren, kommt dieses Attri- but am Land den DIALEKTEN zu. Schüler/innen im ländlichen Umfeld etiket- tieren das HOCHDEUTSCHE vielfach als so etwas wie ihre erste Fremdsprache, als Sprache der Gebildeten bzw. der Bildung oder als Sprache der Schrift – auf jeden Fall als eine exogene Varietät. Auch hier bestehen durchaus Pa- 3. Weil Standard verständlich ist und Dialekt authentisch macht \| 439 rallelen zu Sprachkonzeptionen von Deutschschweizer/inne/n (vgl. Chris- ten & Schmidlin 2019). ten & Schmidlin 2019). 4. Aus verschiedenen Attribuierungen und Bewertungen ergeben sich schließ- lich auch Erwartungen an Varietätenverwendungen. Wenn etwa auf der ei- nen Seite Universitätslehrer/innen den Gebrauch des STANDARDS mit Kom- petenz und Professionalität oder Studierende mit wissenschaftlicher Seriosität assoziieren, so ergibt sich daraus fast zwangsläufig, dass solche Akteure und Akteurinnen für universitäre Lehrveranstaltungen und Prü- fungsgespräche die Verwendung der STANDARDSPRACHE einfordern – wie auch immer sich diese am Ende wirklich anhört. Wird auf der anderen Seite – von Universitätsangehörigen wie auch von Lehrenden und Schü- ler/inne/n an den Schulen gleichermaßen – hervorgehoben, dass regionale NONSTANDARD-Varietäten wichtige identitätsstiftende Funktionen hätten und die Sprecher/innen ,authentischer‘ wirken ließen, so wird damit ihre Verwendung in Unterrichtssituationen legitimiert. Eine ganz eigene Recht- fertigung des NONSTANDARD-Gebrauchs im Unterricht findet sich in Äuße- rungen von Lehrer/inne/n vor allem ländlicher Schulen: Da DIALEKTE so stark in der Alltagskommunikation vieler Schüler/innen verankert und auch durchaus positiv konnotiert seien, sei die Verwendung von NONSTAN- DARD-Varietäten im Unterricht nicht nur üblich, sondern in einem bestimm- ten Rahmen auch angemessen und legitim. 5 Diskussion und Fazit Für die Verhält- nisse an mittel- und norddeutschen Schulen und Universitäten, die weitgehend vom monovarietären Gebrauch von STANDARDNAHEN Varietäten – oder zumindest einem monovarietären Habitus – geprägt sind, mögen sich die drei genannten Untersuchungsfragen hingegen erst gar nicht stellen. 440 \| Philip C. Vergeiner, Elisabeth Buchner, Eva Fuchs, Stephan Elspaß 440 In methodischer Hinsicht zeigt sich der Wert qualitativer Ansätze, die zum einen eine analytische Tiefe erreichen, welche bei rein quantitativen Zugängen nicht möglich wäre, und die zum anderen durchaus eine Generalisierbarkeit der Ergebnisse beanspruchen können, wenn sie sich – wie im vorliegenden Fall – auf eine große Zahl von GP stützen. Dadurch wird natürlich der Wert quantitati- ver Untersuchungen keineswegs in Abrede gestellt; vielmehr können sich der quantitative und der qualitative Ansatz gegenseitig sinnvoll ergänzen – wie in den beiden Projekten, in deren Rahmen der vorliegende Beitrag entstanden ist. Literaturverzeichnis In Christina Cuonz & Rebekka Stud- ler (Hrsg.), Sprechen über Sprache. Perspektiven und neue Methoden der Spracheinstel- lungsforschung, 31–64. Tübingen: Stauffenburg. Dannerer, Monika (2018): Sprachwahl, Sprachvariation und Sprachbewertung an der Universi- tät. In Arnulf Deppermann & Silke Reineke (Hrsg.), Sprache im kommunikativen, interakti- ven und kulturellen Kontext, 169–192. Berlin, Boston: De Gruyter. Dannerer, Monika (2018): Sprachwahl, Sprachvariation und Sprachbewertung an der Universi- tät. In Arnulf Deppermann & Silke Reineke (Hrsg.), Sprache im kommunikativen, interakti- ven und kulturellen Kontext, 169–192. Berlin, Boston: De Gruyter. Elspaß, Stephan & Konstantin Niehaus (2014): The standardization of a modern pluriareal language. Concepts and corpus designs for German and beyond. Orð og tunga 16, 47–67. Elspaß, Stephan & Stefan Kleiner (2019): Forschungsergebnisse zur arealen Variation im Stan- darddeutschen. In Joachim Herrgen & Jürgen Erich Schmidt (Hrsg.), Sprache und Raum – Ein internationales Handbuch der Sprachvariation. Band 4: Deutsch, 159–184. Berlin, Bos- ton: De Gruyter. Errington, Joseph (1999): Ideology. Journal of Linguistic Anthropology 9 (1), 115–117. Garrett, Peter, Nikolas Coupland & Angie Williams (2003): Investigating language attitudes: Social meanings of dialect, ethnicity and performance. Cardiff: University of Wales Press. Gloy, Klaus (1975): Sprachnormen I. Linguistische und soziologische Analysen. Stuttgart: Frommann-Holzboog. Gloy, Klaus (2004): Norm. In Ulrich Ammon, Norbert Dittmar, Klaus J. Mattheier & Peter Trudgill (Hrsg.), Sociolinguistics. An international handbook of the science of language and socie- ty. Volume 1. 2nd completely rev. and extended ed, 392–399. Berlin, New York: De Gruy- ter. Hochholzer, Rupert (2004): Konfliktfeld Dialekt. Das Verhältnis von Deutschlehrerinnen und Deutschlehrern zu Sprache und ihren regionalen Varietäten. Regensburg: Edition Vulpes. Hundt, Markus (2009): Normverletzungen und neue Normen. In Marek Konopka & Bruno Stre- cker (Hrsg.), Deutsche Grammatik – Regeln, Normen, Sprachgebrauch, 117–140. Berlin: De Gruyter. Hundt, Markus, Nicole Palliwoda & Saskia Schröder (2015): Der deutsche Sprachraum aus der Sicht linguistischer Laien – das Kieler DFG-Projekt. In Michael Elmentaler, Markus Hundt & Erich Jürgen Schmidt (Hrsg.), Deutsche Dialekte. Konzepte, Probleme, Handlungsfelder. Akten des 4. Kongresses der Internationalen Gesellschaft für Dialektologie des Deutschen (IGDD) in Kiel, 295–322. Stuttgart: Steiner. Irvine, Judith. T. & Susan Gal (2000): Language Ideology and Linguistic Differentiation. In Paul V. Kroskrity (Hrsg.), Regimes of language. Ideologies, polities, and identities. 1. Aufl., 35– 84. Santa Fe, NM: School of American Research Press. Knöbl, Ralf (2012): Dialekt – Standard – Variation. Formen und Funktionen von Sprachvariation in einer mittelschwäbischen Schulklasse. Heidelberg: Winter. Krefeld, Thomas & Elissa Pustka (2010): Für eine perzeptive Varietätenlinguistik. Literaturverzeichnis Ajzen, Icek (1989): Attitude structure and behavior. In Anthony R. Pratkanis, Steven J. Breckler & Anthony G. Greenwald (Hrsg.), Attitude structure and function, 241–274. Hillsdale: Law- rence Erlbaum. Allport, Gordon W. (1935): Attitudes. In Carl Murchison (Hrsg.), A handbook of social psycholo- gy. Worcester: Clark University Press. Ammon, Ulrich (1972): Dialekt, soziale Ungleichheit und Schule. Weinheim: Beltz. Anders, Christina Ada (2010): Wahrnehmungsdialektologie. Das Obersächsische im Alltagsver- ständnis von Laien. Berlin: De Gruyter. Bartsch, Renate (1987): Sprachnormen. Theorie und Praxis. Tübingen: Niemeyer. Bellamy, John (2012): Language Attitudes in England and Austria. A Sociolinguistic Investiga- tion into Perceptions of High and Low-Prestige Varieties in Manchester and Vienna. Stutt- gart: Steiner. Berger, Peter L. & Thomas Luckmann (2016): Die gesellschaftliche Konstruktion der Wirklich- keit. Eine Theorie der Wissenssoziologie. 26. Aufl. Frankfurt a. M.: Fischer. Beuge, Patrick (2019): Was ist gutes Deutsch? Eine qualitative Analyse laienlinguistischen Sprachnormwissens. Berlin, Boston: De Gruyter. Cavanaugh, Jillian R. (2013): Language ideologies and language attitudes. A linguistic anthro- pological perspective. In Peter Auer, Javier Caro Caro Reina & Göz Kaufmann (Hrsg.), Lan- guage variation – European perspectives IV. Selected papers from the Sixth International Conference on Language Variation in Europe (ICLaVE 6), Freiburg, June 2011, 45–55. Ams- terdam: Benjamins. Christen, Helen & Regula Schmidlin (2019): Die Schweiz. Dialektvielfalt in mehrsprachigem Umfeld. In Rahel Beyer & Albrecht Plewnia (Hrsg.), Handbuch des Deutschen in West- und Mitteleuropa: Sprachminderheiten und Mehrsprachigkeitskonstellationen, 193–244. Tü- bingen: Narr Francke Attempto. Cillia, Rudolf de (2018): Sprache(n) im Klassenzimmer. Varietätengebrauch und Spracheinstel- lungen in der Schule aus Sicht der Lehrer/innen und der Schüler/innen. In Monika Danne- rer & Peter Mauser (Hrsg.), Formen der Mehrsprachigkeit in sekundären und tertiären Bil- dungskontexten. Verwendung, Rolle und Wahrnehmung von Sprachen und Varietäten. Unter Mitarbeit von Philip C. Vergeiner, 67–85. Tübingen: Stauffenburg. Coupland, Nikolas & Adam Jaworski (2004): Sociolinguistic perspectives on metalanguage: Reflexivity, evaluation and ideology. In Adam Jaworski, Dariusz Galasiński & Nikolas Cou- Weil Standard verständlich ist und Dialekt authentisch macht \| 441 pland (Hrsg.), Metalanguage. Social and ideological perspectives, 15–51. Berlin, New York: De Gruyter. pland (Hrsg.), Metalanguage. Social and ideological perspectives, 15–51. Berlin, New York: De Gruyter. Cuonz, Christina (2014): Was kann die diskursive Spracheinstellungs forschung (nicht)? Me- thodologische und epistemologische Überlegungen. In Christina Cuonz & Rebekka Stud- ler (Hrsg.), Sprechen über Sprache. Perspektiven und neue Methoden der Spracheinstel- lungsforschung, 31–64. Tübingen: Stauffenburg. Cuonz, Christina (2014): Was kann die diskursive Spracheinstellungs forschung (nicht)? Me- thodologische und epistemologische Überlegungen. Literaturverzeichnis In Thomas Krefeld & Elissa Pustka (Hrsg.), Perzeptive Varietätenlinguistik, 9–28. Frankfurt a. M.: Lang. Kroskrity, Paul V. (2009): Language Ideologies. In Alessandro Duranti (Hrsg.), A Companion to Linguistic Anthropology. 4. Aufl., 496–517. Malden: Blackwell. Lambert, Wallace E., Richard Hodgson, Robert C. Gardner & Samuel Fillenbaum (1960): Evalua- tional reactions to spoken languages. Journal of Abnormal and Social Psychology 60 (1), 44–51. 442 \| Philip C. Vergeiner, Elisabeth Buchner, Eva Fuchs, Stephan Elspaß 442 Liebscher, Grit & Jennifer Dailey-O’Cain (2009): Language attitudes in interaction. Journal of Sociolinguistics 13 (2), 195–222. Lasagabaster, David (2004): Attitude / Einstellung. In Ulrich Ammon, Norbert Dittmar, Klaus J. Mattheier & Peter Trudgill (Hrsg.), Sociolinguistics. An international handbook of the sci- ence of language and society. Volume 1. 2nd completely rev. and extended ed., 399–405. Berlin, New York: De Gruyter. Mattheier, Klaus J. (1991): Standardsprache als Sozialsymbol. Über kommunikative Folgen gesellschaftlichen Wandels. In Rainer Wimmer (Hrsg.), Das 19. Jahrhundert. Sprachge- schichtliche Wurzeln des heutigen Deutsch, 41–72. Berlin, New York: De Gruyter. Moosmüller, Sylvia (1991): Hochsprache und Dialekt in Österreich. Soziophonologische Unte suchungen zu ihrer Abgrenzung in Wien, Graz, Salzburg und Innsbruck. Wien: Böhlau. Preston, Dennis R. (2009): Language, space and the folk. In Peter Auer & Jürgen Erich Schmidt (Hrsg.), Language and Space. An International Handbook of Linguistic Variation. Volume 1: Theories and Methods, 179–201. Berlin: De Gruyter. Purschke, Christoph & Philipp Stoeckle (2019): Perzeptionslinguistik arealer Sprachvariation im Deutschen. In Joachim Herrgen & Jürgen Schmidt (Hrsg.), Sprache und Raum – Deutsch. Ein internationales Handbuch der Sprachvariation, 844–860. Berlin: De Gruyter. Scharloth, Joachim (2005): Asymmetrische Plurizentrizität und Sprachbewusstsein. Einstellun- gen der Deutschschweizer zum Standarddeutschen. Zeitschrift für Germanistische Lingu- istik 33 (2), 236–267. Schoel, Christiane & Dagmar Stahlberg (2012): Spracheinstellungen aus sozialpsychologischer Perspektive II: Dialekte. In Ludwig M. Eichinger, Albrecht Plewnia, Christiane Schoel & Dagmar Stahlberg (Hrsg.), Sprache und Einstellungen. Spracheinstellungen aus sprach- wissenschaftlicher und sozialpsychologischer Perspektive. Mit einer Sprachstandserhe- bung zum Deutschen von Gerhard Stickel. 1. Aufl, 205–225. Tübingen: Narr Francke At- tempto. Silverstein, Michael (1992): The uses and utility of ideology: Some reflections. Special Issue of Pragmatics 2 (3), 311–323. Soukup, Barbara (2009): Dialect use as interaction strategy. A sociolinguistic study of contex- tualization, speech perception, and language attitudes in Austria. Wien: Braumüller. Steiner, Astrid (2008): Unterrichtskommunikation. Eine linguistische Untersuchung der Ge- sprächsorganisation und des Dialektgebrauchs in Gymnasien der Deutschschweiz. Tübin- gen: Narr. Vergeiner, Philip C. (2019): Kookkurrenz – Kovariation – Kontrast. Literaturverzeichnis Formen und Funktionen individueller Dialekt-/Standardvariation in Beratungsgesprächen. Wien: Peter Lang. Vergeiner, Philip C. (2020): Sprachnormbezogene Akkommodation in der Hochschullehre. Zeitschrift für Angewandte Linguistik 72, 33–62. DOI: https://doi.org/10.1515/zfal-2020- 2027 (letzter Zugriff 02.03.2020). Vergeiner, Philip C. (2021): Bewertungen – Erwartungen – Gebrauch: Sprachgebrauchsnormen zur inneren Mehrsprachigkeit an der Universität. Stuttgart: Steiner. Vergeiner, Philip C., Elisabeth Buchner, Eva Fuchs & Stephan Elspaß (2019): Sprachnormvor- stellungen in sekundären und tertiären Bildungseinrichtungen in Österreich. Zeitschrift für Dialektologie und Linguistik 86 (3), 284–330. f g g Woolard, Kathrvn A. (1992): Language Ideology: Issues and Approaches. Pragmatics 2 (3), 235– 249. Woolard, Kathrvn A. & Bambi B. Schieffelin (1994): Language Ideology. Annual Review of An- thropology 23, 55–82.
https://openalex.org/W4390443638	https://link.springer.com/content/pdf/10.1007/s00590-023-03806-5.pdf	English	null	Effectiveness of permissive weight bearing in surgically treated trauma patients with peri- and intra-articular fractures of the lower extremities: a prospective comparative multicenter cohort study	European journal of orthopaedic surgery & traumatology	2,023	cc-by	6,625	Abstract Purpose The aim of the present study was to investigate the effectiveness of a novel approach involving permissive weight bearing (PWB) in surgically treated trauma patients with peri- and intra-articular fractures of the lower extremities.i Purpose The aim of the present study was to investigate the effectiveness of a novel approach involving permissive weight bearing (PWB) in surgically treated trauma patients with peri- and intra-articular fractures of the lower extremities. Methods Prospective comparative multicenter cohort study in one level 1 trauma center and five level 2 trauma centers. Surgically treated trauma patients with peri- and intra-articular fractures of the lower extremities were included. Permissive weight bearing (PWB) in comparison to restricted weight bearing (RWB) was assessed over a 26-week post-surgery follow- up period. Patients’ self-perceived outcome levels regarding activities of daily living (ADL), quality of life (QoL), pain and weight bearing compliance were used.i Results This study included 106 trauma patients (N = 53 in both the PWB and RWB groups). Significantly better ADL and QoL were found in the PWB group compared to the RWB group at 2-, 6-, 12- and 26-weeks post-surgery. There were no significant differences in postoperative complication rates between the PWB and RWB groups.fii if Conclusion PWB is effective and is associated with a significantly reduced time to full weight bearing, and a significantly better outcome regarding ADL and QoL compared to patients who followed RWB regimen. Moreover, no significant differ- ences in complication rates were found between the PWB and RWB groups. Level of evidence Level II. Registration This study is registered in the Dutch Trial Register (NTR6077). Date of registration: 01-09-2016. Keywords Permissive weight bearing · Restricted weight bearing · Trauma patients · Fractures · Lower extremity · Rehabilitation Keywords Permissive weight bearing · Restricted weight bearing · Trauma patients · Fractures · Lower extremity · Rehabilitation Effectiveness of permissive weight bearing in surgically treated trauma patients with peri‑ and intra‑articular fractures of the lower extremities: a prospective comparative multicenter cohort study Pishtiwan Kalmet1 · Cherelle Maduro1 · Coen Verstappen1 · Guido Meys2 · Yvette van Horn2 · Raoul van Vugt3 · Heinrich Janzing4 · Alexander van der Veen5 · Coen Jaspars6 · Jan Bernard Sintenie7 · Taco Blokhuis1 · Silvia Evers8,9 · Henk Seelen2,8 · Peter Brink1 · Martijn Poeze1,10 Pishtiwan Kalmet1 · Cherelle Maduro1 · Coen Verstappen1 · Guido Meys2 · Yvette van Horn2 · Raoul van Vugt3 · Heinrich Janzing4 · Alexander van der Veen5 · Coen Jaspars6 · Jan Bernard Sintenie7 · Taco Blokhuis1 · Silvia Evers8,9 · Henk Seelen2,8 · Peter Brink1 · Martijn Poeze1,10 Received: 31 August 2023 / Accepted: 2 December 2023 / Published online: 30 December 2023 © The Author(s) 2023 European Journal of Orthopaedic Surgery & Traumatology (2024) 34:1363–1371 https://doi.org/10.1007/s00590-023-03806-5 European Journal of Orthopaedic Surgery & Traumatology (2024) 34:1363–1371 https://doi.org/10.1007/s00590-023-03806-5 European Journal of Orthopaedic Surgery & Traumatology (2024) 34:1363–1371 https://doi.org/10.1007/s00590-023-03806-5 European Journal of Orthopaedic Surgery & Traumatology (2024) 34:1363–1371 https://doi.org/10.1007/s00590-023-03806-5 ORIGINAL ARTICLE ORIGINAL ARTICLE Extended author information available on the last page of the article Introduction The current recommendations regarding postoperative management in surgically treated trauma patients with peri- and intra-articular fractures of the lower extremities is either non-weight bearing or restricted weight bearing for 6–12 weeks, followed by partial weight bearing with a 25% increase in weight every week [1, 3]. The recommendations for aftercare in surgically treated trauma patients with peri- and intra-articular fractures of the lower extremities are still more or less the same as they were during the last 60 years, without any source of evidence being given for the advice of restricted weight bearing [1]. In view of this lack of evidence, many orthopedic and trauma surgeons tend to advise conservatively regarding postopera- tive management and hold on to the prevailing dogmas, i.e., non-weight bearing or restricted weight bearing [2]. There is no consensus from the surgeons in the current postoperative management [4]. Moreover, almost 90% of the surgeons deviate from the current postoperative management protocols because of e.g., type of fracture, (un-) certainty of fixation, clinical experience, or gut feeling [4]. Furthermore, while instructions on rehabilitation provided to patients may be clear, patients’ compliance with a non-weight bearing or restricted weight bearing regimen is poor, so neither (0121 3456789) 3 3456789) 3 European Journal of Orthopaedic Surgery & Traumatology (2024) 34:1363–1371 1364 surgeons nor patients follow the instructions regarding the postoperative management regimen [5, 6]. hospitals underwent the PWB protocol as aftercare reha- bilitation treatment, while the others followed the RWB protocol [1, 2]. The postoperative management of surgically treated peri- and intra-articular fractures of the lower extremities is very important in view of the impact on the patient’s functional outcome. Recent literature has reported composite postop- erative complication rates of up to 37%, with an average of 10–20% for patients with lower extremity fractures [7–11]. In addition, several studies indicate that the postopera- tive management, i.e., early or permissive weight bearing, increases the postoperative complications rates [3, 4]. Surgically treated trauma patients with peri- and intra- articular fractures of the lower extremities (i.e., pelvic fractures, acetabular fractures, distal femur fractures, tibial plateau fractures, pilon fractures, calcaneal fractures and talar fractures) were eligible for inclusion if they were aged 18 years or over. Patients with pathological fractures, shaft fractures treated with intra-medullary nailing, hip fractures treated with prosthesis, or fractures treated with external fixation, and patients with amputations in the area of the lower extremity, were excluded. Introduction Patients with inability to follow instructions (e.g., cognitive dysfunction), due to the consequences of severe neurotrauma or due to concomitant or mental illness were also excluded [16]. p p p Several biomechanical, animal, and clinical studies have found early or permissive weight bearing to be beneficial [2, 3, 12–15]. However, very few clinical studies are avail- able that compared permissive weight bearing (PWB) with restricted weight bearing (RWB) in surgically treated trauma patients with peri- and intra-articular fractures of the lower extremities. Furthermore, despite the generally accepted value of the use of patient-specific outcome measures, no data is available offering insights into patients’ self-per- ceived outcome levels (e.g., regarding activities of daily living (ADL), quality of life or pain) in PWB versus RWB. The aim of the present study was therefore to investi- gate the effectiveness of PWB in surgically treated trauma patients with peri- and intra-articular fractures of the lower extremities, reporting on patients’ self-perceived outcome levels regarding ADL, quality of life, pain, weight bearing or patients’ compliance and postoperative complications, in comparison to RWB, over a 26-week post-surgery follow- up period. Several biomechanical, animal, and clinical studies have found early or permissive weight bearing to be beneficial [2, 3, 12–15]. However, very few clinical studies are avail- able that compared permissive weight bearing (PWB) with restricted weight bearing (RWB) in surgically treated trauma patients with peri- and intra-articular fractures of the lower extremities. Furthermore, despite the generally accepted value of the use of patient-specific outcome measures, no data is available offering insights into patients’ self-per- ceived outcome levels (e.g., regarding activities of daily living (ADL), quality of life or pain) in PWB versus RWB. Study design and participants This prospective comparative multicenter cohort study included surgically treated trauma patients with peri- and intra-articular fractures of the lower extremities. Subjects were consecutively recruited from six hospitals in the Neth- erlands between October 2017 and September 2018. The allocation of the patients to the intervention or control group depended on the regimen adhered to by the hospital in which the patients were surgically treated. During the conceptu- alization of this study design, randomization was not con- sidered feasible because of the nature of the two different interventions. Namely, implementation of these different protocols includes patient instructions as well as physical therapy guidance and nursing staff participation. A mix of treatment protocols on a single ward was therefore consid- ered suboptimal. However, this meant we had to consider that not randomizing the study could introduce observer bias, which may be a study limitation. Patients from two In the RWB group, the patients underwent a non-weight bearing regimen for 6–12 weeks followed by partial weight bearing with a 25% increase in weight loading every week according to the existing (AO) guidelines [1]. Protocols The PWB treatment involves a gradual progression in func- tional activities guided by patients’ subjective experience (pain and confidence to bear weight) and by objective clini- cal symptoms of the patients occurring during the process of rehabilitation, evaluated by the physical therapist during every outpatient physiotherapy session. Clinical symptoms include the evolution of signs of inflammation, neuro- vascular status, weight-bearing tolerance, changes in the alignment of the affected side of the body, and the quality and function of the soft tissue and the joints involved. The progress in therapy is not determined by any predetermined or fixed degree of loading of the affected side in kg or in percentage of bodyweight, as this has proved to be difficult to adhere to. This process enables patients to carry out the activities with normal/optimal motor skills as soon as pos- sible. The approach is guided by the quality of performance and the safety of the activity (e.g., preventing stumbling). The next stage of the treatment is started when the gait pat- tern associated with the current stage of the treatment is optimally executed and can be performed by the patient safely and independently [2]. The aim of the present study was therefore to investi- gate the effectiveness of PWB in surgically treated trauma patients with peri- and intra-articular fractures of the lower extremities, reporting on patients’ self-perceived outcome levels regarding ADL, quality of life, pain, weight bearing or patients’ compliance and postoperative complications, in comparison to RWB, over a 26-week post-surgery follow- up period. Outcome measures and co‑variables The patients’ self-perceived outcome levels, questionnaires related to the activities of daily living (ADL) were assessed as primary outcome measure. ADL was measured with the Lower Extremity Functional Scale (LEFS). The LEFS con- sists of 20 questions about a person’s ability to perform daily tasks. The score for each question ranges from 0 (extreme 1 3 3 European Journal of Orthopaedic Surgery & Traumatology (2024) 34:1363–1371 1365 t-tests were used for normally distributed continuous data, and chi-squared tests for categorical variables. In the case of non-parametric data, the median with the interquartile range was calculated. Furthermore, a linear mixed model was used to identify any differences among the outcome measures over time. This analysis ensured that both random and cluster effects, such as treatment in different hospitals, and fixed effects, such as ASA classification, could be con- sidered and corrected for. Results are presented as either mean (standard deviation) or frequencies and percentages. The level of statistical significance was set at α = 0.05. The data was analyzed blinded by the researchers. difficulty in performing the activity) to 4 (good performance of activity), maximizing the score at 80 points. The lower the score, the greater the disability [17]. The other patients’ self-perceived outcome levels were assessed as secondary outcome measures, using question- naires related to the quality of life and pain score. The qual- ity of life was measured with the Short Form-12 (SF-12) questionnaire. The SF-12 consists of 12 items that assess 8 dimensions of health: physical functioning, role-physical, bodily pain, general health, vitality, social functioning, role- emotional and mental health. The SF-12 measures various aspects of physical and mental health from which a physical composite score (PCS) and a mental composite score (MCS) can be calculated, ranging from 0 to 100 [18]. The intensity of pain was measured with the Numeric Rating Scale (NRS; 0 indicating no pain and 10 worst pain) [19]. All patients’ self-perceived outcome levels were obtained at the follow- up time-points of 2-, 6-, 12- and 26-weeks post-surgery. The other secondary outcome measures were the rehabilitation outcome (i.e., outpatient physiotherapy, time to full weight bearing, completion of rehabilitation within 26 weeks), com- plications during a 26-week post-surgery follow-up and the progression of weight bearing during the first 12 weeks of rehabilitation. Primary outcome measures All patients’ compliance were monitored for 3 months after surgery with the OpenGo insole (Moticon GmbH, Munich, Germany) [20]. The insole incorporates 13 capaci- tive pressure sensors and a 3D accelerometer, measuring peak pressures (in Newton) and mean weight bearing (in Newton). It operates completely wireless. Data is stored on a flash drive. The insole can be placed in any shoe and shoes can be changed at random during the study due to an auto- mated zeroing system [20]. After a 26 wees post-surgery follow-up, the overall response rate for the patient-specific outcome measures at all meas- urement points was 99.8% (N = 1 patient refused to fill out the patient self-perceived outcome questionnaires at week 26). ADL as measured with the LEFS, and quality of life as measured with the SF-12, were both significantly better in the PWB group compared to the RWB group (p < 0.01) (Appendix I). There were no differences in pain score between the PWB and RWB groups (Appendix I). The patient self-perceived outcome levels regarding ADL and quality of life in the PWB and RWB groups are summarized in Figs. 2 and 3. Baseline characteristics, recorded at admission, included age at time of fracture, sex, ASA (American Society of Anesthesiologists) classification (type 1–6) [21], Charlson comorbidity score (classifying prognostic comorbidity, a higher score correlating with additional comorbidities) [22], type of fracture and in-hospital length of stay (in days). Outcome measures and co‑variables Postoperative complications, i.e., superficial wound infections, deep wound infections, non-unions and secondary dislocations, or other additional adverse situations that required medical intervention, were recorded as either present or non-present, along with the type of complica- tion. Removal of implants was only performed in case of functional complaints. Baseline characteristics A total of 106 patients were included in this cohort study, N = 53 in both the PWB and RWB groups (Fig. 1). As the assumption for normality was violated, non-parametric tests were used, and established that the PWB group patients had comparable ASA score (p = 0.14) and fewer comorbidities, as measured with the Charlson score, (p = 0.03) compared to those in the RWB group. No significant differences in sex, age, type of fracture, number of surgical interventions during primary admission or in-hospital length of stay were found between the groups. Characteristics of patients in the PWB and RWB groups are summarized in Table 1. Statistical analysis Of the total patient population, 77.4% (N = 82) achieved full weight bearing within 12 weeks. The number of patients who achieved this was significantly higher in the PWB group than in the RWB group: 98.1% versus 56.6% (p < 0.01). The median time from surgery to ascertain- ment of full weight bearing was significantly shorter in Statistical analysis was performed with IBM SPSS Statis- tics (Version 25.0, Armonk, NY). Descriptive statistics were used to describe the demographic data and baseline charac- teristics for the entire study population. Independent samples 1 3 European Journal of Orthopaedic Surgery & Traumatology (2024) 34:1363–1371 1366 European Journal of Orthopaedic Surgery & Traumatology (2024) 34:1363–1371 1 3 the PWB group than in the RWB group: 4.0 (2.1) weeks versus 12.2 (4.2) weeks (p < 0.01). The incidence of postoperative complications in the total study popula- tion was 16.0%, with no statistical significant differences Fig. 1 CONSORT Flowchart of study patients Table 1 Baseline characteristics and in-hospital outcome of the PWB and RWB groups Abbreviations: PWB permissive weight bearing, RWB restricted weight bearing, N number of subjects, ASA American Society of Anesthesiologists, IQR interquartile range PWB (N = 53) RWB (N = 53) Total (N = 106) p Female, N 27 (50.9%) 27 (50.9%) 54 (50.9%) 1.00 Median age (IQR), years 55.0 (38.5–65.0) 60.0 (47.0–67.0) 58.0 (43.5–66.3) 0.27 ASA, N I, II > II 49 (92.5%) 4 (7.5%) 44 (83.0%) 9 (17%) 93 (87.7%) 13 (12.3%) 0.14 Median Charlson score (IQR) 1 (0–3) 2 (1–3) 2 (0–3) 0.03 Fracture type, N: Pelvic Acetabular Tibial plateau Pilon Calcaneal 7 (13.2%) 5 (9.4%) 16 (30.2%) 17 (32.1%) 8 (15.1%) 1 (1.9%) 3 (5.7%) 28 (52.8%) 12 (22.6%) 9 (17.0%) 8 (7.5%) 8 (7.5%) 44 (41.5%) 29 (27.4%) 17 (16%) 0.18 In-hospital outcome: Two or more procedures (%) Median length of stay (IQR), in days 9 (17.0) 7.0 (2.0–15.5) 8 (15.1) 5.0 (2.0–11.5) 17 (16.0) 6.0 (2.0–14.0) 0.57 0.24 1366 Fig. Discussion The aim of this study was to investigate the effectiveness of a novel approach involving permissive weight bearing (PWB) in surgically treated trauma patients with peri- and intra-articular fractures of the lower extremities. The PWB regimen led to the patients being able to bear full weight on their affected leg much sooner, with a better ADL and quality of life, compared to those who followed the usual RWB regimen. No significant differences between the two treatment regimens were found in either postoperative complication rates or pain levels.i Patients’ self-perceived outcome levels were signifi- cantly better among patients who followed the PWB pro- tocol than among those who followed the RWB protocol. This study found a general improvement in ADL (LEFS) and quality of life (SF-12) for both groups during the 26-week rehabilitation period. In our total population, the mean LEFS 26 weeks post-surgery was 55.2 (14.3). This is in line with earlier studies, which found similar lev- els of ADL in trauma patients after surgery of the lower extremities [23–25]. The mean quality of life for the total population in our study was also in the same range as that reported by other studies [26, 27]. Fig. 2 Patient self-perceived activities of daily living during a 26-week post-surgery follow-up. ADL Activities of Daily Living, LEFS Lower Extremity Functional Scale, PWB permissive weight bearing, RWB restricted weight bearing Fig. 3 Patient self-perceived quality of life during a 26-week post- surgery follow-up. PWB permissive weight bearing, RWB restricted weight bearing Despite the early PWB regimen, the recorded pain lev- els during the 26-week rehabilitation period were higher in the RWB group than in the PWB group, which could be due to the consequences of immobilization [2]. In our study 56.6% of the patients in the RWB group were already bearing full weight within 12 weeks, in contrast to the standard protocol of 12 weeks non-weight-bearing or partial weight bearing [1]. Earlier studies also reported that one-third of patients (due to e.g., cognitive impairment in older patients to follow instructions) did not comply with a non-weight-bearing or restricted weight bearing regime [5, 6]. Despite the willingness to comply, patients often do not follow the weight-bearing restrictions and increase their weight bearing as fracture healing progresses [6]. This is also in line with our data on weight bearing, as measured with the Moticon insoles. Statistical analysis 1 CONSORT Flowchart of study patients Abbreviations: PWB permissive weight bearing, RWB restricted weight bearing, N number of subjects, ASA American Society of Anesthesiologists, IQR interquartile range PWB (N = 53) RWB (N = 53) Total (N = 106) p Female, N 27 (50.9%) 27 (50.9%) 54 (50.9%) 1.00 Median age (IQR), years 55.0 (38.5–65.0) 60.0 (47.0–67.0) 58.0 (43.5–66.3) 0.27 ASA, N I, II > II 49 (92.5%) 4 (7.5%) 44 (83.0%) 9 (17%) 93 (87.7%) 13 (12.3%) 0.14 Median Charlson score (IQR) 1 (0–3) 2 (1–3) 2 (0–3) 0.03 Fracture type, N: Pelvic Acetabular Tibial plateau Pilon Calcaneal 7 (13.2%) 5 (9.4%) 16 (30.2%) 17 (32.1%) 8 (15.1%) 1 (1.9%) 3 (5.7%) 28 (52.8%) 12 (22.6%) 9 (17.0%) 8 (7.5%) 8 (7.5%) 44 (41.5%) 29 (27.4%) 17 (16%) 0.18 In-hospital outcome: Two or more procedures (%) Median length of stay (IQR), in days 9 (17.0) 7.0 (2.0–15.5) 8 (15.1) 5.0 (2.0–11.5) 17 (16.0) 6.0 (2.0–14.0) 0.57 0.24 Table 1 Baseline characteristics and in-hospital outcome of the PWB and RWB groups Abbreviations: PWB permissive weight bearing, RWB restricted weight bearing, N number of subjects, ASA American Society of Anesthesiologists, IQR interquartile range postoperative complications in the total study popula- tion was 16.0%, with no statistical significant differences the PWB group than in the RWB group: 4.0 (2.1) weeks versus 12.2 (4.2) weeks (p < 0.01). The incidence of the PWB group than in the RWB group: 4.0 (2.1) weeks versus 12.2 (4.2) weeks (p < 0.01). The incidence of 1 3 European Journal of Orthopaedic Surgery & Traumatology (2024) 34:1363–1371 1367 Fig. 2 Patient self-perceived activities of daily living during a 26-week post-surgery follow-up. ADL Activities of Daily Living, LEFS Lower Extremity Functional Scale, PWB permissive weight bearing, RWB restricted weight bearing Discussion These measurements showed that there was no significant difference in mean weight bear- ing between the RWB and PWB groups. The difference in peak loading was nearly significant between the RWB group and PWB group: p = 0.05. The patients in the PWB group were bearing full weight 9 weeks earlier than those in the RWB group. The effort to bear weight earlier was not at the expense of longer duration of outpatient physiotherapy. In fact, the RWB group required significantly longer outpa- tient physiotherapy than the PWB group, viz. 41 versus 25 h, respectively. Furthermore, significantly more patients in the PWB group completed the rehabilitation within 26 weeks compared to the RWB group, viz. 65.2% versus 34.8%. Fig. 3 Patient self-perceived quality of life during a 26-week post- surgery follow-up. PWB permissive weight bearing, RWB restricted weight bearing between the PWB group and the RWB group (11.3% [N = 6] versus 20.8% [N = 11], respectively (p = 0.19), see Table 2. Among all documented complications, superficial wound infections did not require surgical intervention. Consequently, N = 3 patients adhering to the PWB regimen required supplementary surgical procedures, in contrast to N = 5 patients who adhered to the RWB regimen. Weight bearing compliance The mean weight bearing and peak loading were not sig- nificantly different between the subjects who followed the PWB or RWB regimens, see Figs. 4 and 5. 1 3 3 European Journal of Orthopaedic Surgery & Traumatology (2024) 34:1363–1371 1368 Table 2 Rehabilitation outcome and postoperative complications in the PWB and RWB groups Abbreviations: PWB permissive weight bearing, RWB restricted weight bearing, N number of subjects, IQR interquartile range, OPD outpatient physiotherapy duration, FWB full weight bearing PWB (N = 53) RWB (N = 53) Total (N = 106) p Prescribed rehabilitation aftercare (%): PWB 6 weeks RWB 8 weeks RWB 12 weeks RWB Rehabilitation outcome: Median OPD (IQR), in hours FWB within 12 weeks (%), N Median time to FWB (IQR), in weeks N who completed rehabilitation within 26 weeks (%) 53 (100) 0 (0) 0 (0) 0 (0) 25 (13.0–46.8) 52 (98.1) 4.0 (2.0–7.0) 30 (65.2) 0 (0) 36 (67.9) 3 (5.7) 14 (26.4) 41 (28.5–57.5) 30 (56.6) 13.0(9.0–15.0) 16 (34.8) 53 (50) 36 (34.0) 3 (2.8) 14 (13.2) 33 (18.5–52.0) 82 (77.4) 8.0 (4.0–13.0) 46 (43.4) – 0.01 < 0.01 < 0.01 < 0.01 Total post-operative complications (%) Non-unions Secondary dislocations Superficial wound infections Deep wound infections Removal of osteosynthesis material 6 (11.3) 0 (0.0) 1 (1.9) 3 (5.7) 1 (1.9) 1 (1.9) 11 (20.8) 1 (1.9) 2 (3.8) 6 (11.3) 1 (1..9) 1 (1.9) 17 (16.0) 1 (0.9) 3 (2.8) 9 (8.5) 2 (1.9) 2 (1.9) 0.19 Table 2 Rehabilitation outcome and postoperative complications in the PWB and RWB groups Abbreviations: PWB permissive weight bearing, RWB restricted weight bearing, N number of subjects, IQR interquartile range, OPD outpatient physiotherapy duration, FWB full weight bearing Fig. 4 Mean weight bearing compliance during a 26-week post-sur- gery follow-up. Abbreviation: PWB Permissive weight bearing, RWB Restricted weight bearing, N number of subjects, SD standard devia- tion, WB Weight bearing in newton on affected leg Fig. 5 Weight bearing compliance expressed in peak loading during a 26-week post-surgery follow-up. Abbreviations: PWB Permissive weight bearing, RWB Restricted weight bearing, N number of sub- jects, SD standard deviation, Peak loading Peak loading in newton on affected leg Fig. 4 Mean weight bearing compliance during a 26-week post-sur- gery follow-up. Abbreviation: PWB Permissive weight bearing, RWB Restricted weight bearing, N number of subjects, SD standard devia- tion, WB Weight bearing in newton on affected leg Fig. Weight bearing compliance Special thanks are also due to the six par- ticipating centers in the Netherlands: Maastricht University Medical Center+, Maastricht; Zuyderland Medical Center, Heerlen; Elkerliek Hospital, Helmond; Catharina Hospital, Eindhoven; Maxima Medical Center, Veldhoven and Viecuri Medical Center, Venlo. We would like to thank MEMIC Maastricht for managing our data safely. Further- more, we want to thank Adelante Rehabilitation Center, Hoensbroek, the Netherlands for taking care of the postoperative management in the intervention group. We would also like to thank the AO foundation for providing us with the Moticon insole material free of charge. Finally, we want to thank all the members of the research group and Joey Jansen to their contributions to this study. Authors’ contribution PK is the main researcher and has conceptual- ized this paper with CM, HS, TB, PB and MP. PK has written the protocol for this study. The idea for the study originated from PK, GM, Yv. H, MP and PB. All authors participated in recruiting and enrolling patients for this study. HS, MP and PB are the supervisors of PK. All authors have read and have approved the final version of the manuscript. Our study, the first large-scale prospective multicenter cohort study comparing PWB with RWB, adds evidence in support of the use of PWB in surgically treated trauma patients with peri- and intra-articular fractures of the lower extremities. This means that our study contests the para- digm of the current RWB guidelines, which have remained unchanged for 60 years. The time has now come to renew the current guidelines in accordance with the most recent evidence. Funding Funding by Netherlands Organization for Health Research and Development (ZonMW, project number 843001807). The insole material for this study was provided by the AO Foundation TK Sys- tem (AOTK Project number: AO516.05). The funders had no role in the design and conduct of the study; in the collection, management, analysis and interpretation of the data; in the preparation, review, or approval of the manuscript; or in the decision to submit the manuscript for publication. When interpreting our data, some limitations have to be considered. Due to practical reasons, this study featured a non-randomized groups design. However, patients were included to the PWB and RWB groups consecutively to avoid selection bias. There were differences regarding the patients’ comorbidities and the different hospitals in which the patients were treated. Weight bearing compliance 5 Weight bearing compliance expressed in peak loading during a 26-week post-surgery follow-up. Abbreviations: PWB Permissive weight bearing, RWB Restricted weight bearing, N number of sub- jects, SD standard deviation, Peak loading Peak loading in newton on affected leg Our study found that there was no statistical significant difference in postoperative complications between the PWB group and the RWB group. Nevertheless, a discernible clinical difference was observed, with a lower incidence of post-operative complications evident in patients sub- jected to the PWB regimen in comparison to those under- going a RWB regimen. One of the key objections often raised against early weight bearing is the possibility of frac- ture displacement [28]. On the other hand, it has often been stated that early weight bearing does not entail an undue risk of postoperative complications [2, 3, 12, 13, 29]. These two views are contradictory, and our study provides evidence in favor of regimes with early weight bearing instead of the standard non-weight-bearing protocols. According to recent literature, a composite postoperative complications rate of up to 27% has been found in surgically treated trauma patients with peri- and intra-articular fractures of the lower extremities [7–11]. Comparison of our complication rates with data published in recent literature shows that we found lower rates of postoperative complication in these patients when they were treated with the PWB regimen. 1 3 1369 European Journal of Orthopaedic Surgery & Traumatology (2024) 34:1363–1371 Over- and under-loading may lead to prolonged and com- plicated recovery [2]. A certain minimum level of loading is required to elicit micro-movements between adjacent bony fracture components, stimulating biological processes that are converted into cellular signals initiating bone remod- eling [27, 30]. This process is described in the literature as the mechanotransduction in bone. Mechanotransduction is continuously present and enables the bone to resist the mechanical impacts caused by daily activities [30]. To opti- mize recovery with the lowest number of complications and better patients’ self-perceived outcome levels, one should apply a treatment that approaches the upper limit of the therapeutic bandwidth regarding weight bearing, yet is safe enough to avoid complications due to overloading. This is the case with the PWB protocol [2].i Acknowledgements First of all, we would like to thank all patients participating in this study. Weight bearing compliance Our statistical analyses took these issues into consideration, thus correcting the presented results for the confounding influence that these factors may have had on the study results. Surgeon-oriented functional outcome scores (e.g., the function of a knee or ankle joint) were not taken into account. Another limitation of the study is that we did not investigate the cost-effectiveness of PWB. Therefore, further data are needed on the cost-effec- tiveness, and long-term patient-reported outcome of the PWB strategy. Availability of data and material The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Declarations Conflict of interest No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article. All authors declare that they have no poten- tial conflicts of interest. Ethics approval and consent to participate The medical ethics com- mittee of Maastricht University Medical Center, Maastricht+, the Netherlands approved this study, reference number: METC 16-4-236. Informed consent to participate was obtained from all patients. 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/. References 16. Kalmet PHS, Meys G, van Horn YY et al (2018) Permissive weight bearing in trauma patients with fracture of the lower extremities: prospective multicenter comparative cohort study. BMC Surg 18:8 1. Ruedi TP, Buckley RE, Moran CG (2018) AO principles of frac- ture management, 3rd edn. Thieme, New York 17. Doyle DJ, Garmon EH. American Society of Anesthesiologists Classification (ASA Class) (2018) StatPearls. Stat-Pearls Publish- ing StatPearls Publishing LLC, Treasure Island, FL 2. Meys G, Kalmet PHS, Sanduleanu S et al (2019) A protocol for permissive weight-bearing during allied health therapy in surgi- cally treated fractures of the pelvis and lower extremities. J Reha- bil Med 51:290–297 18. Charlson ME, Pompei P, Ales KL, MacKenzie CR (1987) A new method of classifying prognostic comorbidity in longitudinal stud- ies: development and validation. J Chronic Dis 40:373–383 3. Haller JM, Potter MQ, Kubiak EN (2013) Weight bearing after a periarticular fracture: what is the evidence? Orthop Clin N Am 44:509–519 19. Binkley JM, Stratford PW, Lott SA, Riddle DL (1999) The Lower Extremity Functional Scale (LEFS): scale development, measure- ment properties, and clinical application. North American Ortho- paedic Rehabilitation Research Network. Phys Ther 79:371–383 4. van der Vusse M, Kalmet PHS, Bastiaenen CHG et al (2017) Is the AO guideline for postoperative treatment of tibial plateau fractures still decisive? A survey among orthopaedic surgeons and trauma surgeons in the Netherlands. Arch Orthop Trauma Surg 137:1071–1075 20. Ware JE, Keller SD (1995) How to score the SF-12 physical and mental health summaries: a user’s manual. New England Medical Centre, Boston 5. Hustedt JW, Blizzard DJ, Baumgaertner MR et al (2012) Is it pos- sible to train patients to limit weight bearing on a lower extremity? Orthopedics 35:e31–e37 21. Breivik H, Borchgrevink PC, Allen SM et al (2008) Assessment of pain. Br J Anaesth 101:17–24 22. Braun BJ, Bushuven E, Hell R et al (2016) A novel tool for con- tinuous fracture aftercare - clinical feasibility and first results of a new telemetric gait analysis insole. Injury 47:490–494 6. Raaben M, Holtslag HR, Leenen LPH et al (2018) Real-time vis- ual biofeedback during weight bearing improves therapy compli- ance in patients following lower extremity fractures. Gait Posture 59:206–210 23. Repo JP, Barner-Rasmussen I, Roine RP et al (2016) Treatment of compound tibia fracture with microvascular latissimus dorsi flap and the Ilizarov technique: a cross-sectional study of long-term outcomes. * Coen Verstappen Coen.Verstappen@mumc.nl Conclusion This prospective, comparative, multicenter study shows that PWB in surgically treated trauma patients with peri- and intra-articular fractures of the lower extremities is effective and is associated with a significant reduction in time to full weight bearing and significantly better outcomes in terms of ADL and quality of life compared to the RWB regime, with a similar complication rate. This PWB protocol contests the paradigm of the current RWB guidelines, which have remained unchanged for 60 years. 1 3 1 3 1370 European Journal of Orthopaedic Surgery & Traumatology (2024) 34:1363–1371 References Eur J Orthop Surg Traumatol 29:1789–1794 Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 15. Smeeing DPJ, Houwert RM, Briet JP et al (2020) Weight-bearing or non-weight-bearing after surgical treatment of ankle fractures: a multicenter randomized controlled trial. Eur J Trauma Emerg Surg 46:121–130 References J Plast Reconstr Aesthet Surg 69:524–532 7. Choo KJ, Morshed S (2014) Postoperative complications after repair of tibial plateau fractures. J Knee Surg 27:11–19 8. Yu X, Pang QJ, Chen L et al (2013) Postoperative complications after closed calcaneus fracture treated by open reduction and inter- nal fixation: a review. J Int Med Res 42:17–25 24. Garner MR, Thacher RR, Ni A et al (2015) Elective removal of implants after open reduction and internal fixation of Tibial Pla- teau fractures improves clinical outcomes. Arch Orthop Trauma Surg 135:1491–1496 i 9. Halvorson JJ, Winter SB, Teasdall RD, Scott AT (2013) Talar neck fractures: a systematic review of the literature. J Foot Ankle Surg 52:56–61 g 25. von Rüden C, Wenzel L, Becker J et al (2019) The pararectus approach for internal fixation of acetabular fractures involving the anterior column: evaluating the functional outcome. Int Orthop 43:1487–1493 10. Schwabe P, Wichlas F, Druschel C et al (2014) Complications after osteosynthetic treatment of acetabular fractures. Orthopade 43:24–34 26. Kerschbaum M, Hausmann N, Worlicek M et al (2017) Patient related outcome of unstable pelvic ring fractures stabilized with a minimal invasive screw-rod system. Health Qual Life Outc 15:248 11. Papakostidis C, Kanakaris NK, Giannoudis PV (2009) Pelvic ring disruptions: treatment modalities and analysis of outcomes. Int Orthop 33:329–338 27. Westerman RW, Hull P, Hendry RG, Cooper J (2008) The physi- ological cost of restricted weight bearing. Injury 39:725–727 12. Kalmet PHS, van Horn YY, Sanduleanu S et al (2019) Patient- reported quality of life and pain after permissive weight bear- ing in surgically treated trauma patients with tibial plateau frac- tures: a retrospective cohort study. Arch Orthop Trauma Surg 139:483–488 28. Black JD, Bhavikatti M, Al-Hadithy N et al (2013) Early weight- bearing in operatively fixed ankle fractures: a systematic review. Foot (Edinb) 23:78–85 29. Williamson M, Iliopoulos E, Jain A et al (2018) Immediate weight bearing after plate fixation of fractures of the tibial plateau. Injury 49:1886–1890 13. Kubiak EN, Beebe MJ, North K et al (2013) Early weight bearing after lower extremity fractures in adults. J Am Acad Orthop Surg 21:727–738 30. Klein-Nulend J, Bacabac RG, Mullender MG (2005) Mechanobi- ology of bone tissue. Pathol Biol 53:576–580 14. Consigliere P, Iliopoulos E, Ads T, Trompeter A (2019) Early ver- sus delayed weight bearing after surgical fixation of distal femur fractures: a non-randomized comparative study. Authors and Affiliations Pishtiwan Kalmet1 · Cherelle Maduro1 · Coen Verstappen1 · Guido Meys2 · Yvette van Horn2 · Raoul van Vugt3 · Heinrich Janzing4 · Alexander van der Veen5 · Coen Jaspars6 · Jan Bernard Sintenie7 · Taco Blokhuis1 · Silvia Evers8,9 · Henk Seelen2,8 · Peter Brink1 · Martijn Poeze1,10 Pishtiwan Kalmet Pishtiwan.Kalmet@mumc.nl * Coen Verstappen Coen.Verstappen@mumc.nl 1 3 European Journal of Orthopaedic Surgery & Traumatology (2024) 34:1363–1371 1371 European Journal of Orthopaedic Surgery & Traumatology (2024) 34:1363–1371 Cherelle Maduro Cherelle.Maduro@mumc.nl Guido Meys G.Meys@adelante-zorggroep.nl Yvette van Horn Y.Horn@adelante-zorggroep.nl Raoul van Vugt r.vanvugt@zuyderland.nl Heinrich Janzing hjanzing@viecuri.nl Alexander van der Veen alexander.vd.veen@catharinaziekenhuis.nl Coen Jaspars C.Jaspars@mmc.nl Jan Bernard Sintenie jb.sintenie@elkerliek.nl Taco Blokhuis Taco.Blokhuis@mumc.nl Silvia Evers s.evers@maastrichtuniversity.nl Henk Seelen h.seelen@maastrichtuniversity.nl Peter Brink p.brink@mumc.nl Martijn Poeze M.Poeze@mumc.nl 1 Maastricht University Medical Center+, Maastricht, The Netherlands 2 Adelante Rehabilitation Center, Hoensbroek, The Netherlands 3 Zuyderland Medical Center, Heerlen, The Netherlands 4 Viecuri Medical Center, Venlo, The Netherlands 5 Catharina Hospital, Eindhoven, The Netherlands 6 Maxima Medical Center, Veldhoven, The Netherlands 7 Elkerliek Hospital, Helmond, The Netherlands 8 Maastricht University, Care and Public Health Research Institute (CAPHRI), Maastricht, The Netherlands 9 Trimbos Institute, Netherlands Institute of Mental Health and Addiction, Utrecht, The Netherlands 10 Nutrim School for Nutrition, Toxicology and Metabolism, Maastricht University, Maastricht, The Netherlands Cherelle Maduro Cherelle.Maduro@mumc.nl Guido Meys G.Meys@adelante-zorggroep.nl Yvette van Horn Y.Horn@adelante-zorggroep.nl Raoul van Vugt r.vanvugt@zuyderland.nl Heinrich Janzing hjanzing@viecuri.nl Alexander van der Veen alexander.vd.veen@catharinaziekenhuis.nl Coen Jaspars C.Jaspars@mmc.nl Jan Bernard Sintenie jb.sintenie@elkerliek.nl Taco Blokhuis Taco.Blokhuis@mumc.nl Silvia Evers s.evers@maastrichtuniversity.nl Henk Seelen h.seelen@maastrichtuniversity.nl Peter Brink p.brink@mumc.nl Martijn Poeze M.Poeze@mumc.nl 1 Maastricht University Medical Center+, Maastricht, The Netherlands 2 Adelante Rehabilitation Center, Hoensbroek, The Netherlands 3 Zuyderland Medical Center, Heerlen, The Netherlands 4 Viecuri Medical Center, Venlo, The Netherlands 5 Catharina Hospital, Eindhoven, The Netherlands 6 Maxima Medical Center, Veldhoven, The Netherlands 7 Elkerliek Hospital, Helmond, The Netherlands 8 Maastricht University, Care and Public Health Research Institute (CAPHRI), Maastricht, The Netherlands 9 Trimbos Institute, Netherlands Institute of Mental Health and Addiction, Utrecht, The Netherlands 10 Nutrim School for Nutrition, Toxicology and Metabolism, Maastricht University, Maastricht, The Netherlands Peter Brink p.brink@mumc.nl Martijn Poeze M.Poeze@mumc.nl 1 Maastricht University Medical Center+, Maastricht, The Netherlands 2 Adelante Rehabilitation Center, Hoensbroek, The Netherlands 3 Zuyderland Medical Center, Heerlen, The Netherlands 4 Viecuri Medical Center, Venlo, The Netherlands 5 Catharina Hospital, Eindhoven, The Netherlands 6 Maxima Medical Center, Veldhoven, The Netherlands 7 Elkerliek Hospital, Helmond, The Netherlands 8 Maastricht University, Care and Public Health Research Institute (CAPHRI), Maastricht, The Netherlands 9 Trimbos Institute, Netherlands Institute of Mental Health and Addiction, Utrecht, The Netherlands 10 Nutrim School for Nutrition, Toxicology and Metabolism, Maastricht University, Maastricht, The Netherlands Cherelle Maduro Cherelle.Maduro@mumc.nl Guido Meys G.Meys@adelante-zorggroep.nl Yvette van Horn Y.Horn@adelante-zorggroep.nl Raoul van Vugt r.vanvugt@zuyderland.nl Heinrich Janzing hjanzing@viecuri.nl Alexander van der Veen alexander.vd.veen@catharinaziekenhuis.nl Coen Jaspars C.Jaspars@mmc.nl Jan Bernard Sintenie jb.sintenie@elkerliek.nl Taco Blokhuis Taco.Blokhuis@mumc.nl Silvia Evers s.evers@maastrichtuniversity.nl Henk Seelen h.seelen@maastrichtuniversity.nl Peter Brink p.brink@mumc.nl Martijn Poeze M.Poeze@mumc.nl 1 Maastricht University Medical Center+, Maastricht, The Netherlands 2 Adelante Rehabilitation Center, Hoensbroek, The Netherlands 3 Zuyderland Medical Center, Heerlen, The Netherlands 4 Viecuri Medical Center, Venlo, The Netherlands 5 Catharina Hospital, Eindhoven, The Netherlands 6 Maxima Medical Center, Veldhoven, The Netherlands 7 Elkerliek Hospital, Helmond, The Netherlands 8 Maastricht University, Care and Public Health Research Institute (CAPHRI), Maastricht, The Netherlands 9 Trimbos Institute, Netherlands Institute of Mental Health and Addiction, Utrecht, The Netherlands 10 Nutrim School for Nutrition, Toxicology and Metabolism, Maastricht University, Maastricht, The Netherlands 10 Nutrim School for Nutrition, Toxicology and Metabolism, Maastricht University, Maastricht, The Netherlands 1 3 1 3 3
https://openalex.org/W4308545571	https://www.researchsquare.com/article/rs-761797/latest.pdf	English	null	The protective effects of melatonin in high glucose environment by alleviating autophagy and apoptosis on primary cortical neurons	Molecular and cellular biochemistry	2,022	cc-by	7,306	The protective effects of melatonin in high glucose environment by alleviating autophagy and apoptosis on primary cortical neurons Lijiao , Xiong First Affiliated Hospital Of Gannan Medical University Song Liu Gannan Medical University Chaoming Liu Gannan Medical University Tianting Guo Ganzhou People's Hospital Zhihua Huang (  zh.huang@gmu.edu.cn ) Gannan Medical University https://orcid.org/0000-0002-2610-1999 Liangdong Li First Affiliated Hospital of Fujian Medical University Research Article Keywords: Cortical neurons, High glucose environment, Melatonin, Autophagy, Apoptosis Posted Date: August 10th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-761797/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License The protective effects of melatonin in high glucose environment by alleviating autophagy and apoptosis on primary cortical neurons Lijiao , Xiong First Affiliated Hospital Of Gannan Medical University Song Liu Gannan Medical University Chaoming Liu Gannan Medical University Tianting Guo Ganzhou People's Hospital Zhihua Huang (  zh.huang@gmu.edu.cn ) Gannan Medical University https://orcid.org/0000-0002-2610-1999 Liangdong Li First Affiliated Hospital of Fujian Medical University Research Article Keywords: Cortical neurons, High glucose environment, Melatonin, Autophagy, Apoptosis Posted Date: August 10th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-761797/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Research Article Page 1/17 Page 1/17 Page 1/17 Abstract Cognitive dysfunction has been regarded as a complication of diabetes. Melatonin shows a neuroprotective effect on various neurological diseases. However, it’s protective effect on cortical neurons in high glucose environment has not been reported. Our present study aims to observe the protective effect of melatonin on rat cortical neurons and its relationship with autophagy in high glucose environment. The rat primary cortical neurons damaged model was induced by high glucose. The CCK-8, flow cytometry, Western Blot and immunofluorescence methods were used to examine the cell viability, apoptosis rate and proteins expression. Our results showed that there were no differences in cell viability, apoptosis rate, and protein expression among the control MLT and mannitol group. The cell viability of the glucose group was significantly lower than that of the control group, and the apoptosis rate of the glucose group was significantly higher than that of the control group. Compared with the glucose group, the glucose + melatonin group showed a significant increase in cell viability and a notable decrease in apoptosis rate. Melatonin concentration of 0.1-1 mmol/L can significantly reduce the injury of cortical neurons by high glucose. Compared with the control group, the glucose group showed a significant reduction of Bcl-2 protein expression, while remarkable elevations of Bax, caspase-3, Beclin-1 and LC3B levels. The neurons pre-administered with melatonin obtained significantly reversed these changes induced by high glucose. The phosphorylation levels of Akt and mTOR in the glucose group were significantly lower than those in the control group, were significantly increased in the glucose + MLT group compared with the glucose group. These data indicated that melatonin has a neuroprotective effect on cortical neurons under high glucose environment, which may work by activating Akt/mTOR pathway and following the down-regulation of autophagy. 1. Introduction The incidence of diabetes has been increasing year by year. Traditionally, diabetes complications mainly affect the kidney, retina, peripheral nerve and cardiovascular system. Recently, diabetes cognitive dysfunction has been regarded as a complication of the central nervous system of diabetes, attracting more and more attention. "Diabetes encephalopathy (diabetic encephalopathy, DE)" was first proposed by Nielon scholars in 1965, means the changes in brain structure, function and metabolic state caused by long-term sustained hyperglycemia, which eventually causes nerve behavior and cognitive function defect. It is mainly manifested in the descent of comprehension ability, memory decline, cognitive dysfunction, accompanied by indifference, delayed behavior, and even the development of dementia. Its pathogenesis is related to glucotoxicity, insulin resistance, apoptosis of nerve cells, vascular diseases, oxidative stress, mitochondrial dysfunction, inflammatory response, amyloid deposition and synaptic changes of nerve cells (1). With high incidence of DE, the search for new effective diagnostic methods and therapeutic measures of DE has important clinical and social application value. At present, DE has been recognized as an independent type of dementia, and the pathological changes of Tau protein hyperphosphorylation and amyloid protein deposition have also been found in the brains of patients with diabetes. DE and Alzheimer's disease (AD), Parkinson's disease (PD), multiple sclerosis and Page 2/17 Page 2/17 huntingtin's disease (HD) all belong to neurodegenerative diseases. Abnormal accumulation of proteins in cells formed in the affected brain regions is a common feature of these diseases (2)。 When cells are stimulated by the outside environment, they form a membrane-like structure wrapped by a double membrane, transport misfolded proteins or abnormal cell components to lysosomal degradation and maintain cell self-renewal, this process known as Autophagy. Autophagy is essential for maintaining neuronal homeostasis by clearing damaged mitochondria and phagocytes, such as HD and PD related proteins (3). Studies have shown that autophagy disorders can lead to degenerative changes in neurons, such as Parkinson's syndrome and amyotrophic lateral sclerosis (4)。Mitochondrial autophagy plays an important role in apoptosis of nerve cells. In the early stage of nerve cell injury, autophagy can remove harmful substances to protect cells from apoptosis. Excessive activation of autophagy may lead to excessive self-digestion, which in turn triggers the apoptosis process to initiate and promote apoptosis (5). 1. Introduction On account of involved in the pathogenesis of cognitive dysfunction, autophagy regulation has been regarded as a target for the therapy of neurodegenerative disease (3-5)。 Studies have shown that the hyperglycemic state of diabetes leads to excessive activation of autophagy, which damages the target organs. For example, in diabetic retinopathy, excessive activation of autophagy by neurons leads to increased apoptosis of retinal nerve cells, while inhibiting their autophagy activity can reduce hyperglycemia toxicity and improve retinal function (6). Animal experiments showed that the cognitive dysfunction of diabetes also has excessive activation of autophagy, and inhibiting the autophagy activity can improve its cognitive function (7)。Therefore, we speculated that the use of drugs or other methods to inhibit neuronal autophagy could delay the occurrence and development of diabetic cognitive dysfunction. Melatonin is a hormone synthesized and secreted by pineal gland, which has the physiological functions of regulating sleep, immune regulation, anti-inflammatory and antioxidant. Melatonin has a neuroprotective effect, which can ameliorate brain tissue edema and injury, inhibit neuronal apoptosis, reduce oxidative stress, prevent Aβ aggregation, and so on. It shows protective effects on PD, stroke, AD and etc in animal model tests (8, 9). Animal experiments show that melatonin can regulate autophagy and play a neuroprotective role in the rat model of ischemia reperfusion injury (10). However, whether there is protective effect of melatonin on cortical neurons in high glucose environment has not been reported. Therefore, in our study, the cortical neurons in the high glucose environment were used as the model to simulate the central neuropathy of diabetes mellitus and investigate the protective effect of melatonin and its related mechanisms. 2.2. Primary culture of cortical neurons from fetal rats 2.2. Primary culture of cortical neurons from fetal rats Experimental protocols for animal studies were approved by the local ethics committee. Primary cortical neurons were isolated and cultured from day E18 Sprague Dawley rat embryos. Anatomical microscope was used to dissect the cortex of rats and put it into Hank's anatomical solution. Cortex tissues were digested in 0.125% trypsin 18 min at 37 ° C water bath, meanwhile oscillation of 2–3 times. DMEM medium containing 10% FBS serum was used to terminate digestion. Centrifuge for 10 min at 1000 rpm/min, and the cells were resuspended with the density of 105/mL and inoculated in the Poly-L-lysine- coated (0.1 g/L) plates, and then cultured in DMEM supplemented with 10% fetal bovine serum, L- glutamine (200 nM), 50 U/mL penicillin and 50 µg/mL streptomycin at 37°C and 5% CO2 incubator. This medium was removed after 4 h and replaced with Neurobasal medium containing 2% B27 supplement. Half the medium was removed every 3 d in culture and replaced with fresh Neurobasal medium with supplements. After 6 d in the supplemented Neurobasal medium, the primary cortical neurons were used for the experiment. 2.1. Materials Female SD rats with pregnancy 18 days weighing 250–280 g were purchased from Hunan Slake Jingda Experimental Animals Limited Liability Company. (license number of animal center: SCXK(Xiang) 2016- Page 3/17 Page 3/17 0002, grade: SPF). Melatonin (M5250) purchased from Sigma-Aldrich, using Dimethyl sulfoxide (DMSO) solution to dissolve it. Dulbecco's modified Eagle's medium (DMEM, C11996600BT), Neurobasal medium (21103-049), B27 supplement (50×) (17504-044), GlutamaxTM-1(100×) (35050-061) were from Gibco. Fetal bovine serum (FBS), trypsin, penicillin-streptomycin and L-glutamine were from Gibco. Poly-L-lysine (molecular weight 15,000–30,000), glucose, Cell Counting kit-8 (CCK8) (CA1210) was purchased from Solarbio. Apoptosis detection kit (556547) was purchased from BD Pharmingen Company. Bcl-2(sc-7382) and Bax (sc-20067) antibody were purchased from Santa Cruz Blotechnology (CA, USA). Caspase-3 (9664S), LC3B (2775S), Beclin-1 (3738S), p-mTOR (5536S), mTOR (2983S), p-Akt (4046S) and Akt (9272S) antibody were purchased from Cell Signaling Technology. p-Beclin-1 (ab183335) antibody was purchased from Abcam (Cambridge, UK). β-tubulin (MA5-11732) antibody was purchased from Invitrogen. Goat anti-rabbit IgG (31460), goat anti-rat IgG (31430), PVDF membrane (88518), protein quantitative reagent (A33972), and enhanced chemiluminescence reagent (34580) were purchased from Thermo Fisher. 2.4. Experimental groups The cells were divided into control group (Ctrl), melatonin group (MLT), Mannitol group (100 mmol/L), Glucose group (100 mmol/L), and Glucose + MLT group. The primary fetal rat cortical neuron cells were mature after 6 days. Melatonin was added for pretreatment for half an hour, and then glucose or mannitol was added for treatment for 48 h. After that, neurons were collected for experimental detection (Fig. 1A). The concentrations of melatonin in MLT and Glucose + MLT groups were 0.1, 0.3, 1, 3, 10 microns /L in the cell viability assessment. The final concentrations of melatonin in the subsequent experiments were 0.1 µmol/L. 2.6. Detection of Apoptotic Cells by Annexin V-FITC/PI Flow Cytometry 2.6. Detection of Apoptotic Cells by Annexin V-FITC/PI Flow Cytometry 1.5×106 cells per pore were plated in 6 well plates. The cells were cultured for 6 days and then treated with drugs for 48 hours. Cells were trypsinized and harvested by centrifugation and then followed the instructions of Annexin V-FITC/PI kit to incubate with Annexin V-FITC and PI for 15 min at room temperature, away from light. Apoptosis rate was examined by flow cytometry in 30 minutes. The experiment was repeated 3 times with 4 duplicate holes in each group. 2.3. Cell immunofluorescent staining Neuronal nuclear protein (NeuN) immunostaining was performed to evaluate the purification rate of cortical neurons. Immunofluorescence of other proteins Bax, Bcl-2, Beclin-1, LC3B and Caspase-3 was performed. The culture medium was removed and the neurons were rinsed for 5 min three times in cold phosphate buffered saline (PBS). Cells were fixed with 4% paraformaldehyde for 30 min, permeabilized in PBS containing 0.2% Triton for 30 min, and blocked with 10% normal goat serum for 1 h. Cells were incubated with mouse anti-Bax, Bcl-2, Beclin-1, LC3B and Caspase-3 primary monoclonal antibody (1:50) for 2 h at 37°C and washed three times in PBS (5 min per wash), then incubated with goat anti-mouse secondary antibody conjugated to FITC (1:500) for 1 h at 37°C and washed three times in PBS (5 min per wash). Cells were incubated with DAPI (1:1000) for 5 min at room temperature and examined for fluorescence using a standard fluorescence photomicroscope (DM IRE2, Leica Microsystems, Cambridge, UK). Page 4/17 Page 4/17 2.5. Cell viability assessment Cell viability was quantified by CCK-8 assay. The cells were inoculated in 96-well plate with a concentration of 1×105/ml, and the treated cells were cultured for 48 h after 7 days. 10 µL CCK-8 was added to each hole, and cultured for 2 h, and the light absorption value was measured at 490 nm on a plate reader, and the neuronal cell viability was calculated as a mean percentage of the control group. All experiments were carried out in quadruplicate. 2.7. Western blot analysis of p-mTOR, mTOR, p-Akt, Akt, p- Beclin-1, Beclin-1, LC3B, Bcl-2, Bax, Caspase-3 and β-tubulin Primary fetal cortical neurons were processed and cells were collected. The protein concentration in each sample was determined by the bicinchoninic acid (BCA) protein assay and equal amounts of protein per lane were loaded onto the gel. Briefly, samples were electrophoresed on 10% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes. After blocking with Tris hydrochloric acid buffered saline (TBS) containing 0.1% Tween 20 and 5% nonfat milk, the membranes were incubated overnight at 4°C with a rabbit polyclonal antibody against p-mTOR, mTOR, p-Akt, Akt, p-Beclin-1, Beclin-1, LC3B, Bcl-2, Bax, Caspase-3 and β-tubulin, diluted 1:2000 in 0.05% BSA. They were then washed and incubated with a horseradish peroxidase-conjugated goat-anti-rabbit or goat-anti-mice secondary antibody IgG (1:5000). Protein bands were visualized by chemiluminescence with a Western Chemiluminescent HRP Substrate kit (Immobilon) and the band images analyzed with Image J software (National Institutes of Health, Page 5/17 Page 5/17 Bethesda, MD, USA). β-actin was used as a control for the protein loading. Each experiment was repeated at least three times. Bethesda, MD, USA). β-actin was used as a control for the protein loading. Each experiment was repeated at least three times. 2.8. Statistical Analysis All statistical analyses were carried out with GraphPad Prism 5.0 software. Data are expressed as mean ± SEM and analyzed by one-way ANOVA followed by post hoc Student–Newman–Keuls test to determine the influence of different groups. P < 0.05 was considered statistically significant. 3.1. Effect of melatonin on the cell viability and apoptosis of cortical neurons in high glucose environment 3.1. Effect of melatonin on the cell viability and apoptosis of cortical neurons in high glucose environment When melatonin concentration was 0.1, 0.3 and 1 µmol/L, the survival rate of cortical neurons was not statistically different from that of the control group (P>0.05). When melatonin concentration was 3 and 10 µmol/L, the cell survival rate was significantly lower than that of the control group, which indicating that 3 and 10 µmol/L MLT had toxic effects on primary cultured cortical neurons (Figure 1B). The survival rate of glucose group was significantly lower than that of the control group (P<0.01). Compared with the glucose group, the cell survival rate was significantly increased with a significant statistical difference (P<0.01), indicating that the high glucose induced cortical neuron injury could be significantly reduced with 0.1-1µmol/L melatonin (Figure 1C). There was no statistical difference in apoptosis rate between the control group, melatonin group and mannitol group (P>0.05). The apoptosis rate in the Glucose group was significantly higher than that in the control group (P<0.05), and significantly lower in the Glucose + MLT group than that in the Glucose group (P<0.05) (Figure 1D-F). This indicated that melatonin concentration of 0.1-1 µM can alleviate the damage of cortical neurons induced by high glucose, while melatonin concentration of 3 and 10 µM MLT can have toxic effects on the original cultured cortical neurons. 3.2. Effects of melatonin on the apoptosis-related proteins expression of Bcl-2, Bax and Caspase-3 on cortical neurons in high-glucose environment There was no significant difference in protein expression of Bcl-2, Bax and Caspase-3 among the control, melatonin and mannitol group by Western Blot and immunofluorescence (P>0.05) (Figure 2A-G). Western Blot and immunofluorescence both showed that the expression of Bcl-2 was significantly decreased while the expression of Bax and Caspase-3 was significantly increased in the glucose group compared with the control group, showing a statistically significant difference (P<0.05). Compared with the glucose group, glucose + MLT group obtained a significantly increase in Bcl-2 protein expression, while significantly decreases in Bax and Caspase-3 expression with statistical significant difference (P<0.05) (Figure 2A-G). These results indicate that MLT regulate the expression of apoptosis relation protein and inhibit cell apoptosis induced by high glucose stimulation. 3.4. Effects of melatonin on the phosphorylation of Akt/mTOR protein on cortical neurons in high-glucose environment by Western Blot The phosphorylation of AKT and mTOR protein in each group was determined by Western Blot, and there was no statistically significant difference among the control, melatonin and mannitol group (P>0.05) (Figure 4A-D). p-Akt/Akt, p-mTOR/mTOR and mTOR were significantly decreased in the glucose group compared with the control group (P<0.05), and were significantly increased in the glucose + MLT group compared with the glucose group (P<0.05) (Figure 4A-E). It suggested that AKT/mTOR activation was significantly inhibited in the glucose group, and melatonin could increase the phosphorylation level of AKT/mTOR in cortical neurons in the high glucose environment. Page 6/17 Page 6/17 3.3. Effects of melatonin on the autophagy related proteins expression of Beclin-1 and LC3B on cortical neurons in high-glucose environment Western Blot and immunofluorescence both showed that there was no statistical difference on the expression of autophagy related proteins Beclin-1 and LC3B among the control, melatonin, mannitol group (Figure 3A-F). Immunofluorescence results showed that Beclin-1 and LC3B expression in the glucose group were significantly higher than the control group. Beclin-1 and LC3B expression were significantly lower in the glucose +MLT group than in the glucose group, and the difference was statistically significant (Figure 3A, B). Western Blot results show that the expression of p-Beclin-1/Beclin- 1, LC3BⅠand LC3BⅡ were significantly increased in the glucose group compared with control group, with a statistically significant difference (P < 0.01). Compared with the glucose group, glucose +MLT showed significant reductions in the expression of p-Beclin-1/Beclin-1, LC3BⅠand LC3BⅡ(P < 0.01) (Figure 3C-F). The results of western Blot and immunofluorescence were consistent. 4. Discussion The pathogenesis of diabetic central neuropathy is not completely clear. The high fatty acids, high blood glucose and abnormal increase of glycation end products caused by hyperglycemia induce oxidative stress. Excess produced or insufficient eliminated ROS will directly cause biological membrane lipid peroxidation, protein and enzyme degeneration, mitochondrial dysfunction, which lead to neuronal cell damage, scabbard film, axon swelling degeneration and even broke off, neurons chromatin dissolved, cytoplasm vacuoles degeneration necrosis, mitochondrial dysfunction, nuclear pyknosis and nerve cells apoptosis (2). Abnormal changes in brain electrophysiology, imaging and pathology caused by diabetic metabolic factors such as glucose and lipid metabolism disorder, insulin resistance, vascular disease and obesity are closely related to the occurrence and development of DE. Melatonin is a strong antioxidant, with small molecular volume and amphiphilic nature, which is very easy to enter the cells, especially the mitochondria, and participate in mitochondrial energy metabolism to reduce mitochondrial damage caused by oxidative stress (11-13). Relevant studies have shown that melatonin can directly interact with electrons or induce the production of antioxidant enzymes to reduce the damage of oxidative respiratory chain, enhance mitochondrial function and reduce oxidative stress Page 7/17 even reduce cell apoptosis (11). In addition, melatonin can help restore the function of islet B cells, improve insulin sensitivity and glucose tolerance, reduce hyperinsulinemia, and reduce insulin resistance. Melatonin may be a potential diabetes treatment drug. even reduce cell apoptosis (11). In addition, melatonin can help restore the function of islet B cells, improve insulin sensitivity and glucose tolerance, reduce hyperinsulinemia, and reduce insulin resistance. Melatonin may be a potential diabetes treatment drug. In the hippocampus and cortex of diabetic rats, lipid peroxides increased, and rats showed obvious cognitive impairment. However, these manifestations were significantly reduced or even disappeared after the treatment with melatonin or antioxidants vitamin E (14, 15). Melatonin can also alleviate peripheral neuralgia caused by diabetes and improve diabetic neuropathy (16). In addition, melatonin has neuroprotective effects. Patients suffered from AD showed a reduction in secretion of melatonin, which slowed down the synthesis of the protein of Aβ protein and delay the progression of this disease (17). Activating the receptor of melatonin presented a protective effect on spinal cord injury (18). In addition, melatonin showed satisfactory neuroprotection and antioxidation effects on cultured primary cortical neurons deprived of oxygen and sugar, and animal brain injury induced by cerebral ischemic stroke (19). 4. Discussion In this study, we found that melatonin could improve the survival rate of cortical neurons and reduce the apoptosis rate of neurons in fetal rats under high-glucose environment, indicating that melatonin could protect neurons in cortex of fetal rats under high glucose environment. The cerebral cortex is an important anatomical basis for learning and memory and it is vulnerable to various adverse factors. The thickness and volume of cerebral cortex were significantly reduced in diabetic patients, which is the structural basis for learning and memory impairment in diabetic patients (20). High glucose environment can cause apoptosis of cortex and hippocampal neurons (21-22). Bax and Bcl-2 family are very important apoptotic regulatory genes. Bax is apoptosis promoting gene, and Bcl-2 is apoptosis inhibiting gene. In addition, Bcl-2 protein family can not only regulate cell apoptosis but also affect other cell processes, such as cell cycle, calcium signaling, glucose stability, autophagy and etc, which play an important role in cell survival and death (23). Caspase-3 is the main executor of apoptosis, and activation of caspase-3 can cause downstream apoptosis cascade reaction. In this study, we found that the high glucose environment can lead to apoptosis of cortical neurons, and regulated the expression of Bax, Caspase-3 and Bcl-2, and the hypertonic state of mannitol had no effect on neuronal apoptosis and protein levels, suggesting that it is high glucose rather than hyperosmosis directly leads to apoptosis and the changes of relevant protein expression in neurons, Melatonin can reverse the change of Bax, caspase-3 and bcl-2 proteins in cortical neurons induced by high-glucose environment and reduce neuron apoptosis. Autophagy is an important degradation pathway for phagocytosis of aging proteins or organelles in the cytoplasm, which is degraded by autophagy lysosome to complete the metabolic needs of the body and the renewal of aging organelles (24).. Different from other cells, neurons don't have the function of re- division, and can't dilute harmful substances in cells by way of division. Therefore, the autophagic lysosome degradation pathway is particularly important in neurons (3). At present, studies have found that the abnormal autophagy function plays an important role in the occurrence and development of nervous system diseases, such as PD and HD. The regulation of autophagy activity can delay the progress of the disease through devouring damaged mitochondria, reducing oxidative stress and Page 8/17 inhibiting apoptosis (4, 5). The excessive activation of autophagy can promote apoptosis. 4. Discussion In addition, autophagy and apoptosis can exist simultaneously and interact with each other (25). Inhibition of autophagy activity in cerebral ischemia reperfusion rats can improve their neural function (26). At present, there are few studies on autophagy in diabetic encephalopathy, and changes in autophagy activity of cells may be one of the important mechanisms of diabetic encephalopathy (7, 27, 28). Many studies have shown that the neuron apoptosis and cognitive decline in diabetic rats are related to the excessive activation of autophagy, and inhibition of autophagy activity can improve the cognitive function of diabetic mice (7, 27, 28). Diabetic retinopathy and diabetic hearing impairment were associated with elevated levels of autophagy in diabetic nerve cells (6, 29). This study showed that the expression of Beclin-1 and LC3B, two landmark molecules of autophagy, could be increased in high glucose environment, suggesting that high glucose environment could induce autophagy. Melatonin can decrease Beclin-1 and LC3B in high glucose environment, and reduce the apoptosis rate of cortical neurons. It suggests that melatonin can protect cortical neurons by down-regulating autophagy activity. This study is similar to the results of other studies (7, 27, 28). However, some studies showed that the autophagy activity of neurons in diabetic neuropathy and diabetic rats with microvascular disease decreases, and the activation of autophagy can reduce the damage of neurons (30-32). We believe that the differences in these findings are related to the degree of autophagy activation. Moderate autophagy activation has neuroprotective effects, but excessive autophagy activation can induce programmed cell death. Akt/mTOR signaling pathway is an important mechanism for the negative regulation of autophagy. Bcl-2 inhibits autophagy initiation by binding to Beclin-1 in the structural domain of BH3 protein, and mTOR is required to participate in the binding of Beclin1 to BH3 protein. Activation of mTOR directly inhibits autophagic initiation, and mTOR regulates autophagic related protein phosphorylation and thus blocks autophagy (33-35). Studies found that diabetic cognitive dysfunction is related to mTOR signaling pathway, and activation of PI3K/Akt/mTOR signaling pathway can improve nerve defects in diabetic cerebral ischemia reperfusion rats and hyperglycemia induced neurotoxicity of PC-12 cells (22, 36). In addition, activation of Akt/mTOR signaling pathway can promote the growth of posterior axons and improve the functional recovery after stroke (37). 4. Discussion It also found that melatonin can regulate AMPK/mTOR signaling pathway and play a protective role in myocardial ischemia reperfusion (13), reduce the neuron apoptosis induced by focal cerebral ischemia in mice via activating the PI3K/Akt signaling pathway (38), and alleviate nerve defects caused by middle cerebral artery occlusion in adult male rats through activating Akt/mTOR signaling pathway (39). Therefore, we believe that melatonin can regulate Akt/mTOR signaling pathway, which has also been demonstrated in this study. In this study, we found that high glucose environment inhibited the Akt/mTOR signaling pathway of cortical neurons, reduced the expression of Bcl-2, and over-activated the autophagy level of cortical neurons. Melatonin can activate Akt/mTOR pathway, up-regulate the expression of Bcl-2, inhibit the over-activation of autophagy and show neuroprotective effects. Page 9/17 To sum up, melatonin can reduce the apoptosis of cortical neurons under the high-sugar environment by activating Akt/mTOR pathway and following the down-regulation of autophagy, which has a neuroprotective effect. Funding This work was supported by the Innovation Team Foundation of Gannan Medical University (No. TD201705). Availability of data and materials The data that support the findings of this study are available from the corresponding author upon reasonable request. Ethics approval and consent to participate Animal experimental protocols were approved by the Gannan Medical University Animal Care and Use Committee. All animal experiments were performed following the Guidelines for the Care and Use of Laboratory Animals of China Medical University. Authors' contributions Liangdong Li and Zhihua Huang participated in the design of this study, and they both performed the statistical analysis. Lijiao Xiong and Song Liu carried out the study and collected important background information. Lijiao Xiong drafted the manuscript. Chaoming Liu and Tianting Guo provided assistances for data acquisition, data analysis and statistical analysis. All authors have read and approved the final manuscript. Competing interests The authors declare that they have no competing interests References 1. Vieira LL, de Lima Soares RG, Da Silva Felipe SM, de Moura FC, de Castro Brito GA, Pacheco C, Soares PM (2017) Physiological targets for the treatment of diabetic encephalopathy. Cent Nerv Syst Agents Med Chem 17(1):78–86 1. Vieira LL, de Lima Soares RG, Da Silva Felipe SM, de Moura FC, de Castro Brito GA, Pacheco C, Soares PM (2017) Physiological targets for the treatment of diabetic encephalopathy. Cent Nerv Sys Agents Med Chem 17(1):78–86 p Soares PM (2017) Physiological targets for the treatment of diabetic encephalopathy. Cent Nerv Syst Agents Med Chem 17(1):78–86 2. Zilliox LA, Chadrasekaran K, Kwan JY, Russell JW (2016) Diabetes and cognitive impairment. Curr Diab Rep 16(9):87 2. Zilliox LA, Chadrasekaran K, Kwan JY, Russell JW (2016) Diabetes and cognitive impairment. Curr Diab Rep 16(9):87 3. Nixon RA (2013) The role of autophagy in neurodegenerative disease. Nat Med 19(8):983–997 4 Jiang PD Mizushima N (2014) Autophagy and human diseases Cell Res 24(1SI):69 79 3. Nixon RA (2013) The role of autophagy in neurodegenerative disease. Nat Med 19(8):983–997 Page 10/17 3. Nixon RA (2013) The role of autophagy in neurodegenerative disease. Nat Med 19(8):983–997 4. Jiang PD, Mizushima N (2014) Autophagy and human diseases. Cell Res 24(1SI):69–79 Page 10/17 4. Jiang PD, Mizushima N (2014) Autophagy and human diseases. Cell Res 24(1SI):69–79 4. Jiang PD, Mizushima N (2014) Autophagy and human diseases. Cell Res 24(1SI):69–79 Page 10/17 Page 10/17 5. Ghavami S, Shojaeid S, Yeganeh B, Ande SR, Jangamreddy JR, Mehrpour M, Christoffersson J, Chaabane W, Moghadam AR, Kashani HH, Hashemi M, Owji AA, Los MJ (2014) Autophagy and apoptosis dysfunction in neurodegenerative disorders. Prog Neurobiol 112(1):24–49 6. Wang W, Wang Q, Wan D, Sun Y, Wang L, Chen H, Liu C, Petersen RB, Li J, Xue W, Zheng L, Huang K (2017) Histone HIST1H1C/H1.2 regulates autophagy in the development of diabetic retinopathy. Autophage 13(5):941–954 7. Ma L, Lv Y, Huo K, Liu J, Shang S, Fei Y, Li Y, Zhao B, Wei M, Deng Y, Qu Q (2017) Autophagy- lysosome dysfunction is involved in A beta deposition in STZ-induced diabetic rats. Behav Brain Res 320:484–493 8. Kim HA, Lee KH, Lee BH (2014) Neuroprotective effect of melatonin against kainic acid-induced oxidative injury in hippocampal slice culture of rats. Int J Mol Sci 15(4):5940–5951 9. References Dehdashtian E, Mehrzadi S, Yousefi B, Hosseinzadeh A, Reiter RJ, Safa M, Ghaznavi H, Naseripour M (2018) Diabetic retinopathy pathogenesis and the ameliorating effects of melatonin; involvement of autophagy, inflammation and oxidative stress. Life Sci 193:20–33 10. Roohbakhsh A, Shamsizadeh A, Hayes AW, Reiter RJ, Karimi G (2018) Melatonin as an endogenous regulator of diseases: The role of autophagy. Pharmacol Res 133:265–276 11. Ganie SA, Dar TA, Bhat AH, Dar KB, Anees S, Zargar MA, Masood A (2016) Melatonin: A potential anti-oxidant therapeutic agent for mitochondrial dysfunctions and related disorders. Rejuvenation Res 19(1):21–40 12. Paradies G, Paradies V, Ruggiero FM, Petrosillo G (2015) Protective role of melatonin in mitochondrial dysfunction and related disorders. Arch Toxicol 89(6):923–939 12. Paradies G, Paradies V, Ruggiero FM, Petrosillo G (2015) Protective role of melatonin in mitochondrial dysfunction and related disorders. Arch Toxicol 89(6):923–939 13. Chen WR, Liu HB, Chen YD, Sha Y, Ma Q, Zhu PJ, Mu Y (2018) Melatonin Attenuates Myocardial Ischemia/Reperfusion Injury by Inhibiting Autophagy Via an AMPK/mTOR Signaling Pathway. Cell Physiol Biochem 47(5):2067–2076 14. Tuzcu M, Baydas G (2006) Effect of melatonin and vitamin E on diabetes-induced learning and memory impairment in rats. Eur J Pharmacol 537(1–3):106–110 14. Tuzcu M, Baydas G (2006) Effect of melatonin and vitamin E on diabetes-induced learning and memory impairment in rats. Eur J Pharmacol 537(1–3):106–110 15. Olajide OJ, Asogwa NT, Moses BO, Oyegbola CB (2017) Multidirectional inhibition of cortico- hippocampal neurodegeneration by kolaviron treatment in rats. Metab Brain Dis 32(4):1147–1161 15. Olajide OJ, Asogwa NT, Moses BO, Oyegbola CB (2017) Multidirectional inhibition of cortico- hippocampal neurodegeneration by kolaviron treatment in rats. Metab Brain Dis 32(4):1147–1161 16. Negi G, Kumar A, Kaundal RK, Gulati A, Sharma SS (2010) Functional and biochemical evidence indicating beneficial effect of Melatonin and Nicotinamide alone and in combination in experimental diabetic neuropathy. Neuropharmacology 58(3):585–592 16. Negi G, Kumar A, Kaundal RK, Gulati A, Sharma SS (2010) Functional and biochemical evidence indicating beneficial effect of Melatonin and Nicotinamide alone and in combination in experimental diabetic neuropathy. Neuropharmacology 58(3):585–592 17. Buendia I, Parada E, Navarro E, Leon R, Negredo P, Egea J, Lopez MG (2016) Subthreshold concentrations of melatonin and galantamine improves pathological AD-Hallmarks in hippocampal organotypic cultures. Mol Neurobiol 53(5):3338–3348 17. Buendia I, Parada E, Navarro E, Leon R, Negredo P, Egea J, Lopez MG (2016) Subthreshold concentrations of melatonin and galantamine improves pathological AD-Hallmarks in hippocampal organotypic cultures. References Mol Neurobiol 53(5):3338–3348 18. Gao Y, Bai C, Zheng D, Li C, Zhang W, Li M, Guan W, Ma Y (2016) Combination of melatonin and Wnt- 4 promotes neural cell differentiation in bovine amniotic epithelial cells and recovery from spinal cord injury. J Pineal Res 60(3):303–312 18. Gao Y, Bai C, Zheng D, Li C, Zhang W, Li M, Guan W, Ma Y (2016) Combination of melatonin and Wnt- 4 promotes neural cell differentiation in bovine amniotic epithelial cells and recovery from spinal cord injury. J Pineal Res 60(3):303–312 Page 11/17 19. Parada E, Buendia I, Leon R, Negredo P, Romero A, Cuadrado A, Lopez MG, Egea J (2014) Neuroprotective effect of melatonin against ischemia is partially mediated by alpha-7 nicotinic receptor modulation and HO-1 overexpression. J Pineal Res 56(2):204–212 20. Geijselaers SL, Sep SJ, Stehouwer CD, Biessels GJ (2015) Glucose regulation, cognition, and brain MRI in type 2 diabetes: a systematic review. Lancet Diabetes Endocrinol 3(1):75–89 20. Geijselaers SL, Sep SJ, Stehouwer CD, Biessels GJ (2015) Glucose regulation, cognition, and brain MRI in type 2 diabetes: a systematic review. Lancet Diabetes Endocrinol 3(1):75–89 21. Qi WW, Zhong LY, Li XR, Li G, Liu ZX, Hu JF, Chen NH. Hyperglycemia induces the variations of 11beta-hydroxysteroid dehydrogenase type 1 and peroxisome proliferator-activated receptor-gamma expression in hippocampus and hypothalamus of diabetic rats. Exp Diabetes Res, 2012, 2012: 107130 22. Wu J, Zhou SL, Pi LH, Shi XJ, Ma LR, Chen Z, Qu ML, Li X, Nie SD, Liao DF, Pei JJ, Wang S (2017) High glucose induces formation of tau hyperphosphorylation via Cav-1-mTOR pathway: A potential molecular mechanism for diabetes-induced cognitive dysfunction. Oncotarget 8(25):40843–40856 23. Strappazzon F, Vietri-Rudan M, Campello S, Nazio F, Florenzano F, Fimia GM, Piacentini M, Levine B, Cecconi F (2011) Mitochondrial BCL-2 inhibits AMBRA1-induced autophagy. EMBO J 30(7):1195– 1208 24. Tooze SA, Codogno P (2011) Compartmentalized regulation of autophagy regulators: fine-tuning AMBRA1 by Bcl-2. EMBO J 30(7):1185–1186 24. Tooze SA, Codogno P (2011) Compartmentalized regulation of autophagy regulators: fine-tuning AMBRA1 by Bcl-2. EMBO J 30(7):1185–1186 25. Yang M, Yang XM, Yin DH, Tang QL, Wang L, Huang C, Li P, Li SS (2018) Beclin1 enhances cisplatin- induced apoptosis via Bcl-2-modulated autophagy in laryngeal carcinoma cells Hep-2. Neoplasma 65(1):42–48 26. References Wang J, Mei Z, Fu Y, Yang S, Zhang S, Huang W, Xiong L, Zhou H, Tao W, Feng Z (2018) Puerarin protects rat brain against ischemia/reperfusion injury by suppressing autophagy via the AMPK- mTOR-ULK1 signaling pathway. Neural Regen Res 13(6):989–998 27. Kong F, Ma L, Guo J, Xu L, Li Y, Qu S (2018) Endoplasmic reticulum stress/autophagy pathway is involved in diabetes-induced neuronal apoptosis and cognitive decline in mice. Clin Sci 132(1):111– 125 28. Li X, Hue Q, Chen L, Liu S, Su S, Tao H, Zhang L, Sun T, He L (2017) HSPB8 Promotes the Fusion of Autophagosome and Lysosome during Autophagy in Diabetic Neurons. Int J Med Sci 14(13):1335– 1341 29. Huang X, Zhang W, Yuan Y, Liu X, Yang C, Li Q, Zeng J, Zhou K, Liang Y (2017) Implication of autophagy and apoptosis in spiral ganglion cells and cochlear nucleus nuerons in diabetes-induced hearing impairment in rats. Biomed Res-India 28(5):2164–2169 29. Huang X, Zhang W, Yuan Y, Liu X, Yang C, Li Q, Zeng J, Zhou K, Liang Y (2017) Implication of autophagy and apoptosis in spiral ganglion cells and cochlear nucleus nuerons in diabetes-induced hearing impairment in rats. Biomed Res-India 28(5):2164–2169 30. Liu S, Chen L, Li X, Hu Q, He L (2018) Lycium barbarum polysaccharide protects diabetic peripheral neuropathy by enhancing autophagy via mTOR/p70S6K inhibition in Streptozotocin-induced diabetic rats. J Chem Neuroanat 89:37–42 30. Liu S, Chen L, Li X, Hu Q, He L (2018) Lycium barbarum polysaccharide protects diabetic peripheral neuropathy by enhancing autophagy via mTOR/p70S6K inhibition in Streptozotocin-induced diabetic rats. J Chem Neuroanat 89:37–42 31. Guan Z, Tao Y, Zhang X, Guo Q, Liu Y, Zhang Y, Wang Y, Ji G, Wu G, Wang N, Yang H, Yu Z, Guo J, Zhou H (2017) G-CSF and cognitive dysfunction in elderly diabetic mice with cerebral small vessel 31. Guan Z, Tao Y, Zhang X, Guo Q, Liu Y, Zhang Y, Wang Y, Ji G, Wu G, Wang N, Yang H, Yu Z, Guo J, Zhou H (2017) G-CSF and cognitive dysfunction in elderly diabetic mice with cerebral small vessel Page 12/17 Page 12/17 disease: Preventive intervention effects and underlying mechanisms[J]. CNS Neurosci Ther 23(6):462–474 disease: Preventive intervention effects and underlying mechanisms[J]. CNS Neurosci Ther 23(6):462–474 32. Areti A, Komirishetty P, Akuthota M, Malik RA, Kumar A. Melatonin prevents mitochondrial dysfunction and promotes neuroprotection by inducing autophagy during oxaliplatin-evoked peripheral neuropathy. References J Pineal Res, 2017, 62(3) 33. Zhang F, Wang J, Chu J, Yang C, Xiao H, Zhao C, Sun Z, Gao X, Chen G, Han Z, Zou W, Liu T (2015) MicroRNA-146a induced by hypoxia promotes chondrocyte autophagy through Bcl-2. Cell Physiol Biochem 37(4):1442–1453 34. Ryskalin L, Limanaqi F, Frati A, Busceti CL, Fornai F. mTOR-related brain dysfunctions in neuropsychiatric disorders. Int J Mol Sci, 2018, 19(8) 35. Kim YC, Guan K (2015) mTOR: a pharmacologic target for autophagy regulation. mTOR: a pharmacologic target for autophagy regulation. J Clin Invest 125(1):25–32 36. Pan Y, Wang N, Xia P, Wang E, Guo Q, Ye Z (2018) Inhibition of Rac1 ameliorates neuronal oxidative stress damage via reducing Bcl-2/Rac1 complex formation in mitochondria through PI3K/Akt/mTOR pathway. Exp Neurol 300:149–166 37. Mao L, Jia J, Zhou X, Xiao Y, Wang Y, Mao X, Zhen X, Guan Y, Alkayed NJ, Cheng J (2013) Delayed administration of a PTEN inhibitor BPV improves functional recovery after experimental stroke. Neuroscience 231:272–281 38. Kilic U, Caglayan AB, Beker MC, Gunal MY, Caglayan B, Yalcin E, Kelestemur T, Gundogdu RZ, Yulug B Yilmaz B, Kerman BE, Kilic E. Particular phosphorylation of PI3K/Akt on Thr308 via PDK-1 and PTEN mediates melatonin's neuroprotective activity after focal cerebral ischemia in mice. Redox Bio, 12: 657–665 39. Koh P (2008) Melatonin prevents ischemic brain injury through activation of the mTOR/p70S6 kinase signaling pathway. Neurosci Lett 444(1):74–78 Figures Figures Page 13/17 Page 13/17 Figure 1 Effects of melatonin on the cell viability and apoptosis of cortical neurons in high glucose environment. (A) Flow chart of the experiment. The primary fetal cortex neurons were cultured and were mature after 6 days. Melatonin was added for pre-administering for half an hour, and then glucose was added for treatment for 48 h. After that, neurons were collected for experimental detection on the eighth day. (B) Cell viability from the six different groups, Control, 0.1μΜ MLT, 0.3μΜ MLT, 1 μΜ MLT, 3 μΜ MLT, 10 μΜ MLT, were expressed as mean ± SEM. (C) Cell viability from the six different groups, Control, Glucose, Glucose+0.1μΜ MLT, Glucose +0.3μΜ MLT, Glucose+1 μΜ MLT, Glucose+3 μΜ MLT and Glucose+10 μΜ MLT, were expressed as mean ± SEM. (D, E) The percentage of surviving and apoptotic from the different five groups, Control, 0.1 μΜ MLT, Mannitol, Glucose and Glucose+0.1 μΜ MLT, were showed also as mean ± SEM. (F) The data of Annexin V/PI double staining collected by flow cytometry were showed as scatter plot. Results demonstrate that MLT protect the primary cultured neurons from subsequent high glucose toxicity injury. (P<0.05, P<0.01, *P<0.001 vs. Control; #P< 0.05, ##P < 0.01, ###P< 0.001 vs. Glucose) Figure 1 Effects of melatonin on the cell viability and apoptosis of cortical neurons in high glucose environment Figure 1 Figure 1 Effects of melatonin on the cell viability and apoptosis of cortical neurons in high glucose environment. (A) Flow chart of the experiment. The primary fetal cortex neurons were cultured and were mature after 6 days. Melatonin was added for pre-administering for half an hour, and then glucose was added for treatment for 48 h. After that, neurons were collected for experimental detection on the eighth day. (B) Cell viability from the six different groups, Control, 0.1μΜ MLT, 0.3μΜ MLT, 1 μΜ MLT, 3 μΜ MLT, 10 μΜ MLT, were expressed as mean ± SEM. (C) Cell viability from the six different groups, Control, Glucose, Glucose+0.1μΜ MLT, Glucose +0.3μΜ MLT, Glucose+1 μΜ MLT, Glucose+3 μΜ MLT and Glucose+10 μΜ MLT, were expressed as mean ± SEM. (D, E) The percentage of surviving and apoptotic from the different five groups, Control, 0.1 μΜ MLT, Mannitol, Glucose and Glucose+0.1 μΜ MLT, were showed also as mean ± SEM. (F) The data of Annexin V/PI double staining collected by flow cytometry were showed as scatter plot. Results demonstrate that MLT protect the primary cultured neurons from subsequent high glucose toxicity injury. (P<0.05, P<0.01, P<0.001 vs. Control; #P< 0.05, ##P < 0.01, ###P< 0.001 vs. Glucose) ( ) Glucose+0.1μΜ MLT, Glucose +0.3μΜ MLT, Glucose+1 μΜ MLT, Glucose+3 μΜ MLT and Glucose+10 μΜ MLT, were expressed as mean ± SEM. (D, E) The percentage of surviving and apoptotic from the different five groups, Control, 0.1 μΜ MLT, Mannitol, Glucose and Glucose+0.1 μΜ MLT, were showed also as mean ± SEM. (F) The data of Annexin V/PI double staining collected by flow cytometry were showed as scatter plot. Results demonstrate that MLT protect the primary cultured neurons from subsequent high glucose toxicity injury. (P<0.05, P<0.01, P<0.001 vs. Control; #P< 0.05, ##P < 0.01, ###P< 0.001 vs. Glucose) Page 14/17 Figure 2 Figure 2 Effects of melatonin on the apoptosis-related proteins expression of Bcl-2, Bax and Caspase-3 on cortical neurons in high-glucose environment. The neuronal cells were cultured in 60 mm dish (4.5×106/well) for Western blot assay, and were cultured on glass sheet in 24 well plates (1×105 /well) for immunofluorescence test. (A-C) Bcl-2, Bax and Caspase-3 localization were showed as fluorescent photo. (D-G) Bcl-2, Bax and Caspase-3 protein level (A) from the different groups, control, 0.1 μΜ MLT, Mannitol, Glucose and Glucose+0.1 μΜ MLT, were expressed as mean ± SEM. Figure 1 Results demonstrate that MLT increases Bcl-2, decreases Bax and Caspase-3 proteins expression in primary cortical neurons injured by high glucose. (P<0.05, P<0.01, P<0.001 vs. Control; #P< 0.05, ##P < 0.01, ###P< 0.001 vs. Glucose) Figure 2 Effects of melatonin on the apoptosis-related proteins expression of Bcl-2, Bax and Caspase-3 on cortical neurons in high-glucose environment. The neuronal cells were cultured in 60 mm dish (4.5×106/well) for Western blot assay, and were cultured on glass sheet in 24 well plates (1×105 /well) for immunofluorescence test. (A-C) Bcl-2, Bax and Caspase-3 localization were showed as fluorescent photo. (D-G) Bcl-2, Bax and Caspase-3 protein level (A) from the different groups, control, 0.1 μΜ MLT, Mannitol, Glucose and Glucose+0.1 μΜ MLT, were expressed as mean ± SEM. Results demonstrate that MLT increases Bcl-2, decreases Bax and Caspase-3 proteins expression in primary cortical neurons injured by high glucose. (P<0.05, P<0.01, P<0.001 vs. Control; #P< 0.05, ##P < 0.01, ###P< 0.001 vs. Glucose) Page 15/17 Figure 3 Effects of melatonin on the autophagy related proteins expression of Beclin-1 a neurons in high-glucose environment. (A-B) Beclin-1 and LC3B localization were photo. (C-F) Beclin-1, p-Beclin-1 and LC3B protein level from the different groups Mannitol, Glucose and Glucose+0.1 μΜ MLT, were expressed as mean ± SEM. R Figure 3 Effects of melatonin on the autophagy related proteins expression of Beclin-1 and LC3B on cortical neurons in high-glucose environment. (A-B) Beclin-1 and LC3B localization were showed as fluorescent photo. (C-F) Beclin-1, p-Beclin-1 and LC3B protein level from the different groups, control, 0.1 μΜ MLT, Mannitol, Glucose and Glucose+0.1 μΜ MLT, were expressed as mean ± SEM. Results demonstrate that Figure 3 Figure 3 Effects of melatonin on the autophagy related proteins expression of Beclin-1 and LC3B on cortical neurons in high-glucose environment. (A-B) Beclin-1 and LC3B localization were showed as fluorescent photo. (C-F) Beclin-1, p-Beclin-1 and LC3B protein level from the different groups, control, 0.1 μΜ MLT, Mannitol, Glucose and Glucose+0.1 μΜ MLT, were expressed as mean ± SEM. Results demonstrate that Page 16/17 Page 16/17 MLT decreases Beclin-1, p-Beclin-1 and LC3B proteins expression in primary cortical neurons injured by high glucose. (P<0.05, P<0.01, P<0.001 vs. Control; #P< 0.05, ##P < 0.01, ###P< 0.001 vs. Glucose) Figure 4 Effects of melatonin on the phosphorylation of AKT/mTOR protein on cortical neurons in high-glucose environment by Western Blot. (A-E) Akt, p-AKT, p-mTOR and mTOR protein level from the different groups, control, 0.1 μΜ MLT, Mannitol, Glucose and Glucose+0.1 μΜ MLT, were expressed as mean ± SEM. Results demonstrate that MLT increases Akt, p-AKT, p-mTOR and mTOR proteins expression in primary cortical neurons injured by high glucose (P<0 05 P<0 01 P<0 001 vs Control; #P< 0 05 ##P < 0 01 Figure 4 Effects of melatonin on the phosphorylation of AKT/mTOR protein on cortical neurons in high-glucose environment by Western Blot. (A-E) Akt, p-AKT, p-mTOR and mTOR protein level from the different groups, control, 0.1 μΜ MLT, Mannitol, Glucose and Glucose+0.1 μΜ MLT, were expressed as mean ± SEM. Results demonstrate that MLT increases Akt, p-AKT, p-mTOR and mTOR proteins expression in primary cortical neurons injured by high glucose. (P<0.05, P<0.01, *P<0.001 vs. Control; #P< 0.05, ##P < 0.01, ###P< 0.001 vs. Glucose) Page 17/17 Page 17/17
https://openalex.org/W2201044037	https://europepmc.org/articles/pmc4696739?pdf=render	English	null	Correlative Analysis of miRNA Expression and Oncotype Dx Recurrence Score in Estrogen Receptor Positive Breast Carcinomas	PloS one	2,015	cc-by	6,606	Rajyasree Emmadi1, Emanuele Canestrari2, Zarema H. Arbieva3, Wenbo Mu4, Yang Dai4, Jonna Frasor5, Elizabeth Wiley1 1 Department of Pathology, University of Illinois at Chicago, Chicago, Illinois, United States of America, 2 Center for Pharmaceutical Biotechnology, University of Illinois at Chicago, Chicago, Illinois, United States of America, 3 Core Genomics Facility, University of Illinois at Chicago, Chicago, Illinois, United States of America, 4 Department of Bioengineering, University of Illinois at Chicago, Chicago, Illinois, United States of America, 5 Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, Illinois, United States of America emmadi@uic.edu * emmadi@uic.edu RESEARCH ARTICLE Abstract Altered expression of miRNAs has been observed in many types of cancer, including breast cancer, and shown to contribute to cancer growth, aggressiveness, and response to thera- pies. In this pilot study, we investigated the possible correlation of miRNAs with risk of recur- rence of estrogen receptor positive, lymph node-negative mammary carcinomas as determined by the Oncotype DX1 Breast Cancer assay. To accomplish this, we extracted RNA from a collection of breast carcinomas that had previously been analyzed by Oncotype DX1. Multiple Let-7 family members were negatively correlated with the recurrence score (RS), which is consistent with their tumor suppressor properties. Additional miRNAs were found to positively correlate with RS, including miR-377-5p, miR-633b, miR-548t and miR- 3648. Pathway analysis of putative and validated targets suggests that these miRNAs may have a diverse range of functions that may contribute to tumor recurrence. Taken together, these findings provide evidence that a miRNA expression signature can be developed to aid existing methods to determine the risk of recurrence for women with estrogen receptor positive breast cancers treated with endocrine therapy. Editor: Nancy Lan Guo, West Virginia University, UNITED STATES Received: August 23, 2015 Accepted: December 2, 2015 Published: December 30, 2015 Copyright: © 2015 Emmadi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Received: August 23, 2015 Accepted: December 2, 2015 Published: December 30, 2015 Copyright: © 2015 Emmadi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: © 2015 Emmadi 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: Emmadi R, Canestrari E, Arbieva ZH, Mu W, Dai Y, Frasor J, et al. (2015) Correlative Analysis of miRNA Expression and Oncotype Dx Recurrence Score in Estrogen Receptor Positive Breast Carcinomas. PLoS ONE 10(12): e0145346. doi:10.1371/journal.pone.0145346 Editor: Nancy Lan Guo, West Virginia University, UNITED STATES Correlative Analysis of miRNA Expression and Oncotype Dx Recurrence Score in Estrogen Receptor Positive Breast Carcinomas Rajyasree Emmadi1, Emanuele Canestrari2, Zarema H. Arbieva3, Wenbo Mu4, Yang Dai4, Jonna Frasor5, Elizabeth Wiley1 Introduction Invasive breast tumors are biologically diverse with different rates of progression, treatment response, and outcomes. The current standard of care requires all newly diagnosed cases of invasive breast cancer to be routinely tested by immunohistochemistry (IHC) for the expres- sion of estrogen receptor alpha (ER), progesterone receptor (PR) and the growth factor recep- tor HER2/neu (ERBB2). ER-positive tumors are treated with endocrine therapy, such as tamoxifen or aromatase inhibitors. However, about one third to one half of patients treated with tamoxifen or other endocrine therapy will develop recurrence, in part due to development of acquired endocrine resistance. The differential responsiveness of ER-positive breast cancers Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the National Institutes of Health (R01 CA130932-05 to JF), and University of Illinois at Chicago Chancellor’s Discovery Fund (JF, EW, and YD). Funding: This work was supported by the National Institutes of Health (R01 CA130932-05 to JF), and University of Illinois at Chicago Chancellor’s Funding: This work was supported by the National Institutes of Health (R01 CA130932-05 to JF), and University of Illinois at Chicago Chancellor’s Discovery Fund (JF, EW, and YD). Discovery Fund (JF, EW, and YD). Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. 1 / 12 PLOS ONE \| DOI:10.1371/journal.pone.0145346 December 30, 2015 miRNA Expression and Oncotype Dx Recurrence Score in Breast Carcinomas to endocrine therapy is explained by the heterogeneity of these tumors, which has been clearly demonstrated by genome-wide expression profiling studies. The two sub-classifications of ER- positive tumors, representing biologically distinct tumor types, luminal A and luminal B, have been identified, with the luminal A tumors having a good response to endocrine therapy and luminal B tumors responding poorly [1, 2]. Based on gene expression studies, the Oncotype DX1 assay was developed through the NSABP clinical trials [3]. Since 2007, the Association for Clinical Oncology (ASCO) Guidelines have used Oncotype DX1 testing for treatment stratification of ER-positive, lymph node nega- tive breast carcinomas [4]. Oncotype DX1 is a reverse transcription polymerase chain reaction (RT-PCR) based assay that is performed on RNA isolated from formalin fixed paraffin embed- ded (FFPE) tumor tissue blocks. Introduction Based on the expression of 21 genes in the tumor, a Recur- rence Score (RS) is issued. The 16 cancer genes included in the assay are associated with proliferation (Ki67, STK15, Survivin, Cyclin B1, MYBL2), estrogen action (ER, PR, Bcl-2, SCUBE2), HER2/cERBB2 action (GRB7, HER2), invasion (Stromelysin 3, Cathepsin L2), or other functions (GSTM1, CD68 and BAG1). Five reference genes (beta-actin, GADPH, RPLPO, GUS and TFRC) are also included in the assay. The recurrence score is represented by a num- ber between 0 and 100, which correlates to a specific likelihood of breast cancer recurrence within 10 years of initial diagnosis. The scores are grouped into 3 risk categories (low: RS 17, intermediate: RS = 18–30, and high: RS>31). Oncologists use the RS to tailor the use of chemo- therapies based on the finding that women with high RS benefit from chemotherapy whereas women with low RS do not [5]. Hence, women at low risk for recurrence may avoid the toxicity of chemotherapy and be successfully managed with hormonal therapy alone. Patients with high RS, on the other hand, are at an increased risk of recurrence and may receive one or both types of therapy. Little is known about what distinguishes the intermediate from the high RS groups so they generally receive similar therapies. In fact, in 2006, the National Cancer Institute (NCI) sponsored a prospective randomized clinical trial, the TAILORx trial, involving hor- mone-receptor-positive, HER2-negative, axillary node-negative breast cancers less than 5.0 cm in size, assigned to one of three treatment arms based on their OncotypeDX RS results. The results of this trial have recently been released, showing that patients with tumors that had favorable gene-expression profiles (RS of 0–10) had very low rates of recurrence at 5 years with endocrine therapy alone. Of note, however, is that new cut-offs were used to designate the three subgroups of RS compared to the original OncotypeDX assay. The new RS ranges were 0–10 (low risk), 11–25 (intermediate risk) and 26 (high risk), in contrast to prior ranges of <18, 18–30 and 31, respectively. The overall sample size of 10,253 women was driven by the need to include a sufficient number of patients in the ‘new’ intermediate risk range of RS of 11–25. PLOS ONE \| DOI:10.1371/journal.pone.0145346 December 30, 2015 miRNA Profiling RNA was extracted from each sample using the FFPE RNA Purification Kit, according to the manufacturer’s instructions (Norgen Biotek Corp., Thorold, ON, Canada). The quality of the total RNA was verified by Agilent 2100 Bioanalyzer profile. Total RNA from each sample was labeled with Hy3 and a reference sample, consisting of equal amounts of total RNA combined from every individual sample, was labeled with Hy5 fluorescent label, using the miRCURY LNAmicroRNA Hi-Power Labeling Kit (Exiqon, Denmark) following the procedure described by the manufacturer. The Hy3-labeled samples and a Hy5-labeled reference sample were mixed pair-wise and hybridized to the miRCURY LNA microRNA Array 6th gen (Exiqon, Denmark), which contains capture probes targeting all microRNAs for human, mouse or rat registered in the miRBASE 16.0. The hybridization was performed using a Tecan HS4800 hybridization station (Tecan, Austria). After hybridization the microarray slides were scanned and stored in an ozone free environment (ozone level below 2.0 ppb) in order to prevent poten- tial bleaching of the fluorescent dyes. The miRCURY LNA microRNA Array slides were scanned using the Agilent G2565BA Microarray Scanner System (Agilent Technologies, Inc., USA) and the image analysis was carried out using ImaGene 9 software (Exiqon, Denmark). The quantified signals were background corrected (Normexp with offset value 10) [16] and normalized using the global Lowess regression algorithm. Introduction In addition to gene expression signatures, recent studies have revealed associations between miRNAs and tumor expression of ER, PR and HER2, as well as breast carcinoma subtypes and tumor grades [6, 7]. In addition to these correlative studies, specific miRNAs have been shown to play key roles in hormone-dependent breast cancer biology. For example, down-regulation of miR-451 can promote breast cancer cell survival and endocrine resistance [8], a miR- NA196a2-TP63 circuit controls breast cancer proliferation and invasiveness properties [9], loss of miR-200c promotes breast cancer cell migration and invasion [10], and down-regulation of miR-29 contributes to progestin-induced stem cell expansion [11]. Let-7 family members have been found to inhibit breast cancer stem cell renewal [12] and have been implicated in tamoxi- fen response through modulation of ER levels [13]. Furthermore, miRNAs that are differen- tially expressed in tamoxifen-sensitive vs. resistant cell lines and tumors have been identified [14, 15]. These findings suggest that miRNAs may function as biomarkers of tumor response to therapy and/or risk of recurrence, as well as important drivers of these phenotypically PLOS ONE \| DOI:10.1371/journal.pone.0145346 December 30, 2015 2 / 12 miRNA Expression and Oncotype Dx Recurrence Score in Breast Carcinomas different tumors. In this study we conducted miRNA profiling in a collection of breast tumors with previous testing for recurrence risk by the Oncotype DX1 assay in an attempt to identify additional miRNAs biomarkers of breast tumor recurrence and poor outcome in patients with ER-positive breast cancers. This is a pilot study with a small sample size. However, cases were selected to span the range of the Oncotype DX1 Recurrence Scores. We acknowledge the limi- tations of the study including retrospective analysis and a small sample size. Patient Cohort and Tumor Characteristics This research was a retrospective, non-interventional analysis, conducted following approval from the Institutional Review Boards (IRBs) of the University of Illinois Cancer Center and Provena Saint Joseph Medical Center in Illinois. The research was judged to qualify for waiver of informed consent based on the provisions under HHS regulations at 45 CFR 46.116(d). The research did not involve any risk to patients, data was anonymized and no patient identifiers were included. Formalin-fixed, paraffin-embedded (FFPE) tumor tissues from twenty-three cases of early stage breast carcinomas representing low, intermediate and high Oncotype DX Recurrence Score were obtained. Patient samples and clinical data were collected and processed in compliance with protocols approved by the University of Illinois Cancer Center and Provena Saint Joseph Medical Center Institutional Review Boards. Tumor tissue was macro-dissected by comparison to an adjacent H&E stained section to ensure that tissue used for miRNA analy- sis contained >70% tumor, in accordance with samples sent for Oncotype DX1 testing. Clini- cal data collected on each patient included information on ER, PR, p53, Ki67 and Her2 status for correlation with study results (Table 1). Data Analysis Principal Components Analysis (PCA) was performed using the top 50 microRNAs that had the largest variation across all samples (Fig 1). A heatmap diagram was generated based on the two-way clustering method with the complete-linkage and Euclidean distance measurement 3 / 12 PLOS ONE \| DOI:10.1371/journal.pone.0145346 December 30, 2015 miRNA Expression and Oncotype Dx Recurrence Score in Breast Carcinomas Table 1. Patient and tumor characteristics. ID RS Group Recurrence Risk (%) ER PR p53 Ki67 Her2 Age Race S2 3 Low 4 81 52 13 0 1.7 54 White S53 6 Low 5 30 75 0 6 0 46 White S70 7 Low 6 97 50 9 16 1.1 45 AA S46 8 Low 6 20 9 0 2 0.2 58 White S30 9 Low 6 93 63 0 8 0 62 White S21 11 Low 8 50 48 0 2 0 73 White S11 12 Low 8 66 57 0 7 0.5 60 White S20 12 Low 8 56 18 1 4 0.6 52 White S7 14 Low 9 29 19 10 23 0.9 57 White S23 14 Low 9 87 85 1 8 1 54 White S63 14 Low 9 72 78 0 7 1.4 52 White S33 16 Low 10 90 68 0 13 0.2 65 White S26 26 Int 17 98 97 13 53 1 46 White S29 26 Int 17 1 2 7 3 1.2 70 White S8 27 Int 17 88 42 16 3 1.12 70 White S16 29 Int 20 83 71 36 17 0 75 White S58 31 High 21 37 42 0 9 0.9 50 White S25 32 High 22 29 86 5 14 1.9 53 White S12 38 High 26 95 91 14 42 0 47 Asian S49 39 High 27 58 62 2 21 1.4 55 White S64 40 High 27 100 37 2 63 1.4 59 White S6 47 High 32 29 52 0 33 0.3 43 White S32 56 High 34 23 2 1 31 1.4 48 White doi:10.1371/journal.pone.0145346.t001 Table 1. Patient and tumor characteristics. (Fig 2). The intermediate RS group was included in the high RS group since the number of sam- ples in the former was small. Student’s t-test was performed for differential expression analysis between low RS and high RS groups. Pearson correlation coefficient was used for correlation tests between the expression value of individual microRNAs and RS. All p-values were adjusted (Fig 2). The intermediate RS group was included in the high RS group since the number of sam- ples in the former was small. Student’s t-test was performed for differential expression analysis between low RS and high RS groups. doi:10.1371/journal.pone.0145346.t001 Pearson correlation coefficient was used for correlation tests between the expression value of individual microRNAs and RS. All p-values were adjusted Fig 1. Principal components analysis. Principal components analysis performed on all samples using the top 50 miRNAs showing the highest standard deviation across all samples. doi:10.1371/journal.pone.0145346.g001 Fig 1. Principal components analysis. Principal components analysis performed on all samples using the top 50 miRNAs showing the highest standard deviation across all samples. Fig 1. Principal components analysis. Principal components analysis performed on all samples using the top 50 miRNAs showing the highest standard deviation across all samples. doi:10.1371/journal.pone.0145346.g001 doi:10.1371/journal.pone.0145346.g001 PLOS ONE \| DOI:10.1371/journal.pone.0145346 December 30, 2015 4 / 12 miRNA Expression and Oncotype Dx Recurrence Score in Breast Carcinomas Fig 2. Heat map of unsupervised hierarchical clustering. Hierarchical clustering performed on all samples using the top 50 miRNAs showing the highest standard deviation across all samples. doi:10.1371/journal.pone.0145346.g002 Fig 2. Heat map of unsupervised hierarchical clustering. Hierarchical clustering performed on all samples using the top 50 miRNAs showing the highest standard deviation across all samples. doi:10.1371/journal.pone.0145346.g002 Fig 2. Heat map of unsupervised hierarchical clustering. Hierarchical clustering performed on all samples using the top 50 miRNAs showing the highest standard deviation across all samples. doi:10.1371/journal.pone.0145346.g002 doi:10.1371/journal.pone.0145346.g002 based on the Benjamini-Hochberg procedure. The threshold for the adjusted p-value <0.05 was used to determine the significant correlation (Table 2). The analysis was carried out using pack- ages in R. Predicted and Validated miRNA Targets The miRNA story is an evolving one and the currently available databases are still incomplete. In order to maximize the information for correlative analysis, we looked at multiple miRNA databases. Predicted miRNA targets were obtained using DIANA-microT-CDS [17, 18]. Vali- dated miRNA targets were obtained using miRWalk with a default p-value of 0.05 [19]. The findings of the predicted miRNA targets and pathways are presented in Fig 3 and validated tar- gets and pathways are presented in Table 3 and Fig 4. Pathway Analysis Predicted targets. miRNA targets were predicted based on DIANA-microT-CDS. The combined effect of groups of miRNAs on pathways was determined based on the union of tar- get genes for enrichment analysis using DIANA miRPath v.2.0 (Vlachos). Heatmaps were gen- erated using the log-transformed enrichment p-values as features for each miRNA. [20]. 5 / 12 PLOS ONE \| DOI:10.1371/journal.pone.0145346 December 30, 2015 miRNA Expression and Oncotype Dx Recurrence Score in Breast Carcinomas Table 2. Differential expression of miRNAs. High vs. Low (T-test) Low vs. Int vs. High (ANOVA) Correlation Analysis miRNA Annotation Log FC Adj. P. Val Adj. P. Val Correlation Coefﬁcient Adj. P.Val hsa-let-7c -1.506 5.04E-03 0.115 -0.7503 9.22E-03 hsa-let-7b -1.716 7.35E-03 0.115 -0.7278 1.36E-02 hsa-let-7a -1.348 2.23E-02 0.115 -0.6774 2.71E-02 hsa-miR-126 -1.108 2.64E-02 hsa-miR-193a-5p -0.976 2.95E-02 hsa-let-7d -0.933 3.68E-02 hsa-miR-126 -0.819 3.68E-02 hsa-miR-26b -1.192 3.68E-02 hsa-miR-30c -1.167 3.68E-02 hsa-miR-3648 0.651 3.68E-02 0.7085 1.91E-02 hsa-miR-492 0.539 3.83E-02 hsa-miR-361-5p 0.340 3.93E-02 hsa-miR-4328 -1.014 3.93E-02 hsa-miR-663b 0.631 3.93E-02 0.6837 2.65E-02 hsa-miR-1914 0.332 3.98E-02 hsa-miR-195 -1.261 3.98E-02 hsa-miR-663 0.416 3.98E-02 hsa-miR-29c -1.314 4.01E-02 hsa-miR-377-5p 0.235 4.60E-02 4.23E-02 0.7886 3.87E-03 hsa-miR-143 -1.144 4.64E-02 hsa-miR-26a -1.032 4.64E-02 hsa-miR-1297 -0.795 4.80E-02 hsa-let-7f -0.962 4.98E-02 hsa-let-7f-1* 0.253 4.98E-02 hsa-miR-4301 -0.765 4.98E-02 hsa-miR-548t 0.6886 2.65E-02 doi:10.1371/journal.pone.0145346.t002 Table 2. Differential expression of miRNAs. High vs. Low (T-test) Low vs. Int vs. High (ANOVA) Correlation Analysis miRNA Annotation Log FC Adj. P. Val Adj. P. Val Correlation Coefﬁcient Adj. P.Val hsa-let-7c -1.506 5.04E-03 0.115 -0.7503 9.22E-03 hsa-let-7b -1.716 7.35E-03 0.115 -0.7278 1.36E-02 hsa-let-7a -1.348 2.23E-02 0.115 -0.6774 2.71E-02 hsa-miR-126 -1.108 2.64E-02 hsa-miR-193a-5p -0.976 2.95E-02 hsa-let-7d -0.933 3.68E-02 hsa-miR-126* -0.819 3.68E-02 hsa-miR-26b -1.192 3.68E-02 hsa-miR-30c -1.167 3.68E-02 hsa-miR-3648 0.651 3.68E-02 0.7085 1.91E-02 hsa-miR-492 0.539 3.83E-02 hsa-miR-361-5p 0.340 3.93E-02 hsa-miR-4328 -1.014 3.93E-02 hsa-miR-663b 0.631 3.93E-02 0.6837 2.65E-02 hsa-miR-1914 0.332 3.98E-02 hsa-miR-195 -1.261 3.98E-02 hsa-miR-663 0.416 3.98E-02 hsa-miR-29c -1.314 4.01E-02 hsa-miR-377-5p 0.235 4.60E-02 4.23E-02 0.7886 3.87E-03 hsa-miR-143 -1.144 4.64E-02 hsa-miR-26a -1.032 4.64E-02 hsa-miR-1297 -0.795 4.80E-02 hsa-let-7f -0.962 4.98E-02 hsa-let-7f-1* 0.253 4.98E-02 hsa-miR-4301 -0.765 4.98E-02 hsa-miR-548t 0.6886 2.65E-02 doi:10.1371/journal.pone.0145346.t002 Fig 3. Pathway analysis for putative miRNA target genes. Pathways showing significant enrichment in putative target genes for miRNAs positively correlated with recurrence score (left) or negatively correlated with recurrence score (right) were clustered. doi:10.1371/journal.pone.0145346.g003 PLOS ONE \| DOI:10 1371/journal pone 0145346 December 30 2015 6 / 12 Fig 3. Pathway analysis for putative miRNA target genes. Pathways showing significant enrichment in putative target genes for miRNAs positively correlated with recurrence score (left) or negatively correlated with recurrence score (right) were clustered. doi:10 1371/journal pone 0145346 g003 Fig 3. Pathway analysis for putative miRNA target genes. Pathways showing significant enrichment in putative target genes for miRNAs positively correlated with recurrence score (left) or negatively correlated with recurrence score (right) were clustered Fig 3. Pathway analysis for putative miRNA target genes. doi:10.1371/journal.pone.0145346.t002 PLOS ONE \| DOI:10.1371/journal.pone.0145346 December 30, 2015 Pathways showing significant enrichment in putative target genes for miRNAs positively correlated with recurrence score (left) or negatively correlated with recurrence score (right) were clustered. Fig 3. Pathway analysis for putative miRNA target genes. Pathways showing significant enrichment in putative target genes for miRNAs positively correlated with recurrence score (left) or negatively correlated with recurrence score (right) were clustered. d i 10 1371/j l 0145346 003 doi:10.1371/journal.pone.0145346.g003 PLOS ONE \| DOI:10.1371/journal.pone.0145346 December 30, 2015 6 / 12 miRNA Expression and Oncotype Dx Recurrence Score in Breast Carcinomas Table 3. Validated gene targets for miR-377-5p and miR-663b using miRWalk. miR-377-5p Gene Name Entrez ID Ensmbl ID LAMA1 284217 ENSG00000101680 CHRD 8646 ENSG00000090539 MITF 4286 ENSG00000187098 NOG 9241 ENSG00000183691 CYP7A1 1581 ENSG00000167910 CDKN1A 1026 ENSG00000124762 IFNG 3458 ENSG00000111537 PAK1 5058 ENSG00000149269 IL1B 3553 ENSG00000125538 PPM1A 5494 ENSG00000100614 NOS2A 4843 ENSG00000007171 PKN1 5585 ENSG00000123143 TNF 7124 ENSG00000232810 SOD1 6647 ENSG00000142168 HMOX1 3162 ENSG00000100292 SOD2 6648 ENSG00000112096 DICER1 23405 ENSG00000100697 COMP 1311 ENSG00000105664 ERBB2 2064 ENSG00000141736 GHRHR 2692 ENSG00000106128 miR-663b Gene Name Entrez ID Ensmbl ID EPHB2 2048 ENSG00000133216 LATS2 26524 ENSG00000150457 KRAS 3845 ENSG00000133703 CCND2 894 ENSG00000118971 TXNIP 10628 ENSG00000117289 doi:10.1371/journal.pone.0145346.t003 Table 3. Validated gene targets for miR-377-5p and miR-663b using miRWalk. Validated targets. Validated miRNA targets were available only for miR-377-5p (miR- 377) and miR-663b using miRWalk. No gene targets were elucidated for miR-3648 and miR- 748t. The gene targets for miR-377-5p and miR-663b were combined and pathway enrichment analysis was performed using the Panther classification system v9.0 and Panther pathway, enriched with a default p-value of 0.05 [21, 22]. Results Genome wide profiling of miRNA expression in 23 human breast tumors was carried out using a locked nucleic acid (LNA) based microRNA array that contained ~2800 capture probes com- plementary to mature microRNAs based on miRBase v16 (Exiqon). All tumors were ER-posi- tive, lymph node-negative, invasive mammary carcinomas. Clinical and laboratory data collected for each patient are shown in Table 1 and include the Recurrence Score (RS) based on the Oncotype DX1 assay and the estimated risk of recurrence (R-Rate). The tumors profiled included 12 low, 4 intermediate and 7 high RS scores. Principal components analysis (PCA), which was conducted for the top 50 miRNAs with the largest variation in expression across all samples, revealed that the tumors with high or low RS largely clustered independently of each other but that tumors with intermediate RS were 7 / 12 PLOS ONE \| DOI:10.1371/journal.pone.0145346 December 30, 2015 miRNA Expression and Oncotype Dx Recurrence Score in Breast Carcinomas Fig 4. Pathway analysis for validated miRNA target genes. Pathways showing enrichment in validated targets genes for miR-377-5p and miR-663b. Fig 4. Pathway analysis for validated miRNA target genes. Pathways showing enrichment in validated targets genes for miR-377-5p and miR-663b. doi:10 1371/journal pone 0145346 g004 Fig 4. Pathway analysis for validated miRNA target genes. Pathways showing enrichment in validated targets genes for miR-377-5p and miR-663b. d miRNA target genes. Pathways showing enrichment in validated targets genes for miR-377-5p and miR-663b. Fig 4. Pathway analysis for validated miRNA target genes. Pathways showing enrichment in validated ta doi:10.1371/journal.pone.0145346.g004 more widely distributed (Fig 1). Of the samples with low RS, one (#S7) was more closely related to the high RS samples than other tumors with low RS. Two-way hierarchical clustering by miRNA expression levels confirmed the relationship between samples and recurrence score groups (Fig 2). To identify specific miRNAs that are associated with RS, two types of analyses were per- formed. First, differential expression of miRNAs between the low and high RS groups was car- ried out using Student’s T test followed by Benjamini Hochberg adjustment for multiple tests. Twenty-five miRNAs were found to be statistically different (P<0.05) between the two groups (Table 2). However, when all samples were analyzed as three groups (low, intermediate, and high) using ANOVA and Benjamini Hochberg adjustment, only three of miRNAs remained as significantly different among the groups. PLOS ONE \| DOI:10.1371/journal.pone.0145346 December 30, 2015 miRNA Expression and Oncotype Dx Recurrence Score in Breast Carcinomas Fig 5. Correlation analysis between miRNA expression level and recurrence score. The relationship between miRNA expression and recurrence score is shown for seven miRNAs with a statistically significant correlation. Fig 5. Correlation analysis between miRNA expression level and recurrence score. The relationship between miRNA expression and recurrence score is shown for seven miRNAs with a statistically significant correlation. doi:10.1371/journal.pone.0145346.g005 miRNAs showing negative correlation with RS comprise several members of the Let-7 fam- ily, which have been found to inhibit breast cancer stem cell renewal [12] and have been impli- cated in Tamoxifen response through modulation of ER levels [13]. However, little is known about the other miRNAs (miR-377-5p, miR-663b, miR-548T and miR-3648) that we found to be higher in breast tumors with high RS. To determine pathways that may be controlled by these miRNAs, DIANA miRPath was first used to identify putative targets (see S1 Data for complete results). The union of the puta- tive targets from Let-7a, 7b and 7c (all negatively correlated with RS) and the union of putative targets from miR-663b, miR-377-5p and miR-3648 (all positively correlated with RS) were sep- arately used for pathway enrichment analysis. The miRNAs were then clustered based on the log-transformed enrichment p-values and visualized in heatmaps. As shown in Fig 3, target genes for miRNAs positively correlated with RS are fairly divergent with chemical carcinogene- sis and xenobiotic metabolism associated miR-3648, and glycan degradation and systemic lupus erythematosus with miR-633b. ErbB signaling, on the other hand, was enriched for both miR-377-5p and miR-633b. In contrast, pathways well characterized in breast cancer progres- sion (PI3K/AKT, MAPK, cytokines, and WNT), are highly enriched for all three Let-7 family members showing positive correlation with RS score. Validated gene targets obtained for miR-377-5p and miR-663b using miRWalk (Table 3) were then used for pathway enrichment analysis using PANTHER 9.0 and showed association with multiple pathways, in particular those involving angiogenesis, EGF receptor signaling, inflammation mediated by chemokines and cytokines, integrin signaling, PI3 kinase, TGF-beta signaling and Ras and p53 pathway feedback loops (Fig 4). Results In the second approach, correlation analysis between miRNA expression levels and the actual RS was conducted. Expression levels for seven miR- NAs were significantly correlated with RS (Fig 5), six of which overlapped with other analyses as indicated in Table 2. 8 / 12 PLOS ONE \| DOI:10.1371/journal.pone.0145346 December 30, 2015 Discussion Several publications have looked at the miRNA expression profiles in breast carcinoma cell lines and tumor tissues. Most of the studies have varied representation of the different subtypes of breast carcinoma (luminal, basal-like, Her2-enriched) and with or without lymph node 9 / 12 PLOS ONE \| DOI:10.1371/journal.pone.0145346 December 30, 2015 miRNA Expression and Oncotype Dx Recurrence Score in Breast Carcinomas involvement. As a result, mixed (non subtype-specific) miRNA signatures emerge and have been reported in ranges of 1–21 miRNAs [23–27]. Commonly reported differential expressions include upregulation of miR-21, miR-155, miR-206, miR-122a and miR-210 and downregulation of let-7, miR-10b, miR-125a, miR-125b and miR-145. Putative or validated target gene and path- way analyses demonstrate involvement of oncogenes and tumor suppressors, such as ErbB2, Akt, NF-κB, Myc, p53, Rb and PTEN with consequent proliferation and cell survival effects as well as inhibition of apoptosis. While cell cycle and proliferation signals would be expected to correlate with outcomes, other signatures described are associated with hypoxia, cytokines, stroma and chromosomal instability. In essence it appears that different reported signatures may represent different aspects of ultimate multifactorial mechanisms. Several of the miRNAs associated with RS in ER+ breast tumors have been described to play a role in cancer progression. For example, Let-7 family members, which in this study were found to be down-regulated in tumors with high recurrence score, have been found to inhibit breast cancer stem cell renewal [12] and have been implicated in tamoxifen response through modulation of ER levels [13]. On the other hand, little is known about the other miRNAs that we find to be higher in breast tumors with high RS. The inferences we have made by correlating the results of our analyses and literature are presented. 1. miRNA profiling captures basic biological relationship between high vs low risk of recurrence. More refined risk prediction is needed—both Oncotype DX1 and the Mamma- Print1 assays have a discordance rate between the assay prediction and clinical-pathologic risk category of approximately 30% [28]. 1. miRNA profiling captures basic biological relationship between high vs low risk of recurrence. More refined risk prediction is needed—both Oncotype DX1 and the Mamma- Print1 assays have a discordance rate between the assay prediction and clinical-pathologic risk category of approximately 30% [28]. 1. miRNA profiling captures basic biological relationship between high vs low risk of recurrence. PLOS ONE \| DOI:10.1371/journal.pone.0145346 December 30, 2015 Discussion More refined risk prediction is needed—both Oncotype DX1 and the Mamma- Print1 assays have a discordance rate between the assay prediction and clinical-pathologic risk category of approximately 30% [28]. 2. The intermediate group does not represent a unique group biologically but appears to be distributed along a continuum of high to low. This is similar to findings reported previ- ously by Ivshina et al, who found 6 markers that could effectively separate grade 1 and grade 3 tumors and could also separate grade 2 tumors into two highly discriminate classes [29]. 2. The intermediate group does not represent a unique group biologically but appears to be distributed along a continuum of high to low. This is similar to findings reported previ- ously by Ivshina et al, who found 6 markers that could effectively separate grade 1 and grade 3 tumors and could also separate grade 2 tumors into two highly discriminate classes [29]. 3. Suppression of the Let-7 family is associated with increased RS score. Of particular inter- est is the reduced expression of Let-7 family members in cases with high RS. Six family members were found to be significantly associated with low risk of recurrence using at least one statistical analysis. The hsa-let-7 family is well established as a tumor suppressor with interactions with cell cycle proliferative genes including Aurora Kinase A (AURKA), which it downregulates [30]. A recent meta-analysis shows a significant correlation between increased AURKA expression and decreased metastasis-free survival in ER-positive breast cancers [31]. The hsa-let-7 family involvement in breast carcinoma is well documented and serves to confirm the validity of our findings. 3. Suppression of the Let-7 family is associated with increased RS score. Of particular inter- est is the reduced expression of Let-7 family members in cases with high RS. Six family members were found to be significantly associated with low risk of recurrence using at least one statistical analysis. The hsa-let-7 family is well established as a tumor suppressor with interactions with cell cycle proliferative genes including Aurora Kinase A (AURKA), which it downregulates [30]. A recent meta-analysis shows a significant correlation between increased AURKA expression and decreased metastasis-free survival in ER-positive breast cancers [31]. The hsa-let-7 family involvement in breast carcinoma is well documented and serves to confirm the validity of our findings. 4. Novel miRNAs involved in breast carcinoma. Acknowledgments This research was supported by the University of Illinois at Chicago through the Chancellor’s Discovery Fund (JF, EW, YD) and the National Institute of Health (R01 CA130932-05 to JF). The research was conducted in collaboration with Provena St. Joseph Medical Center after This research was supported by the University of Illinois at Chicago through the Chancellor’s Discovery Fund (JF, EW, YD) and the National Institute of Health (R01 CA130932-05 to JF). The research was conducted in collaboration with Provena St. Joseph Medical Center after obtaining IRBs from each institution. We thank Dr. J. Natarelli (Provena St. Joseph Medical center, Pathology) for reviewing the cases and selecting tumor tissue blocks for analysis. Author Contributions Conceived and designed the experiments: RE JF YD. Performed the experiments: RE EC ZA WM YD. Analyzed the data: RE JF ZA WM YD. Contributed reagents/materials/analysis tools: RE JF ZA YD W t th RE JF EW YD y J g / / y RE JF ZA YD. Wrote the paper: RE JF EW YD. RE JF ZA YD. Wrote the paper: RE JF EW YD. Discussion The three miRNAs (hsa-miR-377-5p, hsa- miR-663b and hsa-miR-3648) that are positively correlated with high Oncotype DX1 RS scores have not been previously detailed in association with breast carcinoma outcomes. Very little specific data exists regarding the interactions and roles of these miRNAs with regard to ER-positive breast carcinoma. 4. Novel miRNAs involved in breast carcinoma. The three miRNAs (hsa-miR-377-5p, hsa- miR-663b and hsa-miR-3648) that are positively correlated with high Oncotype DX1 RS scores have not been previously detailed in association with breast carcinoma outcomes. Very little specific data exists regarding the interactions and roles of these miRNAs with regard to ER-positive breast carcinoma. Information available via the tools listed suggest that multiple pathways are involved by miR-377-5p and miR-663b, affecting tumor suppressor functions (LATS2, TXNIP, wtKRAS, CDKN1A, PPM1A), cytoskeleton regulation (PAK1, PKN1) and a proinflammatory and reac- tive oxygen species adaptive response (TNF, IFNG, IL1B, HMOX1, SOD1 and SOD2). We infer that multiple factors are involved in separating those tumors that have a high RS score includ- ing specific proliferation advantages as well as suppression of tumor response mechanisms. While these genes and pathways may represent possible therapeutic targets for tumor sup- pression, these miRNAs also are of particular interest as they may provide novel approaches 10 / 12 PLOS ONE \| DOI:10.1371/journal.pone.0145346 December 30, 2015 miRNA Expression and Oncotype Dx Recurrence Score in Breast Carcinomas for specific, targeted, less toxic systemic treatment protocols in the future. Progress in micro- RNA-directed therapeutic approaches offers hope that such strategies might prove useful in reversing endocrine resistance and reducing breast cancer recurrence. for specific, targeted, less toxic systemic treatment protocols in the future. Progress in micro- RNA-directed therapeutic approaches offers hope that such strategies might prove useful in reversing endocrine resistance and reducing breast cancer recurrence. PLOS ONE \| DOI:10.1371/journal.pone.0145346 December 30, 2015 References Cittelly DM, Finlay-Schultz J, Howe EN, Spoelstra NS, Axlund SD, Hendricks P, et al. Progestin sup- pression of miR-29 potentiates dedifferentiation of breast cancer cells via KLF4. Oncogene. 2013 May 16; 32(20):2555–64 doi: 10.1038/onc.2012.275 PMID: 22751119 12. Yu F, Yao H, Zhu P, Zhang X, Pan Q, Gong C, et al. let-7 Regulates Self Renewal and Tumorigenicity of Breast Cancer Cells. Cell 2007; 131(6):1109–1123 PMID: 18083101 13. Zhao Y, Deng C, Lu W, Xiao J, Ma D, Guo M, et al. let-7 MicroRNAs Induce Tamoxifen Sensitivity by Downregulation of Estrogen Receptor α Signaling in Breast Cancer. Mol Med. 2011; 17(11–12)1233– 1241 doi: 10.2119/molmed.2010.00225 PMID: 21826373 14. Miller TE, Ghoshal K, Ramaswamy B, Roy S, Datta J, Shapiro CL, et al. MicroRNA-221/222 confers tamoxifen resistance in breast cancer by targeting p27Kip1. J Biol Chem. 2008 Oct 31; 283 (44):29897–903. doi: 10.1074/jbc.M804612200 PMID: 18708351 15. Manavalan TT, Teng Y, Appana SN, Datta S, Kalbfleisch TS, Li Y, et al. Differential expression of micro- RNA expression in tamoxifen-sensitive MCF-7 versus tamoxifen-resistant LY2 human breast cancer cells. Cancer Letters, 2011; 313(1):26–43. doi: 10.1016/j.canlet.2011.08.018 PMID: 21955614 16. Ritchie ME, Silver J, Oshlack A, Holmes M, Diyagama D, Holloway A, et al. A comparison of back- ground correction methods for two-colour microarrays. Bioinformatics. 2007; 23(20):2700–2707. PMID: 17720982 17. Paraskevopoulou MD, Georgakilas G, Kostoulas N, Vlachos IS, Vergoulis T, Reczko M, at al. DIANA- microT web server v5.0: service integration into miRNA functional analysis workflows. Nucleic Acids Res. 2013 Jul; 41(Web Server issue):W169–73. doi: 10.1093/nar/gkt393 PMID: 23680784 18. Reczko M, Maragkakis M, Alexiou P, Grosse I, Hatzigeorgiou AG. Functional microRNA targets in pro- tein coding sequences. Bioinformatics. 2012; 28(6):771–776. doi: 10.1093/bioinformatics/bts043 PMID: 22285563 19. Dweep H, Sticht C, Pandey P, Gretz N. miRWalk—database: prediction of possible miRNA binding sites by "walking" the genes of 3 genomes, Journal of Biomedical Informatics. 2011; 44: 839–7. doi: 10.1016/j.jbi.2011.05.002 PMID: 21605702 20. Vlachos IS, Kostoulas N, Vergoulis T, Georgakilas G, Reczko M, Maragkakis M, et al. DIANA miRPath v.2.0: investigating the combinatorial effect of microRNAs in pathways. Nucleic Acids Research 2012 Jul; 40(Web server issue): W498–504. doi: 10.1093/nar/gks494 PMID: 22649059 21. Mi H, Muruganujan A, Thomas PD. PANTHER in 2013: modeling the evolution of gene function, and other gene attributes, in the context of phylogenetic trees. Nucl. Acids Res. 2013; 41(D1):D377–D386. 22. Mi H, Thomas PD. Protein Networks and Pathway Analysis. Methods in Molecular biology, 2009; 563 Part 2: 123–140. 23. References 1. Voduc KD, Cheang M, Tyldesley S, Gelmon K, Nielsen TO, Kennecke H. Breast Cancer Subtypes and the Risk of Local and Regional Relapse. J Clin Oncol. 2010; 28:1684–1691. doi: 10.1200/JCO.2009. 24.9284 PMID: 20194857 2. Goldhirsch A, Winer EP, Coates AS, Gelber RD, Piccart-Gebhart M, Thürliman B, et al. Panel mem- bers. Personalizing the treatment of women with early breast cancer: highlights of the St Gallen Interna- tional Expert Consensus on the Primary Therapy of Early Breast Cancer 2013. Annals of Oncology. 2013; 24: 2206–2223 doi: 10.1093/annonc/mdt303 PMID: 23917950 3. Paik S, Shak S, Tang G, Kim C, Baker J, Cronin M, et al. A multigene assay to predict recurrence of tamoxifen-treated, node-negative breast cancer. N Engl J Med. 2004; 351:2817–2826 PMID: 15591335 4. Harris L, Fritsche H, Mennel R, Norton L, Ravdin P, Taube S, et al. American Society of Clinical Oncol- ogy 2007 update of recommendations for the use of tumor markers in breast cancer. J Clin Oncol. 2007; 25:5287–5312 PMID: 17954709 5. Paik S, Tang G, Shak S, Kim C, Baker J, Kim W, et al. Gene expression and benefit of chemotherapy in women with node-negative, estrogen receptor-positive breast cancer. J Clin Oncol. 2006; 24:3726– 3734 PMID: 16720680 6. Lowery AJ, Miller N, Devaney A, McNeill RE, Davoren PA, Lemetre C, et al. MicroRNA signatures pre- dict oestrogen receptor, progesterone receptor and HER2/neu receptor status in breast cancer. Breast Cancer Res. 2009; 11(3):R27 doi: 10.1186/bcr2257 PMID: 19432961 7. Blenkiron C, Goldstein LD, Thorne NP, Spiteri I, Chin S- F, Dunning MJ, et al. MicroRNA expression profiling of human breast cancer identifies new markers of tumor subtype. Genome Biology. 2007; 8: R214 PMID: 17922911 8. Bergamaschi A, Katzenellenbogen BS. Tamoxifen downregulation of miR-451 increases 14-3-3zeta and promotes breast cancer cell survival and endocrine resistance. Oncogene. 2012; 31:39–47 doi: 10.1038/onc.2011.223 PMID: 21666713 9. Kim K, Madak-Erdogan Z, Ventrella R, Katzenellenbogen BS. A MicroRNA196a2* and TP63 Circuit Regulated by Estrogen Receptor-alpha and ERK2 that Controls Breast Cancer Proliferation and Inva- siveness Properties. Horm Cancer 2013 Apr; 4(2):78–91 doi: 10.1007/s12672-012-0129-3 PMID: 23250869 10. Cochrane DR, Spoelstra NS, Howe EN, Nordeen SK, Richer JK 2. MicroRNA-200c mitigates invasive- ness and restores sensitivity to microtubule-targeting chemotherapeutic agents. Mol Cancer Ther. 2009; 8:1055–1066 doi: 10.1158/1535-7163.MCT-08-1046 PMID: 19435871 11 / 12 PLOS ONE \| DOI:10.1371/journal.pone.0145346 December 30, 2015 miRNA Expression and Oncotype Dx Recurrence Score in Breast Carcinomas 11. PLOS ONE \| DOI:10.1371/journal.pone.0145346 December 30, 2015 References Wang V and Wu W. MicroRNA: A New Player in Breast Cancer Development. Journal of Cancer Mole- cules. 2007; 3(5): 133–138. 24. Lu J, Getz G, Miska EA, Alvarez-Saavedra E, Lamb J, Peck D, et al. MicroRNA expression profiles classify human cancers. Nature. 2005; 435:834–838. PMID: 15944708 25. Iorio M, Ferracin M, Liu C-G, Veronese A, Spizzo R, Sabbioni S, et al. MicroRNA Gene Expression Deregulation in Human Breast Cancer. Cancer Res. 2005; 65(16):7065–70. PMID: 16103053 26. Volinia S, Calin GA, Liu CG, Ambs S, Cimmino A, Petrocca F, et al. A microRNA expression signature of human solid tumors defines cancer gene targets. Proc Natl Acad Sci U S A. 2006; 103(7):2257–61. PMID: 16461460 27. Si M-L, Zhu S, Wu H, Lu Z, Wu F and Mo Y-Y. miR-21-mediated tumor growth. Oncogene. 2007; 26:2799–2803. PMID: 17072344 28. Albain KS, Paik S, van’t Veer L. Prediction of adjuvant chemotherapy benefit in endocrine responsive, early breast cancer using multigene assays. The Breast. 2009; S*, S141–S145. doi: 10.1016/S0960- 9776(09)70290-5 PMID: 19914534 29. Ivshina AV, George J, Senko O, Mow B, Putti TC, Smeds J, et al. Genetic reclassification of histologic grade delineates new clinical subtypes of breast cancer. Cancer Res. 2006 Nov 1; 66 (21):10292–301. PMID: 17079448 30. Johnson CD, Esquela-Kerscher A, Stefani G, Byrom M, Kelnar K, Ovcharenko D, et al. The let-7 Micro- RNA Represses Cell Proliferation Pathways in Human Cells. Cancer Res. 2007; 67(16):7713–22 PMID: 17699775 31. Weier H-U G, Mao J-H. Meta-analysis of Aurora Kinase A (AURKA) Expression Data Reveals a Signifi- cant Correlation between Increased AURKA Expression and Distant Metastases in Human ER-positive Breast Cancers. J Data Mining Genomics Proteomics. 2013; 4(1):127. PMID: 24729942 12 / 12 PLOS ONE \| DOI:10.1371/journal.pone.0145346 December 30, 2015
W3177991376.txt	https://library.oapen.org/bitstream/20.500.12657/41356/1/sg_010_DavidHenseler.pdf	fr		Datenschutz bei drohnengestützter Datenbearbeitung durch Private	null	2,020	cc-by	90,859	sui generis Z BEI TÜTZTER EITUNG TE Datenschutz bei drohnengestützter Datenbearbeitung durch Private David Henseler David Henseler Datenschutz bei drohnen gestützter Datenbearbeitung durch Private Dissertation sui generis, Zürich 2020 «Dann aber können wir Erdbewohner, die wir sonst schon allen möglichen Gefahren ausgesetzt sind, aufs neue und dieses Mal von der Luft aus bedroht werden, und zwar hinsichtlich unserer persönlichen und materiellen Rechtsgüter.» (Friedrich Meili, Ballons, Flugmaschinen und die Jurisprudenz, Allgemeine österreichische Gerichts-Zeitung 1909, Nr. 39, S. 3) V Vorwort Die vorliegende Arbeit ist zwischen Januar 2017 und Dezember 2019 entstanden. Während dieser Zeit war ich für zwei Jahre als Assistent und danach als Candoc-Stipendiant an der Universität Zürich angestellt. Den grössten Teil der Schreibarbeit konnte ich während Auslandaufenthalten am Center for IP & IT Law (CiTiP) der KU Leuven (Belgien) sowie am Max-Planck-Institut für ausländisches öffentliches Recht und Völkerrecht in Heidelberg erledigen. Für die Unterstützung durch alle vorgenannten Institutionen bin ich sehr dankbar. Die Arbeit wurde am 1. April 2020 an der Rechtswissenschaftlichen Fakultät der Universität Zürich als Dissertation abgenommen. Lehre und Rechtsprechung wurden bis Ende Oktober 2019 berücksichtigt, vereinzelt darüber hinaus. Mein herzlicher Dank gilt zunächst meiner Doktormutter, Frau Prof. Dr. Regina Kiener. Sie hat mich während der Ausarbeitung dieser Arbeit mit grossem Interesse unterstützt und stand für Fragen und Hinweise stets zur Verfügung. Während der Zeit als Assistent an ihrem Lehrstuhl habe ich sehr viel gelernt. Vor allem die Mitarbeit an Gutachten sowie an verschiedenen Publikationsprojekten habe mich wissenschaftlich ein grosses Stück weitergebracht. Daneben möchte ich mich auch ganz herzlich bei Prof. Dr. Thomas Gächter für die zügige Erstellung des Zweitgutachtens bedanken. Für den immer sehr interessanten Austausch zu verschiedensten Fragen mit Bezug zu Drohnen möchte ich mich bei Rechtsanwalt Dr. Kurt Moll sowie Dr. Maximilian Jablonowski bedanken. Ebenfalls gilt mein Dank dem gesamten Team «Innovation und Digitalisierung» des Bundesamts für Zivilluftfahrt, insbesondere Marcel Kägi und Sandra Bodmer. Den Austausch mit ihnen habe ich stets sehr geschätzt. Für interessante Inputs aus der Sozialgeografie gebührt mein Dank ausserdem Prof. Dr. Francisco Klauser. Für die sorgfältige Lektüre und Korrektur der Arbeit möchte ich meinem geschätzten Kollegen Rechtsanwalt Denis Parvex und insbesondere meinem lieben Bruder Rechtsanwalt Simon Henseler herzlich danken. Des Weiteren gilt mein Dank Herrn Moritz Henseler für die zweimalige und äusserst sorgfältige Durchsicht des Manuskripts. Meine Eltern Brigitte Henseler-Pfenninger und Moritz Henseler haben mir eine universitäre Ausbildung ermöglicht und mich stets bei all meinen Vorhaben unterstützt. Dafür werde ich ihnen immer dankbar sein. Das zentrale Element für den erfolgreichen Abschluss der vorliegenden Arbeit war und ist aber meine Frau Marina. Sie hat mich und meine Launen, meine VI Schreibblockaden und Schreibphasen hautnah miterlebt und mich immer wieder auf die richtige Spur gelenkt. Ohne Marina hätte ich mein Dissertations projekt vielleicht aufgegeben. Ihr ist diese Arbeit gewidmet. Zürich, im April 2020 David Henseler VII Übersicht Vorwort . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V Übersicht . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII Inhaltsverzeichnis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX Literatur- und Quellenverzeichnis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XIX Materialienverzeichnis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . LIX Abkürzungsverzeichnis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . LXIII Einleitung .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Teil 1: Rechtliche, begriffliche und technische Grundlagen . . . . . . . . . . . . 11 Teil 2: Die drohnengestützte Datenbearbeitung im Lichte der Grundrechte . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 Teil 3: Bestandesaufnahme (1): Der E-DSG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 Teil 4: Bestandesaufnahme (2): Die «Drohnenverordnungen» .. . . . . . . 119 Teil 5: Die Durchsetzung von datenschutzrechtlichen Ansprüchen . 157 Zusammenfassung und Schlussbemerkungen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175 IX Inhaltsverzeichnis Vorwort . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V Übersicht . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII Inhaltsverzeichnis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX Literatur- und Quellenverzeichnis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XIX Materialienverzeichnis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . LIX Abkürzungsverzeichnis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . LXIII Einleitung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 § 1 Einführung in das Problemfeld . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 § 2 Gegenstand und Ziel der Arbeit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 I. Forschungsfrage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 II. Eingrenzung des Themas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 III. Relevanz des Themas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1. Anzahl und Verbreitung von Drohnen . . . . . . . . . . . . . . . . . . . . . . . . 3 2. Neue Gefahren durch neue Technologie . . . . . . . . . . . . . . . . . . . . . 4 3. Misstrauen und Ablehnung in der Bevölkerung . . . . . . . . . 5 4. Wirtschaftliches Potenzial . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 IV. Stand der Wissenschaft . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 § 3 Aufbau und Methode .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 I. Gang der Untersuchung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 II. Methode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Teil 1: Rechtliche, begriffliche und technische Grundlagen .. . . . . . 11 § 1 Bemerkungen zu den zentralen Rechtsgrundlagen . . . . . . . . . 11 I. Datenschutzgesetz . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 1. Auslöser und Ziele der Totalrevision . . . . . . . . . . . . . . . . . . . . . . . . . . 11 2. Abstellen auf den E-DSG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 II. Drohnenverordnungen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 1. EU-Recht und dessen Bedeutung für die Schweiz . . . . . . 13 2. Übernahme durch die Schweiz . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 § 2 Begriffe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Drohne . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 15 X 1. Technische Bezeichnungen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 2. Kategorisierung anhand der Konstruktionsweise . . . . . . . 18 2.1 Drehflügler .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 2.2 Starrflügler .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 2.3 Schwingflügler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 3. Zwischenfazit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 4. Regelung in den Drohnenverordnungen . . . . . . . . . . . . . . . . . . . . . . 20 4.1 Drohnendefinition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 4.2 Die drei Drohnenkategorien . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 a Offene Kategorie . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 b Spezielle Kategorie . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 c Zulassungspflichtige Kategorie . . . . . . . . . . . . . . . . . . . . . . . . . . 23 4.3 Produkteklassen innerhalb der offenen Kategorie . 24 5. Zusammenfassung und Stellungnahme . . . . . . . . . . . . . . . . . . . . . . . 26 II. Drohnenpilot und Drohnenbetreiber . . . . . . . . . . . . . . . . . . . . . . . . . . 27 1. Pilot, Fernpilot, Drohnenpilot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 2. Betreiber und Halter .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 III. Daten . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 1. Personendaten im Allgemeinen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 2. Besonders schützenswerte Personendaten .. . . . . . . . . . . . . . . . . 31 2.1 Erfasste Kategorien . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 2.2 Biometrische Daten insbesondere . . . . . . . . . . . . . . . . . . . . . . . . 31 2.3 Verfassungsrechtliche Einordnung . . . . . . . . . . . . . . . . . . . . . . 32 IV. Datenbearbeitung .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 V. Drohnengestützte Datenbearbeitung . . . . . . . . . . . . . . . . . . . . . . . . . . 33 § 3 Einsatzformen von Drohnen in der Schweiz .. . . . . . . . . . . . . . . . . . . . . . I. Nutzung durch Behörden . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Nutzung durch Private . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Kommerzielle Nutzung .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Hobbynutzung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . § 4 Stand der Technik . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. In der Schweiz verbreitete Hobbydrohnen .. . . . . . . . . . . . . . . . . II. Sensoren .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Fotokamera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Videokamera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Mikrofon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Infrarot- und Wärmebildkameras . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. IMSI-Catcher . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Zwischenfazit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 35 37 37 38 39 39 40 40 41 41 42 42 43 XI Teil 2: Die drohnengestützte Datenbearbeitung im Lichte der Grundrechte . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 § 1 Vorbemerkungen ................................................................. § 2 Grundrechtliche Schutzpflichten . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Verpflichtungsdimensionen der Grundrechte . . . . . . . . . . . . 1. Die drei Kategorien der Verpflichtungsdimensionen . . . . 2. Schutz vor Übergriffen Privater .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Grundlage der Schutzpflichten . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. In der Bundesverfassung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. In der Europäischen Menschenrechtskonvention . . . . . . . III. Semantik . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Schutzpflichten und deren Verhältnis zur Lehre der Drittwirkung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Ausprägungen der Schutzpflichten . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Adressaten . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Primäre Verpflichtung des Gesetzgebers . . . . . . . . . . . . . . . . . . . . . 2. Exekutive und Judikative . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Tatbestand . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. Rechtsfolge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX. Schutzberechtigte .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X. Justiziabilität grundrechtlicher Schutzpflichten . . . . . . . . 1. Grundsätzliche Justiziabilität . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Durchsetzungsproblematik vor Bundesgericht . . . . . . . . . . . 3. Praxis des EGMR .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XI. Zwischenfazit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . § 3 Privatsphäre . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Vorbemerkungen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Die einzelnen Aspekte der Privatsphäre . . . . . . . . . . . . . . . . . . . . . . 2. Persönlicher Schutzbereich . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Achtung des Brief-, Post- und Fernmeldeverkehrs . . . . . . 1. Inhalt und sachlicher Schutzbereich .. . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Bei der drohnengestützten Datenbearbeitung durch Private insbesondere . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Achtung der Wohnung .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Inhalt und sachlicher Schutzbereich .. . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Bei der drohnengestützten Datenbearbeitung durch Private insbesondere . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 46 46 46 46 47 47 48 48 49 50 51 51 51 52 53 55 55 55 56 57 57 58 58 58 58 59 59 59 60 60 60 XII IV. Achtung des Privatlebens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 1. Inhalt und sachlicher Schutzbereich .. . . . . . . . . . . . . . . . . . . . . . . . . . . 61 2. Bei der drohnengestützten Datenbearbeitung durch Private insbesondere . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 § 4 Informationelle Selbstbestimmung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Entstehung und Einordnung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Schutzbereich . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Persönlicher Schutzbereich . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Sachlicher Schutzbereich . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Herrschende Lehre und Rechtsprechung .. . . . . . . . . . . . 2.2 Kritische Lehrstimmen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Verfassungsrevision? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 Zwischenfazit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Bei der drohnengestützten Datenbearbeitung durch Private insbesondere . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . § 5 Weitere möglicherweise tangierte Grundrechte . . . . . . . . . . . . . . . . I. Menschenwürde . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Inhalt und Schutzbereich . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Bei der drohnengestützten Datenbearbeitung durch Private insbesondere . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Glaubens- und Gewissensfreiheit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Inhalt und Schutzbereich . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Bei der drohnengestützten Datenbearbeitung durch Private insbesondere . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Meinungsäusserungs- und Versammlungsfreiheit . . . . . 1. Inhalt und Schutzbereich . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Bei der drohnengestützten Datenbearbeitung durch Private insbesondere . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Eigentumsgarantie . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Inhalt und Schutzbereich . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Bei der drohnengestützten Datenbearbeitung durch Private insbesondere . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 62 63 63 63 63 64 64 65 65 66 66 66 66 67 67 67 68 68 68 68 68 69 § 6 Schutzpflichten im Kontext der informationellen Selbstbestimmung .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 I. Anerkannte Schutzpflichtendimension . . . . . . . . . . . . . . . . . . . . . . 69 II. Verfassungsrechtliche Anforderungen an das Datenschutzrecht . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 XIII Teil 3: Bestandesaufnahme (1): Der E-DSG .................................... 73 ................................................................. 73 § 2 Anwendbarkeit des Datenschutzgesetzes . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Verantwortlicher und betroffene Person . . . . . . . . . . . . . . . . . . . . 1. Verantwortlicher . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Betroffene Person . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Sachlicher Geltungsbereich . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Ausnahme: Bearbeitung ausschliesslich zum persönlichen Gebrauch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Die Regelung des E-DSG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Interessenausgleich als normative Grundlage .. . . . . . . . . . . . 3. Anwendungsbeispiele . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Bekanntgabe an Aussenstehende .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Haushaltsausnahme bei der drohnengestützten Datenbearbeitung? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Ausgangsbeispiel: «Nachbarschafts-Drohne» . . . . . . . . . . . . . . 2. Detailprüfung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Zusammenfassung und Fazit .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 § 1 Vorbemerkungen § 3 Zentrale Bestimmungen des E-DSG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Bearbeitungsgrundsätze . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Rechtmässigkeit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Treu und Glauben und Verhältnismässigkeit . . . . . . . . . . . . . . . 2.1 Verhältnismässigkeit .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Treu und Glauben .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Zweckbindung und Erkennbarkeit (Transparenz) .. . . . . . 3.1 Zweckbindung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Erkennbarkeit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Datenrichtigkeit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Einwilligung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Rechtsfolge bei Missachtung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Privacy by Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Die Regelung im Einzelnen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1 Datenschutz durch Technik . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 Beurteilung der Angemessenheit . . . . . . . . . . . . . . . . . . . . . . . . . 1.3 Stand der Technik insbesondere . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Sanktionierung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Bedeutung bei der drohnengestützten Datenbearbeitung durch Private . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 74 75 75 75 76 77 78 78 79 79 80 81 81 82 82 83 83 84 85 85 85 86 87 88 89 89 89 90 91 92 92 XIV III. Informationspflicht . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 1. Voraussetzungen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94 2. Umfang und Modalitäten .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94 3. Zeitpunkt der Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 4. Ausnahmen und Einschränkungen . . . . . . . . . . . . . . . . . . . . . . . . . . 96 4.1 Ausnahmen .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 4.2 Einschränkungen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 5. Sanktionierung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 6. Bedeutung bei der drohnengestützten Datenbearbeitung durch Private . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 IV. Datenschutz-Folgenabschätzung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 1. Voraussetzungen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 2. Sanktionierung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 3. Bedeutung bei der drohnengestützten Datenbearbeitung durch Private . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 V. Auskunftsrecht . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 1. Zentrale Bedeutung des Anspruchs . . . . . . . . . . . . . . . . . . . . . . . . . 100 2. Auskunftsberechtigte und Auskunftsverpflichtete . 101 3. Voraussetzungen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 4. Ausübung des Auskunftsrechts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 4.1 Auskunftsbegehren . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 4.2 Identifikation des Auskunftsberechtigten . . . . . . . . . 102 5. Erteilung der Auskunft . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 5.1 Form und Inhalt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 5.2 Einschränkungen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104 6. Zeitpunkt der Auskunft . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 7. Sanktionierung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 8. Bedeutung bei der drohnengestützten Datenbearbeitung durch Private . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 VI. Rechte bei Persönlichkeitsverletzungen . . . . . . . . . . . . . . . . . . 106 1. Persönlichkeitsverletzung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106 2. Rechtfertigungsgründe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108 3. Rechtsansprüche .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 3.1 Übersicht .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 3.2 Klagen zum Schutz der Persönlichkeit .. . . . . . . . . . . . . . 110 3.3 Die Ansprüche im Einzelnen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112 a Beseitigung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112 b Unterlassung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 c Genugtuung .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114 d Feststellung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 XV 4. Bedeutung bei der drohnengestützten Datenbearbeitung durch Private . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . § 4 Gesamtbetrachtung und Überleitung Teil 4: 116 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 Bestandesaufnahme (2): Die «Drohnenverordnungen» . . . . . . 119 § 1 Vorbemerkungen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 § 2 Identifizierung von Drohnenbetreibern . . . . . . . . . . . . . . . . . . . . . . . . . . 120 I. Registrierung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 1. Zentrale Bedeutung der Registrierung . . . . . . . . . . . . . . . . . . . . . 120 2. Registrierungspflicht für Drohnen und Betreiber . . . . 120 3. Die Kriterien im Einzelnen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121 3.1 Zertifizierung der Konstruktion . . . . . . . . . . . . . . . . . . . . . . . . 121 3.2 Kinetische Energie . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122 a Begriff und Bedeutung .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122 b Regelung in der Durchführungsverordnung .. . . . 123 3.3 Risiken für Privatsphäre und den Schutz von Personendaten . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124 3.4 Ausnahme für «Spielzeugdrohnen» .. . . . . . . . . . . . . . . . . 125 a Begriff . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 b Fähigkeit zur Datenbearbeitung als Ausschlusskriterium? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126 4. Zwischenfazit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128 5. Registrierungsverfahren . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128 6. Anzugebende Informationen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130 7. «Drohnenbetreiberregister» . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 7.1 Ausgestaltung des Registers .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 7.2 Vergleichbare Register . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132 a Luftfahrzeugregister .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133 b Informationssystem Verkehrszulassung . . . . . . . . . 133 7.3 Zwischenfazit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134 8. Datenschutzrechtliche Beurteilung und Überleitung . 135 II. Kennzeichnung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135 1. Arten der Kennzeichnung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135 2. Kennzeichnung mit Betreibernummer . . . . . . . . . . . . . . . . . . . . 136 2.1 Begriff . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136 2.2 Praktische Umsetzung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136 3. Datenschutzrechtliche Beurteilung . . . . . . . . . . . . . . . . . . . . . . . . . . 137 XVI III. Fernidentifizierung .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138 1. Bedeutung und Begriff . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138 2. Erfasste Drohnen .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138 3. Zusatzgerät für die direkte Fernidentifizierung . . . . . . . 139 3.1 Allgemeine Anforderungen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139 3.2 Empfang durch Mobilfunkgeräte insbesondere .. . . . 140 4. Datenschutzrechtliche Beurteilung . . . . . . . . . . . . . . . . . . . . . . . . . . 141 IV. Flugverkehrsmanagement U-Space (UTM) .. . . . . . . . . . . . . . 142 1. Begriff . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142 2. Inhalt und Zweck . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 3. Datenschutzrechtliche Beurteilung . . . . . . . . . . . . . . . . . . . . . . . . . . 143 V. Spezialfall: Selbst gebaute Drohnen . . . . . . . . . . . . . . . . . . . . . . . . . 144 1. Definition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144 2. Anwendbare Vorschriften . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 2.1 Grundsätzliche Zugehörigkeit zur offenen Kategorie . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 2.2 Registrierung und Kennzeichnung .. . . . . . . . . . . . . . . . . . . 145 2.3 Fernidentifizierung .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 3. Datenschutzrechtliche Beurteilung . . . . . . . . . . . . . . . . . . . . . . . . . . 145 VI. Herausforderungen bei der Umsetzung . . . . . . . . . . . . . . . . . . . 146 1. Notwendige Mitwirkung der Drohnenbetreiber . . . . . . . 146 2. Übergangsbestimmungen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147 § 3 Präventive und flankierende Massnahmen . . . . . . . . . . . . . . . . . . . . . 148 I. Information und Ausbildung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148 1. Informationsbeiblatt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148 2. Online-Theorieprüfung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149 3. Staatliche Informationsmassnahmen . . . . . . . . . . . . . . . . . . . . . . . 151 II. Geo-Awareness (Geofencing) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151 1. Begriff und Abgrenzung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151 2. Inhalt und Grenzen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152 3. Datenschutzrechtliche Beurteilung . . . . . . . . . . . . . . . . . . . . . . . . . 153 III. Positionslichter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153 1. Erfasste Drohnen .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153 2. Datenschutzrechtliche Beurteilung . . . . . . . . . . . . . . . . . . . . . . . . . 154 § 4 Gesamtbetrachtung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155 XVII Teil 5: Die Durchsetzung von datenschutzrechtlichen Ansprüchen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157 § 1 Schutzpflicht bei der drohnengestützten Datenbearbeitung durch Private . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157 I. Informations- und Machtungleichgewicht als Ausgangspunkt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157 II. Tatbestand . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158 1. Vorliegen eines Störerdreiecks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158 2. Kenntnis der konkreten Gefährdung . . . . . . . . . . . . . . . . . . . . . . . 158 3. Kompetenz des Bundes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159 III. Adressat der Schutzpflicht . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159 1. Vollzugsbehörden . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160 2. Gesetzgeber . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160 IV. Zwischenfazit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161 § 2 Wahrnehmung der Schutzpflicht de lege ferenda . . . . . . . . . . . . 162 I. Achtung der Ansprüche der Drohnenbetreiber . . . . . . . 162 II. Bestimmung des passenden Gesetzes .. . . . . . . . . . . . . . . . . . . . . . 164 § 3 Vorschläge für eine Gesetzesbestimmung . . . . . . . . . . . . . . . . . . . . . . . 165 I. Zu regelnde Aspekte .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165 II. Kommentierter Formulierungsvorschlag . . . . . . . . . . . . . . . . 166 1. Kompetenz (Abs. 1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166 1.1 Formulierungsvorschlag . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166 1.2 Bemerkungen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166 2. Zweck (Abs. 2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167 2.1 Formulierungsvorschlag . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167 2.2 Bemerkungen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167 3. Inhalt (Abs. 3) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168 3.1 Formulierungsvorschlag . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168 3.2 Bemerkungen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168 4. Datenbekanntgabe (Abs. 4 und 5) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169 4.1 Formulierungsvorschlag . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169 4.2 Bemerkungen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169 5. Zugriff im Abrufverfahren (Abs. 6) .. . . . . . . . . . . . . . . . . . . . . . . . . . . 171 5.1 Formulierungsvorschlag . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171 5.2 Bemerkungen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171 III. Unkommentierter Formulierungsvorschlag . . . . . . . . . . . 172 Zusammenfassung und Schlussbemerkungen § 1 Zusammenfassung .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175 § 2 Schlussbemerkungen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177 XIX Literatur- und Quellenverzeichnis Die angeführten Autorinnen und Autoren werden im Haupttext — sofern bei den einzelnen Publikationen nicht anders angegeben — mit ihrem Nach namen und mit der Seitenzahl, dem Paragraphen oder der sonstigen Fundstelle abgekürzt zitiert. Neuerscheinungen und Neuauflagen wurden bis zum 31. Oktober 2019 berücksichtigt. Internetquellen wurden am 31. Oktober 2019 zuletzt besucht. Abeyratne Ruwantissa, Convention on International Civil Aviation: A Commentary, Cham et al. 2014 Aebi-Müller Regina E., Personenbezogene Informationen im System des zivilrechtlichen Persönlichkeitsschutzes, Unter besonderer Berücksichtigung der Rechtslage in der Schweiz und in Deutschland, Bern 2005 Albers Marion, Informationelle Selbstbestimmung, Baden-Baden 2007 Alexy Robert, Theorie der Grundrechte, Baden-Baden 1985 American Civil Liberties Union (ACLU), Protecting Privacy From Aerial Surveillance: Recommendations for Government Use of Drone Aircraft, New York 2011 Applin Sally A., Deliveries by Drone: Obstacles and Sociability, in: Custers (Hrsg.), The Future of Drone Use: Opportunities and Threats from Ethical and Legal Perspectives, Den Haag 2016, S. 71 ff. Arai-Takahashi Yutaka, The Margin of Appreciation Doctrine and the Principle of Proportionality in the Jurisprudence of the ECHR, Diss. Cambridge 1998, Antwerpen 2002 Arbeitskreis im Erfahrungsaustauschkreis der Zentralstellen der Länder für Sicherheitstechnik (ZLS), Beschlüsse des Arbeitskreises 2.2 «Spielzeug», München 2015 (zit. Arbeitskreis ZLS) Arnosti Dominique C., Dashcam: Risiko oder Garant im (Rechts)Verkehr?, Eine rechtswissenschaftliche Analyse der datenschutzrechtlichen Zulässigkeit von Dashcam-Aufnahmen und deren Verwertbarkeit im Zivilprozess, Zürich 2019Arora Siddhartha, Swiss Commercial Drone Industry: A possibility with potential?, Zürich/St. Gallen 2016 XX Literatur- und Quellenverzeichnis Arterburn David / Olivares Gerardo / Bolte John / Prabhu Raj / Duma Stefan, Task A14: UAS Ground Collision Severity Evaluation 2017-2019 (zit. Arterburn et al.) Article 29 Data Protection Working Party, Opinion 01/2015 on Privacy and Data Protection Issues relating to the Utilisation of Drones, Working Paper 231, 16. Juni 2015 (zit. Article 29 Working Party) Aubert Jean-François / Mahon Pascal, Petit commentaire de la Consti tution fédérale de la Confédération suisse du 18 avril 1999, Zürich/Basel/ Genf 2003 (zit. Bearbeiter, Petit commentaire, Art. xx Cst. N xx) Auer Andreas / Malinverni Giorgio / Hottelier Michel, Droit constitu tionnel suisse, Volume II, Les droits fondamentaux, 3. Aufl., Bern 2013 Austin Reg, Unmanned Aircraft Systems: UAVS Design, Development and Deployment, New York 2010 Aviation Rulemaking Committee, Unmanned Aircraft Systems (UAS) Registration Task Force, Task Force Recommendation Final Report, 21. November 2015 Bacher Bettina, Medienfreiheit und Persönlichkeitsschutz, Zivilrechtliche Auswirkungen der Lösung eines Grundrechtskonflikts, Diss. Freiburg i.Üe. 2015, Basel 2015 Bächli Marc, Das Recht am eigenen Bild: die Verwendung von Personen bildern in den Medien, in der Kunst, der Wissenschaft und in der Werbung aus der Sicht der abgebildeten Person, Diss. Basel 2001, Basel 2002 Baeriswyl Bruno — Die Verselbständigung des Computers — datenschutzrechtliche Aspekte, in: Kündig/Bütschi (Hrsg.), Die Verselbständigung des Computers, Zürich 2008, S. 117 ff. (zit. Baeriswyl, Verselbständigung) — Internet als Fahndungsmittel mit Kollateralschäden, Sicherheit & Recht 1/2010, S. 11 ff. (zit. Baeriswyl, Fahndungsmittel) — Entwicklungen im Datenschutzrecht / Le point sur le droit de la protection des données, SJZ 115/2019, S. 592 ff. (zit. Baeriswyl, Entwicklungen) Literatur- und Quellenverzeichnis XXI Baeriswyl Bruno / Rudin Beat (Hrsg.) — Praxiskommentar zum Informations- und Datenschutzgesetz des Kantons Zürich (IDG), Zürich/Basel/Genf 2012 (zit. PK IDG/ZH-Bear beiter/-in, § xx N xx) — Datenschutzgesetz (DSG), Stämpflis Handkommentar, Bern 2015 (zit. SHK DSG-Bearbeiter/-in, Art. xx N xx) Bajde Domen / Hojer BruUn Maja / Sommer Jannek K. / Waltrop Karen, General Public’s Privacy Concerns Regarding Drone Use in Residential and Public Areas, Empirical research report, University of Southern Denmark/Aalborg University, Mai 2017 (zit. Bajde et al., Privacy Concerns) Bajde Domen / Woermann Niklas / Hojer BruUn Maja / Gahrn-Andersen Rasmus / Sommer Jannek K. / Nojgaard Mikkel / Christensen Sofie H. / Kirschner Heiko / Skaarup Jensen Rune Hagel / Bucher Jan Hendrik, Public reactions to drone use in residential and public areas, University of Southern Denmark/Aalborg University, Dezember 2017 (zit. Bajde et al., Public reactions) Balser Markus, Unerkannte Flugobjekte, SZ vom 19. August 2019, S. 5 Bartsch Ron, Unmanned and Uncontrolled: The Commingling Theory and the Legality of Unmanned Aircraft System Operations, J. Aeronaut Aero space Eng 2015 (1), S. 1 ff. Bartsch Ron / Coyne James / Gray Katherine, Drones in Society: Explor ing the strange new world of unmanned aircraft, Oxford/New York 2017 Baur Georges, Datenschutz-Grundverordnung Schengen-relevant? Vielleicht nicht, aber …, in: Breitenmoser/Lagodny/Uebersax (Hrsg.), Schengen und Dublin in der Praxis: Aktuelle Herausforderungen, Zürich/St. Gallen 2018, S. 165 ff. Belser Eva Maria, Zur rechtlichen Tragweite des Grundrechts auf Datenschutz: Missbrauchsschutz oder Schutz der informationellen Selbst bestimmung?, in: Epiney/Fasnacht/Blaser (Hrsg.), Instrumente zur Umsetzung des Rechts auf informationelle Selbstbestimmung, Zürich/ Basel/Genf 2013, S. 19 ff. (zit. Belser, Tragweite) Belser Eva Maria / Epiney Astrid / Waldmann Bernhard, Datenschutzrecht: Grundlagen und öffentliches Recht, Bern 2011 (zit. Bearbeiter/-in, in: Belser/Epiney/Waldmann) XXII Literatur- und Quellenverzeichnis Belser Eva Maria / Waldmann Bernhard, Grundrechte II: Die einzelnen Grundrechte, Zürich/Basel/Genf 2012 Belser Urs, Das Recht auf Auskunft, die Transparenz der Datenbearbeitung und das Auskunftsverfahren, in: Schweizer (Hrsg.), Das neue Datenschutzgesetz des Bundes, Zürich 1993, S. 55 ff. (zit. Belser U.) Berger Max, Keine Angst vor Dashcams: Video-Aufnahmen als Beweis mittel im Zivilprozess, Jusletter 8. April 2019 Bernauw Kristiaan, Drones: The Emerging Era of Unmanned Civil Aviation, Zbornik Pravnog fakulteta u Zagrebu [= Collected Papers of Zagreb Law Faculty], Vol. 66 (2016), S. 223 ff. Bertolini Andrea, Artificial Intelligence and civil law: liability rules for drones, Study for the JURI committee, Directorate General for Internal Policies of the Union, Brüssel November 2018 Besson Samantha — Les obligations positives de protection des droits fondamentaux — Un essai en dogmatique comparative, ZSR 2003 I, S. 49 ff. (zit. Besson, obligations positives) — Comment humaniser le droit privé sans commodifier les droits de l’homme, in: Werro (Hrsg.), Droit civil et convention européenne des droits de l’homme / Zivilrecht und Europäische Menschenrechtskonvention, Zürich 2006, S. 1 ff. (zit. Besson, droit privé) Bestaoui Sebbane Yasmina, Smart autonomous aircraft: Flight control and planning for UAV, Boca Raton/London/New York 2015 Betschon Stefan, Ein Himmel voller Drohnen, 10. November 2016 Biaggini Giovanni — «Durchnummerieren» der Bevölkerung und Verfassung: Zur Zulässigkeit eines koordinierten eidgenössischen Personenidentifikators im Lichte des verfassungsrechtlichen Persönlichkeitsschutzes, in: Noll/ Olano (Hrsg.), «Im Namen des Obergerichts», Festschrift zur Pensionierung von Frau Dr. Magdalena Rutz, Liestal 2004, S. 71 ff. (zit. Biaggini, «Durchnummerieren») — Bundesverfassung der Schweizerischen Eidgenossenschaft, Kommentar, 2. Aufl., Zürich 2017 (zit. Biaggini, Komm. BV) Bieri Peter, Bearbeitung von Daten über Richterinnen und Richter: Bedarf und Schranken, Diss. Bern 2016, Zürich 2017 Literatur- und Quellenverzeichnis XXIII Biermann Kai / Wiegold Thomas, Drohnen: Chancen und Gefahren einer neuen Technik, Berlin 2015 Bischof Claudia, Drohnen im rechtlichen Praxistest, DuD 3/2017, S. 142 ff. Blue Ribbon Task Force (BRTF), Interim Report on UAS Mitigation at Airports, Juli 2019 Bolt Roger / Bryner Simone, Observationen in der Praxis — Sichtweise Suva, HAVE 2018, S. 209 ff. Boucher Philip, Joint Research Centre, Institute for the Protection and Security of the Citizen, Report EUR 26824, Civil Drones in Society, Luxemburg 2014 Bracken-Roche Ciara, Domestic drones: the politics of verticality and the surveillance industrial complex, Geogr. Helv. 71 (2016), S. 167 ff. Bracken-Roche Ciara / Lyon David / Mansour Mark James / Molnar Adam / Saulnier Alana / Thompson Scott, Surveillance drones: Privacy implications of the spread of unmanned aerial vehicles (UAVs) in Canada, Surveillance Studies Centre, Queen’s University, Kingston 2014 (zit. Bracken-Roche et al.) Brahms Florian / Maslaton Martin, Die gewerbliche Nutzung von Drohnen im Lichte der geplanten Novelle der LuftVO, NVwZ 2016, S. 1125 ff. Breitenmoser Stephan, Der Schutz der Privatsphäre gemäss Art. 8 EMRK, Das Recht auf Achtung des Privat- und Familienlebens, der Wohnung und des Briefverkehrs, Diss. Basel 1986, Basel/Frankfurt a.M. 1986 Brunner Stephan C., Mit rostiger Flinte unterwegs in virtuellen Welten?, Jusletter 4. April 2011 Büchi Roland — Fascination Quadrocopter, Basics — Electronics — Flight Experience, Norderstedt 2011 (zit. Büchi, Quadrocopter) — Das grosse Buch der Drohnen, Grundlagen — Technik — Flugpraxis, Baden-Baden 2018 (zit. Büchi, Buch der Drohnen) Büchler Andrea / Jakob Dominique (Hrsg.), Kurzkommentar ZGB, 2. Aufl., Basel 2018 (zit. KuKo ZGB-Bearbeiter/-in, Art. xx N xx) XXIV Literatur- und Quellenverzeichnis Bull Hans Peter — Informationelle Selbstbestimmung — Vision oder Illusion?, 2. Aufl., Tübingen 2011 (zit. Bull, Selbstbestimmung) — Sinn und Unsinn des Datenschutzes, Tübingen 2015 (zit. Bull, Datenschutz) Bundesamt für Justiz (BJ) — Obligation positive de mise à disposition d’aires de séjour ou de transit en faveur des gens du voyage suisses et portée de l’arzit. 35 Cst., VPB 2017.2, S. 19 ff. (zit. BJ, Obligation positive) — Gesetzgebungsleitfaden, Leitfaden für die Ausarbeitung von Erlassen des Bundes, 4. Aufl., Bern 2019 (zit. BJ, Leitfaden) Bundesamt für Lebensmittelsicherheit und Veterinärwesens (BLV), Erläuterungen zur Lebensmittel- und Gebrauchsgegenständeverord nung (zit. BLV, Erläuterungen LGV) Bundesamt für Zivilluftfahrt (BAZL) — Das Verhältnis zwischen der Schweiz und der Europäischen Agentur für Flugsicherheit (EASA), Bern November 2013 (zit. BAZL, Verhältnis) — BAZL RPAS Working Group, Zivile Drohnen in der Schweiz: Eine neue Herausforderung, Bern 7. Februar 2016 (zit. BAZL, Zivile Drohnen) — U-Space Program Management, Swiss U-Space ConOps, Version 1.0; 29. März 2019 (zit. BAZL, ConOps) — Memorandum of Cooperation established between Swiss FOCA and Skyguide, 20. Dezember 2018 (zit. BAZL, Memorandum) — Swiss U-Space Implementation (zit. BAZL, U-Space) — Übernahme neue europäische Drohnenregulierung (zit. BAZL, Übernahme) — Fernidentifizierung von Drohnen, 16. September 2019 (zit. BAZL, Fern identifizierung) — SUSI’s Remote ID Demonstration of September 16 2019, Report (zit. BAZL, Demo Report) Burger Jodie / Scuderi Eddie, Unmanned & unregulated: where are the privacy protections from drones?, LSJ Issue 46, Juli 2018, S. 88 f. Burkert Herbert, Internationale Grundlagen, in: Rossnagel (Hrsg.), Handbuch Datenschutzrecht, Die neuen Grundlagen für Wirtschaft und Verwaltung, München 2003, S. 85 ff. Literatur- und Quellenverzeichnis XXV Burkert Herbert / Hettich Peter / Thouvenin Florent, Grundlagen: Eine kritische Geschichte des Informationsrechts, in: Gschwend et al. (Hrsg.), Recht im digitalen Zeitalter, Festgabe Schweizer Juristentag 2015 in St. Gallen, Zürich/St. Gallen 2015 Burri Thomas, The Politics of Robot Autonomy, EJRR 2/2016, S. 341 ff. Burris Thomas, Daten «löschen» oder wirksam entfernen? Speichermedien vergessen nicht so schnell — über das Löschen und Wiederherstellen von Daten, digma 2008, S. 110 ff. Büttner Jean-Martin, Der Schweizer Drohnenkrieg, Tages-Anzeiger vom 30. April 2018, S. 1 Bydlinski Franz, Juristische Methodenlehre und Rechtsbegriff, 2. Aufl., Wien/New York 1991 Bygrave Lee A., Data Privacy Law, An International Perspective, Oxford 2014 Calliess Christian — Die grundrechtliche Schutzpflicht im mehrpoligen Verfassungsrechtsverhältnis, JZ 2006, S. 321 ff. (zit. Calliess, Schutzpflicht) — Gewährleistung von Freiheit und Sicherheit im Lichte unterschied licher Staats- und Verfassungsverständnisse, DVBl 2003, S. 1096 ff. (zit. Calliess, Gewährleistung) Calo M. Ryan, The Drone as Privacy Catalyst, 64 Stan. L. Rev. Online, 12. Dezember 2011, S. 29 ff. Canaris Klaus-Wilhelm, Grundrechte und Privatrecht, AcP 184 (1984), S. 201 ff. Cassart Alexandre — Drones: y-a-t’il un télépilote dans l’aéronef?, RDTI 2016 Nr. 1, S. 69 ff. (zit. Cassart, RDTI) — Droit des drones: Belgique, France, Luxembourg, Brüssel 2017 (zit. Cassart, Droit des drones) Cavoukian Ann, Privacy and Drones: Unmanned Aerial Vehicles, Information and Privacy Commissioner of Ontario, Canada, Ontario August 2012 Chadoian Satenig M., Das Fernmeldegeheimnis im Zeitalter der Internetund Mobilfunküberwachung: Eine rechtsvergleichende Untersuchung des schweizerischen und österreichischen Grundrechtsverständnisses im Hinblick auf neuartige technische Überwachungsmassnahmen, Diss. Wien 2011, Baden-Baden und Wien 2015 XXVI Literatur- und Quellenverzeichnis Chang Victoria / Chundury Pramod / Chetty Marshini, «Spiders in the Sky»: User Perceptions of Drones, Privacy and Security, Proceedings of the 2017 Conference on Human Factors in Computing Systems, Denver 2017, S. 6765 ff. Christen Markus / Guillaume Michel / Jablonowski Maximilian / Lenhart Peter / Moll Kurt, TA-SWISS (Hrsg.), Zivile Drohnen — Herausforderungen und Perspektiven, Zürich 2018 (zit. Christen et al.) Clarke Roger, The regulation of civilian drones’ impacts on behavioural privacy, CLSR 30 (2014), S. 286 ff. Clifford Damian, The Legal Limits to the Monetisation of Online Emotions, Diss. Löwen 2019, Löwen 2019 Clothier Reece A. / Greer Dominique A. / Greer Duncan G. / Mehta Amisha M., Risk Perception and the Public Acceptance of Drones, Risk Analysis 35 (2015) Nr. 6, S. 1167 ff. (zit. Clothier et al.) Corcoran Mark, Drone wars: The definition dogfight, ABC News, 1. März 2013 Cottier Bertil, L’ère numérique et le principe de légalité — Frictions et possibilités d’adaptation, in: Epiney/Sangsue (Hrsg.), Digitalisierung und Schutz der Privatsphäre, Zur Steuerungsfähigkeit der «traditionellen» Rechtsgrundsätze: Analyse und Perspektiven, Zürich 2018, S. 25 ff. Cuccuru Pierluigi, Profili di regolazione non-normativa dei droni alla luce della proposta di revisione del regolamento (CE) N. 216/2008 in materia di aviazione civile, in: Palmerini/Biasiotti/Aiello (Hrsg.), Diritto dei Droni: Regole, questioni e prassi, Mailand 2018, S. 333 ff. Custers Bart, Drones Here, There and Everywhere, Introduction and Overview, in: Custers (Hrsg.), The Future of Drone Use: Opportunities and Threats from Ethical and Legal Perspectives, Den Haag 2016, S. 1 ff. Dalamagkidis Konstantinos — Definitions and Terminology, in: Valavanis/Vachtsevanos (Hrsg.), Handbook of Unmanned Aerial Vehicles, Volume I, Heidelberg/New York/ London 2015, S. 43 ff. (zit. Dalamagkidis, Definitions) — Aviation History and Unmanned Flight, in: Valavanis/Vachtsevanos (Hrsg.), Handbook of Unmanned Aerial Vehicles, Volume I, Heidelberg/ New York/London 2015, S. 57 ff. (zit. Dalamagkidis, History) Literatur- und Quellenverzeichnis XXVII Dalamagkidis Konstantinos / Valavanis Kimon P. / Piegl Les A., On Integrating Unmanned Aircraft Systems into the National Airspace System, 2. Aufl., Dordrecht et al. 2012 Daphinoff Eva / Hassler Jannine, Teil 3: Datenschutzrechtliche Fragen im öffentlichen Personenverkehr, in: Furrer/Vasella (Hrsg.), Der öffentliche Personenverkehr — Haftung und Sicherheitsfragen, Bern 2018, S. 229 ff. Däubler Wolfgang / Wedde Peter / Weichert Thilo / Sommer Imke, EU-Datenschutz-Grundverordnung und BDSG-neu, Kompaktkommentar, Frankfurt a.M. 2018 (zit. Bearbeiter/-in, Däubler et al., Art. xx DSGVO N xx) Davola Antonio, L’acquisizione di dati da parte dei privati nelle operazioni con SAPR, in: Palmerini/Biasiotti/Aiello (Hrsg.), Diritto dei Droni: Regole, questioni e prassi, Mailand 2018, S. 137 ff. De Miguel Molina Blanca / Segarra Oña Marrival, The Drone Sector in Europe, in: de Miguel Molina/Santamarina Campos (Hrsg.), Ethics and Civil Drones: European Policies and Prosposals for the Industry, Cham 2018, S. 7 ff. De Terwangne Cécile — L’exception concernant les traitements de données à des fins personnelles et domestiques de la directive 95/46 relative à la protection des données, RDTI 2015 Nr. 1, S. 41 ff. (zit. de Terwangne, RDTI) — Définitions clés et champs d’application du RGDP, in: de Terwangne/ Rosier (Hrsg.), Le Règlement Général sur la Protection des Données (RGPD/GDPR), Analyse approfondie, Brüssel 2018, S. 59 ff. (zit. de Terwangne, champ d’application) De Vries Karin, Right to Respect for Private and Family Life, in: van Dijk et al. (Hrsg.), Theory and Practice of the European Convention on Human Rights, 5. Aufl., Cambridge/Antwerpen/Portland 2018, S. 667 ff. Deleury Stéphane, Les drones menacent toujours plus la sécurité de l’aviation civile en Suisse, 28. Juli 2017 Delise Riccardo, L’evoluzione del quadro regolamentare, in: Palmerini/ Biasiotti/Aiello (Hrsg.), Diritto dei Droni: Regole, questioni e prassi, Mailand 2018, S. 23 ff. Dempsey Paul Stephen, Public International Air Law, 2. Aufl., Montreal 2017 XXVIII Literatur- und Quellenverzeichnis Dettling-Ott Regula — Das bilaterale Luftverkehrsabkommen der Schweiz und der EG, in: Thürer et al. (Hrsg.), Bilaterale Verträge I & II Schweiz EU: Handbuch, Zürich/Basel/Genf 2007, S. 491 ff. (zit. Dettling-Ott, Luftverkehrs abkommen) — H. Luftverkehrsrecht — Teil II: Betrieb der Luftfahrt, in: Müller (Hrsg.), Verkehrsrecht, SBVR IV, Basel 2008, S. 391 ff. (zit. Dettling-Ott, Betrieb) — Exkurs: Organisation des Luftverkehrs in der Schweiz, in: Hobe/von Ruckteschell (Hrsg.), Kölner Kompendium des Luftrechts, Band 1: Grundlagen, Köln 2008, S. 251 ff. (zit. Dettling-Ott, Organisation) — Exkurs: Flugsicherung in der Schweiz, in: Hobe/von Ruckteschell (Hrsg.), Kölner Kompendium des Luftrechts, Band 2: Luftverkehr, Köln 2009, S. 1018 ff. (zit. Dettling-Ott, Flugsicherung) — Exkurs: Der Luftfahrzeughalter in der Schweiz, in: Hobe/von Ruckteschell (Hrsg.), Kölner Kompendium des Luftrechts, Band 2: Luftverkehr, Köln 2009, S. 1118 ff. (zit. Dettling-Ott, Luftfahrzeughalter) Dieckert Ulrich / Eich Stephan (Hrsg.), Drohnen — Technik und Recht: bei gewerblicher und behördlicher Nutzung, Köln 2018 (zit. Bearbeiter/-in, in: Dieckert/Eich) Doman David B. / Oppenheimer Michael W. / Sigthorsson David O., Dynamics and Control of Flapping Wing MAWs, in: Valavanis/Vachtsevanos (Hrsg.), Handbook of Unmanned Aerial Vehicles, Volume I, Heidelberg/ New York/London 2015, S. 329 ff. Döpke Christian F. / Jülicher Tim, Der Defikopter, InTeR 2017, S. 20 ff. DroneRules.eu — Privacy Handbook (zit. DroneRules, Handbook) — Privacy-by-Design Guide, A DronesRules.eu PRO resource for drone manufacturers (zit. DroneRules, Guide) Dubey Jacques — Droits fondamentaux, Volume I: Notion, garantie, restriction et juridiction, Basel 2018 (zit. Dubey, Vol. I) — Droits fondamentaux, Volume II: Libertés, garanties de l’Etat de droit, droits sociaux et politiques, Basel 2018 (zit. Dubey, Vol. II) Ebinger Christoph, Zivilrechtliche Haftung des Luftfrachtführers im Personentransport: Unter besonderer Berücksichtigung der Beförderung von Personen durch ferngesteuerte Luftfahrzeuge, Diss. Zürich 2010, Zürich/Basel/Genf 2012 Literatur- und Quellenverzeichnis XXIX Egli Patricia, Drittwirkung von Grundrechten: Zugleich ein Beitrag zur Dogmatik der grundrechtlichen Schutzpflichten im Schweizer Recht, Diss. Zürich 2002, Zürich 2002 Ehmann Eugen / Martin Selmayr (Hrsg.), Datenschutz-Grundverordnung, Kommentar, 2. Aufl., München 2018 (zit. Bearbeiter/-in, in: Ehmann/ Selmayr, Art. xx DSGVO N xx) Ehrenzeller Bernhard / Schindler Benjamin / Schweizer Rainer J. / Vallender Klaus A. (Hrsg.), Die Schweizerische Bundesverfassung, St. Galler Kommentar, 3. Aufl., Zürich/St. Gallen 2014 (zit. SGK BV-Bearbeiter/-in, Art. xx N xx) Eidgenössische Finanzverwaltung (EFV), Voranschlag 2019 mit inte griertem Aufgaben- und Finanzplan 2020–2022 der Verwaltungsein heiten, Band 2B (EFD, WBF, UVEK), Bern 2018 Eidgenössischer Datenschutz- und Öffentlichkeitsbeauftragter (EDÖB) — 25. Tätigkeitsbericht 2017/18, Bern 2018 (zit. EDÖB, Tätigkeitsbericht 2017/18) — Erläuterungen zu Videoüberwachung in Fahrzeugen (Dashcam) (zit. EDÖB, Dashcams) — Videoüberwachung mit Drohnen durch Private (zit. EDÖB, Drohnen) Eidgenössisches Departement für auswärtige Angelegenheiten (EDA), Aide-mémoire: Kompetenzen und Vorgehen für Beschlüsse der Gemischten Ausschüsse der sektoriellen Abkommen Schweiz–EG, Bern, 6. September 2007 Engberts Bart / Gillissen Edo, Policing from Above: Drone Use by the Police, in: Custers (Hrsg.), The Future of Drone Use: Opportunities and Threats from Ethical and Legal Perspectives, Den Haag 2016, S. 93 ff. Ensthaler Jürgen, Technikrecht: Einige Anmerkungen zum Verhältnis von juristischem und technischem Wissen, InTeR 1/2013, S. 34 ff. Epiney Astrid, Besonders schützenswerte Personendaten, Zu den Anforderungen an die Rechtmässigkeit der Bearbeitung durch öffentliche Organe im Falle des Fehlens einer gesetzlichen Grundlage, in: Rumo-Jungo et al. (Hrsg.), Une empreinte sur le Code civil, Mélanges en l’honneur de Paul-Henri Steinauer, Bern 2013, S. 97 ff. XXX Literatur- und Quellenverzeichnis Epiney Astrid / Frei Nula, Zur Übernahme weiterentwickelten EU-Rechts durch den Bundesrat, Selbstständige Vertragsschlusskompetenzen im Rahmen der Bilateralen Abkommen am Beispiel der Liberalisierung des internationalen Schienenpersonenverkehrs, Jusletter 12. August 2019 Euler Thorsten / Cobus Vanessa / Koelle Marion, Nummernschilder für Drohnen, Gesellschaftliche Vorstellungen zur Regulation von Smart Cams im öffentlichen Raum, DuD 2017, S. 147 ff. Europäische Kommission — Riga declaration on remotely piloted aircraft (drones), «Framing the future of aviation», 6. März 2015 (zit. Europäische Kommission, Riga declaration) — Warsaw declaration, «Drones as leverage for jobs and new business opportunities», 24. November 2016 (zit. Europäische Kommission, Warsaw declaration) — Richtlinie 2009/48/EG über die Sicherheit von Spielzeug, Erläuternde Leitlinien, Version 1.9 vom 10. Februar 2016, Brüssel 2016 (zit. Europäische Kommission, Leitlinien) European Union Aviation Safety Agency (EASA) — Advance Notice of Proposed Amendment 2015-1, Introduction of a regulatory framework for the operation of drones, 31. Juli 2015 (zit. EASA, A-NPA) — Vorschlag für die Erstellung von gemeinsamen Vorschriften für den Betrieb von Drohnen in Europa, September 2015 (zit. EASA, Drohnen in Europa) — Prototype Commission Regulation on Unmanned Aircraft Operations, Explanatory Note, 22. August 2016 (zit. EASA, Explanatory Note) — Notice of Proposed Amendment (NPA) 2017-05 (A), Introduction of a regulatory framework for the operation of drones, 4. Mai 2017 (zit. EASA, NPA) — Opinion No 01/2018, Introduction of a regulatory framework for the operation of unmanned aircraft systems in the ‹open› and ‹specific› categories (zit. EASA, Opinion) — Safe operations of drones in Europe, On Air Newsletter, Issue 20, 28. September 2018 (zit. EASA, On Air) — Acceptable Means of Compliance (AMC) and Guidance Material (GM) to Part-UAS, UAS operations in the ‹open› and ‹specific› categories, Issue 1, 9. Oktober 2019 (zit. EASA, GM Part-UAS) Literatur- und Quellenverzeichnis XXXI — Acceptable Means of Compliance (AMC) and Guidance Material (GM) to Commission Implementing Regulation (EU) 2019/947, Issue 1, 9. Oktober 2019 (zit. EASA, GM Implementing Regulation) — Flying a drone awareness leaflets (zit. EASA, awareness leaflets) — Opinions Information (zit. EASA, Opinions Information) Falcone Marco / Hahn Jörg / Burger Thomas, Galileo, in: Teunissen/ Montenbruck (Hrsg.), Springer Handbook of Global Navigation Satellite Systems, Cham 2017, S. 247 ff. Farber Hillary B., Eyes in the Sky: Constitutional and Regulatory Approaches to Domestic Drone Deployment, Syracuse L. Rev. 2013, S. 1 ff. Feiler Lukas / Forgó Nikolaus / Weigl Michaela, The EU General Data Protection Regulation (GDPR): A Commentary, Woking 2018 Finn Rachel L. / Wright David, Unmanned aircraft systems: Surveillance, ethics and privacy in civil applications, CLSR 28 (2012), S. 184 ff. Fluck Sara, Polizei mahnt — Dutzende private Drohnen über dem Züri- Fäscht, tagesanzeiger.ch, 8. Juli 2019 Flückiger Alexandre — Droits fondamentaux et vidéosurveillance par les particuliers et les autorités des espaces ouverts au public, in: Schwarzenegger/Nägeli (Hrsg.), Drittes Zürcher Präventionsforum: Videoüberwachung als Prävention?, Zürich 2011, S. 195 ff. (zit. Flückiger, vidéosurveillance) — L’autodétermination en matière de données personnelles: un droit (plus si) fondamental à l’ère digitale ou un nouveau droit de propriété?, AJP 2013, S. 837 ff. (zit. Flückiger, autodétermination) Frei Nula, Die Revision des Datenschutzgesetzes aus europarechtlicher Sicht, Jusletter 17. September 2018 Freudiger Patrick, Anstalt oder Aktiengesellschaft?, Zur Bedeutung der Rechtsform bei Ausgliederungen, Diss. Bern 2015, Bern 2016 Froomkin Michael A. / Colangelo P. Zak, Self-Defense Against Robots and Drones, 48 Conn. L. Rev. 2015 Nr. 1, S. 1 ff. Frowein Jochen Abr. / Peukert Wolfgang, Europäische Menschenrechtskonvention, EMRK-Kommentar, 3. Aufl., Kehl am Rhein 2009 Fumagalli Antonio, «Präsenz Schweiz» setzt auf Drohnen, Die PR-Agentur des Bundes will das Land noch stärker als Hochtechnologie-Standort positionieren, NZZ vom 4. Mai 2019, S. 31 XXXII Literatur- und Quellenverzeichnis Gächter Thomas, § 30 Allgemeine Grundrechtslehren, in: Biaggini/Gächter/Kiener (Hrsg.), Staatsrecht, 2. Aufl., Zürich/St. Gallen 2015, S. 437 ff. Gächter Thomas / Egli Philipp, Informationsaustausch im Umfeld der Sozialhilfe, Jusletter 6. September 2010 Gächter Thomas / Werder Gregori, Einbettung ausgewählter Konzepte in das schweizerische Datenschutzrecht, in: Belser/Fasnacht/Blaser (Hrsg.), Instrumente zur Umsetzung des Rechts auf informationelle Selbstbestimmung, Zürich/Basel/Genf 2013, S. 87 ff. Galli Peter / Moser André / Lang Elisabeth / Steiner Marc, Praxis des öffentlichen Beschaffungsrechts, Eine systematische Darstellung der Rechtsprechung des Bundes und der Kantone, 3. Aufl., Zürich 2013 (zit. Galli et al.) Gasser Urs, Das Recht neu codieren, NZZ vom 6. Februar 2016, S. 12 Gättelin Patrick / Müller Reto Patrick, Klare Regeln für Drohnen einsätze, ASMZ 2006/10, S. 44 f. Geiser Thomas / Fountoulakis Christiana (Hrsg.), Basler Kommentar, Zivilgesetzbuch I, Art. 1–456 ZGB, 6. Aufl., Basel 2018 (zit. BSK ZGB IBearbeiter / in, Art. xx N xx) Geiser Thomas / Uttinger Ursula, Keine Selbstjustiz gegen Drohnen, NZZ vom 7. Februar 2018, S. 9 Geiser Thomas / Wolf Stephan (Hrsg.), Basler Kommentar, Zivilgesetzbuch II, Art. 457–977 ZGB und Art. 1–61 SchlT ZGB, 6. Aufl., Basel 2019 (zit. BSK ZGB II-Bearbeiter/-in, Art. xx N xx) Germann Peter, Modellflug und Drohnenbetrieb: der Unterschied, AeroRevue 11/2016, S. 13 Gerschwiler Stefan, § 3 Prinzipien der Datenbearbeitung durch Privatpersonen und Behörden, in: Passadelis/Rosenthal/Thür (Hrsg.), Datenschutzrecht, Beraten in Privatwirtschaft und öffentlicher Verwaltung, Basel 2015, S. 73 ff. Giemulla Elmar, Unbemannte Luftfahrzeugsysteme — Probleme ihrer Einfügung in das zivile und militärische Luftrecht, ZLW 2/2007, S. 195 ff. Giemulla Elmar / Freiherr von Bothmer Bernhard, Ziviler Einsatz von unbemannten Luftfahrzeugen, CP 2013, S. 20 f. Literatur- und Quellenverzeichnis XXXIII Giemulla Elmar / van Schyndel Heiko / Friedl Achim, Gewerblicher und privater Einsatz von Drohnen: Regelung des Betriebs von unbemannten Fluggeräten, Köln 2018 Gierschmann Sibylle / Schlender Katharina / Stentzel Rainer / Veil Winfried (Hrsg.), Kommentar Datenschutz-Grundverordnung, Köln 2018 (zit. Bearbeiter/-in, in: Gierschmann et al., Art. xx DSGVO N xx) Giger Hans, SVG Kommentar, Strassenverkehrsgesetz mit weiteren Erlassen, 8. Aufl., Zürich 2014 (zit. OFK SVG-Giger, Art. xx N xx) Giuriato Luca, Durchzogener Heimelektronik Markt 2017 mit Lichtblicken, GfK Marktbericht Consumer Electronics, 8. Februar 2018 Glaneuski Jason, Unmanned Aircraft System (UAS) Service Demand 2015– 2035, September 2013 Glaus Bruno, Das Recht an der eigenen Stimme: informationelle Selbst bestimmung als Schranke der Medienfreiheit mit allgemeinen Geschäftsbedingungen für das Mediengespräch, Diss. Zürich 1997, Bern 1997 Gnehm Oliver, Das datenschutzrechtliche Auskunftsrecht, in: Epiney/ Nüesch (Hrsg.), Durchsetzung der Rechte der Betroffenen im Bereich des Datenschutzes, Zürich 2015, S. 77 ff. Gogarty Brendan / Hagger Meredith, The Laws of Man over Vehicles Unmanned: The Legal Response to Robotic Revolution on Sea, Land and Air, JLIS 2008, S. 73 ff. Gola Peter (Hrsg.), Datenschutz-Grundverordnung, VO (EU) 2016/679, Kommentar, 2. Aufl., München 2018 (zit. Bearbeiter/-in, in: Gola, Art. xx DSGVO N xx) González Fuster Gloria, The Emergence of Personal Data Protection as a Fundamental Right of the EU, Diss. Brüssel 2013, Cham et al. 2014 Gorkič Primož, The (F)utility of Privacy Laws: The Case of Drones?, in: Završnik (Hrsg.), Drones and Unmanned Aerial Systems: Legal and Social Implications for Security and Surveillance, Cham et al. 2016, S. 69 ff. Grabenwarter Christoph, Die Vorratsdatenspeicherung aus der Perspektive der EMRK, der Grundrechte-Charta und des Verfassungsrechts, in: Stuckenberg/Gärditz (Hrsg.), Strafe und Prozess im freiheitlichen Rechtsstaat: Festschrift für Hans-Ullrich Paeffgen zum 70. Geburtstag am 2. Juli 2015, Berlin 2015, S. 779 ff. XXXIV Literatur- und Quellenverzeichnis Grabenwarter Christoph / Pabel Katharina, Europäische Menschenrechtskonvention: Ein Studienbuch, 6. Aufl., München und Basel und Wien 2016 Graf Martin / Theler Cornelia / von Wyss Moritz, Parlamentsrecht und Parlamentspraxis der Schweizerischen Bundesversammlung, Kommentar zum Parlamentsgesetz (ParlG) vom 13. Dezember 2002, Basel 2014 (zit. Bearbeiter/-in, Komm. ParlG, Art. xx N xx) Granshaw Stuart I., RPV, UAV, UAS, RPAS… or just Drone?, The Photogrammetric Record 33(162), S. 160 ff. Graumann Horst, Die Drohne — Biene, Flugmodell oder …?, in: Benkö/Kröll (Hrsg.), Luft- und Weltraumrecht im 21. Jahrhundert, Liber Amicorum Karl-Heinz Böckstiegel, Köln et al. 2001, S. 62 ff. Grest Lauren, The drone revolution: can privacy and data protection legislation keep up with the technology?, C.T.L.R. 2015, 21(5), S. 120 ff. Griebel Thorsten, Schutz der Persönlichkeitsrechte vor Drohnenauf nahmen, InTeR 2019 S. 106 ff. Griffel Alain, § 31 Schutz von Person und Persönlichkeit, in: Biaggini/Gächter/Kiener (Hrsg.), Staatsrecht, 2. Aufl., Zürich/St. Gallen 2015, S. 468 ff. Groh Benedikt A., Entwicklung eines Rechtsrahmens zum Betrieb ziviler unbemannter Fluggeräte, Diss. Frankfurt (Oder) 2018, München 2019 Grosskopf Lambert, Aktiver Schutz gegen Medien-Drohnen: Was wird verletzt und wie kann es geschützt werden?, CR 11/2014, S. 759 ff. Haas Raphaël, Die Einwilligung in eine Persönlichkeitsverletzung nach Art. 28 AbS. 2 ZGB, Diss. Luzern 2007, Zürich 2007 Häfelin Ulrich / Haller Walter / Keller Helen / Thurnherr Daniela, Schweizerisches Bundesstaatsrecht, 9. Aufl., Zürich/Basel/Genf 2016 (zit. Häfelin et al.) Häfelin Ulrich / Müller Georg / Uhlmann Felix, Allgemeines Verwaltungsrecht, 7. Aufl., Zürich/St. Gallen 2016 Häfliger Markus, Von der Luftwaffe beim Kiffen erwischt, NZZ vom 23. Mai 2004, S. 14 Haller Ursina, Drohnen sollen mit Chip fliegen, nzz.ch, 14. Juli 2016 (zit. Haller U.) Literatur- und Quellenverzeichnis XXXV Haller Walter, Menschenwürde, Recht auf Leben und persönliche Freiheit, in: Merten/Papier (Hrsg.), HGR VII/2, Heidelberg und Zürich/ St. Gallen 2007, S. 199 ff. (zit. Haller W.) Hänni Peter, Staatshaftung bei unterlassener Wahrnehmung von Schutzpflichten: Grundsatzfragen und Rechtsprechung im Bereich von Menschenrechten, in: Rüssli/Hänni/Häggi Furrer (Hrsg.), Staats- und Verwaltungsrecht auf vier Ebenen: Festschrift für Tobias Jaag, Zürich/ Basel/Genf 2012, S. 429 ff. Hänni Peter / Stöckli Andreas, Schweizerisches Wirtschaftsverwaltungsrecht, Bern 2013 Hänsenberger Silvio — Wenn Drohnen vom Himmel fallen — luftrechtliche Haftungsfragen, AJP 2017, S. 163 ff. (zit. Hänsenberger, AJP 2017) — Die zivilrechtliche Haftung für autonome Drohnen unter Einbezug von Zulassungs- und Betriebsvorschriften, Diss. St. Gallen 2018, Berlin 2018 (zit. Hänsenberger, Diss.) Hänsenberger Silvio/Wildhaber Isabelle, Risiko im Anflug? Die Regulierung ziviler Drohnen, sui-generis 2016, S. 82 ff. Harasgama Rehana C., Erfahren — Wissen — Vergessen, Zur zeitlichen Dimension des staatlichen Informationsanspruches, Diss. St. Gallen 2017, Zürich/St. Gallen 2017 Harris David / O’Boyle Michael / Bates Ed / Buckley Carla, Harris, O’Boyle & Warbrick, Law of the European Convention on Human Rights, 4. Aufl., Oxford 2018 (zit. Harris et al.) Harris Matt / Given Peter, Privacy issues and drones: preparing for take- off, P. & D.P. 2014, S. 7 ff. Hassanalian Mostafa / Abdelkefi Abdessattar, Classification, applications, and design challenges of drones: A review, Progress in Aerospace Sciences 2017, S. 99 ff. Hausheer Heinz / Aebi-Müller Regina E., Das Personenrecht des Schweizerischen Zivilgesetzbuches, 4. Aufl., Bern 2016 Haustein Berthold H., Herausforderungen des Datenschutzrechtes vor dem Hintergrund aktueller Entwicklungen in der Robotik, in: Hilgendorf/Günther (Hrsg.), Robotik und Gesetzgebung: Beiträge der Tagung vom 7. bis 9. Mai 2012 in Bielefeld, Baden-Baden 2013, S. 93 ff. XXXVI Literatur- und Quellenverzeichnis Hegetschweiler Marcel, Drohnen im Dienste der Wirtschaft, Zürcher Wirtschaft 5/2019, S. 5 Henseler David — Die rechtliche Einordnung von Drohnen: Eine kritische Würdigung der heutigen Rechtslage und Überlegungen de lege ferenda, in: Canapa/Landolt/Müller (Hrsg.), Sein und Schein der Gesetzgebung: Erwartungen — Auswirkungen — Kritik, Zürich 2018, S. 115 ff. (zit. Henseler, Einordnung) — Von Drohnen und anderen Baupolizisten: Verfassungsrechtliche Überlegungen zum Einsatz von Drohnen zur Baukontrolle, Jusletter 25. Juni 2018 (zit. Henseler, Baupolizist) — Wenn die Digitalisierung Behörden lahmlegt …, Rechtliche Handlungsoptionen bei Untätigkeit einer Bundesbehörde — am Beispiel des Bundesamts für Zivilluftfahrt (BAZL), in: Dal Molin-Kränzlin/Schneuwly/ Stojanovic (Hrsg.), Digitalisierung und Recht, Zürich 2019, S. 183 ff. (zit. Henseler, Digitalisierung) Hertzberg Joachim / Lingemann Kai / Nüchter Andreas, Mobile Roboter: Eine Einführung aus Sicht der Informatik, Berlin/Heidelberg 2012 Hesse David, Surr mich nicht an, derbund.ch, 31. Mai 2017 Hilf Juliane / Umbach Klaus, The Commercial Use of Drones, An Overview of Regulatory Developments in the US and the EU, CRi 3/2015, S. 65 ff. Hodel Alois, Entwicklung der Landwirtschaft — Grosser Umbruch, BlAR 2016 Nr. 2-3, S. 17 ff. Hodgkinson David / Johnston Rebecca, Aviation Law and Drones: Unmanned Aircraft and the Future of Aviation, Oxford 2018 Honsell Heinrich (Hrsg.), Kurzkommentar OR, Basel 2014 (zit. KuKo ORBearbeiter/-in, Art. xx N xx) Honsell Heinrich / Vogt Nedim Peter / Watter Rolf (Hrsg.), Basler Kommentar, Obligationenrecht II, Art. 530–964 OR inkl. Schlussbestimmungen, 5. Aufl., Basel 2016 (zit. BSK OR II-Bearbeiter/-in, Art. xx N xx) Howley Kevin, Drones: Media Discourse and the Public Imagination, New York et al. 2018 Hrubesch-Millauer Stephanie/Bruggisser David, Sachenrechtliche Aspekte zum Einsatz von privaten Drohnen, Jusletter 11. August 2014 Huber Hans, Das Recht im technischen Zeitalter, Berner Rektoratsreden, Bern 1960 (zit. Huber H.) Literatur- und Quellenverzeichnis XXXVII Huber Martin, Drohnenflug bleibt straflos — weil bestimmte Augenzeugen fehlen, tagesanzeiger.ch, 8. Juli 2014 (zit. Huber M.) Huber Rolf, Rechtsprobleme der Personalakte, Diss. Zürich 1984, Zürich 1984 (zit. Huber R.) Husi-Stämpfli Sandra, DSG-Revision: Schritt in die richtige Richtung, digma 2017, S. 50 ff. Huttunen Mikko — Unmanned, Remotely Piloted, or Something Else?, Analysing the Ter minological Dogfight, Air & Space Law 2017, S. 349 ff. (zit. Huttunen, Unmanned) — The U-space Concept, Air & Space Law 2019, S. 69 ff. (zit. Huttunen, U-space) International Civil Aviation Organization (ICAO), Circular 328 AN/190, Unmanned Aircraft Systems (UAS), Montreal 2011 (zit. ICAO) International Transport Forum at the OECD, (Un)certain Skies?, Drones in the World of Tomorrow, Corporate Partnership Board Report, 22. Mai 2018 (zit. ITF/OECD) Isensee Josef — Das Grundrecht auf Sicherheit: Zu den Schutzpflichten des freiheitlichen Verfassungsstaates, Berlin/New York 1983 (zit. Isensee, Sicherheit) — § 191 Das Grundrecht als Abwehrrecht und als staatliche Schutzpflicht, in: Isensee/Kirchhof (Hrsg.), Handbuch des Staatsrechts der Bundesrepublik Deutschland, Band IX, Allgemeine Grundrechtslehren, 3. Aufl., Heidelberg 2011, S. 413 ff. (zit. Isensee, Handbuch, Rn. xx) Jackman Anna, Consumer drone evolutions: trends, spaces, temporalities, threats, Defense & Security Analysis, 24. Oktober 2019 Jöhri Yvonne, § 8 Aufgabe und Bedeutung der öffentlichen Datenschutzbeauftragten, in: Passadelis/Rosenthal/Thür (Hrsg.), Datenschutzrecht, Beraten in Privatwirtschaft und öffentlicher Verwaltung, Basel 2015, S. 245 ff. Joint Authorities for Rulemaking on Unmanned Systems (JARUS) — UAS Operational Categorization, Version 1.0, 21. Juni 2019 (zit. JARUS, Categorization) — Recommendations for Unmanned Aircraft Systems (UAS) Category A & Category B Operations, Version 1.0, 11. Juli 2019 (zit. JARUS, Operations) XXXVIII Literatur- und Quellenverzeichnis Jones Kevin D. / Platzer Max F., Flapping-Wing Propelled Micro Air Vehicles, in: Valavanis/Vachtsevanos (Hrsg.), Handbook of Unmanned Aerial Vehicles, Volume III, Heidelberg/New York/London 2015, S. 1359 ff. Josipovic Neven, Europäische Regulierung des Betriebs unbemannter Luftfahrzeuge, NVwZ 2019, S. 438 ff. Juniper Adam, Das Drohnen Praxisbuch, Kauf — Selbstbau — Foto & Video — Rennen, Heidelberg 2018 Kaiser Stefan A., Legal Aspects of Unmanned Aerial Vehicles, ZLW 3/2006, S. 343 ff. Kälin Walter / Künzli Jörg, Universeller Menschenrechtsschutz, Der Schutz des Individuums auf globaler und regionaler Ebene, 4. Aufl., Basel 2019 Karjoth Günther, Fähigkeiten der Gesichtserkennung, digma 2019, S. 6 ff. Karlen Peter — Das Grundrecht der Religionsfreiheit in der Schweiz, Diss. Zürich 1987, Zürich 1988 (zit. Karlen, Religionsfreiheit) — Schweizerisches Verwaltungsrecht, Zürich 2018 (zit. Karlen, Verwaltungsrecht) Karpenstein Ulrich / Mayer Franz C. (Hrsg.), Konvention zum Schutz der Menschenrechte und Grundfreiheiten: Kommentar, 2. Aufl., München 2015 (zit. Karpenstein/Mayer-Bearbeiter/-in, Art. xx EMRK N xx) Keller Benjamin, Ausbildung von Drohnen-Besatzungen in der Schweizer Luftwaffe, ASMZ 2013/7, S. 32 f. Kettiger Daniel — Das gerichtliche Verbot als Instrument zur Abwehr ziviler Drohnen, Jusletter 11. April 2016 (zit. Kettiger, Abwehr) — Rechtliche Rahmenbedingungen des Einsatzes von Drohnen zum Erheben von Geobasisdaten, cadastre Nr. 20, April 2016, S. 24 ff. (zit. Kettiger, Geobasisdaten) Kiener Regina — Die «Rote Zora», die Zuhälterbande und die Polizei: Wie das Zürcher Kassationsgericht (sinngemäss) Schutzpflichten anerkannte, in: Jositsch/ Schwarzenegger/Wohlers (Hrsg.), Festschrift für Andreas Donatsch, Zürich/Basel/Genf 2017, S. 619 ff. (zit. Kiener, «Rote Zora») — Zugang zur Justiz, ZSR 138 II, S. 5 ff. (zit. Kiener, Zugang) Literatur- und Quellenverzeichnis XXXIX — V.2 Grundrechte in der Bundesverfassung, in: Hertig/Schindler/Diggelmann (Hrsg.), Verfassungsrecht der Schweiz / Droit constitutionnel suisse, 2. Aufl., im Erscheinen (zit. Kiener, Grundrechte in der BV) — V.6 Grundrechtsschranken, in: Hertig/Schindler/Diggelmann (Hrsg.), Verfassungsrecht der Schweiz / Droit constitutionnel suisse, 2. Aufl., im Erscheinen (zit. Kiener, Grundrechtsschranken) Kiener Regina / Kälin Walter / Wittenbach Judith, Grundrechte, 3. Aufl., Bern 2018 Kiener Regina / Leuzinger Andreas, Verdeckte polizeiliche Massnahmen, in: Kiener/Bühler/Schindler (Hrsg.), Sicherheits- und Ordnungsrecht des Bundes, SBVR III/2, Basel 2018, S. 470 ff. Kiener Regina / Rütsche Bernhard / Kuhn Mathias, Öffentliches Verfahrensrecht, 2. Aufl., Zürich/St. Gallen 2015 Klauser Francisco / Pedrozo Silvana, Big data from the sky: popular perceptions of private drones in Switzerland, Geogr. Helv. 72 (2017), S. 231 ff. Klauser Francisco / Pauschinger Dennis / Pedrozo Silvana / Stuber Lea / Placi Rahel, Professional Drone Usage in Switzerland: Results of a Quantitative Survey of Public and Private Drone Users, MAPS Working Paper 2/2017, Neuenburg 2017 Klein Hans H., Die grundrechtliche Schutzpflicht, DVBl 1994, S. 489 ff. Kley Andreas, Der Grundrechtskatalog der nachgeführten Bundesverfassung — ausgewählte Neuerungen, ZBJV 1999, S. 301 ff. Klitou Demetrius, Privacy-Invading Technologies and Privacy by Design, Safeguarding Privacy, Liberty and Security in the 21st Century, Den Haag 2014 Koelle Marion / Brück Yvonne / Cobus Vanessa / Heuten Wilko / Boll Susanne, Respektvolle tragbare Kameras?, Technische Gestaltung einer sozialakzeptablen Nutzung von Datenbrillen und Smart Cams, DuD 2017, S. 152 ff. (zit. Koelle et al.) Kokott Juliane / Sobotta Christoph, The distinction between privacy and data protection, IDPL 2013 (4), S. 222 ff. XL Literatur- und Quellenverzeichnis Koops Bert-Jaap / Leenes Ronald, Privacy Regulation Cannot Be Hardcoded. A critical comment on the ‹privacy by design› provision in dataprotection law, International Review of Law, Computers & Technology Vol. 28 (2014), S. 159 ff. Kornmeier Claudia, Der Einsatz von Drohnen zur Bildaufnahme: Eine luftverkehrsrechtliche und datenschutzrechtliche Betrachtung, Diss. Freiburg i.Br. 2011, Berlin 2012 Kramer Ernst A., Juristische Methodenlehre, 6. Aufl., München und Wien und Bern 2019 Krumm Malte — Der neue europäische Rechtsrahmen für unbemannte Luftfahrzeuge — Anpassungsbedarf im deutschen Luftverkehrsrecht?, EuZW 2019, S. 114 ff. (zit. Krumm, Anpassungsbedarf) — Luftverkehrsrecht: Neuer europäischer Rechtsrahmen für unbemannte Luftfahrzeuge tritt in Kraft, EuZW 2019, S. 484 (zit. Krumm, Rechts rahmen) Kühling Jürgen, Datenschutz und die Rolle des Rechts, in: Stiftung Datenschutz (Hrsg.), Zukunft der informationellen Selbstbestimmung, Berlin 2016, S. 49 ff. Kühling Jürgen / Buchner Benedikt (Hrsg.), Datenschutz-Grundverordnung, Kommentar, München 2017 (zit. Bearbeiter/-in, in: Kühling/ Buchner, Art. xx DSGVO N xx) Kühling Jürgen / Sivridis Anastasios / Schwuchow Mathis / Thorben Burghardt, Das datenschutzrechtliche Vollzugsdefizit im Bereich der Telemedien — ein Schreckensbericht, DuD 6/2009, S. 335 ff. (zit. Kühling et al.) Kunz Peter V., Staatsbeteiligungen und ausgewählte Verantwortlichkeiten, GesKR 2018, S. 156 ff. Künzli Jörg, Zwischen Rigidität und Flexibilität: Der Verpflichtungsgrad internationaler Menschenrechte, Diss. Bern 1999, Berlin 2001 Kutschera Wolfgang, Rechtliche Grundlagen für die Verwendung von Drohnen in Österreich, Diss. Linz 2016, Linz 2016 Labiod Houda / Afifi Hossam / De Santis Costantino, WiFi, Bluetooth, Zigbee and WiMax, Dordrecht 2007 Literatur- und Quellenverzeichnis XLI Landrock Holm / Baumgärtel Anne, Die Industriedrohne — der fliegende Roboter, Professionelle Drohnen und ihre Anwendung in der Industrie 4.0, Wiesbaden 2018 Langner Dirk / Klindt Thomas / Schucht Carsten, Kapitel C. VI. Technische Sicherheitsvorschriften und Normen, in: Dauses/Ludwigs (Hrsg.), Handbuch des EU-Wirtschaftsrechts, 47. Ergänzungslieferung, März 2019, München 2019 Laumanns Horst W., Drohnen: seit 1990, Stuttgart 2012 Lavallée Chantal, The New EU Policy on Civil Drones, A Paradigm Shift for European Airspace, PRIO Policy Brief 5/2019, S. 1 ff. Lavrysen Laurens — Human Rights in a Positive State, Rethinking the Relationship between Positive and Negative Obligations under the European Convention on Human Rights, Cambridge/Antwerpen/Portland 2017 (zit. Lavrysen, Positive State) — System of Restrictions, in: van Dijk et al. (Hrsg.), Theory and Practice of the European Convention on Human Rights, 5. Aufl., Cambridge/Antwerpen/Portland 2018, S. 307 ff. (zit. Lavrysen, Restrictions) Lee Isabella, DJI Maintains 74% of Market Share, Three Key Insights from the Skylogic 2018 Drone Market Report, 19. September 2018 Lee Yeon-Hee, Neue Kriterien für einen Eingriff in die informationelle Selbstbestimmung, Diss. Hamburg 2016, Hamburg 2016 Leenes Ronald / Palmerini Erica / Koops Bert-Jaap / Bertolini Andrea / Salvini Pericle / Lucivero Federica, Regulatory challenges of robo tics: some guidelines for addressing legal and ethical issues, Law, Innovation and Technology, Vol. 9 Nr. 1, S. 1 ff. (zit. Leenes et al.) Leissler Günther, Drohnen und Datenschutz: ein europäisches Problem, ecolex 2015, S. 535 ff. Lenk Klaus, Der Staat am Draht: Electronic Government und die Zukunft der öffentlichen Verwaltung — eine Einführung, Berlin 2004 Leutert Stefan, Polizeikostentragung bei Grossveranstaltungen: eine Studie unter Berücksichtigung der Grundrechte, des Polizeirechts und des Abgaberechts, Diss. Zürich 2005, Zürich 2005 Lynskey Orla, The Foundations of EU Data Protection Law, Diss. Cambridge 2009, Oxford 2015 XLII Literatur- und Quellenverzeichnis Mahn-Gauseweg Carolin, Automated Warfare — Operationen unbemannter Systeme: Eine technische Systematisierung, in: Frau (Hrsg.), Drohnen und das Recht, Tübingen 2014, S. 1 ff. Mahon Pascal, Droit constitutionnel, Volume II, Droits fondamentaux, 3. Aufl., Basel/Neuenburg 2015 Malgieri Gianclaudio, La titolarità dei dati trattati per mezzo dei droni tra privacy e proprietà intellettuale, in: Palmerini/Biasiotti/Aiello (Hrsg.), Diritto dei Droni: Regole, questioni e prassi, Mailand 2018, S. 163 ff. Marsch Nikolaus, Das europäische Datenschutzgrundrecht, Grundlagen — Dimensionen — Verflechtungen, München 2018 Marte Daniel A. / Anania Emily C. / Rice Stephen / Mehta Rian / Milner Mattie N. / Winter Scott R. / Walters Nathan W. / Capps John / Ragbir Nadine K., What type of person supports 24/7 police drones over neighbourhoods? A regression analysis, JUAS 2018 Vol. 4 Nr. 1, S. 61 ff. (zit. Marte et al.) Marti Gian Andrea, Die Schweiz wappnet sich gegen Drohnen, NZZ vom 21. März 2018, S. 17 Martin Céline, Grundrechtskollisionen, Diss. Basel 2007, Basel 2007 Martini Mario, Neue Freunde und Helfer?, Drohnen als Mittel der Beobachtung von Grossveranstaltungen und Versammlungen, DÖV 18/2019, S. 732 ff. Masutti Anna, The Regulation of Unmanned Air System Use in Common Airspace, JLIS Special Edition: The Law of Unmanned Vehicles 2011/2012, S. 212 ff. Masutti Anna / Tomasello Filippo, International Regulation of NonMilitary Drones, Cheltenham/Northampton 2018 Matter Livia, Gesichtserkennung auf dem Vormarsch, digma 2019, S. 14 ff. Matthias Andreas, Automaten als Träger von Rechten. Plädoyer für eine Gesetzesänderung, Diss. Berlin 2007, Berlin 2008 Mauchle Yves, Die regulatorische Antwort auf FinTech: Evolution oder Revolution?, Eine Verortung aktueller Entwicklungen, SZW 2017, S. 810 ff. Maurer-Lambrou Urs / Blechta Gabor P., Datenschutzgesetz Öffentlichkeitsgesetz, Basler Kommentar, 3. Aufl., Basel 2014 (zit. BSK DSG-Bearbeiter/-in, Art. xx N xx) Literatur- und Quellenverzeichnis XLIII McNally Maya, Recht der Sicherung und der Finanzierung von Luftfahrzeugen, Unter besonderer Berücksichtigung des Übereinkommens über internationale Sicherungsrechte an beweglicher Ausrüstung und des Protokolls betreffend Besonderheiten der Luftfahrzeugausrüstung (Kapstadt 2011), Diss. Luzern 2009, Zürich/St. Gallen 2009 Medici Gabriela Naemi, Migrantinnen als Pflegehilfen in Schweizer Privathaushalten: Menschenrechtliche Vorgaben und staatliche Handlungspflichten, Diss. Zürich 2015, Zürich/Basel/Genf 2015 Mégret Frédéric, Nature of Obligations, in: Moeckli/Shah/Sivakumaran (Hrsg.), International Human Rights Law, 3. Aufl., Oxford 2018, S. 86 ff. Meier Philippe, Protection des données: Fondements, principes généraux et droit privé, Bern 2011 Meier-Hayoz Arthur / Forstmoser Peter / Sethe Rolf, Schweizerisches Gesellschaftsrecht, 12. Aufl., Bern 2018 Meili Andreas, Freiheit zwischen Gucklöchern und Scheinwerfern — Eine Standortbestimmung zum Persönlichkeitsschutz im digitalen Zeitalter, NZZ vom 24. August 2015, S. 46 Meili Katja, Grundlagen und Grundsätze der Luftfahrthaftpflicht, Zürich 2014 Mendes de Leon Pablo / Scott Benjamyn Ian, An Analysis of Unmanned Aircraft Systems Under Air Law, in: Završnik (Hrsg.), Drones and Unmanned Aerial Systems: Legal and Social Implications for Security and Surveillance, Cham et al. 2016, S. 185 ff. Mihalyi Jessica S., Risiken und Rechtspflichten: Das Zusammenspiel von 3D-Druck-Technologie und IT-Sicherheitsrecht, InTeR 2017, S. 193 ff. Moeckli Daniel, V.10 Persönlichkeitsschutz, in: Hertig/Schindler/Diggelmann (Hrsg.), Verfassungsrecht der Schweiz / Droit constitutionnel suisse, 2. Aufl., S. 477 ff., im Erscheinen Mohler Markus H.F. — Grundzüge des Polizeirechts in der Schweiz, Basel 2012 (zit. Mohler, Polizeirecht) — Staatliches Gewaltmonopol, Sicherheit & Recht 3/2012, S. 152 ff. (zit. Mohler, Gewaltmonopol) XLIV Literatur- und Quellenverzeichnis Moor Pierre / Bellanger François / Tanquerel Thierry, Droit administratif, Volume III: L’organisation des activités administratives. Les biens de l’Etat, 2. Aufl., Bern 2018 Mowbray Alastair R., The Development of Positive Obligations under the European Convention on Human Rights by the European Court of Human Rights, Oxford/Portland 2004 Müller Georg — Die Drittwirkung der Grundrechte, Überblick über den Stand der Diskussion in Lehre und Rechtsprechung, ZBl 1978, S. 233 ff. (zit. Müller G., Drittwirkung) — Rechtskenntnis und Gesetzessprache, in: Müller et al. (Hrsg.), Staats organisation und Staatsfunktionen im Wandel, Festschrift für Kurt Eichenberger zum 60. Geburtstag, Basel/Frankfurt a.M. 1982, S. 152 ff. (zit. Müller G., Rechtskenntnis) — § 204 Schutzwirkung der Grundrechte, in: Merten/Papier (Hrsg.), Handbuch der Grundrechte in Deutschland und Europa, Band VII/2, Grundrechte in der Schweiz und in Liechtenstein, Heidelberg 2007, S. 59 ff. (zit. Müller G., HdG VII/2) Müller Georg / Uhlmann Felix, Elemente einer Rechtssetzungslehre, 3. Aufl., Zürich 2013 Müller Jörg Paul — Die Grundrechte der Verfassung und der Persönlichkeitsschutz des Privatrechts, Diss. Bern 1964, Bern 1964 (zit. Müller J.P., Diss.) — Elemente einer schweizerischen Grundrechtstheorie, Bern 1982 (zit. Müller J.P., Elemente) — Verwirklichung der Grundrechte nach Art. 35 BV: Der Freiheit Chancen geben, Bern 2018 (zit. Müller J.P., Verwirklichung) Müller Lucien — Videoüberwachung in öffentlich zugänglichen Räumen — insbesondere zur Verhütung und Ahndung von Straftaten, Diss. St. Gallen 2011, Zürich/ St. Gallen 2011 (zit. Müller L., Diss.) — Private Videoüberwachung in öffentlich zugänglichen Räumen — Datenschutzrechtliche Aspekte, Sicherheit & Recht 2/2012, S. 63 ff. (zit. Müller L., Sicherheit & Recht 2/2012) Literatur- und Quellenverzeichnis XLV Murswiek Dietrich — Die staatliche Verantwortung für die Risiken der Technik: Verfassungsrechtliche Grundlagen und immissionsschutzrechtliche Ausformung, Berlin 1985 (zit. Murswiek, Verantwortung) — Freiheit und Freiwilligkeit im Umweltrecht, JZ 1988, S. 985 ff. (zit. Murswiek, Freiheit) Nadakavukaren Schefer Krista, Ein völkerrechtlicher Schutz der kollektiven Privatsphäre?, Der Schutz der Privatsphäre und die Anonymität im Zeitalter kommerzieller Drohnen, ZSR 2014 I, S. 259 ff. Niggli Marcel Alexander / Wiprächtiger Hans (Hrsg.), Basler Kommentar, Strafrecht (StGB/JStGB), 4. Aufl., Basel 2019 (zit. BSK StGB-Bearbeiter/-in, Art. xx N xx) Noll Peter, Gesetzgebungslehre, Reinbek bei Hamburg 1973 Nussbaum Hans Georg, Polizeiliche Massnahmen im Bereich des Zollwesens, in: Kiener/Bühler/Schindler (Hrsg.), Sicherheits- und Ordnungsrecht des Bundes, SBVR III/2, S. 232 ff. Oesch Matthias, Europarecht, Band I: Grundlagen, Institutionen, Verhältnis Schweiz–EU, 2. Aufl., Bern 2019 Paal Boris P. / Pauly Daniel A. (Hrsg.), Datenschutz-Grundverordnung, Beck’sche Kompakt-Kommentare, 2. Aufl., München 2018 (zit. Bearbeiter/-in, in: Paal/Pauly, Art. xx DSGVO N xx) Palmerini Erica, I Droni per uso civile nella prospettiva giuridica: Appunti per una sistemazione concettuale e normativa, in: Palmerini/Biasiotti/ Aiello (Hrsg.), Diritto dei Droni: Regole, questioni e prassi, Mailand 2018, S. 3 ff. Pärli Kurt, Observationen von Versicherten — Der Gesetzgeber auf Abwegen, recht 2018, S. 120 ff. Pauner Cristina / Viguri Jorge, A Legal Approach to Civilian Use of Drones in Europe. Privacy and Personal Data Protection Concerns, Democrazia e Sicurezza 2015 Nr. 3, S. 85 ff. Pedrazzini Mario M. — Die Grundlagen des Datenschutzes im Privatbereich: die Grundzüge und der Geltungsbereich des Bundesgesetzes, in: Schweizer (Hrsg.), Das neue Datenschutzrecht des Bundes, Zürich 1993, S. 19 ff. (zit. Pedrazzini, Grundlagen) XLVI Literatur- und Quellenverzeichnis — Der Rechtsschutz der betroffenen Personen gegenüber privaten Bearbeitern (Klagen, vorsorgliche Massnahmen, Gerichtsstand), in: Schweizer (Hrsg.), Das neue Datenschutzrecht des Bundes, Zürich 1993, S. 81 ff. (zit. Pedrazzini, Rechtsschutz) Pedrozo Silvana, Swiss military drones and the border space: a critical study of the surveillance exercised by border guards, Geogr. Helv. 72 (2017), S. 97 ff. Pedrozo Silvana / Klauser Francisco, Drones d’utilité publique: Une acceptabilité controversée, EspacesTemps.net, 13. Juli 2018 Perritt Henry H. / Sprague Eliot O. — Drones, Vanderbilt Journal of Entertainment and Technology Law 3/2015, S. 673 ff. (zit. Perritt / Sprague, JETLaw) — Law Abiding Drones, STLR 2015, S. 385 ff. (zit. Perritt / Sprague, STLR) — Domesticating Drones: The Technology, Law, and Economics of Unmanned Aircraft, Oxford/New York 2017 (zit. Perritt/Sprague, Domes ticating Drones) Peter James T., Das Datenschutzgesetz im Privatbereich: Unter besonderer Berücksichtigung seiner motivationalen Grundlage, Diss. Zürich 1994, Zürich 1994 Pétermann Nathanaël, Les obligations positives de L’Etat dans la juris prudence de la Cour européenne des droits de l’homme: Théorie générale, incidences législatives et mise en œuvre en droit suisse, Diss. Lausanne 2014, Bern 2014 Peterson Mark Edward, The UAV and the Current and Future Regulatory Construct for Integration into the National Airspace System, Journal of Air Law and Commerce 2006, S. 521 ff. Plath Kai-Uwe (Hrsg.), DSGVO/BDSG, Kommentar zu DSGVO, BDSG und den Datenschutzbestimmungen von TMG und TKG, 3. Aufl., Köln 2018 (zit. Bearbeiter/-in, in: Plath, Art. xx DSGVO N xx) Plücken Milan A., Unbemannte Luftfahrzeugsysteme: Zulassungsvorgaben und -vorschriften der ICAO bzw. der EU, Diss. Köln 2016, Köln 2017 Poledna Thomas / Codoni Stefano, Sanktionierung von Informationsrechtsverletzungen, in: Baeriswyl/Rudin (Hrsg.), Perspektive Datenschutz, Praxis und Entwicklungen in Recht und Technik, Zürich 2002, S. 265 ff. Literatur- und Quellenverzeichnis XLVII Poltier Etienne, Droit des marchés publics, Bern 2014 Popper Ben, Our favorite drone crash-landed at the White House, 26. Januar 2015 Post CH AG — Weitere Drohnenflüge in Lugano, Medienmitteilung vom 4. Oktober 2017 (zit. Post, Medienmitteilung 4. Oktober 2017) — Drohnen, Längst keine Vision mehr (zit. Post, Drohnen) — Abgestürzte Post-Drohne ist geborgen, Medienmitteilung vom 28. Januar 2019 (zit. Post, Medienmitteilung 28. Januar 2019) — Post nimmt Drohnenflüge im Dienste der Medizin wieder auf, Mitteilung vom 12. April 2019 (zit. Post, Mitteilung 12. April 2019) Powers Caitlin / Mellinger Daniel / Kumar Vijay, Quadromotor Kinematics and Dynamics, in: Valavanis/Vachtsevanos (Hrsg.), Handbook of Unmanned Aerial Vehicles, Volume I, Heidelberg/New York/London 2015, S. 307 ff. Preznuk David, The Drone Age: A Primer for Individuals and the Enterprise, Haymarket 2016 Pugin Catherine, Ferngelenkte fliegende Kisten: Kurzfassung der TASWISS-Studie «Zivile Drohnen — Herausforderungen und Perspektiven», Bern 2018 Pulver Bernhard, Die Verbindlichkeit staatlicher Schutzpflichten — am Beispiel des Arbeitsrechts, AJP 2005, S. 413 ff. Pytlik Zillig Lisa M. / Duncan Brittany / Elbaum Sebastian / Detweiler Carrick, A Drone by Any Other Name, IEEE Technology and Science Magazine, März 2018, S. 80 ff. (zit. Pytlik Zillig et al.) Rao Bharat / Gopi Ashwin G. / Maione Romana, The societal impact of commercial drones, Technology in Society 45 (2016), S. 83 ff. Rebane Gerli, EASA Basic Regulation, Improving the Performance of the European Aviation Safety System, ZLW 2019, S. 51 ff. Rehbinder Manfred, Rechtssoziologie, 8. Aufl., München 2014 Reimers Andreas, Multikopter-Einsatz am Beispiel Rangierbahnhof Limmat tal SBB, cadastre Nr. 19, Dezember 2015, S. 14 ff. Rensmann Thilo, Wertordnung und Verfassung, Das Grundgesetz im Kontext grenzüberschreitender Konstitutionalisierung, Tübingen 2007 XLVIII Literatur- und Quellenverzeichnis Revnivykh Sergey / Bolkunov Alexey / Serdyukov Alexander / Montenbruck Oliver, GLONASS, in: Teunissen/Montenbruck (Hrsg.), Springer Handbook of Global Navigation Satellite Systems, Cham 2017, S. 219 ff. (zit. Revnivykh et al.) Rhinow René / Schefer Markus / Uebersax Peter, Schweizerisches Verfassungsrecht, 3. Aufl., Basel 2016 Rice Stephen / Tamilselvan Gajapriya / Winter Scott R. / Milner Mattie N. / Anania Emily C. / Sperlak Lauren / Marte Daniel A., Public perception of UAS privacy concerns: a gender comparison, JUVS 2018 Vol. 6 Nr. 2, S. 83 ff. (zit. Rice et al.) Riedi Severin, Die technischen Normen der Internationalen Organisation für Zivilluftfahrt (ICAO), Diss. Bern 2014, Bern 2015 Roma Alfredo, Remotely Piloted Aircraft Systems: Privacy and Data Protection Implications, ASJ XXIII (2014) Nr. 1, S. 22 ff. Rose Eugen, Datenbrillen, Drohnen, Dashcams …, Smart Cams im öffentlichen Raum allein durch Rechtsprechung nicht beherrschbar, DuD 2017, S. 137 ff. Rosenthal David — Der Entwurf für ein neues Datenschutzgesetz, Jusletter 27. November 2017 (zit. Rosenthal, Entwurf) — Controller oder Processor: Die datenschutzrechtliche Gretchenfrage, Jusletter 17. Juni 2019 (zit. Rosenthal, Controller) Rosenthal David / Jöhri Yvonne, Handkommentar zum Datenschutz gesetz sowie weiteren, ausgewählten Bestimmungen, Zürich 2008 (zit. Handkomm. DSG-Bearbeiter/-in, Art. xx N xx) Rothen Eduard, Haftpflicht- und Versicherungsrecht — Gegenverkehrs kollisionen, in: Schaffhauser (Hrsg.), Jahrbuch zum Strassenverkehrsrecht 2015, Bern 2015, S. 49 ff. Rudin Beat, Datenschutzreform in der Schweiz: EU-Nachvollzug — idealistisch und realpolitisch, in: Breitenmoser/Lagodny/Uebersax (Hrsg.), Schengen und Dublin in der Praxis: Aktuelle Herausforderungen, Zürich/St. Gallen 2018, S. 215 ff. Rudin Beat / Baeriswyl Bruno (Hrsg.), Praxiskommentar zum Informations- und Datenschutzgesetz des Kantons Basel-Stadt (IDG), Zürich/ Basel/Genf 2014 (zit. PK IDG/BS-Bearbeiter/-in, Art. xx N xx) Literatur- und Quellenverzeichnis XLIX Ruegg Jean / Flückiger Alexandre / November Valérie / Klauser Francisco, Vidéosurveillance et risques dans l’espace à usage public: Représentations des risques, régulation sociale et liberté de mouve ment, Genf 2006 (zit. Ruegg et al.) Ruijten Peter A. M. / Cuijpers Raymond H., If Drones Could See: Investigating Evaluations of a Drone with Eyes, in: Shuzhi et al. (Hrsg.), 10th International Conference on Social Robotics 2018, Cham 2018, S. 65 ff. Rupp Martin, Die grundrechtliche Schutzpflicht des Staates für das Recht auf informationelle Selbstbestimmung im Pressesektor, Diss. Saar brücken 2013, Saarbrücken 2013 Saeed Saim, Europe buys Chinese drones, even as US expresses data concerns, EU countries are using commercial Chinese drones and experts say that rings alarm bells, 9. September 2019 Sägesser Thomas (Hrsg.), Die Bundesbehörden: Bundesversammlung — Bundesrat — Bundesgericht, Kommentar, Beiträge und Materialien zum 5. Titel der schweizerischen Bundesverfassung, Bern 2000 (zit. Bearbeiter/-in, in: Sägesser, Art. xx BV N xx) Saladin Peter, Grundrechte im Wandel, Die Rechtsprechung des Schweizerischen Bundesgerichts zu den Grundrechten in einer sich ändernden Umwelt, 3. Aufl., Bern 1982 Salter Michael, Toys for the Boys? Drones, Pleasure and Popular Culture in the Militarisation of Policing, Crit Crim 22 (2014), S. 163 ff. Sarrión Esteve Joaquín, El régimen jurídico de la utilización de los drones. Una aproximación multinivel a la legislación euoropea y española, REJP Nr. 12, Juni 2017, S. 103 ff. Sasse Christoph, Sinn und Unsinn des Datenschutzes, Karlsruhe/Heidelberg 1975 Savary Fiona, Regulierung dominanter Internetplattformen: Eigenheiten, Herausforderungen und Lösungsansätze im Licht von Ökonomie, Innovation und Recht, Diss. St. Gallen 2017, Zürich/St. Gallen 2017 Schaffhauser René / Peter Erich, Die Betriebsgefahr: Konkretes Schädigungspotential — abstrakt bemessen?, in: Schaffhauser (Hrsg.), Jahrbuch zum Strassenverkehrsrecht 2014, Bern 2014, S. 35 ff. Schaub Hans-Peter / Leuzinger Lukas, Die Stimmbeteiligung an der Glarner Landsgemeinde, LeGes 29 (2018) 1, Nr. 2 L Literatur- und Quellenverzeichnis Schefer Markus — Die Kerngehalte von Grundrechten: Geltung, Dogmatik, inhaltliche Ausgestaltung, Bern 2001 (zit. Schefer, Kerngehalte) — Gefährdung von Grundrechten: Eine grundrechtsdogmatische Skizze, in: Sutter-Somm et al. (Hrsg.), Risiko und Recht, Festgabe zum Schweizerischen Juristentag 2004, Basel/Genf/ München 2004, S. 441 ff. (Schefer, Gefährdung) Scheidt David H., Command and Control of Autonomous Unmanned Vehic les, in: Valavanis/Vachtsevanos (Hrsg.), Handbook of Unmanned Aerial Vehicles, Volume III, Heidelberg/New York/London 2015, S. 1273 ff. Scheurer Martin, Spielerisch selbstbestimmt, Rechtskonforme Einwilligungserklärungen in Zeiten ubiquitärer Digitalisierung, Diss. Passau 2019, Berlin 2019 Schladebach Marcus, Luftrecht, 2. Aufl., Tübingen 2018 Schmid Alain / Schmidt Kirsten Johanna / Zech Herbert, Rechte an Daten — zum Stand der Diskussion, sic! 2018, S. 627 ff. Schmid Alexander, IT- und Rechtssicherheit automatisierter und vernetzter cyber-physischer Systeme, Event Data Recording und integrierte Produktbeobachtung als Massnahmen der IT-Risikominimierung am Beispiel automatisierter und vernetzter Luft- und Strassenfahrzeuge, Diss. Passau 2018, Berlin 2019 Schneider-Marfels Jascha / Kaufmann Sebastian, Abschuss von zivilen Drohnen unter dem Aspekt des Persönlichkeitsschutzes: Wie weit muss man es sich gefallen lassen, von einem solchen Flugobjekt überwacht, gefilmt und ausspioniert zu werden?, medialex 3/2018, S. 2 ff. Schrepfer Thomas W., Datenschutz und Verfassung, Bern/Frankfurt a.M./ New York 1985 Schubert Francis, Le droit aérien, Genf/Zürich/Basel 2017 Schucan Jürg, Datenbanken und Persönlichkeitsschutz, Diss. Zürich 1977, Zürich 1977 Schucht Carsten, Moderne Mobilität im Fokus des europäischen Technikrechts: Herausforderungen, Schnittstellen und Weichenstellungen, EuZW 2018, S. 141 ff. Literatur- und Quellenverzeichnis LI Schwartmann Rolf / Jaspers Andreas / Thüsing Gregor / Kugelmann Dieter (Hrsg.), DS-GVO/BDSG, Heidelberger Kommentar, Heidelberg 2018 (zit. Bearbeiter/-in, in: Schwartmann et al., Art. xx DSGVO N xx) Schweizer Michael, Recht am Wort, Schutz des eigenen Wortes im System von Art. 28 ZGB, Diss. Luzern 2011, Bern 2012 (zit. Schweizer M.) Schweizer Rainer J. / Rechsteiner David, § 2 Grund- und menschenrechtlicher Datenschutz, in: Passadelis/Rosenthal/Thür (Hrsg.), Datenschutzrecht, Beraten in Privatwirtschaft und öffentlicher Verwaltung, Basel 2015, S. 41 ff. Schweizerische Sicherheitsuntersuchungsstelle (SUST) — Grenchen Flugplatz SO, Summarischer Bericht 17. Oktober 2018 (zit. SUST, Bericht Grenchen) — Rund 500 m nordöstlich der Landiwiese, Zürichsee ZH, Summarischer Bericht vom 11. April 2019 (zit. SUST, Bericht Zürichsee) Schwenk Walter / Giemulla Elmar, Handbuch des Luftverkehrsrechts, 5. Aufl., Köln 2019 Senn Carlo, Die Blutprobe kommt auf dem Luftweg, Der Bund, 6. Juni 2018, S. 15 Severoni Cecilia — La disciplina normativa attuale degli aeromobili a pilotaggio remoto, Diritto dei trasporti 2016 I, S. 65 ff. (zit. Severoni, Diritto dei transporti) — Il regime di responsabilità per l’esercizio dei mezzi a pilotaggio remoto, in: Palmerini/Biasiotti/Aiello (Hrsg.), Diritto dei Droni: Regole, questioni e prassi, Mailand 2018, S. 69 ff. (zit. Severoni, responsabilità) Siemen Birte, Datenschutz als europäisches Grundrecht, Diss. Kiel 2004, Berlin 2006 Simitis Spiros / Hornung Gerrit / Spieker genannt Döhmann Indra (Hrsg.), NomosKommentar, Datenschutzrecht, DSGVO mit BDSG, Baden- Baden 2019 (Bearbeiter/-in, in: Simitis et al., Art. xx DSGVO N xx) Simon Jürg W., Amtshilfe: Allgemeine Verpflichtungen, Schranken und Grundsätze, Diss. Bern 1991, Chur/Zürich 1991 Singh Adi / Carthew James / Xiong Weifeng, A Zero-Cost Solution for Remote Identification and Tracking of sUAS in Low Altitude Flights, A Ford Motor Company Whitepaper, März 2018 LII Literatur- und Quellenverzeichnis Single European Sky ATM Research Programme (SESAR) — European Drones Outlook Study: Unlocking the value for Europe, November 2016 (zit. SESAR, Outlook Study) — European ATM Master Plan: Roadmap for the safe integration of drones into all classes of airspace, 8. März 2018 (zit. SESAR, Master Plan) Solmecke Christian / Nowak Fabian, Zivile Drohnen — Probleme ihrer Nutzung, MMR 7/2014, S. 431 ff. Solove Daniel J. — Understanding Privacy, Cambridge (MA)/London 2008 (zit. Solove, Privacy) — Nothing to Hide: The False Tradeoff between Privacy and Security, New Haven/London 2011 (zit. Solove, Nothing to Hide) — Privacy y Design: 4 Key Points, PinG 2015, S. 191 f. (zit. Solove, Privacy by Design) Solove Daniel J. / Schwartz Paul M., Privacy, Information, and Technology, 3. Aufl., New York 2011 Spies Axel, USA: Schon über 400’000 Hobby-Drohnen online registriert, ZD 2016, S. 201 f. Sprecher Franziska, Datenschutz und Big Data im Allgemeinen und im Gesundheitsrecht im Besonderen, ZBJV 2018, S. 482 ff. Sprecher Franziska / Lienhard Andreas / Tschannen Pierre / Tschentscher Axel / Zeller Franz, Die staatsrechtliche Rechtsprechung des Bundesgerichts in den Jahren 2017 und 2018, ZBJV 2018, S. 641 ff. (zit. Sprecher et al.) Städele Julius Ph., Völkerrechtliche Implikationen des Einsatzes bewaffneter Drohnen, Diss. Bielefeld 2014, Berlin 2014 Steiger Martin, Regulierung von Drohnen im zivilen Behördeneinsatz in der Schweiz, Sicherheit & Recht 3/2014, S. 169 ff. (zit. Steiger, Regulierung) Steiger Martin, Sicherheitsesoterik statt Menschenrechte, digma 2015, S. 134 ff. (zit. Steiger, Sicherheitsesoterik) Steinauer Paul-Henri / Fountoulakis Christiana, Droit des personnes physiques et de la protection de l’adulte, Bern 2014 Literatur- und Quellenverzeichnis LIII Stinner Julia — Informationelle Selbstbestimmung und Grenzen rechtlicher Regulierung, in: Stiftung Datenschutz (Hrsg.), Zukunft der informationellen Selbstbestimmung, Berlin 2016, S. 151 ff. (zit. Stinner, Selbstbestimmung) — Staatliche Schutzpflichten im Rahmen informationstechnischer Systeme, Diss. Bonn 2017, Baden-Baden 2018 (zit. Stinner, Schutzpflichten) Stöcker Claudia / Bennett Rohan / Nex Francesco / Gerke Markus / Zevenbergen Jaap, Review of the Current State of UAV Regulations, Remote Sens. 2017 Nr. 45, S. 1 ff. (zit. Stöcker et al.) Straub Sebastian, Beobachtung aus der Luft — Grenzen des staatlichen und zivilen Drohneneinsatzes, Oldenburg 2018 Stumvoll Konstantin, Die Drohne — Das unbemannte Luftfahrzeug im Völkerrecht, Diss. Bonn 2014, Hamburg 2015 Sydow Gernot (Hrsg.), Europäische Datenschutzgrundverordnung, Handkommentar, Baden-Baden 2018 (zit. Bearbeiter/-in, in: Sydow, Art. xx DSGVO N xx) Taeger Jürgen / Gabel Detlev (Hrsg.), DSGVO — BDSG, Kommentar, 3. Aufl., Frankfurt a.M. 2019 (zit. Bearbeiter/-in, in: Taeger/Gabel, Art. xx DSGVO N xx) Tamò-Larrieux Aurelia, Designing for Privacy and its Legal Framework, Data Protection by Design and Default for the Internet of Things, Diss. Zürich 2018, Cham 2018 Tegler Eric, What Happens When a Drone Crashes Into Your Face?, An 18-month-long study attempts to find out., 22. August 2019 Tercier Pierre, Le nouveau droit de la personnalité, Zürich 1984 Thomasen Kristen M.J. — Beyond Airspace Safety: A Feminist Perspective on Drone Privacy Regulation, 22. März 2018 (zit. Thomasen, Beyond Airspace Safety) — Flying Between the Lines: Drone Laws and the (Re)Production of Public Spaces, in: Hilgendorf/Seidel (Hrsg.), Robotics, Autonomics, and the Law, Baden-Baden 2018, S. 205 ff. (zit. Thomasen, Between the Lines) Tombal Thomas, Les droits de la personne concernée dans la RGDP, in: de Terwangne/Rosier (Hrsg.), Le Règlement Général sur la Protection des Données (RGPD/GDPR), Analyse approfondie, Brüssel 2018, S. 407 ff. LIV Literatur- und Quellenverzeichnis Trüeb Hans Rudolf, Bundesgesetz über das öffentliche Beschaffungswesen (BöB), in: Oesch/Weber/Zäch (Hrsg.), Wettbewerbsrecht II, Kommentar, Zürich 2011 (zit. Trüeb, Art. xx BöB N xx) Tschannen Pierre, Staatsrecht der Schweizerischen Eidgenossenschaft, 4. Aufl., Bern 2016 Tschentscher Axel / Lienhard Andreas / Sprecher Franziska, Öffentliches Recht: Verfassungsrecht, Verwaltungsrecht, öffentliches Verfah rensrecht, 2. Aufl., Zürich/St. Gallen 2019 Unabhängiges Datenschutzzentrum Saarland, 25. Tätigkeitsbericht 2013/2014, Saarbrücken, April 2015 Unmanned Airspace, US, Swiss flight trials «show multiple UTM providers can share remote ID services via ASTM standards», 19. September 2019 Unruh Peter, Zur Dogmatik der grundrechtlichen Schutzpflichten, Berlin 1996 Uriante Landa Iñaki, Ámbito de aplicación material, in: Piñar Mañas (Hrsg.), Reglamento General de Protección de Datos, Hacia un nuevo modelo europeo de privacidad, Madrid 2016, S. 63 ff. Uschkereit Tim / Zdanowiecki Konrad, Rechtsrahmen für den Betrieb ziviler Drohnen, NJW 2016 Heft 7, S. 444 ff. Valavanis Kimon P. / Vachtsevanos George J., UAV Autonomy: Introduction, in: dies. (Hrsg.), Handbook of Unmanned Aerial Vehicles, Volume III, Heidelberg/New York/London 2015, S. 1723 ff. Van Gyseghem Jean-Marc, Les catégories particulières de données à caractère personnel, in: de Terwangne/Rosier (Hrsg.), Le Règlement Général sur la Protection des Données (RGPD/GDPR), Analyse approfondie, Brüssel 2018, S. 255 ff. Vasella David — Zur Freiwilligkeit und zur Ausdrücklichkeit der Einwilligung im Datenschutzrecht, Jusletter 16. November 2015 (zit. Vasella, Jusletter) — A‑3548/2018: Entscheid des BVGer i.S. Helsana+, 29. März 2019 (zit. Vasella, Helsana+) — Auftragsbearbeitung im Privatbereich, Zu Begriff, Anforderungen und Rechtsfolgen der arbeitsteiligen Datenbearbeitung, digma 2019, S. 110 ff. (zit. Vasella, Auftragsbearbeitung) Literatur- und Quellenverzeichnis LV Vasella David / Sievers Jacqueline, Der «Swiss Finish» im Vorentwurf des DSG, digma 2017, S. 44 ff. Vergouw Bas / Nagel Huub / Bondt Geert / Custers Bart, Drone Tech nology: Types, Payloads, Applications, Frequency Spectrum Issues and Future Developments, in: Custers (Hrsg.), The Future of Drone Use: Opportunities and Threats from Ethical and Legal Perspectives, Den Haag 2016, S. 21 ff. (zit. Vergouw et al.) Vivet Laura / Smith Lauren, Drones and Privacy by Design: Embedding Privacy Enhancing Technology in Unmanned Aircraft, Future of Privacy Forum Report, 2. August 2016 Vladeck David C., Machines without Principles: Liability Rules and Artificial Intelligence, Wash. L. Rev. 2014, S. 117 ff. Voigt Paul / von dem Bussche Axel, The EU General Data Protection Regulation (GDPR): A Practical Guide, Cham 2017 Voland Thomas / Qiu Stephan, Nochmals: «Abgehobene Mobilität», Zum neuen europäischen Rechtsrahmen für Drohnen, insbesondere für «fliegende Autos», NZV 2019, S. 344 ff. Waldmann Bernhard / Belser Eva Maria / Epiney Astrid (Hrsg.), Basler Kommentar, Bundesverfassung, Basel 2015 (zit. BSK BV-Bearbeiter/-in, Art. xx N xx) Waldmeier Stefanie-Daniela, Informationelle Selbstbestimmung — ein Grundrecht im Wandel?, Diss. Zürich 2015, Zürich 2015 Wang Yang / Xia Huichuan / Yao Yaxing / Huang Yun, Flying Eyes and Hidden Controllers: A Qualitative Study of People’s Privacy Perceptions of Civilian Drones in the US, Proceedings on Privacy Enhancing Technologies 2016 Nr. 3, S. 172 ff. (zit. Wang et al.) Weber Rolf H., Zivile Drohneneinsätze — Herausforderungen für das Wirtschafts- und Datenschutzrecht, in: Widmer (Hrsg.), Der rasante technologische Wandel — Überforderung der Gesellschaft?, Zürich/St. Gallen 2017, S. 1 ff. Weber Rolf H. / Laux Christian / Oertly Dominic, Datenpolitik als Rechts thema: Agenda für Open Government Data, Zürich/Basel/Genf 2016 Weber Rolf H. / Oertly Dominic, Datenschutzrechtliche Problemfelder von zivilen Drohneneinsätzen, Jusletter 26. Oktober 2015 LVI Literatur- und Quellenverzeichnis Weber-Dürler Beatrice, Grundrechtseingriffe, in: Zimmerli (Hrsg.), Die neue Bundesverfassung: Konsequenzen für Praxis und Wissenschaft, BTJP 1999, Bern 2000, S. 131 ff. Weiss Andreas, A. Einführung, Begrifflichkeiten und Marktentwicklung, in: Hilber (Hrsg.), Handbuch Cloud Computing, Köln 2014, S. 1 ff. Weissenberger Philippe, Kommentar Strassenverkehrsgesetz und Ordnungsbussengesetz, Mit Änderungen nach Via Sicura, 2. Aufl., Zürich/ St. Gallen 2014 (zit. Komm. SVG-Weissenberger, Art. xx N xx) Wermelinger Amédéo, Informationelle Amtshilfe: Verunmöglicht Datenschutz eine effiziente Leistungserbringung durch den Staat?, Analyse des eidgenössischen und des luzernischen Rechts, ZBl 2004, S. 173 ff. Werro Franz / Schmidlin Irène, La protection de la personnalité et les medias: une illustration de la rencontre du droit civil et du droit constitutionnel, in: Werro (Hrsg.), Droit civil et convention européenne des droits de l’homme / Zivilrecht und Europäische Menschenrechtskonvention, Zürich 2006, S. 161 ff. Widmer Lüchinger Corinne / Oser David (Hrsg.), Basler Kommentar, Obligationenrecht I, Art. 1–529 OR, 7. Aufl., Basel 2019 (zit. BSK OR I-Bear beiter/-in, Art. xx N xx) Widmer Michael, § 5 Rechte der Datenschutzsubjekte, in: Passadelis/ Rosenthal/Thür (Hrsg.), Datenschutzrecht, Beraten in Privatwirtschaft und öffentlicher Verwaltung, Basel 2015, S. 149 ff. Wiederkehr René — Die Kerngehaltsgarantie am Beispiel kantonaler Grundrechte, Zugleich ein Beitrag zu den Grundrechten als Staatsaufgaben und zu den Grundrechtverwirklichungsbestimmungen, Diss. St. Gallen 1999, Bern 2000 (zit. Wiederkehr, Diss.) — Bundesgericht, I. öffentlich-rechtliche Abteilung, Urteil vom 3.5.2000, Ruth Gonseth c. Stadtrat Liestal, Regierungsrat und Verwaltungsgericht des Kantons Basel-Landschaft, BGE 126 II 300 ff., Verwaltungsgerichtsbeschwerde., Bemerkungen, AJP 2001, S. 215 ff. (zit. Wiederkehr, AJP 2001) Winter Scott R. / Rice Stephen / Tamilselvan Gajapriya / Tokarski Russell, Mission-based citizen views on UAV usage and privacy: an affective perspective, JUVS 2016 Vol. 4 Nr. 2, S. 125 ff. (zit. Winter et al.) Literatur- und Quellenverzeichnis LVII Wright David / Finn Rachel L., Making Drones More Acceptable with Privacy Impact Assessments, in: Custers (Hrsg.), The Future of Drone Use: Opportunities and Threats from Ethical and Legal Perspectives, Den Haag 2016, S. 325 ff. Xenos Dimitris, The Positive Obligations of the State under the European Convention on Human Rights, Abingdon 2012 Zhang WEIxuan / Mueller Mark W. / D’Andrea Raffaello, A controllable flying vehicle with a single moving part, 2016 IEEE International Conference on Robotics and Automation, 9. Juni 2016 LIX Materialienverzeichnis I. Bundesbehörden Anfrage 18.1044, Leutenegger Oberholzer Susanne (Drohnen) Bericht des Bundesrates über die Evaluation des Bundesgesetzes über den Datenschutz vom 9. Dezember 2011, BBl 2012 335 ff. (zit. Bericht Evaluation DSG) Bericht 2016 über die Luftfahrtpolitik der Schweiz (Lupo 2016) vom 24. Fe bruar 2016, BBl 2016 1847 ff. (zit. Bericht Lupo 2016) Botschaft des Bundesrates an die Bundesversammlung zum Entwurf eines Bundesgesetzes über die Luftfahrt vom 28. März 1945, BBl 1945 I 341 ff. (zit. Botschaft LFG) Botschaft über die Änderung des Schweizerischen Zivilgesetzbuches (Persönlichkeitsschutz: Art. 28 ZGB und 49 OR) vom 5. Mai 1982, BBl 1982 II 636 ff. (zit. Botschaft Persönlichkeitsschutz) Botschaft zum Bundesgesetz über den Datenschutz (DSG) vom 23. März 1988, BBl 1988 II 413 ff. (Botschaft DSG 1988) Botschaft über eine neue Bundesverfassung vom 20. November 1996, BBl 1997 I 1 ff. (zit. Botschaft BV) Botschaft zur Genehmigung der sektoriellen Abkommen zwischen der Schweiz und der EG vom 23. Juni 1999, BBl 1999 6128 ff. (zit. Botschaft sektorielle Abkommen) Botschaft zur Änderung des Bundesgesetzes über den Datenschutz (DSG) und zum Bundesbeschluss betreffend den Beitritt der Schweiz zum Zusatzprotokoll vom 8. November 2001 zum Übereinkommen zum Schutz des Menschen bei der automatischen Verarbeitung personenbezogener Daten bezüglich Aufsichtsbehörden und grenzüberschreitende Datenübermittlung vom 19. Februar 2003, BBl 2003 2101 ff. (zit. Botschaft Revision DSG 2003) Botschaft zur Änderung der Militärgesetzgebung (Militärgesetz und Bundesgesetz über die militärischen Informationssysteme) vom 7. März 2008, BBl 2008 3213 ff. (zit. Botschaft MIG) LX Materialienverzeichnis Botschaft zum Bundesbeschluss über den Einsatz der Armee im Assistenzdienst zur Unterstützung der zivilen Behörden anlässlich der FussballEuropameisterschaft 2008 (UEFA EURO 2008) vom 13. September 2006, BBl 2006 8179 ff. (zit. Botschaft EURO 2008) Botschaft zur Teilrevision 1 des Luftfahrtgesetzes vom 20. Mai 2009, BBl 2009 4915 ff. (zit. Botschaft Teilrevision 1 LFG) Botschaft zum Nachrichtendienstgesetz vom 19. Februar 2014, BBl 2014 2105 ff. (zit. Botschaft NDG) Botschaft zum Bundesgesetz über die Totalrevision des Bundesgesetzes über den Datenschutz und die Änderung weiterer Erlasse zum Datenschutz vom 15. September 2017, BBl 2017 6941 ff. (zit. Botschaft Totalrevision DSG) Botschaft zu Via sicura, Handlungsprogramm des Bundes für mehr Sicherheit im Strassenverkehr vom 20. Oktober 2010, BBl 2010 8447 ff. (zit. Botschaft Via sicura) Frage 15.5039, Rusconi Pierre (Regolamentazione droni) Interpellation 14.4223, Schläfli Urs (Private Drohnenflüge und ihre Folgen) Interpellation 16.3837, Tornare Manuel (Zivile Drohnen. Kritische Infrastrukturen besser schützen) Interpellation 16.4032, Leutenegger Oberholzer Susanne (Drohnen zum Zweiten. Zivilrechtliche Beurteilung) Interpellation 18.3397, Jositsch Daniel (Regelung für den Privatgebrauch von Drohnen) Jahresbericht 2007 der Geschäftsprüfungskommissionen und der Geschäftsprüfungsdelegation der eidgenössischen Räte vom 25. Januar 2008, BBl 2008 5061 ff. (zit. Jahresbericht GPK/GPDel 2007) Motion 16.3310, Leutenegger Oberholzer Susanne (Drohnen. Bevölkerung vor Gefährdungen schützen) Motion 18.3371, Candinas Martin (Sicherheit und Ordnung beim Betrieb von Drohnen) Motion 18.4207, Reynard Mathias (Vernetztes Spielzeug. Für die Sicherheit unserer Kinder) Parlamentarische Initiative 14.413, Vischer Daniel (Grundrecht auf informationelle Selbstbestimmung) Materialienverzeichnis LXI Postulat 13.3977, Tornare Manuel (Zivile Drohnen. Gesetzgebung anpassen) Postulat 14.4137, FDP-Liberale Fraktion (Videoaufnahmen durch Private. Die Privatsphäre besser schützen) Postulat 14.4284, Comte Raphaël (Videoaufnahmen durch Private. Die Privat sphäre besser schützen) Postulat 18.3245, Guhl Bernhard (Identifikation von Drohnen und ähnlichen Flugkörpern) Übersicht Kompatibilität des E-DSG mit dem EU-Recht vom 5. August 2019, erstellt durch das Bundesamt für Justiz, Direktionsbereich Öffentliches Recht, Fachbereich Rechtsetzungsprojekte und -methodik (zit. BJ, Kompatibilitätstabelle) Verfügung über eine temporäre Änderung der Luftraumstruktur der Schweiz während der EURO 2008 vom 3. Juni 2008, BBl 2008 4447 ff. (zit. BAZL, Verfügung) II. Weitere Behörden und Gremien Anfrage Fehr Düsel Nina, KR Nr. 150/2017 (Registrierungspflicht für Drohnen) Beschluss des Regierungsrats des Kantons Zürich, Sitzung vom 27. September 2017 (RRB 874/2017) Vorschlag für eine Verordnung des europäischen Parlaments und des Rates zur Festlegung gemeinsamer Vorschriften für die Zivilluftfahrt und zur Errichtung einer Agentur der Europäischen Union für Flugsicherheit sowie zur Aufhebung der Verordnung (EG) Nr. 216/2008 des Europäischen Parlaments und des Rates, Dokument ST_14991_2015_INIT, vom 8. Dezember 2015 (zit. Rat der Europäischen Union, Vorschlag 2015) Vorschlag für eine Verordnung des europäischen Parlaments und des Rates zur Festlegung gemeinsamer Vorschriften für die Zivilluftfahrt und zur Errichtung einer Agentur der Europäischen Union für Flugsicherheit sowie zur Aufhebung der Verordnung (EG) Nr. 216/2008 des Europäischen Parlaments und des Rates, Dokument ST_14469_2016_ADD_1, vom 21. November 2016 (zit. Rat der Europäischen Union, Vorschlag 2016) LXIII Abkürzungsverzeichnis ABl. Abs. ACLU AcP AeroRevue AEUV Amtsblatt der Europäischen Union Absatz / Absätze American Civil Liberties Union Archiv für die civilistische Praxis (Tübingen) Die meistgelesene Aviatikzeitschrift der Schweiz (Brugg) Vertrag über die Arbeitsweise der Europäischen Union, ABl. 2012 C 326/01 AG Aktiengesellschaft AGer Amtsgericht Air & Space Law — Zeitschrift für internationale Aspekte des Luft- und Weltraumrechts (Alphen aan den Rijn) AJP Aktuelle Juristische Praxis (Zürich) AltlV Verordnung über die Sanierung von belasteten Stand orten vom 26. August 1998 (Altlasten-Verordnung, SR 814.680) a.M. anderer Meinung / am Main Amtl. Bull. NR Amtliches Bulletin der Bundesversammlung, Nationalrat Amtl. Bull. SR Amtliches Bulletin der Bundesversammlung, Ständerat A-NPA Advance Notice of Proposed Amendment Art. Artikel AS Amtliche Sammlung des Bundesrechts ASJ The Aviation & Space Journal (Bologna) ASMZ Allgemeine Schweizerische Militärzeitschrift (Volketswil) ASTRA Bundesamt für Strassen ATM Air Traffic Management (= Flugverkehrsmanagement) ATSG Bundesgesetz über den Allgemeinen Teil des Sozialversicherungsrechts vom 6. Oktober 2000 (SR 830.1) Aufl. Auflage BAKOM Bundesamt für Kommunikation BAZL Bundesamt für Zivilluftfahrt BBl Bundesblatt der Schweizerischen Eidgenossenschaft BDSG Bundesdatenschutzgesetz vom 30. Juni 2017 (BGBl. I S. 2097) LXIV Abkürzungsverzeichnis Beschluss Nr. 1/2019 — Beschluss Nr. 1/2019 des Luftverkehrsausschusses Europäische Union/Schweiz zur Ersetzung des Anhangs des Abkommens vom 21. Juni 1999 zwischen der Schweizerischen Eidgenossenschaft und der Europäischen Gemeinschaft über den Luftverkehr vom 22. August 2019 (AS 2019 S. 2979 ff.) BGBl. Bundesgesetzblatt (Deutschland) BGE Entscheidungen des Schweizerischen Bundesgerichts (Amtliche Sammlung) BGer Bundesgericht / Entscheidung des Schweizerischen Bundes gerichts (nicht amtlich publiziert) BJ Bundesamt für Justiz BlAR Blätter für Agrarrecht (Bern) BLV Bundesamt für Lebensmittelsicherheit und Veterinärwesen BöB Bundesgesetz über das öffentliche Beschaffungswesen vom 16. Dezember 1994 (SR 172.056.1) BR Bundesrat, Bundesrätin BRTF Blue Ribbon Task Force BS Kanton Basel-Stadt BSK Basler Kommentar BTJP Berner Tage für die juristische Praxis BV Bundesverfassung der Schweizerischen Eidgenossenschaft vom 18. April 1999 (SR 101) BVerfGE Entscheidungen des Bundesverfassungsgerichts (Tübingen) BVerfGer Bundesverfassungsgericht (Deutschland) BVGE Entscheidungen des Bundesverwaltungsgerichts (Amtliche Sammlung) BVGer Bundesverwaltungsgericht / Entscheidung des Bundesverwaltungsgerichts (nicht amtlich publiziert) BVLOS Beyond Visual Line of Sight (= ausserhalb direkter Sicht) bzw. beziehungsweise c. contre ca. circa cadastre Fachzeitschrift für amtliche Vermessung (Wabern) CCTV Closed Circuit Television CE Conformité Européenne CLSR Computer Law & Security Review, The International Journal of Technology Law and Practice (Amsterdam) Conn. L. Rev. Connecticut Law Review (Hartford) ConOps Concept of Operations Abkürzungsverzeichnis CP LXV Crisis Prevention, Fachmagazin für die Bereiche Innere Sicher heit, Bevölkerungsschutz und Katastrophenhilfe (Bonn) CR Computer und Recht (Köln) CRi Computer law review international (Köln) Crit Crim Critical Criminology (Dordrecht) Cst. Constitution fédérale de la Confédération suisse (= BV) C.T.L.R. Computer and Telecommunications Law Review (London) Defense & Security Analysis — Journal on defense theory and analysis (London) Delegierte Verordnung — Delegierte Verordnung (EU) 2019/945 der Kommission vom 12. März 2019 über unbemannte Luftfahrzeug systeme und Drittlandbetreiber unbemannter Luftfahrzeugsysteme, ABl. 2019 L 152/1 Democrazia & Sicurezza — Democracy and Security Review (Rom) Der Bund Schweizer Tageszeitung aus der Bundesstadt (Bern) ders. derselbe dies. dieselbe / dieselben digma Zeitschrift für Datenrecht und Informationssicherheit (Zürich) Diritto dei trasporti — Rivista semestrale (Rom) Diss. Dissertation d.h. das heisst DJI Da-Jiang Innovations Science and Technology Ltd. DÖV Die öffentliche Verwaltung: Zeitschrift für Öffentliches Recht und Verwaltungswissenschaft (München) DSG Bundesgesetz über den Datenschutz vom 19. Juni 1992 (Datenschutzgesetz; SR 235.1) DSGVO Verordnung (EU) 2016/679 des europäischen Parlaments und des Rates vom 27. April 2016 zum Schutz natürlicher Personen bei der Verarbeitung personenbezogener Daten, zum freien Datenverkehr und zur Aufhebung der Richtlinie 95/46/EG (Datenschutz-Grundverordnung), Abl. 2016 L 119/1 DuD Datenschutz und Datensicherheit (Berlin) Durchführungsverordnung — Durchführungsverordnung (EU) 2019/947 der Kommission vom 24. Mai 2019 über die Vorschriften und Verfahren für den Betrieb unbemannter Luftfahrzeuge, ABl. 2019 L 152/45 DVBl Deutsches Verwaltungsblatt (Köln) E Entwurf E. Erwägung LXVI EASA Abkürzungsverzeichnis European Union Aviation Safety Agency (= Europäische Agentur für Flugsicherheit) EASA-Grundverordnung — Verordnung (EU) 2018/1139 des Europäischen Parlaments und des Rates zur Festlegung gemeinsamer Vorschriften für die Zivilluftfahrt und zur Errichtung einer Europäischen Agentur für Flugsicherheit vom 4. Juli 2018, ABl. 2018 L 212/1 ebd. ebenda EBG Eisenbahngesetz vom 20. Dezember 1957 (SR 742.101) ecolex Fachzeitschrift für Wirtschaftsrecht (Wien) EDA Eidgenössisches Departement für auswärtige Angelegen heiten EDI Eidgenössisches Departement des Innern EDÖB Eidgenössischer Datenschutz- und Öffentlichkeitsbeauftragter E-DSG Entwurf zum Bundesgesetz über den Datenschutz (BBl 2017 S. 7206 ff.), Fassung gemäss Beschluss des NR vom 25. September 2019 EFV Eidgenössische Finanzverwaltung EG Europäische Gemeinschaft(en) / Erwägungsgrund EGMR Europäischer Gerichtshof für Menschenrechte EJRR European Journal of Risk Regulation (Cambridge, Gross britannien) EKMR Europäische Kommission für Menschenrechte E-LFG Revisionsentwurf zu einigen Artikeln des LFG (vgl. BBl 2017 S. 7142), Fassung gemäss Beschluss des NR vom 25. September 2019 EMRK Konvention zum Schutze der Menschenrechte und Grundfreiheiten vom 4. November 1950 (Europäische Menschenrechtskonvention; SR 0.101) EnG Energiegesetz vom 30. September 2016 (SR 730.0) engl. englisch et al. et alii / aliae etc. et cetera ETH Eidgenössische Technische Hochschule EU Europäische Union EuGH Gerichtshof der Europäischen Gemeinschaften EuZW Europäische Zeitschrift für Wirtschaftsrecht (München) f./ff. folgende Abkürzungsverzeichnis FAV FIFG FMG Fn. FOCA g GDPR GE Geogr. Helv. GeoIG GesKR GfK GG ggf. GK gl.M. GLONASS GM GNSS GPDel GPK GPS GR GRCh GSG Handkomm. HAVE HGR h.L. Hrsg. ICAO LXVII Verordnung über Fernmeldeanlagen vom 25. November 2015 (SR 784.101.2) Bundesgesetz über die Förderung der Forschung und der Innovation vom 14. Dezember 2012 (SR 420.1) Fernmeldegesetz vom 30. April 1997 (SR 784.10) Fussnote(n) Federal Office of Civil Aviation (= BAZL) Gramm General Data Protection Regulation (= DSGVO) Kanton Genf Geographica Helvetica: Swiss journal of geography (Göttingen/Zürich) Bundesgesetz über die Geoinformation vom 5. Oktober 2007 (Geoinformationsgesetz; SR 510.62) Gesellschafts- und Kapitalmarktrecht (Zürich) Gesellschaft für Konsumentenforschung Grundgesetz für die Bundesrepublik Deutschland vom 23. Mai 1949 (BGBl. S. 1) gegebenenfalls Grosse Kammer gleicher Meinung Globalnaja nawigazionnaja sputnikowaja sistema (= Globales Satellitennavigationssystem) Guidance Material Globale Navigationssatellitensysteme Geschäftsprüfungsdelegation Geschäftsprüfungskommission(en) Global Positioning System Kanton Graubünden Charta der Grundrechte der Europäischen Union (Grund rechtecharta), ABl. 2000 C 364/1 Bundesgesetz über die von der Schweiz als Gaststaat gewährten Vorrechte, Immunitäten und Erleichterungen sowie finanziellen Beiträge vom 22. Juni 2007 (Gaststaatgesetz; SR 192.12) Handkommentar Haftung und Versicherung (Zürich) Handbuch der Grundrechte herrschende Lehre Herausgeber International Civil Aviation Organization LXVIII Abkürzungsverzeichnis ICAO-Übereinkommen — Übereinkommen über die internationale Zivilluft fahrt, abgeschlossen in Chicago am 7. Dezember 1944, in Kraft getreten für die Schweiz am 4. April 1947 (SR 0.748.0) IDG/BS Gesetz über die Information und den Datenschutz [des Kantons Basel-Stadt] vom 9. Juni 2010 (SG 153.260) IDG/ZH Gesetz über die Information und den Datenschutz [des Kantons Zürich] vom 12. Februar 2007 (LS 170.4) IDPL International Data Privacy Law (Oxford) IEEE Institute of Electrical and Electronics Engineers IMSI International Mobile Subscriber Identity InTeR Zeitschrift zum Innovations- und Technikrecht (Frankfurt a.M.) IT Informationstechnik ITF/OECD International Transport Forum at the OECD i.V.m. in Verbindung mit IVZV Verordnung über das Informationssystem Verkehrszulassung vom 30. November 2018 (SR 741.58) J. Aeronaut Aerospace Eng — Journal of Aeronautics & Aerospace Engineering (Brüssel) JARUS Joint Authorities for Rulemaking on Unmanned Systems JETLaw Vanderbilt Journal of Entertainment & Technology Law (Nashville) JLIS Journal of Law, Information and Science (Hobart) JUAS Journal of Unmanned Aerial Systems (Ottawa) Jusletter Die grösste Schweizer Universalzeitschrift der Schweiz (Bern) Jusletter IT Die Zeitschrift für IT und Recht (Bern) JUVS Journal of Unmanned Vehicle Systems (Ottawa) JZ JuristenZeitung (Tübingen) Kap. Kapitel kg Kilogramm KGer Kantonsgericht KMU Kleine und mittlere Unternehmen Komm. Kommentar KR Kantonsrat KuKo Kurzkommentar LeGes Mitteilungsblatt der Schweizerischen Gesellschaft für Gesetzgebung und der Schweizerischen Evaluationsgesellschaft (Bern) LFG Bundesgesetz über die Luftfahrt vom 21. Dezember 1948 (Luftfahrtgesetz; SR 748.0) Abkürzungsverzeichnis LFV LGV lit. LS LSJ Ltd. LU Lupo LVA CH–EU MA MAPS MAW m.E. medialex MIG MMR m.w.H. N NDG NJW NPA NR Nr. Nrn. NSW NVwZ NZV NZZ OECD OFK LXIX Verordnung über die Luftfahrt vom 14. November 1973 (Luftfahrtverordnung; SR 748.01) Lebensmittel und Gebrauchsgegenständeverordnung vom 16. Dezember 2016 (SR 817.02) litera Loseblattsammlung des Kantons Zürich Law Society of NSW Journal (Sydney) Limited Kanton Luzern Luftfahrtpolitik Abkommen zwischen der Schweizerischen Eidgenossenschaft und der Europäischen Gemeinschaft über den Luftverkehr, abgeschlossen am 21. Juni 1999, in Kraft getreten am 1. Juni 2002 (SR 0.748.127.192.68) Massachusetts Maison d’analyse des processus sociaux (Universität Neuenburg) Mission Adaptive Wing meines Erachtens Zeitschrift für Medienrecht Bundesgesetz über die militärischen Informationssysteme vom 3. Oktober 2008 (SR 510.10) MultiMedia und Recht (München) mit weiterem Hinweis / mit weiteren Hinweisen Note, Randnote Bundesgesetz über den Nachrichtendienst vom 25. September 2015 (Nachrichtendienstgesetz; SR 121) Neue Juristische Wochenschrift (München) Notice of Proposed Amendment Nationalrat Nummer Nummern New South Wales Neue Zeitschrift für Verwaltungsrecht (München) Neue Zeitschrift für Verkehrsrecht (München) Neue Zürcher Zeitung (Zürich) Organisation for Economic Co-operation and Development Orell Füssli Kommentar LXX OR Abkürzungsverzeichnis Bundesgesetz betreffend die Ergänzung des Schweizerischen Zivilgesetzbuches (Fünfter Teil: Obligationenrecht) vom 30. März 1911 (SR 220) OV-UVEK Organisationsverordnung für das Eidgenössische Departement für Umwelt, Verkehr, Energie und Kommunikation vom 6. Dezember 1999 (SR 172.217.1) P. & D.P. Privacy & Data Protection (London) ParlG Bundesgesetz über die Bundesversammlung (Parlamentsgesetz; SR 171.10) PBG/LU Planungs- und Baugesetz [des Kantons Luzern] vom 7. März 1989 (SRL 735) PinG Privacy in Germany: Datenschutz und Compliance (Berlin) PK Praxiskommentar PolG/ZH Polizeigesetz [des Kantons Zürich] vom 23. April 2007 (LS 550.1) PolR Oberes Fricktal — Polizeireglement Oberes Fricktal der Gemeinden Bözen, Densbüren, Effingen, Eiken, Elfingen, Frick, Gansingen, Gipf-Oberfrick, Herznach, Hornussen, Kaisten, Laufenburg, Mettauertal, Oberhof, Oeschgen, Schwaderloch, Sisseln, Ueken, Wittnau, Wölflinswil, Zeihen vom 1. Januar 2016 PRIO Peace Research Institute Oslo Prof. Professor Progress in Aerospace Sciences — An International Review Journal in Aero space Sciences (Amsterdam) QR-Code Quick Response Code RaLA Règlement concernant l’exécution de la loi fédérale sur l’aviation du 9 novembre 1951 (rs/GE H 3 05.02) RDTI Revue du droit des technologies de l’information (Louvain-la-Neuve) recht Zeitschrift für juristische Weiterbildung und Praxis (Bern) REJP Revista de la Escuela Jacobea de Posgrado (Xalapa) Remote Sens. Open access journal about the science and application of remote sensing technology (Basel) RGDP Règlement Général sur la Protection des Données (= DSGVO) Risk Analysis An international journal published on behalf of the Society for Risk Analysis (Oxford/Dordrecht) Abkürzungsverzeichnis RLG LXXI Bundesgesetz über Rohrleitungsanlagen zur Beförderung flüssiger oder gasförmiger Brenn- oder Treibstoffe (Rohrleitungsgesetz; SR 746.1) Rn. Randnummer RPAS Remotely Piloted Aircraft System RPV Remotely Piloted Vehicle RRB Regierungsratsbeschluss Rs. Rechtssache / Rechtssachen rs/GE Recueil systématique genevois RVOG Regierungs- und Verwaltungsorganisationsgesetz vom 21. März 1997 (SR 172.010) S. Seite SA Société anonyme (= AG) SAPR Sistemi Aeromobili a Pilotaggio Remoto (= RPAS) SBB Schweizerische Bundesbahnen SBVR Schweizerisches Bundesverwaltungsrecht (Basel) SchifffahrtsVO LU — Verordnung über die Schifffahrt [des Kantons Luzern] vom 18. Februar 2011 (SRL 787) SD Secure Digital [Memory Card] SERA Standardised European Rules of the Air (in Verordnung (EU) Nr. 923/2012) SESAR Single European Sky ATM Research Programme SEV Sammlung Europäischer Verträge SG Systematische Gesetzessammlung des Kantons Basel-Stadt SGK St. Galler Kommentar SHK Stämpflis Handkommentar SI-Einheit Einheit gemäss dem «Système international d’unités» (= Internationales Einheitensystem) sic! Zeitschrift für Immaterialgüter-, Informations- und Wettbewerbsrecht (Zürich) Sicherheit & Recht — Juristische Fachzeitschrift für Sicherheitsfragen in den Bereichen Polizei, Militär, Umwelt und Technik (Zürich/ St. Gallen) SJZ Schweizerische Juristen-Zeitung (Zürich) SNF Schweizerischer Nationalfonds SO Kanton Solothurn sog. sogenannt LXXII Abkürzungsverzeichnis Spielzeugrichtlinie — Richtlinie 2009/48/EG des Europäischen Parlaments und des Rates über die Sicherheit von Spielzeug vom 18. Juni 2009, ABl. 2009 L 170/1 SR Systematische Sammlung des Bundesrechts / Ständerat SRF Schweizer Radio und Fernsehen SRL Systematische Rechtssammlung des Kantons Luzern Stan. L. Rev. Online — Stanford Law Review Online (Stanford) StGB Schweizerisches Strafgesetzbuch vom 21. Dezember 1937 (SR 311.0) STLR The Columbia Science & Technology Law Review (New York) sUAS small Unmanned Aircraft System sui-generis Die juristische Open-Access-Zeitschrift SUSI Swiss U-Space Implementation SUST Schweizerische Sicherheitsuntersuchungsstelle SVG Strassenverkehrsgesetz vom 19. Dezember 1958 (SR 741.01) Syracuse L. Rev. — Syracuse Law Review (Syracuse) SZ Süddeutsche Zeitung (München) SZW Schweizerische Zeitschrift für Wirtschafts- und Finanzmarktrecht (Zürich) TA-SWISS Stiftung für Technologiefolgen-Abschätzung Technology in Society — International journal (Amsterdam) u.a. unter anderem u.U. unter Umständen UA Unmanned Aircraft UAS Unmanned Aircraft System UAV Unmanned Aerial Vehicle UEFA Union of European Football Associations UIDG Bundesgesetz über die Unternehmens-Identifikationsnummer vom 18. Juni 2010 (SR 431.03) UIDV Verordnung über die Unternehmens-Identifikationsnummer vom 26. Januar 2011 (SR 431.031) UK United Kingdom UNO-Pakt II Internationaler Pakt über bürgerliche und politische Rechte (SR 0.103.2) USA United States of America USB Universal Serial Bus USG Bundesgesetz über den Umweltschutz vom 7. Oktober 1983 (Umweltschutzgesetz; SR 814.01) U-Space Urban Space Abkürzungsverzeichnis UTM UVEK LXXIII Unmanned Aircraft Systems Traffic Management Eidgenössisches Departement für Umwelt, Verkehr, Energie und Kommunikation UWG Bundesgesetz gegen den unlauteren Wettbewerb vom 19. Dezember 1986 (SR 241) v.a. vor allem VDSG Verordnung zum Bundesgesetz über den Datenschutz vom 14. Juni 1993 (SR 235.11) VFAV Verordnung des BAKOM über Fernmeldeanlagen vom 26. Mai 2016 (SR 784.101.21) Verordnung (EG) Nr. 216/2008 — Verordnung (EG) Nr. 216/2008 des Europäischen Parlaments und des Rates zur Festlegung gemeinsamer Vorschriften für die Zivilluftfahrt und zur Errichtung einer Europäischen Agentur für Flugsicherheit vom 20. Fe bruar 2008, ABl. 2008 L 79/1 (nicht mehr in Kraft; aufgehoben durch EASA-Grundverordnung) Verordnung (EG) Nr. 765/2008 — Verordnung (EG) Nr. 765/2008 des Europäischen Parlaments und des Rates über die Vorschriften für die Akkreditierung und Marktüberwachung im Zusammenhang mit der Vermarktung von Produkten und zur Aufhebung der Verordnung (EWG) Nr. 339/93 des Rates vom 9. Juli 2008, ABl. 2008 L 218/30 Verordnung (EU) Nr. 923/2012 — Durchführungsverordnung (EU) 923/2012 der Kommission zur Festlegung gemeinsamer Luftverkehrsregeln und Betriebsvorschriften für Dienste und Verfahren der Flugsicherung und zur Änderung der Durchführungsverordnung (EG) 1035/2011 vom 26. September 2012, ABl. 2012 L 281/1 VGer Verwaltungsgericht vgl. vergleiche VLK Verordnung des UVEK über Luftfahrzeuge besonderer Kategorien vom 24. November 1994 (SR 748.941) Vol. Volume VPB Verwaltungspraxis der Bundesbehörden (Bern) VSFD Verordnung über den Flugsicherungsdienst vom 18. Dezember 1995 (SR 748.132.1) VSS Verordnung des EDI über die Sicherheit von Spielzeug vom 15. August 2012 (Spielzeugverordnung; SR 817.023.11) VSZV Verordnung über die Sicherheitsuntersuchung von Zwischenfällen im Verkehrswesen vom 17. Dezember 2014 (SR 742.161) LXXIV VVEA Abkürzungsverzeichnis Verordnung über die Vermeidung und die Entsorgung von Abfällen vom 4. Dezember 2015 (SR 814.600) Wash. L. Rev. Washington Law Review (Seattle) WiMax Worldwide Interoperability for Microwave Access WLAN Wireless Local Area Network WPJ World Policy Journal (Durham, North Carolina) z.B. zum Beispiel ZBJV Zeitschrift des Bernischen Juristenvereins (Bern) ZBl Schweizerisches Zentralblatt für Staats- und Verwaltungsrecht (Zürich) ZD Zeitschrift für Datenschutz (München) ZG Zollgesetz vom 18. März 2005 (SR 631.0) ZGB Schweizerisches Zivilgesetzbuch vom 10. Dezember 1907 (SR 210) ZH Kanton Zürich Ziff. Ziffer zit. zitiert ZLS Zentralstellen der Länder für Sicherheitstechnik ZLW Zeitschrift für Luft- und Weltraumrecht (Köln) ZPO Schweizerische Zivilprozessordnung vom 19. Dezember 2008 (Zivilprozessordnung; SR 272) ZSR Zeitschrift für Schweizerisches Recht (Basel) Zürcher Wirtschaft — Mitgliederzeitung des Zürcher KMU- und Gewerbe verbands (Zürich) 1 Einleitung § 1 Einführung in das Problemfeld Zivile Drohnen breiten sich in der Schweiz immer weiter aus; Behörden wie Private nutzen diese Technologie immer häufiger. Die digitale Transformation nimmt vermehrt auch den Luftraum ein.1 Drohnen bilden gewissermassen «den materiellen, auf die Welt zugreifenden Arm der Digitalisierung»; «sie haben das Potenzial, die ‹ikonische Maschine› der Digitalisierung zu werden, wie dies einst die Dampfmaschine gewesen ist.»2 Diese technische Entwicklung muss — früher oder später — vom Recht erfasst werden. Dies kann durch Subsumtion der Technik unter bestehende Regelwerke oder durch die Schaffung spezifischer Rechtssätze und Normen geschehen.3 Mithin stellen sich die Fragen, wie das Recht mit dem Einsatz neuer Technik umgeht4 und ob die Wirksamkeit bestehender Rechtsnormen angesichts technologischer Veränderungen sichergestellt werden kann.5 Der Umgang des Schweizer Rechts mit Drohnen war denn auch bereits Thema von zahlreichen parlamentarischen Vorstössen, sowohl auf Bundes- wie auch auf kantonaler Ebene.6 Dies ist Ausdruck davon, dass sich in der Rechtswissenschaft durch Drohnen zahlreiche (neue) Fragen ergeben, die noch nicht analysiert und beantwortet wurden. Die vorliegende Dissertation nimmt sich einer dieser rechtlichen Herausforderungen an. Namentlich sollen die wesentlichen Aspekte im Schnittstellenbereich der aufkommenden Drohnentechnologie und des ebenfalls immer zentraleren Rechtsgebiets des Datenschutzes beleuchtet werden. 1 Vgl. SESAR, Master Plan, S. 3; Bericht Lupo 2016, S. 1856. 2 Christen et al., S. 227; vgl. Palmerini, S. 6. 3 Vgl. allgemein Huber H., S. 14 ff.; Magnin, S. 134; spezifisch für die Luftfahrt Bericht Lupo 2016, S. 1925; für das Datenschutzrecht Haustein, S. 93. 4 Vgl. Kornmeier, S. 3; Gasser, S. 12. 5 Vgl. Burkert/Hettich/Thouvenin, S. 49; Mihalyi, S. 193. 6 Vgl. etwa Postulat Tornare (13.3977); Interpellation Schläfli (14.4223); Frage Rusconi (15.5039); Interpellation Eichenberger-Walther (17.4064); Motion Leutenegger Oberholzer (16.3310); Interpellation Tornare (16.3837); Interpellation Leutenegger Oberholzer (16.4032) sowie z.B. im Kanton Zürich Anfrage Fehr Düsel (Nr. 150/2017) und die Antwort des Regierungsrats dazu (RRB 874/2017). 2 Einleitung Das Thema ist von besonderer Brisanz, da Drohnenpiloten heute oftmals nicht oder nur mit sehr grossem Aufwand identifizierbar sind.7 Diese Anony mität kann dazu führen, dass bestehende Rechtsdurchsetzungsmechanismen an ihre Grenzen stossen oder gar vollkommen ins Leere laufen. Denn die im Sinne des Datenschutzrechts betroffene Person befindet sich in einer Situa tion der Machtlosigkeit; zwischen dem Drohnenpiloten und ihr besteht ein Informations- und dadurch ein Machtgefälle. Aus den Problemstellungen, die sich daraus ergeben, ist das vorliegende Projekt entstanden. § 2 Gegenstand und Ziel der Arbeit I. Forschungsfrage Der Arbeit liegt die folgende Forschungsfrage zugrunde: Wie kann das Recht auf informationelle Selbstbestimmung (Art. 13 Abs. 2 BV; Art. 8 EMRK) — konkretisiert im Datenschutzrecht — bei der drohnengestützten Datenbearbeitung durch Private durchgesetzt werden? Das Ziel der Dissertation besteht darin, eine Möglichkeit aufzuzeigen, wie die Identifizierung der für eine drohnengestützte Datenbearbeitung verantwortlichen Person gewährleistet werden kann. Denn nur so wissen die Betroffenen, gegenüber wem sie ihre datenschutzrechtlichen Ansprüche geltend machen können. II. Eingrenzung des Themas Da Drohnen, wie erwähnt, in verschiedensten Rechtsgebieten neue Fragen aufwerfen, muss der Gegenstand dieser Arbeit klar umgrenzt werden. Der Fokus soll einzig auf zivilen Drohnen liegen, die militärische Nutzung wird nicht behandelt. Ebenfalls wird nicht die Nutzung ziviler Drohnen durch Behörden erörtert;8 vielmehr geht es um die drohnengestützte Datenbearbeitung durch Private. Dabei ist nochmals zwischen der kommerziellen und der hobbymässigen Drohnennutzung zu unterscheiden; oftmals ist in diesem Zusammenhang von «Hobbydrohnen» die Rede.9 Bei dieser liegt der Schwerpunkt des vorliegenden Projekts. 7 Article 29 Working Party, S. 7; Geiser/Uttinger, S. 9; Griebel, S. 113; Leissler, S. 535; Nadakavukaren Schefer, S. 286; Perritt/Sprague, STLR, S. 406 f.; vgl. auch Interpellation Jositsch (18.3397); Büttner, S. 1. 8 Darauf soll einzig im Rahmen der bisherigen Einsatzformen in der Schweiz (S. 35 ff.) kurz eingegangen werden. 9 Statt vieler Christen et al., S. 70, S. 85 und S. 104 f.; vgl. Bracken-Roche, S. 168. § 2 Gegenstand und Ziel der Arbeit 3 Bei der Drohnennutzung durch Private akzentuiert sich das Problem der Anonymität.10 Setzen Behörden Drohnen ein, so sind diese entweder sichtund zuordenbar (z.B. Feuerwehr bei Bränden, Polizei bei Verkehrsunfällen) oder aber gewollt verdeckt (Observationen im Bereich Staatsschutz, Grenzkontrollen etc.).11 In diesen Fällen ist auch die datenschutzrechtliche Pro blematik anders gelagert, da die Behörden entweder vollkommen offen agieren oder die Verborgenheit geradezu Zweck des Einsatzes ist. Deshalb soll der Fokus auf der drohnengestützten Datenbearbeitung durch Private liegen. Als letzte Eingrenzung ist darauf hinzuweisen, dass nicht die Datenbearbeitung durch autonom operierende Drohnen behandelt wird.12 Vollständig autonome Drohnensysteme werden denn auch in absehbarer Zeit nicht bestehen.13 In den hier besprochenen Fällen existiert mithin in jedem Fall eine natürliche (oder juristische) Person, welche für die drohnengestützte Datenbearbeitung verantwortlich14 ist.15 III. Relevanz des Themas 1. Anzahl und Verbreitung von Drohnen Das Bundesamt für Zivilluftfahrt (BAZL) hat in seinem bisher einzigen Bericht zu zivilen Drohnen vom 7. Februar 2016 festgehalten, dass sich «[mindestens] 20’000 zivile unbemannte Luftfahrzeuge, respektive ‹Drohnen›», im Schweizer Himmel bewegen — «Tendenz steigend».16 Gestützt auf eine Schätzung, basierend auf Zahlen des Detailhandels, ging das BAZL sodann im Oktober 2017 von ungefähr 100’000 in der Schweiz verkauften Drohnen aus.17 Der 10 Darauf wurde auch in der soziologischen Forschung hingewiesen; vgl. BrackenRoche, S. 170 m.w.H.: «The mutual glance, the reciprocity of eye-to-eye contact is, according to some, a basic constituent of society.» Ähnlich auch Geiser/Uttinger, S. 9: «Wer Vorgänge in der Öffentlichkeit wahrnehmen will, soll sich auch selber zu erkennen geben. Das ist bei einer Drohne nicht gewährleistet.» 11 Vgl. zu diesen Anwendungsfeldern hinten, S. 35 ff. 12 Ausführlich zum Thema autonomer Entscheidungsprozesse und deren Abgrenzung zur Automation Hänsenberger, Diss., S. 44 ff. Vgl. auch Bartsch, S. 3. 13 Hänsenberger, Diss., S. 48 m.w.H.; Vladeck, S. 121 f.; Perritt/Sprague, JETLaw, S. 689; vgl. auch Burri, S. 343 ff. 14 Zum Verantwortlichen im Sinne des Datenschutzrechts ausführlich hinten, S. 74. 15 Vgl. zum Thema der Verantwortlichkeit von «Automaten» (verstanden als Computer, Roboter etc.) Matthias, passim, v.a. S. 237 ff. Vgl. sodann Solove, Nothing to Hide, S. 185 f.; Haustein, S. 100 f.; Lenk, S. 34. 16 BAZL, Zivile Drohnen, S. 1. Im Jahr 2014 waren es wohl mehrere Tausend (vgl. Steiger, Regulierung, S. 170). 17 BAZL, E-Mail vom 23. Oktober 2017 (beim Autor einsehbar); diese Zahl nennen auch Christen et al., S. 106 m.w.H. 4 Einleitung Bundesrat hielt Ende August 2018 dagegen fest, dass «Drohnen gegenwärtig nicht registriert werden18 und im Verkauf frei erhältlich [sind]. Dadurch ist ihre Zahl so wenig bekannt wie die Art des Einsatzes. (…) Auch die verfügbaren aktuellen Verkaufszahlen aus der Schweiz liefern keine zuverlässige Basis für genauere Schätzungen (…)».19 Tatsache ist, dass die Anzahl Drohnen in der Schweiz und in Europa in den nächsten Jahren und Jahrzehnten weiter zunehmen wird.20 Gemäss Marktforschungsinstitut GfK gingen in der Schweiz im Jahr 2017 73’200 Drohnen über den Ladentisch; der Absatz stieg in den letzten Jahren stetig an.21 Diese Zunahme wird die gesellschaftliche Bedeutung von Drohnen nochmals erhöhen.22 Gemäss einer Schätzung des Präsidenten des Schweizerischen Verbands Ziviler Drohnen (SVZD)23 vom Mai 2019 führen Privatpersonen in der Schweiz wöchentlich ca. 2’500 Drohnenflüge durch.24 2. Neue Gefahren durch neue Technologie Drohnen ermöglichen es, einen (Luft-)Raum einzunehmen, der bisher nur sehr wenig genutzt wurde.25 Dabei werden physische Grenzen überflogen, was die Abgrenzung zwischen Privatem und Öffentlichem zunehmend schwie- 18 Gemäss Art. 2 Abs. 1 i.V.m. Art. 1 VLK werden Drohnen nicht in das Luftfahrzeugregister eingetragen. Vgl. ausführlich zur zukünftigen Registrierung von Drohnen hinten, S. 120 ff. 19 Antwort des BR vom 29. August 2018 auf Anfrage Leutenegger Oberholzer (18.1044) (Hervorhebung hinzugefügt). Vgl. in diesem Zusammenhang auch die Frage Leuten egger Oberholzer (18.5399) sowie Landrock/Baumgärtel, S. 61, die darauf hinweisen, dass «fundierte Zahlen über den Drohnenmarkt noch kaum existieren». 20 In Deutschland sollen im August 2019 rund 500’000 Drohnen in privater und kommerzieller Nutzung betrieben worden sein (Balser, S. 5). Vgl. zur erwarteten Entwicklung der Nachfrage in Europa SESAR, Outlook Study, S. 17 (starke Zunahme bis 2025; bis sieben Millionen verkaufte Drohnen für Freizeitaktivitäten bis 2050); Klauser et al., S. 21. 21 Giuriato; vgl. auch Pugin, S. 5, die von rund 22’000 jährlich verkauften Drohnen spricht. 22 Vgl. SESAR, Outlook Study, S. 14 ff.; Klauser et al., S. 21 f. Kaiser, S. 359, wies bereits 2006 auf die absehbare Zunahme von Klein- und Kleinstdrohnen und das damit einhergehende Gefährdungspotenzial für die Privatsphäre hin. 23 Der SVZD ist der Dachverband des Schweizer Drohnengewerbes; vgl. die Webseite www.drohnenverband.ch. 24 Vgl. Hegetschweiler, S. 5. Über die Dauer und den Ort der einzelnen Flüge machte der Präsident des SVZD keine Angaben. Ihm zufolge kommen KMUs schätzungsweise auf rund 200 Flüge pro Woche. 25 Thomasen, Between the Lines, S. 207 und S. 218 ff.; Palmerini, S. 18. Vgl. Klauser/ Pedrozo, S. 236 f. § 2 Gegenstand und Ziel der Arbeit 5 riger macht.26 Zudem werden diese Grenzen immer häufiger auch wort wörtlich durchschaut: Moderne Sensoren27 können Gespräche im Innern von Gebäuden verfolgen oder (Körper-)Wärme hinter Mauern erkennen und registrieren.28 Gemäss der grössten bisher in der Schweiz durchgeführten Studie (Universität Neuenburg, 2017) sind alle in der Schweiz eingesetzten Drohnen mit mindestens einem Sensor bestückt; ca. 80 Prozent davon sind mit einer Fotooder Videokamera ausgestattet.29 Diese Sensoren sind es denn auch, welche die Datenbearbeitung — mit Drohnen als Plattformen30 — überhaupt ermöglichen und diese Technologie in den Fokus der datenschutzrechtlichen Diskussion rücken.31 Die immer günstiger werdende Drohnentechnologie «will make private spaces easier accessible to surveillance equipment and increase the number of intolerable privacy intrusions.»32 3. Misstrauen und Ablehnung in der Bevölkerung Auch oder gerade wegen dieser Datenbearbeitungsfähigkeiten werden Drohnen von der (Schweizer) Bevölkerung noch immer mit einigem Misstrauen betrachtet.33 Ähnliches wurde auch in anderen Ländern beobachtet. Eine Studie in Dänemark mit dem Titel «Public reactions to drone use in residential and public areas» kam u.a. zu folgendem Schluss: «When seeing a drone, bystanders (…) try to ascertain its purpose and legitimacy by either trying to interpret its flight pattern, lights, or design; or by looking for a pilot to hold accountable.»34 26 Rao/Gopi/Maione, S. 88; vgl. Burger/Scuderi, S. 88; Thomasen, Between the Lines, S. 208 ff. 27 Ausführlich dazu hinten, S. 40 ff. 28 Article 29 Working Party, S. 3; ACLU, S. 4 f.; Hodgkinson/Johnston, S. 15; Palmerini, S. 18; vgl. Bartsch, S. 6. Froomkin/Colangelo, S. 35, weisen in diesem Zusammenhang zudem auf die schnelle Datenübertragung hin. 29 Klauser et al., S. 8. 30 Vgl. Dieckert/Eich, in: dies., S. 2 («Schwebeplattform»); Custers, S. 11. 31 Article 29 Working Party, S. 7; Calo, passim, v.a. S. 32; Finn/Wright, S. 187 und 191 f.; Nadakavukaren Schefer, S. 266 ff.; Weber/Oertly, Rn. 1. 32 Kaiser, S. 360; ähnlich Bernauw, S. 242. Vgl. auch Aebi-Müller, Rn. 510. 33 Klauser/Pedrozo, S. 234 f.; Geiser/Uttinger, S. 9; vgl. auch Bajde et al., Privacy Concerns, S. 14; Christen et al., S. 135 m.w.H.; Chang/Chundury/Chetty, S. 6765 ff.; Rose, S. 140 m.w.H. 34 Bajde et al., Public reactions, S. 5; vgl. auch Bracken-Roche et al., S. 42 ff. (Kanada); Chang/Chundury/Chetty, S. 6770; Rice et al., S. 15; Rao/Gopi/Maione, S. 88 mit Fn. 42; Wang et al., S. 178 f. (alle Vereinigte Staaten von Amerika); Euler/Cobus/ Koelle, S. 150 f. (Deutschland); Marte et al., passim (mit Fokus auf Drohnenüber wachung durch die Polizei). 6 Einleitung Die gesellschaftliche Akzeptanz von Drohnen und deren Nutzung — auch durch Private — ist ein zentraler Aspekt dieser technologischen Entwicklung.35 Die Achtung der Grundrechte, und primär der Privatsphäre sowie der informationellen Selbstbestimmung, muss garantiert sein.36 Dem hat auch der Gesetzgeber im Hinblick auf allfällige zukünftige Normen zu Drohnen Beachtung zu schenken.37 Es ist davon auszugehen, dass die gesellschaftliche Skepsis gegenüber Drohnen anhält, zumindest solange die Anonymität der Drohnenpiloten Bestand hat und das damit verbundene Gefühl der Machtlosigkeit der betroffenen Personen nicht abgemildert werden kann. Die zunehmende Verbreitung von Drohnen und dem damit einhergehenden Informationsbedarf bzw. der damit verbundenen Verunsicherung wurde im November 2017 augenscheinlich. Das BAZL hielt damals auf seiner Webseite fest: «Der Informationsbedarf zu Drohnen ist sehr gross. Aufgrund von personellen Engpässen ist das BAZL momentan nicht mehr in der Lage, generelle Anfragen telefonisch oder per E-Mail zu beantworten.»38 Per Januar 2019 wurden innerhalb des Bundesamts sechs zusätzlichen Vollzeitstellen im Bereich Drohnen und Digitalisierung geschaffen, «um die zunehmende Arbeitslast abzufedern».39 4. Wirtschaftliches Potenzial Unter dem Titel der Relevanz ist schliesslich kurz auf das wirtschaftliche Potenzial der Drohnen hinzuweisen. Gemäss einer Studie der EU wird der europäische Drohnenmarkt ab 2035 schätzungsweise zehn Milliarden Euro pro Jahr ausmachen; ab 2050 sodann über 15 Milliarden Euro.40 In der Schweiz bemisst sich der zivile Drohnenmarkt auf etwa 300 Millionen Franken, wobei für die nächsten zehn Jahre ein jährliches Wachstum von 15 bis 35 EASA, A-NPA, S. 7; Winter et al., S. 131 ff.; ITF/OECD, S. 21; Masutti/Tomasello, S. 226; ausführlich Christen et al., S. 191 ff. Dies hat auch der Mediensprecher des BAZL im Mai 2019 festgehalten (vgl. Hegetschweiler, S. 5). 36 Europäische Kommission, Riga declaration, S. 4; vgl. auch Europäische Kommission, Warsaw declaration, S. 1; Giemulla/van Schyndel/Friedl, S. 6; Martini, S. 736 f.; Euler/Cobus/Koelle, S. 151 in fine. 37 Vgl. Geiser/Uttinger, S. 9; SESAR, Outlook Study, S. 10; EASA, Opinion, S. 9; Ensthaler, S. 34. Ängste und Unsicherheiten in der Gesellschaft werden denn auch regelmässig in politischen Vorstössen aufgegriffen; vgl. dazu die Hinweise vorne, Fn. 6. 38 Diese Information war mindestens bis am 14. März 2019 online einsehbar; zu welchem Zeitpunkt sie entfernt wurde, ist dem Autor nicht bekannt. 39 EFV, S. 278. 40 SESAR, Outlook Study, S. 3, S. 29 f. Vgl. zur kommerziellen Drohnennutzung auch SESAR, Master Plan, S. 19 f.; sowie Perritt/Sprague, Domesticating Drones, S. 295 ff.; mit Blick auf die Schweiz sodann ausführlich Arora, passim; Christen et al., S. 106 ff. sowie S. 115 ff. § 2 Gegenstand und Ziel der Arbeit 7 20 % prognostiziert wird.41 Diese Zahlen sind jedoch mit einer gewissen Vorsicht zu geniessen. Die bisher veröffentlichten Studien zum Wachstums potenzial des Drohnenmarkts präsentieren teilweise sehr unterschiedliche Resultate.42 Eine genaue Aussage ist deshalb nicht möglich. Die Tendenz steht aber auch hier — wie bei der Anzahl Drohnen — auf Wachstum.43 IV. Stand der Wissenschaft Bis anhin war die Identifizierung von Drohnenpiloten im Zusammenhang mit der Durchsetzung von datenschutzrechtlichen Ansprüchen wissenschaftlich nicht aufgearbeitet. Zu rechtlichen Fragen im Zusammenhang mit Drohnen wurden in der Schweiz aber bereits einige wenige Beiträge veröffentlicht. Zunächst ist auf den ausführlichen Artikel von Steiger44 hinzuweisen. Dieser befasst sich mit verschiedenen Aspekten der Drohnennutzung durch zivile Behörden. Auch aus haftungsrechtlicher sowie regulatorischer Sicht wurden Drohnen bereits beleuchtet.45 Des Weiteren wurde auch aus sachenrecht licher sowie zivilprozessualer Sicht erörtert, wie gegen Drohnen vorgegangen werden könnte.46 Haftungsrechtliche Fragen im Zusammenhang mit autonomen Drohnen wurden sodann ausführlich von Hänsenberger analysiert.47 Soweit ersichtlich wurden in der Schweiz bisher erst drei wissenschaftliche Beiträge veröffentlicht, welche sich des Themas Drohnen und Datenschutz annehmen.48 Diesen ist jedoch gemeinsam, dass sie sich nicht mit der Problematik der Identifikation des Drohnenpiloten bzw. der Durchsetzung der (grundrechtlichen) Ansprüche auf Datenschutz auseinandersetzen. In Deutschland wurde im Jahr 2012 die Dissertation von Kornmeier mit dem Titel «Der Einsatz von Drohnen zur Bildaufnahme» veröffentlicht. Die Autorin befasst sich aber darin ebenfalls nicht mit der Frage der Identifizierung 41 Fumagalli, S. 31 m.w.H. 42 Vgl. Christen et al., S. 115; Landrock/Baumgärtel, S. 62, je m.w.H. Dies ist auch auf die sehr unterschiedlichen (Welt-)Regionen oder Anwendungsfelder, die jeweils in die Analyse miteinbezogen wurden, zurückzuführen. 43 Ausführlich zum Ganzen Grohe, S. 28 ff. mit zahlreichen Hinweisen. 44 Steiger, Regulierung, S. 169 ff. 45 Hänsenberger, AJP 2017, sowie ausführlich ders., Diss. (Haftungsfragen); Hänsenberger/Wildhaber (regulatorische, luftrechtliche Aspekte). 46 Vgl. Hrubesch-Millauer/Bruggisser; Schneider-Marfels/Kaufmann (sachenrechtliche Aspekte); Kettiger, Abwehr (gerichtliches Verbot nach 258 ff. ZPO). 47 Hänsenberger, Diss. Vgl. ausführlich zum europarechtlichen Haftungsregime die Studie von Bertolini, passim. 48 Nadakavukaren Schefer, Weber/Oertly und Weber. Vgl. zum deutschen Recht den Artikel von Giebel. 8 Einleitung der Drohnenpiloten. Nur sehr kurz angesprochen wird das Thema Datenschutz im Werk von Kutschera («Rechtliche Grundlagen für die Verwendung von Drohnen in Österreich»).49 Insgesamt kann festgehalten werden, dass primär im spezifischen Gebiet der Literatur zu Drohnen und Datenschutz in der Schweiz sehr wenig Referenzmaterial zur Verfügung steht. Auch die Rechtsprechung in diesem Bereich ist äusserst spärlich. Als eines der ersten Gerichte überhaupt befasste sich das Kantonsgericht Luzern mit der Problematik, dass die Gemeinde Horw für die Wahrnehmung ihrer Kompetenzen im Bereich der Baupolizei auf Drohnenbilder abgestellt hatte; dabei musste das Gericht auch datenschutzrechtliche Erwägungen anstellen.50 Eine Rechtssache, in der es um datenschutzrechtliche Aspekte privater Drohnennutzung geht, existiert — soweit ersichtlich — in der Schweiz bis anhin nicht. Auch die sozialwissenschaftliche Forschung zu zivilen Drohnen wurde erst in den letzten Jahren als eigenständiges Forschungsfeld entdeckt.51 Aus Schweizer Sicht sind primär zwei wichtige Projekte in diesem Bereich zu erwähnen. Einerseits das SNF-Projekt «Power and Space in the Drone Age» unter der Leitung von Prof. Klauser der Universität Neuenburg,52 andererseits das TA-SWISS-Projekt53 von Christen et al. § 3 Aufbau und Methode I. Gang der Untersuchung Damit die eingangs gestellte Forschungsfrage beantwortet werden kann, müssen zunächst die zentralen Begriffe — Drohne, Daten, Datenbearbeitung sowie drohnengestützte Datenbearbeitung — definiert werden. Im Zusammenhang mit Drohnen sind auch der Stand der Technik und deren Einsatzformen in der Schweiz kurz darzustellen. Davor soll jedoch auf die fluide 49 Vgl. Kutschera, S. 49 ff. 50 KGer LU, 7H 17 49 vom 18. April 2018; vgl. auch BVGer, A-2482/2007 vom 26. Juni 2007 E. 5, wo eine kurze Auseinandersetzung mit Drohneneinsätzen und damit einher gehender Datenerhebung durch das Grenzwachtkorps im Lichte von Art. 13 BV und Art. 8 Ziff. 1 EMRK zu finden ist. 51 Christen et al., S. 133. 52 Vgl. die Publikationen von Klauser et al.; Klauser/Pedrozo und Pedrozo/Klauser. Vgl. auch die in Deutschland durchgeführten Studien von Euler/Cobus/Koelle und Koelle et al. 53 Technologiefolgen-Abschätzung ist eine im Bundesgesetz über die Förderung der Forschung und der Innovation (FIFG) verankerte Aufgabe (Art. 11 Abs. 2 lit. c FIFG); die TA-SWISS nimmt diese seit 1992 wahr (vgl. www.ta-swiss.ch — Mandat und Organisation). § 3 Aufbau und Methode 9 Gesetzgebung in den Bereichen Datenschutz und Drohnen sowie die daraus erwachsenden Konsequenzen für die vorliegende Arbeit eingegangen werden (Teil 1: Rechtliche, begriffliche und technische Grundlagen).54 In einem weiteren Schritt wird die drohnengestützte Datenbearbeitung im Lichte der Grundrechte betrachtet. Die Ausführungen zu den Schutzpflichten sind dabei von zentraler Bedeutung. Sie dienen als Massstab für die darauffolgende Analyse der Gesetzgebung im Zusammenhang mit der drohnengestützten Datenbearbeitung durch Private. Die Abhandlungen zu den durch Drohnen möglicherweise tangierten Grundrechte erlauben es, zu bestimmen, in welchen grundrechtlich geschützten Bereichen sich Schutzpflichten aktualisieren (Teil 2: Die drohnengestützte Datenbearbeitung im Lichte der Grundrechte).55 Die erarbeiteten grundrechtlichen Anforderungen sollen sodann auf das materielle Datenschutzrecht, namentlich den E-DSG, angewandt werden. Im Sinne einer Bestandesaufnahme soll überprüft werden, ob und welche datenschutzrechtlichen Ansprüche bei der drohnengestützten Datenbearbeitung durch Private entstehen können und wie es um deren Durchsetzung bestellt ist (Teil 3: Bestandesaufnahme (1): Der E-DSG).56 Die Bestandesaufnahme wird sodann im Luftfahrtrecht weitergeführt. Die neuen europäischen Vorschriften zu Drohnen sollen analysiert und deren datenschutzrechtliche Bedeutung herausgeschält werden. Ebenfalls soll analysiert werden, inwiefern die «Drohnengesetzgebung» die Durchsetzung datenschutzrechtlicher Ansprüche ermöglicht oder zumindest begünstigt (Teil 4: Bestandesauf nahme (2): Die «Drohnenverordnungen»).57 Der fünfte Teil baut auf den beiden Bestandesaufnahmen auf und stellt die dort erarbeiteten Erkenntnisse den Resultaten des zweiten Teils gegenüber. Dies ermöglicht zu beurteilen, ob (weitergehende) Schutzpflichten bestehen und wie diese wahrgenommen werden können. Daraus resultieren konkrete Vorschläge an den Gesetzgeber zur Ermöglichung der Durchsetzung von datenschutzrechtlichen Ansprüchen bei der drohnengestützten Datenbearbeitung durch Private (Teil 5: Die Durchsetzung von datenschutz rechtlichen Ansprüchen).58 Abschliessend folgt eine Zusammenfassung der gewonnenen Erkenntnisse.59 54 Hinten, S. 11 ff. 55 Hinten, S. 45 ff. 56 Hinten, S. 73 ff. 57 Hinten, S. 119 ff. 58 Hinten, S. 157 ff. 59 Hinten, S. 175 ff. 10 Einleitung II. Methode Die vorliegende Dissertation ist eine rechtswissenschaftliche Arbeit. Ihre Erkenntnisse gewinnt sie — der rechtswissenschaftlichen Tradition folgend — primär aus den einschlägigen Rechtssätzen und Materialien, der juristischen Literatur sowie der Judikatur. Dabei wird das Recht mit den Mitteln der grammatikalischen, historischen, systematischen und teleologischen Auslegung analysiert.60 Beim Verfassen dieser Dissertation werden die Bundesverfassung, der Revisionsentwurf zum Bundesgesetz über den Datenschutz,61 die europäischen Verordnungen zu Drohnen62 sowie die Luftfahrtgesetzgebung in der Schweiz63 im Vordergrund stehen. Erkenntnisse aus anderen Wissenschaftsdisziplinen, wie z.B. der Sozialoder Wirtschaftsgeografie oder der Soziologie, werden insofern in die Arbeit miteinbezogen, als dies für das Verständnis der rechtlichen Ausführungen dienlich ist. Ausserdem ist es unumgänglich, dass Erkenntnisse aus verschiedenen Bereichen der Ingenieurswissenschaften Eingang in die Untersuchung finden, namentlich bei den technischen Möglichkeiten von Drohnen,64 deren aerodynamischer Konstruktionsweise65 sowie bei der Beurteilung der von Drohnen ausgehenden Verletzungsgefahr.66 60 Ausführlich zu den einzelnen Auslegungsmethoden Kramer, S. 60 ff. und spezifisch zur Verfassungsauslegung Tschannen, § 4 Rn. 1 ff. Zum sog. «pragmatischen Methoden pluralismus» des Bundesgerichts vgl. Häfelin/Müller/Uhlmann, Rn. 178 ff.; sowie Kramer, S. 132 ff. mit zahlreichen Hinweisen auf die Rechtsprechung. 61 Weshalb auf der Basis des Entwurfs des neuen Datenschutzgesetzes gearbeitet wird, soll sogleich (S. 11 ff.) erläutert werden. 62 Vgl. dazu und deren Bedeutung für die Schweiz hinten, S. 13 ff. 63 Luftfahrtgesetz (LFG), die Luftfahrtverordnung (LFV) sowie die Verordnung des UVEK über Luftfahrzeuge besonderer Kategorien (VLK). 64 Vgl. dazu hinten, S. 40 ff. 65 Vgl. dazu hinten, S. 18 ff. 66 Vgl. dazu hinten, S. 122 ff. 11 Teil 1: Rechtliche, begriffliche und technische Grundlagen § 1 Bemerkungen zu den zentralen Rechtsgrundlagen Wie bei den Ausführungen zur Methode erwähnt,67 basiert diese Arbeit auf dem Entwurf des neuen Bundesgesetzes über den Datenschutz sowie den drei europäischen Verordnungen zu Drohnen. Nachfolgend soll zu beiden Rechtsmaterien kurz ausgeführt werden, weshalb diese Vorgehensweise gewählt wurde. I. Datenschutzgesetz 1. Auslöser und Ziele der Totalrevision Die schweizerische Datenschutzgesetzgebung wird momentan totalrevidiert.68 Der Entwurf zum neuen DSG wurde in der Herbstsession 2019 vom Nationalrat als Erstrat beraten. Der Ständerat befasste sich sodann am 18. Dezember 2019 mit der Vorlage.69 Der Zeitpunkt des Inkrafttretens des revidierten Datenschutzgesetzes ist noch unklar.70 Die Revisionsbestrebungen sind durch Entwicklungen auf europäischer Ebene ausgelöst geworden. Namentlich sollte damit den neu erlassenen Rechtsakten auf Ebene der Europäischen Union sowie des Europarats71 67 Vgl. vorne, S. 10. 68 Vgl. ausführlich zu den verschiedenen Etappen des noch laufenden Revisionsprozesses Curia Vista, 17.059. 69 Aus zeitlichen Gründen konnten die Beschlüsse des SR nicht mehr in die vorliegende Untersuchung einfliessen. 70 Vgl. Art. 69 E-DSG. 71 Namentlich wurde das Übereinkommen zum Schutz des Menschen bei der automatischen Verarbeitung personenbezogener Daten (SEV 108; auch Konvention 108) vom Europarat überarbeitet. Das Änderungsprotokoll liegt seit dem 10. Oktober 2018 vor; der Bundesrat hat dieses am 30. Oktober 2019 unterzeichnet. Vgl. Botschaft Total revision DSG, S. 6966; Bygrave, S. 32 ff. 12 Teil 1: Rechtliche, begriffliche und technische Grundlag Rechnung getragen werden. Die EU hat im Jahr 2016 ihre Datenschutzgesetzgebung auf eine neue Grundlage gestellt und insbesondere die Verordnung (EU) 2016/679 zum Schutz natürlicher Personen bei der Verarbeitung personenbezogener Daten (DSGVO) erlassen.72 Ein weiterer Grund für die Revision des Schweizer Datenschutzrechts waren sodann die Schwächen des «alten» Datenschutzgesetzes von 1992, welche aufgrund der rasanten technologischen Entwicklung entstanden waren.73 Wie noch aufgezeigt wird,74 sind die Regelungen der Datenschutzgesetzgebung und des Persönlichkeitsschutzes gemäss ZGB Ausdruck staatlicher Schutzpflichten.75 Die Totalrevision des DSG hatte mithin ebenfalls zum Ziel, den Schutz grundrechtlich relevanter Ansprüche im Kontext der informa tionellen Selbstbestimmung effektiver zu gestalten. Dies sollte insbesondere durch eine Verbesserung der Transparenz der Datenbearbeitung sowie der Kontrollmöglichkeiten der betroffenen Personen geschehen.76 Es lässt sich — gerade im Datenschutzrecht — eine «zunehmende Sensibilisierung des Grundrechtsverständnisses» beobachten.77 Die Einflüsse der Regelungen der Europäischen Union sind im Entwurf zum neuen DSG klar ersichtlich. Dies ist nicht überraschend, denn die Schweiz beabsichtigt, eine Bestätigung über ein angemessenes Datenschutzniveau zu erhalten (sog. Angemessenheitsbeschluss).78 Diese ermöglicht den freien Datenaustausch zwischen der Schweiz und der EU.79 72 Die DSGVO gilt seit dem 25. Mai 2018. Daneben wurde die Richtlinie (EU) 2016/680 zum Schutz natürlicher Personen bei der Verarbeitung personenbezogener Daten im Bereich des Strafrechts (Schengen-relevant; notifiziert am 1. August 2016) erlassen. Die dadurch notwendig gewordenen Anpassungen wurden bereits in Kraft gesetzt (vgl. BBl 2018 6003 ff.; AS 2019 625 ff.). Für die vorliegende Untersuchung ist die Richtlinie unbeachtlich, da sie die Strafverfolgung betrifft. 73 Vgl. zu den Zielen der Revision Botschaft Totalrevision DSG, S. 6943 und S. 6970 ff. 74 Ausführlich hinten, S. 69 ff. 75 Biaggini, Komm. BV, Art. 13 N 16; vgl. auch BGE 138 II 346, passim, v.a. E. 8.2. 76 Botschaft Totalrevision DSG, S. 6943; Amtl. Bull. NR 2019 S. 1774 (Votum Jauslin), S. 1781 (Votum Fluri) und S. 1782 (Votum BR Keller-Sutter); vgl. auch Aebi-Müller, Rn. 564 sowie Rn. 591 ff.; Savary, S. 217. 77 Stinner, S. 63. Vgl. dazu z.B. EG 73 und 104 DSGVO, wo die Menschenrechte explizit erwähnt werden. Vgl. auch Tamò-Larrieux, S. 77 mit Fn. 33. 78 Teilweise auch Äquivalenzbeschluss oder Adäquanzentscheidung. Vgl. Art. 45 DSGVO und dazu statt aller Schantz, in: Simitis et al., Art. 45 DSGVO N 3 ff.; Baur, S. 172 f. Vgl. sodann BJ, Kompatibilitätstabelle (Vergleich E-DSG mit DSGVO, SEV 108 etc., auch mit Blick auf den Angemessenheitsbeschluss). 79 Ausführlich dazu Botschaft Totalrevision DSG, S. 6964. Vgl. auch Rosenthal, Entwurf, Rn. 4 ff.; Savary, S. 217; Rudin, S. 222 ff. § 1 Bemerkungen zu den zentralen Rechtsgrundlagen 2. 13 Abstellen auf den E-DSG Vor diesem Hintergrund erweist es sich als nicht sinnvoll, für die vorliegende Auseinandersetzung auf das noch in Kraft stehende, aber «alte» DSG abzustellen. Nachfolgend wird deshalb mit dem Entwurf des Datenschutzgesetzes in der Version gemäss Beschluss des Nationalrats vom 25. September 2019 gearbeitet.80 Die selbstverständlich möglichen Änderungen in der weiteren Beratung des Entwurfs dürften — wenn überhaupt — nur sehr beschränkten Einfluss auf die zentralen Bestimmungen des neuen Datenschutzgesetzes und deren Anwendbarkeit auf die drohnengestützte Datenbearbeitung durch Private haben. Literatur und Rechtsprechung zum heute geltenden DSG können an vielen Stellen noch immer verwendet werden, da sich die Grundbegriffe und -prinzipien des Datenschutzrechts weder in der Schweiz noch in der EU im Grundsatz verändert haben bzw. verändern werden.81 II. Drohnenverordnungen 1. EU-Recht und dessen Bedeutung für die Schweiz Auch im Bereich der Luftfahrt ergaben sich unlängst auf europäischer Ebene zentrale Veränderungen.82 Die neue EASA-Grundverordnung ist am 11. Sep tember 2018 in Kraft getreten83 und enthält die gemeinsamen Vorschriften für die Zivilluftfahrt innerhalb der EU. Die Grundverordnung ist von zentraler Bedeutung, da sie — anders als das bisherige Recht84 — grundsätzlich alle Drohnen erfasst und mithin einen einheitlichen europäischen Rechtsrahmen schafft.85 80 Vgl. Fahne mit den Beschlüssen des Nationalrats vom 25. September 2019. 81 Vgl. dazu im Einzelnen die Auseinandersetzung mit den Bestimmungen des E-DSG hinten, S. 74 ff. 82 Diese wurden aber nicht nur mit Blick auf die Drohnentechnologie erlassen; vgl. zum Hintergrund der revidierten EASA-Grundverordnung Rebane, S. 51 ff. 83 Vgl. Art. 141 EASA-Grundverordnung. 84 Vor Inkrafttreten der EASA-Grundverordnung war die Regulierung unbemannter Luftfahrzeuge mit einem Gewicht von weniger als 150 kg Sache der Mitgliedstaaten (vgl. Anhang II lit. i i.V.m. Art. 4 Verordnung [EG] Nr. 216/2008). Vgl. dazu Masutti, S. 213 und S. 215; Voland/Qui, S. 346. 85 Verordnungen der EU haben gemäss Art. 288 Abs. 2 AEUV allgemeine Geltung. Sie sind in allen ihren Teilen verbindlich und gelten unmittelbar in jedem Mitgliedstaat. Sie müssen — anders als Richtlinien — nicht erst in nationales Recht umgesetzt werden (Oesch, Rn. 423 f.; Hänsenberger, Diss., S. 196; Ebinger, S. 5). 14 Teil 1: Rechtliche, begriffliche und technische Grundlag Ausnahmen vom Geltungsbereich sind u.a. für das Militär, Blaulichtorganisationen sowie für Forschungs- und Versuchszwecke vorgesehen.86 Diese sind für die vorliegende Untersuchung nicht weiter von Bedeutung, da sie primär Anwendungen erfassen sollen, «die unter der Kontrolle und Verantwortung eines Mitgliedstaats im öffentlichen Interesse von einer mit hoheitlichen Befugnissen ausgestatteten Stelle oder in deren Auftrag durchgeführt werden».87 Gestützt auf die EASA-Grundverordnung hat die EU-Kommission die Durchführungsverordnung sowie die Delegierte Verordnung erlassen.88 Diese Regelwerke sind per 1. Juli 2019 in Kraft getreten.89 Darin wird die Grundverordnung konkretisiert;90 sie enthalten ausführliche Vorschriften zu verschiedensten Aspekten der Drohnenfliegerei. 2. Übernahme durch die Schweiz Die Schweiz hat diese luftfahrtrechtlichen Vorschriften gestützt auf das Luftverkehrsabkommen Schweiz–EU (LVA CH–EU) übernommen bzw. wird diese noch übernehmen.91 Dies geschieht im Einklang mit dem Grundgedanken des Abkommens, namentlich der dynamischen Rechtsentwicklung und deren Übernahme.92 Mit der Revision des Anhangs zum LVA CH–EU durch den zuständigen Gemischten Ausschuss werden die dort genannten Regelwerke der EU für die Schweiz verbindlich.93 Die entsprechenden Beschlüsse des Gemischten Ausschusses werden gemäss der Vorgabe in Art. 22 Abs. 3 86 Vgl. Art. 2 Abs. 3 und Anhang I EASA-Grundverordnung; Lavallée, S. 2 f.; vgl. Dempsey, S. 63 f. 87 Art. 2 Abs. 3 lit. a EASA-Grundverordnung. Kritisch zur Formulierung der Bestimmung und der daraus möglicherweise resultierenden unsicheren Rechtslage — v.a. bei nichtmilitärischen Anwendungen — Masutti/Tomasello, S. 52 f.; vgl. auch Voland/Qui, S. 346. 88 Vgl. Art. 57 f., Art. 61 und Art. 128 EASA-Grundverordnung; Art. 290 f. AEUV; Josipovic, S. 440; Voland/Qui, S. 347. 89 Art. 23 Abs. 1 Durchführungsverordnung (sie gilt ab dem 1. Januar 2021); Art. 42 Delegierte Verordnung; Krumm, Rechtsrahmen, S. 484. 90 Die grundlegenden Anforderungen an Konstruktion, Herstellung, Instandhaltung, Betrieb, Organisationen, Betreiber und Fernpiloten, Registrierung etc. von Drohnen sind in Anhang IX EASA-Grundverordnung enthalten. Vgl. zu diesen im Einzelnen Josipovic, S. 440 f. 91 Vgl. Art. 23 LVA CH–EU und ausführlich dazu Dettling-Ott, Luftverkehrsabkommen, Rn. 9 ff. und Rn. 53 f. Vgl. zur Bedeutung von EU-Recht für die Regulierung von Drohnen in der Schweiz auch Hänsenberger, Diss., S. 200 und S. 234 f. 92 Vgl. dazu Epiney/Frei, Rn. 1 mit Fn. 1 sowie Rn. 21. Kritisch zur «pauschalen Übernahme europäischer Vorschriften» Christen et al., S. 181 und 225. 93 Art. 2 LVA CH–EU. Vgl. allgemein zur Übernahme des EU-Rechts im Bereich der Luftfahrt Botschaft sektorielle Abkommen, S. 6150 f. § 2 Begriffe 15 LVA CH–EU in der AS sowie im Amtsblatt der EU publiziert.94 Die EASA- Grundverordnung gilt dementsprechend für die Schweiz seit dem 1. September 2019;95 die Delegierte Verordnung soll per 1. Juli 2020 in Kraft treten. Die Durchführungsverordnung soll dagegen erst ein halbes Jahr später, namentlich ab dem 1. Januar 2021, anwendbar sein. Dies hat die EU-Kommission im April 2020 wegen der Coronakrise entschieden. Aus diesem Grund bleibt auch die bisherige Schweizer Drohnenregulierung bis Ende 2020 in Kraft.96 Die europarechtlichen Definitionen und Vorgaben sind bzw. werden somit bald auch in der Schweiz unmittelbar anwendbar.97 Der Gemischte Ausschuss hat die Kompetenz, den Wortlaut einzelner Bestimmungen so anzupassen, dass deren Anwendbarkeit für die Schweiz sichergestellt werden kann. Davon hat er z.B. mit Bezug auf Art. 132 Abs. 1 EASA-Grundverordnung Gebrauch gemacht: Der dortige Verweis auf die DSGVO ist unter LVA CH–EU «als Bezugnahme auf die einschlägigen nationalen Rechtsvorschriften zu verstehen».98 Aufgrund der bereits erfolgten sowie der geplanten Übernahme des euro päischen Rechtsrahmens zu Drohnen rechtfertigt es sich, in der vorliegenden Untersuchung darauf abzustellen. § 2 Begriffe I. Drohne Der Begriff der Drohne ist für das Verständnis der Arbeit zentral.99 Dabei haben die allermeisten Menschen eine relativ klare Vorstellung davon, was darunter zu verstehen ist.100 In der Gesellschaft und in den Medien101 ist er 94 Vgl. zur Veröffentlichungspflicht EDA, S. 8. 95 Beschluss Nr. 1/2019, AS 2019 S. 2979 ff.; vgl. auch ABl. 2019 L 226/1 vom 30. August 2019. 96 So BAZL, Übernahme. Der entsprechende Beschluss des Gemischten Ausschusses war im ersten Quartal 2020 geplant. Wann dieser nun erfolgen wird, ist noch nicht bekannt. 97 Zur «alten» EASA-Grundverordnung BAZL, Verhältnis, S. 6; EDA, S. 5; vgl. BSK BVKern, Art. 87 N 26; Giemulla, S. 197. 98 Beschluss Nr. 1/2019, AS 2019 S. 2985; vgl. auch ABl. 2019 L 226/1 vom 30. August 2019, S. 5. 99 Zum Ursprung des Begriffs vgl. Laumanns, S. 4; Kornmeier, S. 11; Bartsch/Coyne/ Gray, S. 25. Zur Geschichte der Drohnenfliegerei vgl. Dalamagkidis, History, S. 58 ff.; Peterson, S. 535 ff. 100 Vgl. dazu z.B. die Untersuchung zur gesellschaftlichen Akzeptanz von Drohnen von Klauser/Pedrozo, passim. 101 Vgl. statt vieler die Artikel von Betschon; Haller U. sowie kritisch Hesse. Vgl. auch Stumvoll, S. 5. 16 Teil 1: Rechtliche, begriffliche und technische Grundlag denn auch bereits weit verbreitet. Ein Hinweis auf die Verbreitung des Begriffs findet sich auch in einem Urteil des Bundesgerichts aus dem Jahr 2011. Es hatte sich mit dem Vorbringen eines Beschwerdeführers zu befassen, er sei «betroffen gewesen von einer fünfwöchigen Untersuchung seiner Räume mit der Unterstützung von Drohnen».102 Aus rechtswissenschaftlicher Optik sind der Begriff und vor allem seine Definition jedoch nicht klar und einfach.103 Aus diesem Grund soll nachfolgend zunächst eine Annäherung anhand verschiedener (technischer) Bezeichnungen dafür vorgenommen werden (sogleich). Danach wird eine Kate gorisierung anhand der aerodynamischen Konstruktionsweise präsentiert (S. 18 ff.). Des Weiteren wird die Drohnen-Definition gemäss den vorerwähnten EU-Rechtsakten und der darin vorgesehenen Einteilung in Risikokategorien vertieft (S. 20 ff.), bevor eine Zusammenfassung und eine Stellungnahme folgen (S. 26). 1. Technische Bezeichnungen Für Drohnen werden in der (technischen) Literatur verschiedene englische Bezeichnungen verwendet.104 Neben Unmanned Aircraft (kurz UA)105 ist auch UAV (Unmanned Aerial Vehicle)106 weit verbreitet.107 Im Deutschen werden diese Begriffe regelmässig mit «unbemanntes Luftfahrzeug» übersetzt.108 Als Luftfahrzeuge gelten gemäss der Definition in Art. 1 Abs. 2 LFG «Fluggeräte, welche sich durch Einwirkungen der Luft, jedoch ohne die Wirkung von Luft gegen den Boden (Luftkissenfahrzeuge), in der Atmosphäre halten können.»109 102 BGer, 6B_251/2011 vom 5. Juli 2011 E. 3. Drohnen wurden auch in weiteren Urteilen bereits erwähnt; vgl. BGer, 1C_444/2017 vom 20. August 2018 E. 3.3; BGer, 8C_437/2016 vom 10. Januar 2017 E. 2.1; BGer, 1A.365/1999 vom 12. April 2002, passim. 103 Vgl. Christen et al., S. 37 ff.; Mendes de Leon/Scott, S. 186 f.; Granshaw, passim; Salter, S. 163 mit FN 1. 104 Neben den hier erläuterten Begriffen werden in der Literatur teilweise noch weitere Bezeichnungen verwendet; eine vollständige Übersicht kann vorliegend nicht präsentiert werden. Weiterführend Christen et al., S. 37 m.w.H. 105 Dalamagkidis, Definitions, S. 44. 106 Mahn-Gauseweg, S. 2; Steiger, Regulierung, S. 171. 107 So z.B. Kornmeier, S. 10 m.w.H. 108 Stumvoll, S. 5; vgl. Kornmeier, S. 10. Ausführlich zur Bedeutung des Begriffs «unbemannt» in diesem Zusammenhang Henseler, Einordnung, S. 119 f. m.w.H. 109 Vgl. dazu und zur Abgrenzung von Flugkörpern McNally, S. 22 f.; Henseler, Einordnung, S. 118 f. § 2 Begriffe 17 Das Akronym UAS steht für Unmanned Aircraft System110 und stellt die umfassendste Bezeichnung dar.111 Durch die Verwendung des Ausdrucks «System» wird hervorgehoben, dass ein unbemanntes Luftfahrzeug notwendigerweise als Gesamtsystem zum Einsatz kommt.112 Dabei werden etwa auch die Bodenkontrollstation, die Kommunikationstechnologie, Start- und Landeelemente, die Ladung (sog. payload) etc. miteinbezogen.113 Auch das Bodenpersonal wird teilweise zum System gezählt.114 Ein UAS kann auch mehrere Bodenkontrollstationen sowie Start- und Landeelemente umfassen.115 Bei Sicherheitsfragen116 ist u.U. nicht nur das Luftfahrzeug an sich, sondern das System insgesamt massgebend.117 Oftmals sind UA, UAV und UAS aber austauschbar.118 Eng verwandt mit UAS ist die Bezeichnung RPAS (Remotely Piloted Aircraft System).119 Sie stellt explizit auf die Fernsteuerung durch einen sich nicht an Bord befindlichen Piloten ab.120 Somit sind autonom fliegende Luftfahrzeuge nicht erfasst, weshalb RPAS eine Teilmenge der UAS darstellen.121 110 Stumvoll, S. 6 f.; Kornmeier, S. 10; Steiger, Regulierung, S. 171; Kutschera, S. 35; vgl. ICAO, S. x. Städele, S. 25, gibt dagegen «Unmanned Aerial System» an. 111 Steiger, Regulierung, S. 171. 112 Städele, S. 25. 113 Steiger, Regulierung, S. 171; Kornmeier, S. 10; Austin, S. 3; vgl. Dalamagkidis, Definitions, S. 45. 114 So z.B. Stumvoll, S. 6. 115 Dalamagkidis, Definitions, S. 45; Kornmeier, S. 10. 116 Im englischen Sprachgebrauch werden in der Luftfahrt zwei «Arten» von Sicherheit unterschieden, namentlich «Safety» und «Security». Erstere beschreibt die betriebliche Sicherheit, Letztere äussere, nicht betriebsbedingte Gefahren. Auf Deutsch werden üblicherweise die Begriffe «Flugsicherheit» bzw. «Luftsicherheit» verwendet (Schwenk/Giemulla, Kap. 11 Rn. 2; vgl. auch EG 59 EASA-Grundverordnung). In der Textstelle oben wird Sicherheit umfassend verstanden. 117 BAZL, Zivile Drohnen, S. 7; Dalamagkidis/Valavanis/Piegl, S. 2. Diesem Ansatz folgen zu Recht auch die europäischen Verordnungen, vgl. dazu ausführlich hinten, S. 21 ff. 118 Dalamagkidis, Definitions, S. 45; vgl. Kornmeier, S. 11. 119 Abeyratne, S. 117; Kutschera, S. 36; Steiger, Regulierung, S. 171; BAZL, Zivile Drohnen, S. 5. 120 «Unbemannte und doch pilotengesteuerte Luftfahrzeuge» (BAZL, Zivile Drohnen, S. 7); vgl. ICAO, Ziff. 3.2. 121 Hilf/Umbach, S. 65; vgl. SESAR, Master Plan, S. 4; anders Steiger, Regulierung, S. 171, der RPAS und UAS als Synonyme bezeichnet. 18 2. Teil 1: Rechtliche, begriffliche und technische Grundlag Kategorisierung anhand der Konstruktionsweise Teilweise sind Begriffe wie Multikopter bzw. Multirotor122 anzutreffen, wenn von Drohnen die Rede ist.123 Bezeichnet wird damit aber lediglich eine Bauart derselben; sie ist anhand der aerodynamischen Konstruktion von anderen Arten abzugrenzen.124 Die am weitesten verbreiteten Konstruktionsweisen sollen nachfolgend dargestellt werden.125 2.1 Drehflügler Multikopter sind wie Helikopter den Drehflüglern (engl. rotorcraft) zuzurechnen.126 Diese verfügen über einen127 oder mehrere Rotoren als Antrieb und zur Stabilisierung.128 Quadrokopter gehören in diese Gruppe, wobei bei der Bezeichnung auf die Anzahl Propeller abgestellt wird.129 Die Hälfte derselben dreht in die jeweils entgegengesetzte Richtung der gegenüberliegenden Rotoren; unterschiedliche Drehzahlen ermöglichen das Manövrieren.130 Bei den auf dem (Schweizer) Markt erhältlichen Drohnenmodellen sind Quadrokopter die vorherrschende Konfiguration.131 122 Diese beiden Begriffe sind Synonyme (vgl. dazu sogleich, 2.1 Drehflügler). 123 Vgl. Büchi, Buch der Drohnen, S. 12; Kornmeier, S. 11 f.; Steiger, Regulierung, S. 172. 124 Ausführlich zum nachfolgenden Preznuk, S. 17 ff.; Hassanalian/Abdelkefi, S. 101 ff. (je mit zahlreichen Illustrationen). Vgl. auch Kettiger, Abwehr, Rn. 4, der nur zwei Arten unterscheidet, namentlich «Multikopter» und «Tragflächenflieger-Drohnen». Es existieren auch Mischformen der hier dargestellten Konstruktionsarten, vgl. dazu die Hinweise bei Hänsenberger, Diss., S. 17 mit Fn. 112. 125 Vereinzelt werden zusätzliche Kategorien wie z.B. Luftschiffe diskutiert; vgl. dazu die Hinweise bei Henseler, Einordnung, S. 123. 126 Innerhalb der Kategorie der Drehflügler werden Trag- und Hubschrauber unterschieden (vgl. Art. 2 Abs. 1 und Anhang LFV; BGer, 2C_807/2016 vom 17. Juli 2017 E. 5.1). 127 Als Beispiel kann hier der «Monospinner», entwickelt an der ETH Zürich, genannt werden (vgl. dazu Zhang/Mueller/D’Andrea, passim). 128 Brahms/Maslaton, S. 1126; Steiger, Regulierung, S. 172; Vergouw et al., S. 24. 129 Ein Multirotor mit sechs Rotoren ist somit ein Hexakopter, ein solcher mit acht Rotoren ein Oktokopter etc. Vgl. dazu Weber, S. 3 m.w.H. in Fn 9 und ausführlich (aus technischer Optik) Büchi, Quadrocopter, S. 89 ff. 130 Biermann/Wiegold, S. 91 f.; Büchi, Buch der Drohnen, S. 16; Juniper, S. 18; Hänsenberger, Diss., S. 17 f.; Powers/Mellinger/Kumar, S. 310. 131 Preznuk, S. 23; Hodgkinson/Johnston, S. 11; Landrock/Baumgärtel, S. 6; vgl. Steiger, Regulierung, S. 172. Vgl. zu den in der Schweiz verbreiteten Drohnenmodellen hinten, S. 39. § 2 Begriffe 19 2.2 Starrflügler Starrflügler (engl. fixed-wing), wozu insbesondere Flugzeuge zu zählen sind, nutzen ihre Tragflächen zur Generierung von Auftrieb.132 Der Vortrieb wird durch Luftschrauben (Propeller) oder ein Strahltriebwerk erzeugt;133 die Lenkung erfolgt mittels Höhen- und Seitenruder.134 2.3 Schwingflügler Schwingflügler (engl. flapping-wing; Ornithopter) erzeugen — wie beim Flügelschlag von Vögeln oder Insekten — durch Dreh- oder Schlagbewegungen der Tragflächen den erforderlichen Auf- und Vortrieb.135 Aufgrund der Schwierigkeit, eine ausreichende Schlagfrequenz der Flügel zu erreichen, fristet diese Konstruktionsart ein Nischendasein.136 3. Zwischenfazit Die Darstellung der verwendeten Begriffe zeigt auf, dass die Bezeichnung von unbemannten Luftfahrzeugen weder einheitlich noch einfach verständlich ist. Die verbreitete Verwendung des Terms «Drohne» hängt wohl auch mit diesen Schwierigkeiten zusammen.137 Aus sprachlichen Gründen und aufgrund der weiten Verbreitung des Begriffs in der Gesellschaft,138 den Medien und der Literatur139 sollte m.E. primär von «Drohne» die Rede sein.140 In der Bevölkerung wird diese Bezeichnung wohl meist verstanden, 132 Schwenk/Giemulla, Kap. 7 Rn. 18; Dalamagkidis, Definitions, S. 47. Anschaulich zur Funktionsweise Juniper, S. 18. 133 Brahms/Maslaton, S. 1126; vgl. Juniper, S. 18. 134 Vergouw et al., S. 24. 135 Henseler, Einordnung, S. 123; Vergouw et al., S. 25; ausführlich Doman/Oppen heimer/Sighthorsson, passim. 136 Austin, S. 69; Jones/Platzer, S. 1359 ff.; Hänsenberger, Diss., S. 18 f. 137 Vgl. Kornmeier, S. 14. 138 Die EASA hielt etwa unlängst fest: «Everybody calls them drones» (EASA, On Air). 139 Sarrión Esteve, S. 105, befürwortet die Verwendung des Begriffs explizit auch in der akademischen Literatur. 140 A.M. Plücken, S. 36 f., der festhält, dass «[der Begriff] keinen eindeutigen Aufschluss darüber [gibt], worauf er sich bezieht (fliegendes Objekt oder System), welche luftrechtliche Einordnung damit einhergeht (aircraft oder nicht) und welche weiteren Eigenschaften das damit bezeichnete Objekt oder System (Grad der Autonomie) aufweist». Vgl. auch Granshaw, S. 168, wonach der Begriff nur verwendet werden sollte, «when conversing with a non-scientific audience»; ähnlich Cassart, Droit des drones, Rn. 12. Vgl. dagegen Hänsenberger, Diss., S. 11 und passim; Schladebach, § 6 N 49 in fine. 20 Teil 1: Rechtliche, begriffliche und technische Grundlag möglicherweise aber einzig mit Multikoptern assoziiert.141 Die Bekanntheit des Terms ist denn auch bei den wenigen Gerichtsverfahren im Zusammenhang mit Drohnen auffallend: Die Richter gehen nicht darauf ein und setzen ein Verständnis desselben voraus.142 Auch dieser Umstand spricht dafür, den Drohnenbegriff im rechtswissenschaftlichen Kontext zu benützen. Eine negative Konnotation desselben, der gegen eine solche Verwendung sprechen würde, konnte in Studien dazu nicht nachgewiesen werden. Die Terminologie «did not impact support for the technology, [but] it did impact familiarity.»143 Ausserdem hatte sie «minimal affect [recte: effect] on public perceptions, which contrasts with widely held industry beliefs and the results from analyses of media articles.»144 Relativierend ist anzumerken, dass die Studien in englischer Sprache in den USA (Pytlik Zillig et al.) bzw. in Australien (Clothier et al.) durchgeführt wurden. 4. Regelung in den Drohnenverordnungen 4.1 Drohnendefinition Gemäss EASA-Grundverordnung ist ein unbemanntes Luftfahrzeug (d.h. eine Drohne) «ein Luftfahrzeug, das ohne einen an Bord befindlichen Pilo ten autonom oder ferngesteuert betrieben wird oder dafür konstruiert ist».145 Auch Flugmodelle werden von dieser Definition erfasst,146 wobei sie als Unterkategorie von Drohnen zu qualifizieren sind.147 Dieser Ansatz ist mit der Übernahme der EASA-Grundverordnung auch in der Schweiz verbindlich geworden. Vor Übernahme der EU-Vorschriften wurden im Schweizer Recht Drohnen und Flugmodelle als «Modellluftfahrzeuge» zu- 141 Dies hängt mit der weiten Verbreitung dieses Typs zusammen; vgl. dazu Büchi, Buch der Drohnen, S. 12; Christen et al., S. 38 sowie vorne, S. 18. 142 In der Schweiz KGer LU, 7H 17 49 vom 18. April 2018 sowie die Hinweise in Fn. 102. In Deutschland z.B. VGer Braunschweig, 5 A 192/15 vom 7. September 2016, wo neben «Flugdrohne» auch der Begriff «Quadrocopter» erwähnt wird. Anders noch AGer Potsdam, 37 C 454/13 vom 16. April 2015, Tenor Ziff. 1, wo der Begriff Flugdrohne mit «funkgesteuertem Fluggerät» umschrieben wird. 143 Pytlik Zillig et al., S. 87 und S. 89. Zur (ursprünglich) negativen Konnotation des Drohnenbegriffs aus medien- und kulturwissenschaftlicher Sicht Howley, S. xiii ff. 144 Clothier et al., S. 1180. 145 Art. 3 Ziff. 30 EASA-Grundverordnung; vgl. auch die gleichlautende Definition in Art. 3 Ziff. 1 Delegierte Verordnung. 146 EASA, NPA, S. 9; Christen et al., S. 48; kritisch Schubert, S. 228; Germann, S. 13. 147 Flugmodell (Modellflugzeuge, Modellhelikopter usw.) lassen sich nicht ohne Weiteres von Drohnen abgrenzen. Vgl. dazu auch hinten, S. 25 f. § 2 Begriffe 21 sammengefasst, was in der Lehre kritisiert wurde.148 Die Verwendung des Oberbegriffs der «unbemannten Luftfahrzeuge» anstelle von «Modellluftfahrzeuge» ist deshalb ausdrücklich zu begrüssen. Da das Schweizer Luftfahrtrecht «unbemannte Luftfahrzeuge» bereits kennt, diese aber nicht klar von anderen Luftfahrzeugen abgrenzt, wird die EASA-Grundverordnung dazu führen müssen, die Begrifflichkeiten im Schweizer Recht zu überdenken und klarer zu definieren.149 Des Weiteren findet sich in Art. 2 Ziff. 1 Durchführungsverordnung eine Legaldefinition von «unbemanntes Luftfahrzeugsystem».150 Dabei handelt es sich um «ein unbemanntes Luftfahrzeug sowie die Ausrüstung für dessen Fernsteuerung». Diese Ausrüstung umfasst «alle Instrumente, Ausrüstungen, Mechanismen,151 Geräte, Zubehörteile, Software oder Zusatzteile, die für den sicheren Betrieb eines unbemannten Luftfahrzeugs erforderlich sind und die keine Teile152 sind und nicht an Bord des unbemannten Luftfahrzeugs mitgeführt werden».153 Es soll mithin das «Gesamtsystem» erfasst werden.154 4.2 Die drei Drohnenkategorien Die EASA-Grundverordnung teilt alle Drohnen bzw. Drohnenoperationen in drei risikobasierte Betriebskategorien ein, namentlich in die offene, die spezielle und die zulassungspflichtige Kategorie.155 Die Anforderungen an die Drohne und das zugehörige Gesamtsystem steigen von einer Kategorie zur nächsten anhand des damit verbundenen Risikos an.156 Dabei werden u.a. die kinetische Energie des Luftfahrzeugs,157 dessen Grösse und Ausstattung, die Anzahl an Personen im überflogenen Gebiet sowie die Gestal148 Christen et al., S. 49; Henseler, Einordnung, S. 126; zum deutschen Recht Giemulla, S. 203. 149 Vgl. dazu Art. 51 Abs. 3 LFG, Art. 2a LFV und v.a. Art. 1 und Art. 14 ff. VLK. 150 Vgl. auch die gleichlautende Definition in Art. 3 Ziff. 3 Delegierte Verordnung. 151 Der in der englischen Fassung enthaltene Ausdruck «mechanisms» ist hier wohl unbeholfen übersetzt worden. Es wäre korrekter, von «Vorrichtungen» zu sprechen. 152 In der englischen Sprachfassung der Delegierten Verordnung wird klar, dass hier (Bestand-)Teile der Drohne selber nicht erfasst werden sollen. Die Übersetzung ins Deutsche ist auch hier nicht optimal gelungen. 153 Art. 3 Ziff. 2 Delegierte Verordnung. 154 Vgl. dazu bereits vorne, S. 17. 155 Art. 3 Durchführungsverordnung; vgl. Schubert, S. 224 m.w.H. 156 Vgl. EG 32 EASA-Grundverordnung; EASA, Explanatory Note, S. 3; Palmerini, S. 16; Severoni, responsabilità, S. 75; Voland/Qui, S. 347. 157 Dieses Kriterium wird im Zusammenhang mit der Registrierungspflicht ausführlich diskutiert (hinten, S. 122 ff.). 22 Teil 1: Rechtliche, begriffliche und technische Grundlag tung des Luftraums, die Dichte an weiteren (bemannten und unbemannten) Luftfahrzeugen darin sowie die nutzbaren Flugsicherungsdienste158 betrachtet.159 a Offene Kategorie Die offene Kategorie ist als Grundkategorie160 auf pilotengesteuerte Drohnenmodelle ausgerichtet, von welchen ein geringes Betriebsrisiko ausgeht.161 Es ist keine vorgängige Bewilligung vorausgesetzt; das Risiko wird durch Verhaltensregeln und Betriebsbeschränkungen minimiert. So müssen Drohen dieser Kategorie grundsätzlich mit ununterbrochenem Sichtkontakt gesteuert werden162 und dürfen nicht schwerer als 25 kg sein.163 Die offene Kategorie ist in drei Unter- bzw. Einsatzkategorien (A1 bis A3) eingeteilt,164 wobei dem Umstand entscheidende Bedeutung zukommt, ob und wie nahe eine Drohne an Menschen heranfliegt.165 Je nach Unterkategorie kommen unterschiedliche Voraussetzungen an die technische Ausstattung und die Kenntnisse des Piloten zur Anwendung. Ausserdem sind jeweils andere Betriebsbeschränkungen vorgesehen.166 Für die vorliegende Untersuchung sind primär Drohnen dieser Kategorie von Bedeutung, denn diese kleinen, meist in Serie produzierten Drohnen sind generell in Europa und der Schweiz am weitesten verbreitet.167 Hinzu kommt, dass Private vor allem Drohnen in dieser Kategorie betreiben.168 158 «Flugsicherungsdienst» umfasst als Oberbegriff verschiedene Aufgaben wie das Luftraum-Management oder den Flugverkehrsdienst. In der Schweiz werden diese von Skyguide erfüllt. Vgl. ausführlich dazu hinten, S. 142 ff. 159 Josipovic, S. 439 m.w.H. 160 Vgl. JARUS, Categorization, S. 17 sowie die Ausführungen zur speziellen Kategorie sogleich, Rn. 57. 161 Vgl. Art. 4 Abs. 1 Durchführungsverordnung; Masutti/Tomasello, S. 79; Severoni, responsabilità, S. 75; vgl. JARUS, Categorization, S. 17. 162 Art. 4 Abs. 1 lit. d Durchführungsverordnung. 163 Art. 4 Abs. 1 lit. b Durchführungsverordnung; vgl. EASA, Explanatory Note, S. 10; EASA, Drohnen in Europa, S. 4; Hänsenberger, Diss., S. 216. 164 Art. 4 Abs. 2 und Anhang Teil A Durchführungsverordnung. Vgl. dazu Josipovic, S. 439; Hänsenberger, Diss., S. 219 f. 165 Voland/Qui, S. 347; vgl. EASA, Opinion, S. 21; Josipovic, S. 441; JARUS, Categorization, S. 20. 166 UAS.OPEN.020, UAS.OPEN.030 und UAS.OPEN.0 40 Anhang Teil A Durchführungsverordnung; vgl. Voland/Qui, S. 344; JARUS, Categorization, S. 20. 167 Vgl. Josipovic, S. 439; de Miguel Molina/Segarra Oña, S. 12 f. mit Tabelle 2. 168 Vgl. ausführlich dazu hinten, S. 39. § 2 Begriffe b 23 Spezielle Kategorie Die spezielle Kategorie169 baut gemäss Art. 5 Abs. 1 Durchführungsverordnung auf den Vorschriften der offenen Kategorie auf. Die dort geltenden Anforderungen sind grundsätzlich anwendbar. Werden diese nicht erfüllt, so ist eine Betriebsgenehmigung nötig.170 Das Verfahren basiert auf einer vom Betreiber171 der Drohne durchgeführten Risikobewertung,172 welche der zuständigen Behörde — zusammen mit dem Gesuch — einzureichen ist.173 Diese entscheidet über die Genehmigung und ob diese «für einen in Bezug auf Zeit, Ort oder beides einmaligen oder mehrmaligen Betrieb gilt».174 Vereinfachungen und Ausnahmen zur Genehmigungspflicht sind vorgesehen, wenn der Drohnenbetrieb innerhalb eines Standardszenarios erfolgt175 oder der Betreiber eine freiwillige Zertifizierung176 erhalten hat. Der Betrieb von autonomen Drohnen fällt grundsätzlich in die spezielle Kategorie.177 c Zulassungspflichtige Kategorie In die Kategorie «zulassungspflichtig»178 fallen diejenigen Drohnenoperatio nen, von denen ein grosses Risiko ausgeht,179 z.B. weil Menschen befördert 169 In der englischen Fassung der Durchführungsverordnung wird diese Kategorie als «specific» bezeichnet. Aus diesem Grund ist auch in der deutschsprachigen Literatur teilweise von der «spezifischen Kategorie» die Rede, so z.B. bei Hänsenberger, Diss., S. 220 ff. und passim. 170 Gemäss Art. 12 Durchführungsverordnung ist die Betriebsgenehmigung bei der zuständigen Behörde einzuholen. In der Schweiz handelt es sich dabei um das BAZL (vgl. Hänsenberger, Diss., S. 221). 171 Vgl. zum Begriff hinten, S. 27 f. 172 Masutti/Tomasello, S. 79; vgl. Hänsenberger, Diss., S. 221. Ausführlich zu Inhalt und Methodik der Risikobewertung Josipovic, S. 441 f. 173 Art. 5 Abs. 2 Durchführungsverordnung verlangt zudem, dass der Antrag «geeignete Massnahmen zur Risikominderung» darlegt. 174 Art. 5 Abs. 4 lit. a Durchführungsverordnung; vgl. Krumm, Anpassungsbedarf, S. 117. 175 Vgl. Art. 5 Abs. 5 Durchführungsverordnung; Voland/Qui, S. 348. Standardszenarien enthalten Voraussetzungen und damit verbundene Anforderungen für bestimmte Betriebsarten (Josipovic, S. 438). 176 Das sog. «Betreiberzeugnis für Leicht-UAS» (Art. 2 Ziff. 9 Durchführungsverordnung) erlaubt es dem Betreiber, eigene Drohneneinsätze zu genehmigen (vgl. dazu und zu den einzelnen Voraussetzungen Josipovic, S. 438; Voland/Qui, S. 348). 177 Vgl. UAS.SPEC.050 Abs. 1 lit. b Anhang Teil A Durchführungsverordnung; JARUS, Categorization, S. 24; Hänsenberger, Diss., S. 221. 178 In der englischen Fassung der Durchführungsverordnung wird diese Kategorie als «certified» bezeichnet. Aus diesem Grund ist auch in der deutschsprachigen Literatur teilweise von der «zertifizierten Kategorie» die Rede, so z.B. bei Hänsenberger, Diss., S. 223 ff. und Krumm, Anpassungsbedarf, S. 117. 179 Masutti/Tomasello, S. 79; JARUS, Categorization, S. 17. 24 Teil 1: Rechtliche, begriffliche und technische Grundlag oder gefährliche Güter transportiert werden.180 Das Zulassungsverfahren und die zu erfüllenden Anforderungen entsprechen deshalb grundsätzlich denjenigen, die auf die bemannte Luftfahrt zur Anwendung kommen.181 4.3 Produkteklassen innerhalb der offenen Kategorie Innerhalb der offenen Kategorie werden fünf Produkteklassen unterschieden, namentlich die Klassen C0 bis C4.182 Die Produkteklassen widerspiegeln das von der Drohne ausgehende Risiko anhand der jeweiligen Produkteeigenschaften.183 Von Drohnen der Klasse C0 geht das geringste und von solchen der Klassen C3 und C4 das höchste Risiko aus.184 Die Einteilung bezweckt die rasche und unkomplizierte Erkennbarkeit der auf die jeweilige Drohne anwendbaren Vorschriften.185 Ein entsprechendes Klassen-Identifizierungskennzeichen ist aus diesem Grund auf der Drohne anzubringen.186 Die folgende tabellarische Übersicht fasst die für die vorliegende Untersuchung zentralen Elemente zusammen: 180 Vgl. Art. 6 Abs. 1 lit. b Ziff. ii und iii Durchführungsverordnung; Art. 40 Delegierte Verordnung; EASA, GM Implementing Regulation, S. 8. 181 Krumm, Anpassungsbedarf, S. 117; Masutti/Tomasello, S. 79 f.; Palmerini, S. 16; Severoni, responsabilità, S. 75; Voland/Qui, S. 348; JARUS, Categorization, S. 17 und S. 43. Vgl. zu einzelnen Aspekten des Zulassungsverfahrens Huttunen, U-space, S. 72 ff. 182 Vgl. Anhang Delegierte Verordnung (Teil 1 bis 5). 183 Vgl. dazu die Tabelle auf S. 25. 184 Eine detaillierte Darstellung der Anforderungen und Vorschriften für die verschiedenen Produkteklassen würden den Rahmen der vorliegenden Untersuchung sprengen. Vgl. dazu Teile 1 bis 5 Anhang Delegierte Verordnung. 185 Krumm, Anpassungsbedarf, S. 118. 186 Anhang Delegierte Verordnung (Teile 1 bis 5); vgl. zu dieser Kennzeichnung auch hinten, S. 135. 25 § 2 Begriffe Klasse Startmasse187 Ausgewählte technische Anforderungen C0188 < 250 g — Höchstgeschwindigkeit < 19 m/s (68,4 km/h) — max. Flughöhe über Grund: 120 m C1 < 80 Joule oder — Höchstgeschwindigkeit < 19 m/s (68,4 km/h) < 900 g — max. Flughöhe über Grund: 120 m — Lichter189 — direkte Fernidentifizierung190 C2 < 4 kg — max. Flughöhe über Grund: 120 m — Lichter — direkte Fernidentifizierung C3 < 25 kg — max. Flughöhe über Grund: 120 m — Lichter — direkte Fernidentifizierung [C4 < 25 kg — max. Flughöhe über Grund: 120 m] Tabelle: Übersicht ausgewählter Produkteeigenschaften nach Drohnenklasse191 Die Produkteklasse C4 ist hier einzig der Vollständigkeit halber aufgeführt. Sie ist gemäss EASA explizit für unbemannte Luftfahrzeuge vorgesehen, die hauptsächlich für Freizeitzwecke verwendet werden. Es sollen demnach primär Flugmodelle erfasst werden.192 Für diese bestehen weniger strenge technische Anforderungen als für die anderen Produkteklassen; jedoch dürfen 187 Die «höchstzulässige Startmasse» (engl. maximum take-off mass [MTOM]) ist «die vom Hersteller oder Erbauer festgelegte höchstzulässige Masse des unbemannten Luftfahrzeugs, einschliesslich Nutzlast und Kraftstoff, mit der bzw. dem das unbemannte Luftfahrzeug betrieben werden kann» (Art. 2 Ziff. 22 Durchführungsverordnung; vgl. die Konkretisierungen in EASA, GM Implementing Regulation, S. 7). Nachfolgend soll vereinfachend der Begriff «Startmasse» verwendet werden, wenn die «höchstzulässige Startmasse» gemeint ist. 188 Vgl. zu den Anforderungen je Klasse im Einzelnen Teile 1 bis 5 Anhang Delegierte Verordnung. Auf Verweise im Einzelnen soll — im Sinne der Übersichtlichkeit der Tabelle — verzichtet werden. 189 Vgl. dazu ausführlich hinten, S. 153 ff. 190 Vgl. dazu ausführlich hinten, S. 138 ff. 191 Eigene Darstellung nach EASA, Opinion, S. 21. 192 EASA, Opinion, S. 14. Vgl. zu diesem Kriterium auch BAZL, Zivile Drohnen, S. 9. Kritisch zur «etwas schematisch[en]» Abgrenzung nach Verwendungszweck Schladebach, § 6 N 48; ähnlich Henseler, Einordnung, S. 127 f. Die subjektiven Nutzungs absichten verändern das eingesetzte Luftfahrzeug technisch nicht. 26 Teil 1: Rechtliche, begriffliche und technische Grundlag sie über «keine automatischen Steuerungsmodi» verfügen.193 Selbst wenn unbemannte Luftfahrzeuge der Klasse C4 Daten bearbeiten können,194 so stellen sie aus datenschutzrechtlicher Optik ein geringes Risiko dar. Aufgrund der komplexeren Steuerung derselben wird sich der Pilot primär darauf konzentrieren müssen.195 Diese Produkteklasse soll deshalb nachfolgend nicht weiter thematisiert werden. 5. Zusammenfassung und Stellungnahme Für die hier interessierenden Fragestellungen ist von untergeordneter Bedeutung, ob eine klare Abgrenzung zwischen Flugmodellen und Drohnen möglich ist bzw. eine eindeutige Definition dieser beiden Luftfahrzeuge formuliert werden kann. Aus datenschutzrechtlicher Sicht spielt es keine Rolle, ob Daten mit einer Drohne oder einem Flugmodell bearbeitet werden.196 Mit Blick auf die direkte Anwendbarkeit der Definitionen der EASA- Grundverordnung, der Durchführungsverordnung und der Delegierten Verordnung197 wäre eine Verwendung des Begriffs «Drohne» in der Schweizer Gesetzgebung aus Gründen der Rechtssicherheit problembehaftet. Trotzdem soll dieser Bezeichnung für die vorliegende Untersuchung der Vorzug gegeben werden. Dafür spricht deren Bekanntheit und Beliebtheit198 sowie die Komplexität der technischen Begrifflichkeiten.199 Inhaltlich ist damit jeweils ein «unbemanntes Luftfahrzeug» bzw. ein «unbemanntes Luftfahrzeugsystem» gemäss den vorne zitierten Definitionen gemeint.200 In Übereinstimmung mit dem Fokus der Untersuchung werden primär Drohnen der offenen Kategorie behandelt.201 Sollen auch die spezielle und/oder die zulassungspflichtige Kategorie erfasst werden, so wird dies im Text explizit erwähnt. 193 Teil 5 Ziff. 3 Anhang Delegierte Verordnung (engl. no automatic control modes). Vgl. zu diesem Abgrenzungskriterium auch Hänsenberger, Diss., S. 76 m.w.H.; EASA, NPA, S. 9. 194 Vgl. in diesem Kontext BAZL, Zivile Drohnen, S. 9. 195 Allgemein zur Steuerung von Drohnen Büchi, Buch der Drohnen, S. 85 ff.; Hänsenberger, Diss., S. 31 ff. 196 Mutatis mutandis aus haftungsrechtlicher Optik Hänsenberger, Diss., S. 76. Vgl. zu diesem Argument auch die Ausführungen zur Registrierungspflicht hinten, S. 120 ff., v.a. S. 124 f. 197 Vgl. zur direkten Anwendbarkeit vorne, S. 14 f. 198 Vgl. vorne, S. 19 f.; für die USA Perritt/Sprague, STLR, S. 386 mit Fn. 1. 199 Vgl. vorne, S. 16 f. 200 Vgl. vorne, S. 20 f. 201 Vgl. dazu vorne, S. 22. § 2 Begriffe 27 II. Drohnenpilot und Drohnenbetreiber 1. Pilot, Fernpilot, Drohnenpilot Ein Pilot ist, nach allgemeinem Verständnis, diejenige Person, welche die Flugsteuerung eines Luftfahrzeugs bedient.202 Im Rahmen der EASA-Grundverordnung wird für den Piloten einer Drohne der Begriff des «Fernpiloten» eingeführt und wie folgt definiert: «‹Fernpilot› bezeichnet eine natürliche Person, die für die sichere Durchführung des Fluges eines unbemannten Luftfahrzeugs verantwortlich ist, wobei der Fernpilot entweder die Flugsteuerung manuell vornimmt oder, wenn das unbemannte Luftfahrzeug automatisch fliegt,203 dessen Kurs überwacht und in der Lage bleibt, jederzeit einzugreifen und den Kurs zu ändern.»204 Für die vorliegende Untersuchung werden Pilot und Drohnenpilot synonym verwendet, wenn der «Fernpilot» gemäss zitierter Definition gemeint ist. Dies dient sowohl der Klarheit wie der Einfachheit des Textes. Ist der Pilot eines bemannten Luftfahrzeugs gemeint, so wird dies im Text explizit erwähnt. 2. Betreiber und Halter Neben dem Piloten kennt die Durchführungsverordnung den «Betreiber eines unbemannten Luftfahrzeugsystems» (auch «UAS-Betreiber»). Dabei handelt es sich «um eine juristische oder natürliche Person, die ein oder mehrere UAS betreibt oder zu betreiben [beabsichtigt]».205 Mit der Definition wird zum Ausdruck gebracht, dass sich für Betreiber bereits vor dem eigentlichen Betrieb einer Drohne Pflichten ergeben.206 Im Folgenden sollen jeweils «Betreiber» bzw. «Drohnenbetreiber» verwendet werden, wenn der «Betreiber eines unbemannten Luftfahrzeugsystems» gemeint ist. 202 Vgl. z.B. ICAO, S. ix f. (Definitionen «Flying pilot» und «pilot [to]»). Das können auch mehrere Personen gleichzeitig sein, weshalb die ICAO zusätzlich den Begriff des «Pilot-in-command» kennt (vgl. ebd., S. x); vgl. dazu auch Groh, S. 162. Klar zu eng ist die Definition des Duden online, wonach ein Pilot jemand ist, «der (berufsmässig) ein Flugzeug steuert»; sie klammert alle anderen pilotengesteuerten Luftfahrzeuge aus. 203 Vgl. zur Automation und deren Abgrenzung zur Autonomie die Hinweise vorne, Fn. 12. 204 Art. 3 Ziff. 31 EASA-Grundverordnung. Vgl. zur nicht immer einfachen Abgrenzung zwischen der manuellen Flugsteuerung und dem automatischen (bzw. autonomen) Flug Huttunen, Unmanned, S. 361 f. 205 Art. 2 Ziff. 2 Durchführungsverordnung; in der deutschen Fassung ist von «zu betreiben gedenkt» die Rede; «zu betreiben beabsichtigen» ist jedoch die passendere Übersetzung des englischen «intending to operate». Vgl. auch Art. 3 Ziff. 13 EASA-Grundverordnung (Definition des «Luftfahrzeugbetreibers»). 206 So etwa die Registrierungs- und Kennzeichnungspflicht; vgl. dazu ausführlich hinten, S. 120 ff. bzw. S. 138 ff. 28 Teil 1: Rechtliche, begriffliche und technische Grundlag Da der Fokus der Untersuchung auf der drohnengestützten Datenbearbeitung durch (nicht kommerziell operierende) Private liegt, besteht regelmässig Personalunion zwischen dem Piloten und dem Betreiber der betreffenden Drohne.207 Von diesem Verständnis soll nachfolgend ausgegangen werden: Ist vom Drohnenbetreiber bzw. vom Betreiber die Rede, so ist der Drohnenpilot (im Sinne der vorangehenden Ausführungen) miterfasst bzw. dieselbe Person gemeint; andernfalls wird dies explizit erwähnt. Zum Begriff des Betreibers drängen sich aus Sicht des Schweizer Rechts einige Hinweise auf. Dieser wird hierzulande primär im Infrastrukturrecht verwendet.208 Im Luftfahrtrecht (und im Schweizer Verkehrsrecht insge samt) ist dagegen der Terminus des Halters (Luftfahrzeughalter, Motor fahrzeughalter etc.) verbreitet. Eine Legaldefinition des Luftfahrzeughalters existiert nicht; gemäss Bundesgericht beurteilt sich die Haltereigenschaft nach den gleichen Kriterien wie bei Motorfahrzeugen.209 Demnach ist Halter «derjenige, auf dessen eigene Rechnung und Gefahr der Betrieb des [Luft-] Fahrzeugs erfolgt und der zugleich über dieses und allenfalls über die zum Betrieb erforderlichen Personen die tatsächliche, unmittelbare Verfügung besitzt».210 Die Betreiberdefinition gemäss Durchführungsverordnung enthält das Element der tatsächlichen und unmittelbaren Verfügungsgewalt nicht; der Halter ist folglich nicht notwendigerweise mit dem Betreiber identisch.211 Sind der Drohnenpilot und der Drohnenbetreiber die gleiche Person, so ist für diese auch die Haltereigenschaft zu bejahen. Somit erübrigen sich für den Zweck der vorliegenden Untersuchung weitere Ausführungen dazu. Ins gesamt soll der Terminologie der Drohnenverordnungen gefolgt werden, selbst wenn diese — wie aufgezeigt — im Schweizer Luftfahrtrecht (bisher) nicht verwendet wird. 207 Vgl. JARUS, Operations, S. 28. 208 Vgl. etwa Art. 2 lit. a und Art. 40d Eisenbahngesetz (EBG); Art. 16, Art. 19 Abs. 5 Energiegesetz (EnG); Art. 2 Abs. 4 Rohrleitungsgesetz (RLG); BGE 126 III 113 (Skilift); BGE 143 I 395 (Photovoltaikanlage). 209 Ausführlich dazu Komm. SVG-Weissenberger, Art. 58 N 3 ff. mit zahlreichen Hinweisen. 210 BGE 129 III 102 E. 2.1 m.w.H.; 129 III 410 E. 4; Dettling-Ott, Luftfahrzeughalter, Rn. 223; Meili K., S. 59. 211 So aber — jedoch vor Inkrafttreten der Durchführungsverordnung — Dettling-Ott, Luftfahrzeughalter, Rn. 223 und ihr folgend Meili K., S. 59. § 2 Begriffe 29 III. Daten 1. Personendaten im Allgemeinen Grundsätzlich können alle Angaben, d.h. jegliche Art von Information oder Aussage,212 wie z.B. Details, Einzelheiten, Fakten, Grössen, Masse, Messwerte oder Zahlen als Daten gelten.213 Im Rahmen des Datenschutzrechts ist der Begriff der Daten jedoch enger zu verstehen: umfasst werden nämlich einzig Personendaten.214 Um Sachdaten — verstanden als alle anderen Daten — kümmert sich der Datenschutz nicht.215 Art. 13 Abs. 2 BV spricht denn auch von «persönlichen Daten». Dabei handelt es sich um Angaben, die einen hinreichend engen Bezug zu einer natürlichen oder juristischen Person aufweisen.216 Art. 4 lit. a E-DSG definiert Personendaten — und Daten217 — als alle An gaben, die sich auf eine bestimmte oder bestimmbare natürliche Person beziehen.218 Bestimmt ist eine Person, wenn sich ohne Weiteres aus der Angabe selbst ergibt, dass es sich um eine ganz konkrete Person handelt.219 Als Beispiel können hier die Angaben auf einem Personalausweis angeführt werden.220 Bestimmbar ist eine Person, «wenn sie direkt oder indirekt identifiziert werden kann, z.B. über den Hinweis auf Informationen, die sich aus den Umständen oder dem Kontext ableiten lassen (Identifikationsnummer, Standortdaten, spezifische Aspekte, die ihre physische, physiologische, genetische, psychische, wirtschaftliche, kulturelle oder gesellschaftliche Identität betreffen). Die Identifizierung kann über eine einzige Information möglich sein (Telefonnummer, Hausnummer, AHV-Nummer, Fingerab 212 BSK DSG-Blechta, Art. 3 N 6; Handkomm. DSG-Rosenthal, Art. 3 N 8. Ausführlich zum Informationsbegriff Karg, in: Simitis et al., Art. 4 Nr. 1 DSGVO N 25 ff. 213 Belser, in: Belser/Epiney/Waldmann, § 1 Rn. 46. Für eine technische Definition des Datenbegriffs und eine Abgrenzung desselben von «Information» vgl. Albers, S. 89; Weber/Laux/Oertly, S. 11. 214 Vgl. Biaggini, Komm. BV, Art. 13 N 12; BSK BV-Diggelmann, Art. 13 N 33. 215 Bull, Datenschutz, S. 15; Schmid/Schmidt/Zech, S. 628; vgl. Sasse, S. 45. 216 Biaggini, Komm. BV, Art. 13 N 12; vgl. Müller/Schefer, S. 167; BSK BV-Diggelmann, Art. 13 N 33; Auer/Malinverni/Hottelier, Rn. 388; BSK DSG-Blechta, Art. 3 N 7. 217 Im datenschutzrechtlichen Kontext werden die Termini Daten und Personendaten als Synonyme verwendet (vgl. dazu Art. 3 lit. a DSG sowie Botschaft Totalrevision DSG, S. 7019). 218 Juristische Personen sind vom E-DSG nicht mehr erfasst. Vgl. zur Begründung Botschaft Totalrevision DSG, S. 6972 und S. 7019; kritisch Amtl. Bull. NR 2019 S. 1775 f. (Votum Rutz) und S. 1783 f. (Votum Rutz). 219 Botschaft DSG 1988, S. 444; BSK DSG-Blechta, Art. 3 N 9. 220 So BGE 138 II 346 E. 6.1. 30 Teil 1: Rechtliche, begriffliche und technische Grundlag drücke) oder über den Abgleich verschiedener Informationen (Adresse, Geburtsdatum, Zivilstand).»221 Für die Bestimmbarkeit reicht gemäss bundesgerichtlicher Rechtsprechung jedoch nicht jede theoretische Möglichkeit der Identifizierung.222 In einem vielbeachteten Grundsatzurteil führte das Bundesgericht diesbezüglich aus: «Ist der Aufwand derart gross, dass nach der allgemeinen Lebens erfahrung nicht damit gerechnet werden muss, dass ein Interessent diesen auf sich nehmen wird, liegt keine Bestimmbarkeit vor. Die Frage ist abhängig vom konkreten Fall zu beantworten, wobei insbesondere auch die Möglichkeiten der Technik mit zu berücksichtigen sind, so z.B. die im Internet verfügbaren Suchwerkzeuge. Von Bedeutung ist indessen nicht nur, welcher Aufwand objektiv erforderlich ist, um eine bestimmte Information einer Person zuordnen zu können, sondern auch, welches Interesse der Daten bearbeiter oder ein Dritter an der Identifizierung hat.»223 Die Angaben müssen sich zudem nach dem Wortlaut der Legaldefinition auf eine Person beziehen. Vorausgesetzt ist demnach, dass eine Angabe in Verbindung mit einer Person steht und — im weitesten Sinn — «eine Aussage zur Person trifft».224 Thematisch sind die Angaben nicht eingeschränkt; sämtliche Angaben, die sich auf eine Person beziehen, sind umfasst.225 Der Begriff des Personendatums ist folglich sehr weit gefasst; unabhängig davon, ob er im Verfassungs- oder Gesetzeskontext Verwendung findet.226 Insgesamt kann festgehalten werden, dass er mit Blick auf natürliche Per sonen227 im verfassungsrechtlichen Sinn mit der einfachgesetzlichen Legal definition desselben gemäss DSG bzw. E-DSG deckungsgleich ist.228 221 Botschaft Totalrevision DSG, S. 7019; vgl. auch Botschaft DSG 1988, S. 444; BGE 136 II 508 E. 3.2; BSK DSG-Blechta, Art. 3 N 7; Aebi-Müller, Rn. 547; Flückiger, vidéosurveillance, S. 197. Aus rechtsvergleichender Perspektive ausführlich Bygrave, S. 133 ff. 222 Zur Frage, aus wessen Perspektive die Bestimmbarkeit zu beurteilen ist, BGE 136 II 508 E. 3.4; Handkomm. DSG-Rosenthal, Art. 3 N 20; Gerschwiler, Rn. 3.30. 223 BGE 138 II 346 E. 6.1 (Google Street View); vgl. auch BGE 136 II 508 E. 3.2. 224 Karg, in: Simitis et al., Art. 4 Nr. 1 DSGVO N 27 (Hervorhebung im Original); Handkomm. DSG-Rosenthal, Art. 3 N 13. 225 Vgl. BSK DSG-Blechta, Art. 3 N 6 f.; Karg, in: Simitis et al., Art. 4 Nr. 1 DSGVO N 27; detailliert zu diesem Aspekt Handkomm. DSG-Rosenthal, Art. 3 N 14. 226 Bieri, Rn. 626; Blonski, S. 52; BSK DSG-Blechta, Art. 3 N 7 («ausserordentlich weit»). Vgl. auch Bygrave, S. 135; Lee Y., S. 190 mit zahlreichen Hinweisen in Fn. 844. 227 Vgl. vorne, S. 29, sowie hinten, S. 63. 228 Bieri, Rn. 626; SGK BV-Schweizer, Art. 13 N 75; vgl. BVGE 2013/50 E. 9.2; Dubey, Vol. II, Rn. 1850; Belser, in: Belser/Epiney/Waldmann, § 1 Rn. 47; Blonski, S. 52; Moeckli, Rn. 38; Schweizer/Rechsteiner, Rn. 2.6. Ebenso Karg, in: Simitis et al., Art. 4 Nr. 1 DSGVO N 1 m.w.H. zu Art. 4 Ziff. 1 DSGVO und Art. 7 und 8 GrCH sowie Art. 2 Abs. 1 i.V.m. Art. 1 Abs. 1 GG; Kornmeier, S. 116; vgl. Kokott/Sobotta, S. 223. § 2 Begriffe 31 Auch der EGMR hat im Rahmen des Rechts auf Achtung des Privatlebens gemäss Art. 8 EMRK entschieden, dass der Begriff «Personendaten» alle Informationen umfasse, die sich auf eine bestimmte oder bestimmbare Person beziehen.229 2. Besonders schützenswerte Personendaten 2.1 Erfasste Kategorien Einen erhöhten Schutz lässt auch das Datenschutzgesetz einer bestimmten Kategorie von Personendaten zukommen, namentlich den sog. besonders schützenswerten Personendaten.230 Als solche gelten gemäss Art. 4 lit. c E-DSG Daten über religiöse, weltanschauliche oder politische Ansichten oder Tätigkeiten, über die Gesundheit, die Intimsphäre, die Rassenzugehörigkeit, aber auch genetische und biometrische Daten231 und solche über verwaltungs- und strafrechtliche Verfolgungen oder Sanktionen sowie über Massnahmen der sozialen Hilfe.232 2.2 Biometrische Daten insbesondere Die biometrischen Daten sollen kurz vertieft werden. Unter Biometrie wird die automatisierte Analyse und Erkennung von Menschen anhand ihrer körperlichen Merkmale, z.B. die Fingerbild-, Hand-, Stimm-, Iris-, Retina- oder auch die Gesichtserkennung verstanden.233 Besonders schützenswert sind biometrische Daten, wenn sie «eine natürliche Person eindeutig identifi zieren» (Art. 4 lit. c Ziff. 4 E-DSG).234 229 EGMR, Amann c. Schweiz, Nr. 27798/95 (2000) Ziff. 65; EGMR (GK), Rotaru c. Rumänien, Nr. 28341/95 (2000) Ziff. 43; Kokott/Sobotta, S. 223 und 225. Ähnlich auch EuGH, Rs. C-92/09 und C-93/09 vom 9. November 2010, Schecke und Eifert, Ziff. 52. 230 Vgl. statt aller BSK DSG-Blechta, Art. 3 N 27; Handkomm. DSG-Rosenthal, Art. 3 N 42. 231 Genetische und biometrische Daten sind nur dann besonders schützenswert, wenn sie eine «natürliche Person eindeutig identifizieren». So soll z.B. die Forschung nicht allzu stark eingeschränkt werden (vgl. Botschaft Totalrevision DSG, S. 7020; Amtl. Bull. NR 2019 S. 1784 [Votum Romano]) 232 Eine Vertiefung aller in Art. 4 lit. c aufgelisteten Kategorien kann die vorliegende Arbeit nicht leisten. Vgl. dazu im Einzelnen SHK DSG-Rudin, Art. 3 N 25 ff.; BSK DSGBlechta, Art. 3 N 32 ff. 233 Petri, in: Simitis et al., Art. 4 Nr. 14 DSGVO N 1; Kampert, in: Sydow, Art. 4 DSGVO N 183; Ernst, in: Paal/Pauly, Art. 4 DSGVO N 99; Müller L., Sicherheit & Recht 2/2012, S. 64. Als weitere körperliche Merkmale kommen z.B. der Tastendruck oder Venenscans in Betracht (Petri, in: Simitis et al., Art. 4 Nr. 14 DSGVO N 10; Matter, S. 14). Detailliert zu weiteren biometrischen Merkmalen Blonski, S. 16 ff. 234 Eindeutig identifiziert ist eine natürliche Person dann, wenn die Bearbeitung die konkrete Zuordnung zu einer bestimmten Person ermöglicht und mithin deren Identi fizierung verifiziert (vgl. Petri, in: Simitis et al., Art. 4 Nr. 14 DSGVO N 9; ausführlich Arning/Rothkegel, in: Taeger/Gabel, Art. 4 DSGVO N 323 f.). 32 Teil 1: Rechtliche, begriffliche und technische Grundlag Biometrische Daten sind Personendaten, «die durch ein spezifisches tech nisches Verfahren zu den physischen, physiologischen oder verhaltens typischen Merkmalen eines Individuums gewonnen werden und die eine eindeutige Identifizierung der betreffenden Person ermöglichen oder bestätigen. Diese Daten müssen zwingend auf einem spezifischen technischen Verfahren beruhen, das die eindeutige Identifizierung oder Authentifizierung einer Person erlaubt.»235 Ein solches «spezifisches technisches Verfahren» liegt bei Fotos nur dann vor, wenn ein Lichtbild einer Person «unmittelbar zum Zwecke der Identifizierung oder Authentifizierung» erstellt wird.236 Somit liegen «bei gewöhnlichen Fotografien» grundsätzlich keine biometrischen Daten vor.237 Ebenso verhält es sich bei Ton- und Bildaufnahmen handelsüblicher Videokameras oder Mikrofone. 2.3 Verfassungsrechtliche Einordnung Art. 13 BV kennt die Kategorie der besonders schützenswerten Personendaten nicht explizit.238 In diesem Zusammenhang spricht das Bundesgericht teilweise von sensiblen Informationen,239 verwendet aber auch (im verfassungsrechtlichen Kontext) den Terminus der besonders schützenswerten Personendaten.240 Unabhängig von der konkreten Bezeichnung im Einzelfall bestehen bei Daten, welche persönlichkeitsnah sind und mit deren Bearbeitung für die betroffene Person ein erhöhtes Risiko verbunden ist, regelmässig strengere verfassungsrechtliche Anforderungen, und zwar bezüglich der Verhältnismässigkeit, des öffentlichen Interesses sowie der gesetzlichen Grundlage.241 So erfordert die Bearbeitung solcher Daten eine Grundlage in einem 235 Botschaft Totalrevision DSG, S. 7020; vgl. Matter, S. 14. In der Legaldefinition von Art. 4 Nr. 14 DSGVO werden ein «spezielles technisches Verfahren» und «die eindeutige Identifizierung» denn auch explizit erwähnt. Vgl. ausführlich dazu Kampert, in: Sydow, Art. 4 DSGVO N 184 f. 236 Petri, in: Simitis et al., Art. 4 Nr. 14 DSGVO N 8; vgl. Weichert, in: Kühling/Buchner, Art. 4 Nr. 14 DSGVO N 3; spezifisch zur Gesichtserkennung Karjoth, S. 7 ff. 237 Botschaft Totalrevision DSG, S. 7020. Vgl. EG 51 DSGVO; Kampert, in: Sydow, Art. 4 DSGVO N 184; Petri, in: Simitis et al., Art. 4 Nr. 14 DSGVO N 8; Ernst, in: Paal/Pauly, Art. 4 DSGVO N 103; van Gyseghem, Rn. 8. 238 Biaggini, Komm. BV, Art. 13 N 12; SGK BV-Schweizer, Art. 13 N 77. 239 Vgl. BGE 145 IV 42 E. 4.2; 138 II 346 E. 8.2; 122 I 360 E. 5d. Vgl. auch SGK BV-Schweizer, der von «sensitiven Personendaten» spricht. 240 Vgl. etwa BGer, 1C_447/2016, 1C_448/2016, 1C_449/2016 vom 31. August 2017 E. 5.3. 241 Biaggini, Art. 13 N 12; Kiener/Kälin/Wyttenbach, § 14 N 56; Belser, in: Belser/ Epiney/Waldmann, § 6 Rn. 91. § 2 Begriffe 33 formellen Gesetz (Art. 36 Abs. 1 BV).242 Gleich wie das Bundesgericht erkennt auch der EGMR sensiblen Daten eine besondere Schutzwürdigkeit zu.243 IV. Datenbearbeitung Der Term Datenbearbeitung umfasst jeden Umgang mit (Personen-)Daten,244 namentlich das Beschaffen, Erheben, Sammeln, Verarbeiten, Speichern, Aufbewahren und Archivieren, Verbreiten sowie das Bekannt-245 und Weitergeben.246 Daneben werden auch das Löschen oder Vernichten von Daten erfasst.247 Dabei ist unerheblich, welche Verfahren zur Anwendung kommen und welche Hilfsmittel oder Methoden eingesetzt werden.248 Auch heute noch nicht bekannte Technologien und Bearbeitungsweisen fallen darunter.249 Insgesamt kann in Bezug auf den Begriff der Bearbeitung konstatiert werden, dass dieser umfassend zu verstehen ist. Im Anwendungsbereich von Art. 13 Abs. 2 BV und des E-DSG kann von demselben Verständnis ausgegangen werden.250 V. Drohnengestützte Datenbearbeitung Es stellt sich folglich die Frage, ob und welche Art von Personendaten beim Einsatz von Drohnen durch Private bearbeitet werden. Dabei werden regelmässig keine hochtechnischen Sensoren zum Einsatz kommen, sondern vorwiegend eine Foto- und/oder Videokamera. Eine Ausstattung mit solcher Technik ist heute serienmässig der Fall.251 242 BGE 143 I 253 E. 6.3; 122 I 360 E. 5b; Blonski, S. 53. 243 Vgl. EGMR, I c. Finnland, Nr. 20511/03 (2008) Ziff. 38 und 40; EGMR, Avilkina u.a. c. Russland, Nr. 1585/09 (2013) Ziff. 45; Grabenwarter/Pabel, § 22 N 45 und N 60; de Vries, S. 673. 244 SGK BV-Schweizer, Art. 13 N 74; Aebi-Müller, Rn. 549; Biaggini, Komm. BV, Art. 13 N 11; vgl. Meier, Rn. 30; Kiener/Kälin/Wyttenbach, § 14 N 57; Griffel, § 31 Rn. 46. 245 Neu wird in Art. 4 lit. e E-DSG auch das Bekanntgeben definiert. Namentlich handelt es sich dabei um das Übermitteln oder Zugänglichmachen von Personendaten. Vgl. dazu auch § 3 Abs. 5 und 6 IDG/ZH. 246 Biaggini, Komm. BV, Art. 13 N 11; SGK BV-Schweizer, Art. 13 N 74; Kiener/Kälin/ Wyttenbach, § 14 N 57; Tschentscher/Lienhard/Sprecher, Rn. 267; Rhinow/ Schefer/Uebersax, Rn. 1378; BGE 137 I 167 E. 3.2; 128 II 259 E. 3.2; 122 I 360 E. 5a. 247 Waldmeier, S. 37; Meier, Rn. 30; Blonski, S. 56; vgl. § 3 Abs. 5 IDG/ZH. 248 Vgl. Art. 4 lit. d E-DSG; SGK BV-Schweizer, Art. 13 N 74; Blonski, S. 56; BSK DSGBlechta, Art. 3 N 71. 249 PK IDG/ZH-Rudin, § 3 N 32. 250 Bieri, Rn. 626; SGK BV-Schweizer, Art. 13 N 74; vgl. BGE 144 I 126 E. 4.1; Kiener/ Kälin/Wyttenbach, § 14 N 57. 251 Dieckert, in: Dieckert/Eich, S. 408. Ausführlich zu den Sensoren hinten, S. 40 ff. 34 Teil 1: Rechtliche, begriffliche und technische Grundlag Gemäss Lehre und Rechtsprechung stellen «Bilddaten in Form einer eine bestimmte Person zeigenden Fotografie» ein Personendatum dar.252 Auch ein Bild eines Grundstücks oder eines Hauses, welches mit einer Drohne auf genommen wird, ist regelmässig als Personendatum zu qualifizieren.253 Gleiches gilt grundsätzlich für Bilder von privaten Gärten, Höfen, Balkonen oder Hausfassaden.254 Werden Personen auf einem Privatgrundstück fotografiert oder gefilmt, so ist eine Identifikation derselben meist durch die Anschrift am Briefkasten bzw. an der Türklingel möglich. Bei Bildern von Grundstücken an sich kommen zudem eine Internetsuche oder «allgemeinzugängliche Zusatzinformationen wie z.B. eine elektronische Eigentümerabfrage» in Betracht.255 Mit einer Drohne erstellte Foto- und Videoaufnahmen, welche Personen abbilden, stellen potenziell besonders schützenswerte Personendaten dar,256 da auf einem Bild — je nach Auflösung — z.B. die Rassenzugehörigkeit und/ oder die religiöse Ansicht bzw. die Weltanschauung einer Person erkennbar ist.257 Biometrische Daten dürften bei einer drohnengestützten Datenbearbeitung durch Private dagegen regelmässig nicht vorliegen, da kein spezifisches technisches Verfahren zur Anwendung kommt.258 Die Bearbeitung im Sinne des Datenschutzrechts erfolgt durch die Aufnahme von Fotos und Videos mittels technischer Hilfsmittel. Dies stellt klarer weise ein Bearbeiten im Sinne des Datenschutzrechts dar; dass die Technik an der Drohne befestigt bzw. darin verbaut ist, ändert daran nichts.259 Insge252 BGE 127 III 481 E. 3a/bb; vgl. BGE 138 II 346 E. 8; 136 III 401 E. 5.2.1; Botschaft DSG 1988, S. 444; Malgieri, S. 196; Meier, Rn. 30. Zum Recht am eigenen Bild ausführlich Bächli, S. 59 ff. Zum Recht an der eigenen Stimme vgl. BGE 110 II 411 E. 3b; zum Recht am eigenen Wort Glaus, passim, v.a. S. 37 ff. und zur Umsetzung desselben im zivilrecht lichen Persönlichkeitsschutz Schweizer M., Rn. 69 ff. Vgl. sodann BSK ZGB I-Meili, Art. 28 N 19 und 22 m.w.H. 253 KGer LU, 7H 17 49 vom 18. April 2018 E. 3.4 f.; Weber/Oertly, Rn. 19; Henseler, Baupolizist, Rn. 32; vgl. BGE 138 II 346 E. 6.1; Arnosti, Rn. 34; BSK BV-Diggelmann, Art. 13 N 33; Moeckli, Rn. 38. 254 BGE 138 II 346 E. 6.3 mit Hinweis auf Müller L., Diss., S. 49. Vgl. KGer LU, 7H 17 49 vom 18. April 2018 E. 3.5; Moeckli, Rn. 38. 255 Vgl. nur KGer LU, 7H 17 49 vom 18. April 2018 E. 3.5 m.w.H. 256 Es ist grundsätzlich möglich, dass auch Tonaufnahmen besonders schützenswerte Personendaten darstellen (etwa die Aufnahme eines Raucherhustenanfalls; vgl. Art. 4 lit. c Ziff. 2 E-DSG). Darauf soll nicht weiter eingegangen werden. 257 Vgl. Malgieri, S. 181 f.; KGer LU, 7H 17 49 vom 18. April 2018 E. 3.5; Flückiger, vidéosurveillance, S. 197. Vgl. Müller L., Sicherheit & Recht 2/2012, S. 66 f. mit zahlreichen Hinweisen. 258 Zum Terminus der biometrischen Daten vorne, S. 31 f. 259 Vgl. KGer LU, 7H 17 49 vom 18. April 2018 E. 3.4. § 3 Einsatzformen von Drohnen in der Schweiz 35 samt kann deshalb konstatiert werden, dass bei Foto- und/oder Videoaufnahmen (inkl. Tonaufnahmen) durch Private, welche mithilfe von Drohnen erstellt werden, eine Bearbeitung von Personendaten (und möglicherweise von besonders schützenswerten Personendaten) im Sinne des E-DSG sowie der BV vorliegt. § 3 Einsatzformen von Drohnen in der Schweiz Die möglichen Anwendungsfelder von Drohnen sind sehr breit.260 Aus diesem Grund ist es schwierig, sich ein umfassendes Bild über die bisherigen und zukünftigen Einsatzformen von Drohnen in der Schweiz zu verschaffen.261 Es ist zudem vorstellbar, dass durch Drohnen in Zukunft Dienstleistungen angeboten werden können, die heute noch gar nicht existieren. Die nachfolgende Darstellung erhebt denn auch keinen Anspruch auf Vollständigkeit. I. Nutzung durch Behörden Während längerer Zeit wurden Drohnen fast ausschliesslich von Behörden eingesetzt. Namentlich militärische Drohnen stehen bereits seit den 1990erJahren im Einsatz, oftmals auch zur Unterstützung von zivilen Einsatzkräften.262 So wurden Drohnen des Militärs etwa 1994 bei der Waldbrandbekämpfung eingesetzt.263 Vorwiegend kommen die Drohnen der Luftwaffe aber zu Aufklärungs-, Such- und Überwachungszwecken zum Einsatz: Das Grenzwachtkorps (GWK)264 setzt diese seit 2001 regelmässig bei der Grenzkontrolle ein.265 Namentlich kam das Aufklärungsdrohnensystem 95 (ADS 95 Ranger) zum 260 Vgl. dazu statt vieler SESAR, Outlook Study, S. 8 f.; Roma, S. 22. 261 Eine ausführliche Darstellung dazu mit Fokus auf die Schweiz findet sich bei Christen et al., S. 80 ff., jeweils mit einer Darstellung der aktuellen Lage (2018) sowie einer Zukunftsprognose (2025). 262 Pedrozo/Klauser m.w.H. 263 Steiger, Regulierung, S. 169 mit Hinweis auf Lucien Trueb, Militärische Drohnen für die Waldbrandbekämpfung, NZZ vom 25. Mai 1994, S. 75. 264 Das GWK ist ein uniformierter und bewaffneter Verband (Art. 91 Abs. 2 ZG) innerhalb der Eidgenössischen Zollverwaltung. Vgl. zur Verwendung von Drohnen durch das GWK Kiener/Leuzinger, Rn. 141 mit Fn. 365 sowie BVGer, A-2482/2007 vom 26. Juni 2007. 265 Keller, S. 32; vgl. Gättelin/Müller, S. 44 f.; Steiger, Regulierung, S. 171. Zu den verschiedenen Zwecken des Einsatzes von Drohnen und anderen Luftfahrzeugen durch das GWK vgl. Nussbaum, Rn. 68 ff. 36 Teil 1: Rechtliche, begriffliche und technische Grundlag Einsatz;266 dieses ist u.a. mit Wärmebild- und Infrarotkameras ausgestattet.267 Im November 2019 wurden die Drohnen dieses Typs indes ausgemustert.268 Grössere — auch mediale — Aufmerksamkeit wurde Drohneneinsätzen aber erst anlässlich der Fussball-Europameisterschaft 2008 zuteil.269 Zur Unterstützung der Polizeikräfte während dieses Grossanlasses setzte die Luftwaffe rund um die Stadien der Schweizer Austragungsorte Basel, Bern und Zürich Drohnen ein.270 Seither ist das Interesse an Drohneneinsätzen bei verschiedenen Behörden gewachsen. Polizeikorps aus mehreren Kantonen setzen zunehmend (kleine) Drohnen ein.271 Diese kommen primär für Luftaufnahmen (etwa bei Verkehrsunfällen) oder für die Überwachung aus der Luft zum Zug.272 Daneben haben auch andere Blaulichtorganisationen die neue Technologie für sich entdeckt.273 So besitzt z.B. die Feuerwehr Rapperswil-Jona eigene Drohnen, welche bei Brandfällen oder zur Personensuche eingesetzt werden.274 Seit dem 1. September 2017 darf der Nachrichtendienst des Bundes Vorgänge und Einrichtungen an öffentlichen und allgemein zugänglichen Orten beobachten und in Bild und Ton festhalten. Dazu kann er u.a. «Fluggeräte» einsetzen, womit auch Drohnen erfasst sind.275 Seit kurzer Zeit dürfen sodann im Bereich der Sozialversicherungen Drohnen eingesetzt werden. Dieser Einsatz ist jedoch auf die Standortbestimmung beschränkt und umfasst 266 Keller, S. 32; Pedrozo, S. 101; vgl. Häfliger, S. 14. 267 Pedrozo, S. 101; Botschaft MIG, S. 3271. Vgl. auch Art. 2 Abs. 1 Verordnung über den Einsatz von Bildaufnahme-, Bildaufzeichnungs- und anderen Überwachungsgeräten durch die Eidgenössische Zollverwaltung (SR 631.053). 268 Ab Mitte 2020 sollen dann die neu beschafften Drohnen des Typs Hermes 900 eingesetzt werden. Während der Übergangszeit werden Helikopter die Überwachungsaufgaben übernehmen. Vgl. SRF Online, 16. September 2019, Schweizer Armee fehlen die Drohnen für den Grenzschutz. 269 Vgl. Steiger, Regulierung, S. 171 m.w.H. 270 Vgl. dazu Botschaft EURO 2008, S. 8189 f.; BAZL, Verfügung, S. 4447. 271 Klauser et al., S. 6; Steiger, Regulierung, S. 171; vgl. Klitou, S. 63; Martini, S. 733. Zum Kanton Zürich vgl. BGE 136 I 87 E. 8 sowie § 32b f. PolG/ZH. 272 Vgl. Klauser et al., S. 6; Landrock/Baumgärtel, S. 37. 273 Gemäss Klauser et al., S. 4, waren 11 % der 400 Studienteilnehmer aus öffentlichen Einrichtungen von Feuerwehren und anderen Notfalldiensten (ohne Polizei). Vgl. zum Einsatz von sog. «Defikoptern» in Deutschland Döpke/Jülicher, passim. 274 Stadtverwaltung Rapperswil-Jona, E-Mail vom 25. September 2017 (beim Autor einsehbar). 275 Art. 14 Abs. 1 NDG. Vgl. Steiger, Sicherheitsesoterik, S. 134; Kiener/Leuzinger, Rn. 140; Botschaft NDG, S. 2150. § 3 Einsatzformen von Drohnen in der Schweiz 37 keine drohnengestützte Observation.276 Als weitere behördliche Einsatzfelder sind die Erfüllung von Baupolizeiaufgaben277 oder die Erhebung von Geodaten278 zu nennen. II. Nutzung durch Private 1. Kommerzielle Nutzung Die Breite der möglichen Einsatzgebiete von Drohnen führt dazu, dass vermehrt auch Private auf Drohnen setzen.279 In der Schweiz werden sie momentan — im kommerziellen Bereich — für Luftaufnahmen (Foto und Video), Vermessungen, zur Kontrolle und Instandhaltung von Infrastrukturen (wie Brücken, Hochspannungsleitungen, Gleisen etc.)280 sowie in der Landwirtschaft eingesetzt. Private Sicherheitsfirmen setzen Drohnen sodann für Überwachungszwecke ein,281 und auch für die Forschung werden sie in der Schweiz verwendet.282 Grosse Aufmerksamkeit erlangte das Projekt der Post CH AG (Post), welche vom BAZL eine Ausnahmebewilligung für Drohnenflüge ohne direkten Augenkontakt erhielt.283 Bei diesen Drohnenflügen in Lugano werden Medikamente und Laborproben zwischen zwei Spitälern hin- und hertransportiert.284 Ähnliche Projekte wurden im Juni 2018 in Bern und im Dezember desselben Jahres in Zürich lanciert.285 Die Flüge in Zürich waren jedoch problemanfällig: Nach einem ersten Absturz einer Postdrohne am 25. Januar 276 Art. 43a f. ATSG; vgl. dazu Amtl. Bull. SR 2018 S. 236 (Votum Bischof); Pärli, S. 121; Bolt/Bryner, S. 211; unklar dagegen Amtl. Bull. NR 2018 S. 369 (Votum BR Berset); vgl. sodann BGer, 1C_389/2018, 1C_543/2018, 1C_649/2018 vom 8. August 2019 E. 5.3 und 5.4.2 (zur Publikation vorgesehen). 277 Vgl. dazu KGer LU, 7H 17 49 vom 18. April 2018. Vgl. auch § 203 Abs. 5 Satz 2 PBG/LU, wonach im Rahmen der Baukontrolle «nach Vorankündigung Flugaufnahmen von Grundstücken» erstellt werden dürfen (vgl. kritisch zu dieser Bestimmung Henseler, Baupolizist, Rn. 41 ff.). 278 Reimers, S. 14 ff.; vgl. Kettige r, Geobasisdaten, S. 24. Vgl. die Legaldefinition des Begriffs in Art. 3 Abs. 1 lit. a Geoinformationsgesetz (GeoIG). 279 Vgl. Mendes de Leon/Scott, S. 185 f.; ausführlich zu verschiedensten Anwendungsfeldern auf der ganzen Welt Kornmeier, S. 30 ff. 280 Vgl. Daphinoff/Hassler, Rn. 760 f. 281 Vgl. zu diesen Einsatzgebieten Klauser et al., S. 3 f.; aus europäischer Perspektive SESAR, Master Plan, S. 6 sowie ausführlich SESAR, Outlook Study, S. 52 ff. 282 Vgl. bspw. Schaub/Leuzinger, Rn. 20 mit Fn. 4, zum Drohneneinsatz bei der Erhebung der Stimmbeteiligung an der Glarner Landsgemeinde. 283 Vgl. dazu Art. 17 Abs. 1 und Art. 18 Abs. 1 lit. b VLK. 284 Vgl. Post, Medienmitteilung 4. Oktober 2017. 285 Senn, S. 15; Post, Drohnen. 38 Teil 1: Rechtliche, begriffliche und technische Grundlag 2019 über dem Zürichsee wurden die Flüge während ungefähr elf Wochen unterbrochen.286 Kurz nach der Wiederaufnahme derselben am 15. April 2019 stürzte eine weitere Drohne ab — diesmal über einem Waldstück unweit von dort spielenden Kindern. Die Drohnenflüge wurden daraufhin bis auf Weiteres eingestellt.287 In der Landwirtschaft wurden Drohnen bereits mehrfach zur Rettung von Rehkitzen in Feldern eingesetzt;288 die Jungtiere werden mit einer Wärme bildkamera an der Drohne aufgespürt und danach in Sicherheit gebracht.289 In dieser Branche wird die Drohnentechnologie auch zum Ausbringen von Pestiziden und zur Überwachung des Wachstums der Kulturen eingesetzt;290 des Weiteren können Drohnen auch die Bestäubung bestimmter Pflanzen arten übernehmen.291 2. Hobbynutzung Auch im Bereich der nicht kommerziellen Nutzung haben sich Drohnen in den letzten Jahren stark ausgebreitet.292 Hobbynutzer293 setzen Drohnen in verschiedensten Bereichen der Freizeitgestaltung ein, so u.a. für Foto- und Videoaufnahmen. Daneben kommen sie auch der Freude am Fliegen willen zum Einsatz294 oder werden für das sog. FPV-Racing eingesetzt (FPV = «First Person View»).295 Dabei werden die Drohnen anhand der Videoaufnahmen gesteuert, welche in Echtzeit auf eine Virtual-Reality-Brille übertragen werden.296 286 Post, Medienmitteilung 29. Januar 2019; Post, Mitteilung 12. April 2019; vgl. zur Ursache des Absturzes SUST, Bericht Zürichsee, v.a. S. 12. 287 Vgl. SRF Online, 9. Mai 2019, Erneut eine Post-Drohne in Zürich abgestürzt. 288 Hodel, S. 23 und S. 44 f. 289 Zur Wärmebildkamera hinten, S. 42. 290 Eines der führenden Unternehmen im Bereich der Agrardrohnen ist die senseFly SA mit Sitz in Lausanne (vgl. www.sensefly.com). 291 Dropcoter, ein Unternehmen mit Sitz in Kalifornien, hat z.B. Mandel- und Kirschbäume mittels Drohnen bestäubt (sUAS News, 4. Juli 2018, Dropcopter releases pollination results). 292 Vgl. dazu die Ausführungen vorne, S. 3 f. Zur zunehmenden Verbreitung von «Spielzeugdrohnen» Schucht, S. 143. 293 Gemäss Schladebach, § 6 N 50, «zumeist erwachsene Männer». 294 Perritt/Sprague, Domesticating Drones, S. 29 m.w.H. 295 Ungefähr «Ich-Perspektive». Vgl. zu diesen «Renndrohnen» Juniper, S. 32 f. 296 Ausführlich dazu Büchi, Buch der Drohnen, S. 60 ff. § 4 Stand der Technik 39 § 4 Stand der Technik I. In der Schweiz verbreitete Hobbydrohnen Wie einleitend ausgeführt, kann (momentan) keine exakte Angabe über die Anzahl in der Schweiz verkauften und/oder geflogenen Drohnen gemacht werden.297 Aus denselben Gründen kann auch keine genaue Angabe zu den in der Schweiz verbreiteten Drohnenmodellen gemacht werden. Anhand von Zahlen der Marktforschung und Studien zur Marktentwicklung lässt sich aber festhalten, dass Hobbydrohnen grundsätzlich dem Typ «Quadrokopter» zuzuordnen sind.298 Diese sind regelmässig unter 25 kg schwer und ferngesteuert.299 Der Marktführer im Segment dieser Hobbydrohnen ist der chinesische Hersteller DJI; seine Modelle «Mavic» und «Phantom» sind nicht nur in der Schweiz sehr beliebt.300 Gemäss einer Studie von Skylogic Research, einer auf Drohnen spe zialisierten Forschungseinrichtung, vom September 2018 verfügt DJI im privaten und kommerziellen Sektor gesamthaft über einen Marktanteil von 74 %.301 Die mögliche Flugzeit von Hobbydrohnen ist beschränkt.302 Bei Multikoptern mit einem Gewicht von bis zu 10 kg und einer Ladung von bis zu 1 kg (wenn diese selbst Akku verbraucht, wie z.B. eine Kamera), liegt die Flugzeit im Allgemeinen zwischen 7 und 30 Minuten.303 Kleinere Drohnen (mit bis zu 2 kg Gewicht) können Flugzeiten von lediglich 15 Minuten erreichen.304 Die genaue Flugzeit hängt zudem stark vom jeweils verwendeten Modell ab. Auch die maximale Traglast hängt vom jeweiligen Modell und dessen Grösse ab. Die Akkulaufzeit wird umso kürzer, je schwerer die Zuladung im Einzelfall ist.305 Dieser Umstand setzt der Grösse und vor allem dem Gewicht der Ladung klare Grenzen.306 297 Vgl. vorne, S. 3 f. 298 Vgl. BAZL, Zivile Drohnen, S. 22; Christen et al., S. 104 f.; Preznuk, S. 23; Hodgkinson/Johnston, S. 11; Landrock/Baumgärtel, S. 6; Steiger, Regulierung, S. 172. 299 EASA, Explanatory Note, S. 12 m.w.H.; vgl. Thomasen, S. 2. 300 Vgl. Christen et al., S. 105 und S. 117; de Miguel Molina/Segarra Oña, S. 10; Hänsenberger, Diss., Fn. 1212, 1553 und 1590; Martini, S. 735 mit Fn. 35; Vergouw et al., S. 29; Perritt/Sprague, Domesticating Drones, S. 29. Popper bezeichnet das Modell «Phantom» sehr anschaulich als «the Kleenex of drones». 301 Lee I. m.w.H.; Saeed. 302 Christen et al., S. 35; vgl. Graumann, S. 66. 303 Landrock/Baumgärtel, S. 8 und 63; Vergouw et al., S. 29. Vgl. auch den Produktbeschrieb der Drohne Phantom 4 Advanced auf der Herstellerseite. 304 Christen et al., S. 77; vgl. Büchi, Buch der Drohnen, S. 194 f. 305 Ausführlich zu den in Drohnen verwendeten Akkus Eich, in: Dieckert/Eich, S. 207 ff. 306 Vergouw et al., S. 35. Vgl. Applin, S. 74 m.w.H.; Engberts/Gillissen, S. 99. 40 Teil 1: Rechtliche, begriffliche und technische Grundlag II. Sensoren Die hiervor dargestellten Einsatzformen von Drohnen weisen eine zentrale Gemeinsamkeit auf, namentlich das Sammeln bzw. Bearbeiten von Sach- und Personendaten.307 Diese können sodann gezielt weiterverarbeitet (d.h. z.B. gespeichert, ausgewertet und versendet) werden. Für die vorliegende Ausein andersetzung kommt somit den Sensoren besondere Bedeutung zu; sie sind grundsätzlich geeignet, Personendaten zu bearbeiten.308 Im Vordergrund stehen dabei Foto- und Videokameras: Diese sind erstens bei privaten Drohnen am weitesten verbreitet und zweitens leicht und kostengünstig erhältlich.309 Der Vollständigkeit halber werden nachfolgend aber auch weitere — für Private eher schwer erhältliche — Sensoren kurz angesprochen.310 Vorauszuschicken ist, dass zur Verbreitung der einzelnen Sensoren keine gesicherten Zahlen existieren. Dies liegt u.a. daran, dass Drohnen oftmals mit verschiedenen Sensoren bestückt werden und gleichzeitig mehrere solche tragen können.311 Ebenfalls fehlen — zumindest im Bereich der Hobbynutzung — wissenschaftliche Erhebungen dazu. Nachfolgend soll deshalb auf die erste Erhebung zur professionellen Drohnennutzung in der Schweiz, bei welcher private Unternehmungen sowie Behörden teilnahmen, verwiesen werden.312 Daraus lassen sich interessante Erkenntnisse zur Art und Häufigkeit bestimmter Sensoren gewinnen. 1. Fotokamera Die am weitesten verbreiteten Sensoren sind Fotokameras. Gemäss Klauser et al. sind knapp über 40 % aller Drohnen damit bestückt.313 Drohnen verfügen oftmals über leistungsfähige Kameras, welche hochauflösende Bilder aufnehmen können.314 Diese werden primär zur Erstellung von Luftbildaufnahmen eingesetzt.315 307 Vgl. Landrock/Baumgärtel, S. 49. 308 Zur Bedeutung von Sensoren für (autonome) Drohneneinsätze Hänsenberger, Diss., S. 20. Vgl. auch Bestaoui Sebbane, S. 78 f. 309 Vgl. dazu Dieckert, in: Dieckert/Eich, S. 408. 310 Für eine ausführliche Darstellung weiterer Sensoren Eich, in: Dieckert/Eich, S. 319 f.; Hänsenberger, Diss., S. 27 ff. 311 Vgl. Hänsenberger, Diss., S. 27 ff.; Abeyratne, S. 120. 312 Klauser et al., S. 8. 313 Klauser et al., S. 8; vgl. auch Landrock/Baumgärtel, S. 16. 314 Eich, in: Dieckert/Eich, S. 317; Grosskopf, S. 759; Martini, S. 735; Büchi, Buch der Drohnen, S. 51 f. Zu den technischen Ausgestaltungen moderner Kameras vgl. Hertz berg/Lingemann/Nüchter, S. 55 ff. 315 Zu den verschiedenen Einsatzformen vorne, S. 35 ff. § 4 Stand der Technik 2. 41 Videokamera Auch (hochauflösende) Videokameras sind bei weiteren ca. 40 % aller Drohnen zu finden. Deren Bildauflösung wird bei gleichbleibendem oder sogar abnehmendem Energieverbrauch stetig besser.316 Es können zwei Arten unterschieden werden: Bei einem Grossteil findet eine Liveübertragung des aufgenommenen Bildmaterials statt; teilweise wird dieses aber lediglich gespeichert und kann erst später gesichtet werden.317 Die Videoaufnahmen erfolgen weitgehend automatisiert. Vom Boden aus können die Einstellungen in Echtzeit überprüft und nötigenfalls angepasst werden.318 Die vorangehend beschriebenen Drohnenmodelle,319 welche bei Privaten weit verbreitet sind, werden serienmässig mit Foto- und Videokamera ausgestattet.320 Die technischen Fortschritte beschränken sich jedoch nicht nur auf die Bildauflösung. Auch die Reichweite und die Lichtempfindlichkeit, insbesondere die gleichzeitige Darstellung von hellen und dunklen Stellen (sog. Restlichtverstärkung) sowie das Autotracking (automatische Kameranachführung) sind hier zu erwähnen. Insgesamt kann so eine immer grössere Fläche in immer besserer Auflösung erfasst werden; Drohnen ermöglichen zudem ganz neue Perspektiven.321 3. Mikrofon Neben Foto- und Videokameras tragen Hobbydrohnen teilweise auch ein Mikrofon.322 Es ermöglicht grundsätzlich die Aufnahme von Geräuschen und Gesprächen. Je nach Qualität des verwendeten Mikrofons können gut verständliche Audioaufnahmen von Gesprächen auch aus einiger Entfernung entstehen.323 Aufgrund der Geräuschkulisse, welche durch handels übliche Drohnen verursacht wird,324 ist damit ein Abhören bzw. Aufnehmen von Konversationen nicht oder kaum möglich. 316 Vgl. Perritt/Sprague, JETLaw, S. 685; Martini, S. 735 m.w.H. 317 Klauser et al., S. 8; vgl. Kaiser, S. 358. 318 Eich, in: Dieckert/Eich, S. 316. 319 Vgl. vorne, S. 39. 320 Vgl. Vergouw et al., S. 30; Schmid, S. 77. 321 Zum Ganzen Scholz, in: Simitis et al., Anhang 1 zu Art. 6 DSGVO N 14. 322 Vergouw et al., S. 30; Wright/Finn, S. 328; vgl. auch Gorkič, S. 69. 323 Ausführlich dazu mit Fokus auf «CCTV Microphones» Klitou, S. 116 ff., v.a. S. 119 f. 324 Vgl. EASA, Opinion, S. 14 und S. 38; JARUS, Categorization, S. 12; Hänsenberger, Diss., S. 208; Brahms/Maslaton, S. 1128. 42 4. Teil 1: Rechtliche, begriffliche und technische Grundlag Infrarot- und Wärmebildkameras Weniger weit verbreitet sind Infrarot- und Wärmebildkameras.325 Mit dieser Technik sind Aufnahmen von Bildern auch in der Nacht, bei Nebel oder bei Rauchbildung möglich.326 Im Bereich der behördlichen Drohnennutzung werden solche Kameras vom GWK eingesetzt.327 Hobbynutzer werden da gegen regelmässig keine solchen Kameras verwenden. Infrarot- und Wärmebildkameras erzeugen durch Infrarot-Scheinwerfer ein sichtbares Bild; die Infrarotenergie von Objekten (z.B. Menschen und Tieren) kann so aufgenommen werden.328 Sie enthalten in jedem Pixel eine Temperaturinformation, die anstelle von Grauwerten als Falschfarben Temperaturunterschiede interpretierbar macht.329 5. IMSI-Catcher Ein noch seltener vorkommender Sensor ist der sog. IMSI-Catcher. Dabei handelt es sich — in den Worten des Bundesgerichts — um «ein tragbares Gerät, das vereinfacht gesagt eine Mobilfunkantenne simuliert und die in der Umgebung des Catchers betriebenen Mobiltelefone dadurch so steuert, dass sie sich statt an der nächstgelegenen Mobilfunkantenne beim IMSI-Catcher anmelden. (…) Mit dem Einsatz von IMSI-Catchern ist es auch möglich, den Datenverkehr selbst abzugreifen, d.h. Telefongespräche mitzuhören oder andere übermittelte Daten zu analysieren. Der IMSI-Catcher erlaubt sodann die genaue Standortermittlung eines eingeschalteten Mobiltelefons.»330 IMSI-Catcher kommen in der Schweiz bei der Strafverfolgung (Art. 269bis und Art. 273 StPO)331 oder im nachrichtendienstlichen Bereich (Art. 28 NDG)332 zum Einsatz. Eine private Nutzung derselben ist nicht gänzlich auszuschliessen, denn ISMI-Catcher sind online für jedermann erhältlich.333 Auch das 325 Gemäss Klauser et al., S. 8, werden solche bei weniger als 10 % der befragten Unternehmen und Organisationen verwendet. 326 Wright/Finn, S. 328. Vgl. zum Einsatz von Infrarotkameras bereits EKMR, Kröcher und Möller c. Schweiz, Nr. 8463/78 (1981), v.a. Ziff. 9. 327 Vgl. dazu vorne, S. 35 f. 328 Glaneuski, S. 48; Müller L., Diss., S. 15 m.w.H. 329 Eich, in: Dieckert/Eich, S. 320; vgl. Hänsenberger, Diss., S. 28. 330 BGer, 1B_251/2017 vom 21. Februar 2018 E. 6.2 mit zahlreichen Hinweisen; vgl. auch BGer, 1B_199/2018 vom 18. Oktober 2018 E. 3.5. 331 BGE 144 IV 370 E. 2; BGer, 1B_252/2017 vom 21. Februar 2018 E. 6; BGer, 1B_696/2012 vom 11. Dezember 2012 E. 4.2 sowie die Hinweise in Fn. 330 hiervor. 332 Vgl. dazu Kiener/Leuzinger, Rn. 130. 333 Vgl. z.B. den Produktbeschrieb eines IMSI-Catchers auf der Webseite des Herstellers. § 4 Stand der Technik 43 Gewicht ist bei einem sog. «IMSI-Catcher mini» kein Hindernis, da dieser nur ca. 0,6 kg wiegt.334 Hobbydrohnen sind grundsätzlich in der Lage, ein solches Gerät zu tragen.335 Die Nutzung eines IMSI-Catchers setzt jedoch eine Bewilligung des BAKOM voraus, welche ausschliesslich an Behörden erteilt wird.336 Das «Inverkehrbringen und Anpreisen» von IMSI-Catchern ist zudem gemäss Art. 179sexies StGB strafbar.337 III. Zwischenfazit Aufgrund der grossen Vielfalt von Drohnenmodellen und verfügbaren Sensoren kann die vorliegende Arbeit keine abschliessende Darstellung derselben leisten. Die Ausführungen in diesem Kapitel haben aber zweierlei aufgezeigt: Erstens sind die von Privaten verwendeten Drohnen relativ klein und leicht. Zweitens sind diese mindestens in einer Weise zu einer Datenbearbeitung befähigt. Die serienmässig angebrachte Foto- und/oder Videotechnik ist soweit ausgereift, dass damit auch aus grösserer Entfernung338 hochauf lösendes Bildmaterial erstellt werden kann, welches regelmässig als Personen daten zu qualifizieren ist.339 334 Das Modell von Intercept wiegt gemäss Angaben auf der Webseite nur gerade 0,58 kg. Ein weiteres online erhältliches Modell wiegt gemäss Angaben auf der Webseite dagegen um die 25 kg. 335 Vgl. dazu vorne, S. 39. 336 Vgl. Art. 32a Fernmeldegesetz (FMG); Art. 26 bis 28 Verordnung über Fernmelde anlagen (FAV); Art. 4 f. Verordnung des BAKOM über Fernmeldeanlagen (VFAV). Auf Einzelheiten des Bewilligungsverfahrens wird nicht eingegangen. 337 Vgl. BSK StGB-Ramel/Vogelsang, Art. 179 sexies N 1a ff. 338 So können z.B. aus zwei Kilometer Entfernung Autokennzeichen erkannt werden (Dieckert, in: Dieckert/Eich, S. 408 mit Fn. 110). Vgl. auch Wright/Finn, S. 328 m.w.H. 339 Ausführlich dazu vorne, S. 29 ff. und insbesondere S. 33 ff. 45 Teil 2: Die drohnengestützte Datenbearbeitung im Lichte der Grundrechte § 1 Vorbemerkungen Nachfolgend soll die drohnengestützte Datenbearbeitung im Lichte der durch die BV sowie die EMRK geschützten Grundrechte analysiert werden.340 Diese Analyse beginnt mit einer eingehenden Diskussion der Dogmatik der grundrechtlichen Schutzpflichten (S. 46 ff.). Die Aufarbeitung dieser Grundlagen ist wesentlich für die später vorzunehmende Prüfung bestehender Schutzpflichten und ihrer Wahrnehmung im Kontext der Durchsetzung daten schutzrechtlicher Ansprüche bei der drohnengestützten Datenbearbeitung durch Private.341 Des Weiteren sind diejenigen Grundrechte zu vertiefen, welche durch die drohnengestützte Datenbearbeitung tangiert werden können. Dabei werden zunächst das Recht auf Privatsphäre (Art. 13 BV; Art. 8 EMRK) und namentlich dessen Teilgehalte des Schutzes des Privatlebens, der Wohnung sowie des Fernmeldeverkehrs behandelt (S. 58 ff.). Im Anschluss daran soll vertieft auf das für die vorliegende Arbeit zentrale Grundrecht der informationellen Selbstbestimmung eingegangen werden (S. 62 ff.). Sodann sind die Menschenwürde (Art. 7 BV; Art. 3 und 7 EMRK), die Glaubens- und Gewissensfreiheit (Art. 15 BV; Art. 9 EMRK), die Meinungsäusserungs- (Art. 16 BV; Art. 10 EMRK) und die Versammlungsfreiheit (Art. 22 BV; Art. 11 EMRK) sowie die Eigentumsgarantie (Art. 26 BV) kurz in ihren Bezügen zur drohnengestützten Datenbearbeitung darzustellen (S. 66 ff.). Weitere Grundrechte werden aufgrund fehlender Relevanz für die Forschungsfrage nicht behandelt. 340 Auf die in den UNO-Pakten verankerten Rechte soll vorliegend nicht vertieft eingegangen werden; ihnen kommt regelmässig keine über die in der BV und EMRK enthaltenen Garantien hinausgehende Bedeutung zu (vgl. etwa BGE 144 I 126 E. 4.1; Schweizer/Rechsteiner, Rn. 2.44 ff.). 341 Diese Prüfung erfolgt zu Beginn von Teil 5 dieser Untersuchung; vgl. hinten, S. 158 ff. 46 Teil 2: Die drohnengestützte Datenbearbeitung im Lichte der Grundrechte Im abschliessenden Abschnitt dieses 2. Teils werden sodann die grundrechtlichen Schutzpflichten im Bereich der informationellen Selbstbestimmung herausgearbeitet und wird aufgezeigt, welche Anforderungen sich daraus mit Blick auf das Datenschutzrecht und insbesondere die drohnengestützte Datenbearbeitung durch Private ergeben (S. 69 ff.). § 2 Grundrechtliche Schutzpflichten I. Verpflichtungsdimensionen der Grundrechte 1. Die drei Kategorien der Verpflichtungsdimensionen Den Grundrechten kommen verschiedene Verpflichtungsdimensionen zu.342 Diese können unterschiedlich kategorisiert werden.343 In der Schweizer Doktrin werden dabei regelmässig drei Kategorien genannt, namentlich staatliche Unterlassungs-, Leistungs- und Schutzpflichten.344 Auch Letztere sind heute sowohl in der Rechtsprechung345 als auch in der Lehre346 anerkannt.347 2. Schutz vor Übergriffen Privater Schutzpflichten aktualisieren sich dort, wo es um «die Vermeidung nicht staatlicher Übergriffe» geht.348 Als nicht staatliche «Gefahrenquellen»349 sind 342 Rhinow/Schefer/Uebersax, Rn. 981 und Rn. 1002; Tschannen, § 7 Rn. 8 f.; Kälin/ Künzli, Rn. 3.68 ff.; Häfelin/Haller/Keller/Thurnherr, Rn. 256 ff.; vgl. Grabenwarter/Pabel, § 19 Rn. 1. 343 Eine Übersicht der verschiedenen Kategorisierungen kann und soll die vorliegende Untersuchung nicht leisten. Vgl. dazu z.B. ausführlich Medici, S. 8 f. m.w.H. 344 Dubey, Vol. I, Rn. 69 ff.; Kiener/Kälin/Wyttenbach, § 4 N 2 ff.; Kälin/Künzli, Rn. 3.69 ff.; vgl. auch Mégret, S. 97 ff. und bereits Müller J.P., Elemente, S. 46 ff. 345 BGE 140 II 315 E. 4.8; 126 II 300 E. 5a; 119 Ia 28 E. 2; vgl. auch BGE 138 II 229 E. 3.2.2; 138 I 246 E. 3.2.2. 346 Auer/Malinverni/Hottelier, Rn. 132; Besson, obligations positives, S. 70 ff.; Biaggini, Komm. BV, Art. 35 N 7; Egli, S. 155 ff.; Gächter, § 30 Rn. 46 f.; Häfelin/Haller/ Keller/Thurnherr, Rn. 266; Hänni, S. 432; Kiener, «Rote Zora», S. 624 ff.; Kiener/ Kälin/Wyttenbach, § 4 N 17 ff.; Mahon, Petit commentaire, Art. 35 Cst. N 13; Rhinow/Schefer/Uebersax, Rn. 1173; Tschentscher/Lienhard/Sprecher, Rn. 122 ff.; BSK BV-Waldmann, Art. 35 N 40. Skeptisch zur «Konstruktion» der grundrechtlichen Schutzpflichten dagegen Tschannen, § 7 N 70 ff. 347 Vgl. Biaggini, Komm. BV, Art. 35 N 7; Medici, S. 11 m.w.H. 348 Kiener/Kälin/Wyttenbach, § 4 N 17; vgl. Isensee, Handbuch, Rn. 192; Medici, S. 11 ff. 349 Egli, S. 286. § 2 Grundrechtliche Schutzpflichten 47 primär «andere Private»350 zu qualifizieren.351 Des Weiteren sind auch Naturund Umweltkatastrophen352 sowie ausländische Träger staatlicher Gewalt erfasst.353 Für die vorliegende Forschungsarbeit können die beiden letztgenannten Gefährdungsquellen ausser Acht gelassen werden. Stehen Übergriffe Privater im Fokus, so entsteht das für grundrechtliche Schutzpflichten typische Dreiecksverhältnis «beeinträchtigter Grundrechts träger» — «Verursacher eines Übergriffs» — Staat354 bzw. «Staat — Störer — Opfer».355 II. Grundlage der Schutzpflichten 1. In der Bundesverfassung Die verfassungsrechtliche Grundlage grundrechtlicher Schutzpflichten ist in Art. 35 Abs. 1 BV zu erblicken.356 Gemäss dieser Bestimmung müssen die Grundrechte in der gesamten Rechtsordnung zur Geltung kommen. Ferner «[kann] die grundrechtliche Schutzpflicht des Staates bei Rechtsverhältnissen unter Privaten als Korrelat zum Gewaltmonopol des Staates gesehen werden».357 Die Bürger verzichten auf (private) Gewalt zugunsten eines Rechts auf wirksamen staatlichen Schutz.358 Auf weitere Ausführungen zur theo retischen Herleitung staatlicher Schutzpflichten wird verzichtet.359 350 BGE 119 Ia 28 E. 2; Egli, S. 287; Kiener/Kälin/Wyttenbach, § 4 N 17. Dabei sind natürliche und juristische Personen erfasst (statt aller Mahon, S. 46). Vgl. Müller J.P., Diss., S. 161 f.; Murswiek, Freiheit, S. 987 ff.; Unruh, S. 22 f. 351 Zur Bedrohung der Grundrechte durch nichtstaatliche Machtträger und der Forderung nach einer «Drittwirkung» der Grundrechte bereits Saladin, S. 308. 352 Kiener/Kälin/Wyttenbach, § 4 N 17; Schefer, Gefährdung, S. 446 mit zahlreichen Hinweisen in Fn. 22; vgl. BGE 140 II 315 E. 4.8. 353 Egli, S. 294 ff. m.w.H.; Schefer, Gefährdung, S. 446; ders., Kerngehalte, S. 260 ff.; vgl. BGE 123 II 511. 354 Medici, S. 10. Anschaulich auch Calliess, Schutzpflicht, S. 326 f. 355 Isensee, Handbuch, Rn. 5; Stinner, S. 63 («Grundrechtsdreieck»). Vgl. Rupp, S. 57 («Störerdreieck»). 356 BGE 126 II 300 E. 5a; Egli, S. 167; Kiener, «Rote Zora», S. 623; Rhinow/Schefer/Uebersax, Rn. 1140 ff. und Rn. 1173 ff. Vgl. BGE 139 I 114 E. 4 (Art. 35 Abs. 1 und 2 BV); Schefer, Kerngehalte, S. 253 f. (Art. 35 Abs. 1 und 3 BV); Mahon, S. 49 (einzig Art. 35 Abs. 3 BV). Vgl. sodann Bacher, Rn. 162 ff. für eine Übersicht und Diskussion der in der Schweizer Lehre vertretenen Ansichten. 357 Müller J.P., Verwirklichung, S. 72; Isensee, Handbuch, Rn. 102 und Rn. 186; Egli, S. 187 ff. Zur Sicherheit als Staatsaufgabe und deren Bedeutung im vorliegenden Zusammenhang auch Schefer, Kerngehalte, S. 239; Bacher, Rn. 153 ff. 358 Alexy, S. 415; Bacher, Rn. 152; Isensee, Handbuch, Rn. 186. 359 Vgl. dazu ausführlich Bacher, Rn. 147 ff. mit zahlreichen Hinweisen; Besson, obligations positives, S. 71 f.; aus deutschrechtlicher Sicht Rupp, S. 28 ff. 48 2. Teil 2: Die drohnengestützte Datenbearbeitung im Lichte der Grundrechte In der Europäischen Menschenrechtskonvention Auch aus der EMRK ergeben sich grundrechtliche Schutzpflichten.360 Diese werden teilweise aus Art. 1 EMRK abgeleitet.361 Sie können sich aber auch direkt aus jeder Konventionsgarantie ergeben:362 «In principle, positive obli gations can be claimed everywhere.»363 Der Gerichtshof hielt fest, er sei nicht gehalten, «to develop a general theory of the positive obligations which may flow from the Convention.»364 Auf eine eingehende Darstellung der Schutz��pflichten unter der Geltung der EMRK sowie deren Begründung wird im Rahmen dieser Untersuchung verzichtet.365 III. Semantik Die zur Bezeichnung von Schutzpflichten verwendeten Termini sind (noch) nicht einheitlich.366 Die Ausdrücke «grundrechtliche Schutzpflicht»367 und «staatliche Schutzpflicht»368 werden nebeneinander und synonym gebraucht.369 Dieser Begriffsverwendung soll auch in der vorliegenden Arbeit gefolgt werden. 360 Besson, droit privé, S. 17; Egli, S. 235 ff.; Lavrysen, Restrictions, S. 322; Rensmann, S. 216; Werro/Schmidlin, S. 184 ff. Zur Anerkennung derselben im Kontext von Art. 8 EMRK Breitenmoser, S. 34 f. 361 In diesem Sinne Pétermann, S. 31 f. m.w.H; vgl. Grabenwarter/Pabel, § 19 N 3. 362 Mowbray, passim, v.a. S. 7 ff.; Lavrysen, Restrictions, S. 322. Aus der Rechtsprechung z.B. EGMR (GK), Söderman c. Schweden, Nr. 5786/08 (2013) Ziff. 80 mit zahlreichen Hinweisen. 363 Xenos, S. 4. 364 EGMR, Plattform «Ärzte für das Leben» c. Österreich, Nr. 10126/82 (1988) Ziff. 31; vgl. Lavrysen, Restrictions, S. 322. Xenos, S. 5, bedauert das Fehlen eines «coherent methodological framework in the planning of the application and development of positive obligations». 365 Ausführlich dazu Rensmann, S. 220 ff. Vgl. zur Praxis des EGMR zu den einzelnen EMRK-Garantien Karpenstein/Mayer-Johann, Art. 1 EMRK N 7. Zur Verwirklichung der Schutzpflichten gemäss EMRK in der Schweiz ausführlich Pétermann, passim. 366 Vgl. die Hinweise bei Biaggini, Komm. BV, Art. 35 N 7. 367 Vgl. z.B. Kiener/Kälin/Wyttenbach, § 4 N 25; Biaggini, Komm. BV, Art. 35 N 7; Egli, S. 160. 368 Vgl. BSK BV-Waldmann, Art. 35 N 43; Kiener/Kälin/Wyttenbach, § 5 N 13 und § 11 N 25. So auch der Titel des Werks von Stinner, Schutzpflichten. 369 Das Adjektiv «staatlich» hebt den Adressaten hervor, das Adjektiv «grundrechtlich» den Gegenstand, den Rechtsgrund und den Begünstigten (so treffend Isensee, Handbuch, Rn. 191; vgl. auch Bacher, Rn. 145). § 2 Grundrechtliche Schutzpflichten 49 Die Bezeichnungen «positive Schutzpflichten»370 bzw. «positive Verpflichtungen»371 sind ebenfalls anzutreffen; diese sind wohl an die Begriffsverwendung des EGMR372 sowie an die französisch- und englischsprachigen Lehre373 angelehnt. Nachfolgend soll deshalb einzig von grundrechtlichen bzw. staatlichen Schutzpflichten die Rede sein. IV. Schutzpflichten und deren Verhältnis zur Lehre der Drittwirkung Schutzpflichten sind von der eng verwandten Lehre der Drittwirkung374 der Grundrechte abzugrenzen.375 Dabei ist anhand ihrer Perspektiven vorzugehen: Während sich die Drittwirkung mit dem Verhältnis zwischen Privaten befasst, fokussiert die Schutzpflichtenlehre auf den Staat und «seine Rolle bei der Ausgestaltung der Beziehungen unter Privaten».376 Georg Müller sieht in der Drittwirkung der Grundrechte eine «Modalität der Schutzpflicht: Der Staat hat die Grundrechte vor Beeinträchtigungen durch übermächtige Private zu schützen».377 Liegt der Fokus auf dem Staat als Adressat der grundrechtlichen Schutzpflichten und obliegt ihm somit die Pflicht, «à veiller [au respect des droits fondamentaux] dans les relations interindividuelles»,378 so rückt die Dritt370 Z.B. bei Schefer, Kerngehalte, S. 236; BGE 139 IV 121 E. 4.6 f. Da es sich bei Schutzpflichten um positive Pflichten handelt, ist diese Bezeichnung redundant. 371 Z.B. bei Kiener/Kälin/Wyttenbach, § 4 N 20, § 11 N 26, § 15 N 12. 372 Vgl. statt vieler Besson, obligations positives, S. 50; Rensmann, S. 216, je m.w.H. 373 Vgl. z.B. Mahon, S. 49; Xenos, passim. 374 Teilweise ist auch der Begriff der Horizontalwirkung anzutreffen, so z.B. bei BSK BV-Waldmann, Art. 35 N 29 sowie Bacher, Rn. 138; vgl. Kiener/Kälin/Wyttenbach, § 4 N 81. Das Bundesgericht verwendet diesen Term in seiner publizierten Rechtsprechung nur selten (vgl. BGE 111 II 245 E. 4b; 114 Ia 329 E. 2b; aus der neuen Rechtsprechung BGE 143 I 217 E. 5.2). Dagegen benutzt das Höchstgericht regelmässig den Ausdruck «Drittwirkung» (aus der neueren Rechtsprechung statt vieler BGE 144 I 50 E. 4.1; 142 I 49 E. 4.5.1; 139 I 306 E. 4.6). Aus diesem Grund wird letzterem Begriff hier der Vorzug gegeben. 375 Biaggini, Komm. BV, Art. 35 N 7, erstes Lemma; Besson, obligations positives, S. 66; Tschentscher/Lienhard/Sprecher, Rn. 125; Müller L., Diss., S. 309. 376 Rhinow/Schefer/Uebersax, Rn. 1181; BJ, Obligation positive, S. 38; Saladin, S. 310. Auf eine weitere Unterscheidung der direkten und indirekten (bzw. der mittelbaren und unmittelbaren) Drittwirkung wird vorliegend verzichtet, vgl. dazu ausführlich Bacher, Rn. 139 ff. mit zahlreichen Hinweisen und bereits Müller G., Drittwirkung, S. 240 ff. 377 Müller G., HdG VII/2, § 204 Rn. 42; so auch Müller J.P., Verwirklichung, S. 128 f.; Tschentscher/Lienhard/Sprecher, Rn. 125 («Sonderfall der Schutzpflicht»); Egli, S. 198 («Unterfall der Lehre der grundrechtlichen Schutzpflichten»). 378 Besson, obligations positives, S. 69; vgl. Kiener, Grundrechte in der BV, Rn. 32; Harris et al., S. 26 f.; Lavrysen, Positive State, S. 13. 50 Teil 2: Die drohnengestützte Datenbearbeitung im Lichte der Grundrechte wirkung in den Hintergrund. Dies gilt umso mehr, wenn — wie in der vorliegenden Untersuchung — deliktisches und nicht vertragliches Handeln der Privaten diskutiert wird.379 Zusammenfassend kann festgehalten werden, dass sich die Untersuchung nicht mit der Drittwirkung von Grundrechten befasst; dieser Begriff wird deshalb im Folgenden nicht verwendet. V. Ausprägungen der Schutzpflichten Schutzpflichten haben entweder präventiven oder kurativen Charakter.380 Präventive Massnahmen zielen darauf ab, einen drohenden Übergriff in eine grundrechtlich geschützte Rechtsposition zu verhindern bzw. dessen Risiko zu mindern.381 In dieser Ausprägung der Schutzpflicht ist ein staatliches Eingreifen bereits dann gefordert, wenn erst die Gefahr eines Übergriffs droht.382 Kurativ bedeutet demgegenüber, dass der Staat im Nachhinein dem Opfer beisteht, z.B. indem er — auf dem Weg der Gesetzgebung — eine möglichst vollständige Wiedergutmachung der bereits eingetretenen Grundrechtseinschränkung ermöglicht.383 Das staatliche Eingreifen zielt darauf ab, eine andauernde oder die Folgen einer bereits eingetretenen Beeinträchtigung zu beseitigen oder zumindest die Grundrechtswidrigkeit festzustellen, um dem Opfer dadurch eine gewisse Genugtuung zu verschaffen.384 Stellt der Staat dem Betroffenen eines Übergriffs durch einen Privaten die rechtlichen Möglichkeiten für ein (gerichtliches) Vorgehen gegen den Verursacher zur Verfügung, so ist dies ebenfalls Ausdruck einer kurativen Massnahme. Ebenso verhält es sich, wenn tatsächliche Hindernisse beseitigt werden, welche den grundsätzlich zur Verfügung stehenden Rechtsschutz gegenüber Dritten erschweren oder gänzlich vereiteln.385 379 Vgl. Tschentscher/Lienhard/Sprecher, Rn. 126. Das so verstandene Deliktsrecht umfasst «unerlaubte Handlungen» von Privaten, wie z.B. bei Persönlichkeitsverletzungen (vgl. zu den Begriffen statt aller KuKo OR-Schönenberger, Vor Art. 41-61 N 1 f.). 380 Statt vieler Kälin/Künzli, Rn. 3.107. 381 Kiener/Kälin/Wyttenbach, § 4 N 20; Müller G., HdG VII/2, § 204 Rn. 7; Rupp, S. 33. 382 Vgl. Schefer, Gefährdung, S. 447 f. 383 Kälin/Künzli, Rn. 3.70; Kiener/Kälin/Wyttenbach, § 4 N 20; Schefer, Kerngehalte, S. 318; Schefer, Gefährdung, S. 447. Rupp, S. 33, spricht in diesem Zusammenhang von «repressiven» Instrumenten. 384 Kälin/Künzli, Rn. 3.107; Schefer, Gefährdung, S. 447; vgl. auch Schefer, Kern gehalte, S. 257 f. 385 Vgl. Müller G., HdG VII/2, § 204 Rn. 7; Rupp, S. 33 mit Hinweis auf die Rechtsprechung des BVerfGer. § 2 Grundrechtliche Schutzpflichten 51 VI. Adressaten 1. Primäre Verpflichtung des Gesetzgebers Als Adressat der Schutzpflichten hat «der Staat in all seinen Erscheinungsformen zu gelten».386 Die Wahrnehmung derselben obliegt jedoch primär dem Gesetzgeber.387 Er «ist (…) verpflichtet, die Rechtsordnung so auszugestalten, dass die Grundrechte auch tatsächlich ausgeübt werden können».388 Des Weiteren ist er gehalten, die Regelungen bei Änderungen der Verhältnisse «nachzubessern» und an neue Gefährdungen anzupassen.389 Dem Gesetzgeber kommt demnach eine «Beobachtungs- und Prüfungspflicht» mit Blick auf die bereits erlassenen Normen zu.390 2. Exekutive und Judikative Daneben können sich auch im Bereich der Rechtsanwendung oder im Rahmen einfachen Verwaltungshandelns Schutzpflichten ergeben und faktische Massnahmen erforderlich machen;391 auch die Judikative und die Exekutive werden somit in die Pflicht genommen.392 Die Exekutive übernimmt im Rahmen ihrer Zuständigkeit die Durchsetzung der erforderlichen Schutzmassnahmen, wobei die öffentliche Sicherheit und Ordnung im Vordergrund steht.393 Im Rahmen des Verwaltungshandelns können z.B. technisches Versagen, fehlende Mittel oder un genügende Ausbildung dazu führen, dass grundrechtliche Schutzpflichten verletzt werden.394 Die Judikative verwirklicht grundrechtliche Schutzpflichten bei der Rechtsanwendung im Einzelfall, also in ihrem angestammten Tätigkeitsbereich. Das Gericht hat das Recht verfassungskonform auszulegen und die 386 Egli, S. 316. Vgl. Isensee, Handbuch, Rn. 219 lit. d; Schefer, Kerngehalte, S. 236; Unruh, S. 24; für die EMRK Pétermann, S. 116 m.w.H. 387 BGE 126 II 300 E. 5b; Kiener/Kälin/Wyttenbach, § 4 N 21; Egli, S. 317; Unruh, S. 23; Rupp, S. 32; Pétermann, S. 117 f. 388 BGE 139 I 114 E. 4, unter expliziter Bezugnahme auf Art. 35 Abs. 1 und 2 BV. 389 Isensee, Handbuch, Rn. 219 lit. e; Bacher, Rn. 257. Spezifisch in Bezug auf neue Technologien Cottier, S. 39. 390 Isensee, Handbuch, Rn. 287. 391 Kiener/Kälin/Wyttenbach, § 4 N 21; Kiener, Grundrechte in der BV, Rn. 30. 392 Egli, S. 315 f.; Unruh, S. 20, je mit zahlreichen Hinweisen. Vgl. auch Biaggini, Komm. BV, Art. 35 N 7. 393 Isensee, Handbuch, Rn. 267; vgl. Mohler, Polizeirecht, Rn. 298 f. 394 Pétermann, S. 118. 52 Teil 2: Die drohnengestützte Datenbearbeitung im Lichte der Grundrechte legislatorische Umsetzung und Konkretisierung der Schutzpflichten zu prüfen sowie ggf. auf dem Wege der Rechtsfortbildung zu ergänzen.395 VII. Tatbestand Schutzpflichten aktualisieren sich nur unter spezifischen Voraussetzungen.396 Zunächst ist vorausgesetzt, dass diejenige Person, deren Schutz infrage steht, vom Geltungsbereich eines Grundrechts erfasst und der Schutzbereich desselben durch das staatliche Unterlassen berührt ist.397 Zudem wird verlangt, dass der Staat bzw. die zuständige Behörde von der konkreten — d.h. realen und unmittelbar drohenden — Gefährdungslage weiss oder bei Anwendung pflichtgemässer Aufmerksamkeit und genügender Sorgfalt hätte wissen müssen.398 Der Staat verletzt seine Schutzpflichten, wenn er bzw. die zuständige Behörde nicht all jene Schutzmassnahmen ergreift, welche «im Rahmen der verfügbaren Mittel vernünftigerweise» zur Verfügung stehen und welche eine Gefahrenabwehr realistischerweise bewirken können.399 Er muss also «faktisch und rechtlich» in der Lage sein, die Grundrechtsbeeinträchtigung zu verhindern.400 Die vorerwähnte Gefährdungslage muss konkret sein, mithin eine gewisse Qualität aufweisen. Gefordert ist das Erreichen einer bestimmten Schwelle401 bzw. eines gewissen Masses.402 Damit von einer «realen und unmittelbar drohenden Gefährdungslage» gesprochen werden kann, muss die rechtlich relevante Gefährdung so geartet sein, dass «bei ungehindertem Ablauf des zu erwartenden Geschehens mit hinreichender Wahrschein395 SGK BV-Schweizer, Art. 13 N 84; Kiener/Kälin/Wyttenbach, § 14 N 66. Ausführlich zu diesen Pflichten Bacher, Rn. 30 ff. und Rn. 285 ff., je m.w.H. 396 Kiener, «Rote Zora», S. 629 f.; Egli, S. 284 ff., je m.w.H. Vgl. EGMR (GK), Osman c. Ver einigtes Königreich, Nr. 23452/94 (1998) Ziff. 115; EGMR, Nencheva u.a. c. Bulgarien, Nr. 48609/06 (2013) Ziff. 108. 397 Grabenwarter/Pabel, § 19 N 5; Kiener, «Rote Zora», S. 629; Kiener/Kälin/ Wyttenbach, § 4 N 31. 398 Kälin/Künzli, Rn. 3.110; Kiener, «Rote Zora», S. 630; Kiener/Kälin/Wyttenbach, § 4 N 31. 399 Kiener/Kälin/Wyttenbach, § 4 N 31; BSK BV-Waldmann, Art. 35 N 52. 400 Kälin/Künzli, Rn. 3.70 (vgl. auch Rn. 3.108); vgl. sodann BSK BV-Waldmann, Art. 35 N 51 und 55; Isensee, Handbuch, Rn. 219 lit. c. 401 Egli, S. 299 m.w.H. Zum Begriff der Gefahrenschwelle Murswiek, Verantwortung, S. 86; Unruh, S. 76. 402 Wiederkehr, AJP 2001, S. 217; vgl. auch Besson, obligations positives, S. 76 («gravité»); Martin, S. 105 f. § 2 Grundrechtliche Schutzpflichten 53 lichkeit»403 ein Übergriff in die grundrechtlich geschützte Rechtsposition derjenigen Person erfolgt, deren Schutz infrage steht.404 Dabei ist für den Erlass gesetzlicher Schutzmassnahmen ein weniger strenger Massstab anzusetzen (blosse Gefährdung kann ausreichen) als für eine direkte Intervention durch Einsatzkräfte wie die Polizei (ernsthafte und qualifizierte Gefahr vorausgesetzt).405 Insgesamt kann der Tatbestand für das Aufleben von Schutzpflichten im vorliegenden Zusammenhang als die reale und unmittelbare Gefährdung der «tatsächlichen Grundlagen eines Grundrechts durch Private, sodass seine Ausübung nicht mehr oder nur noch begrenzt gewährleistet ist»,406 umschrieben werden. VIII. Rechtsfolge Steht im Grundsatz fest, dass eine Schutzpflicht besteht, so gilt es, nach der angebrachten Schutzmassnahme und ihrer konkreten Verwirklichungsweise zu fragen. Die zuständige staatliche Behörde ist gehalten, die Gefährdungslage nach deren Art und Intensität sowie deren Materialisierungswahrscheinlichkeit zu beurteilen und entsprechende Schutzmassnahmen zu treffen.407 Dabei ist nach den üblichen Verhältnismässigkeitskriterien vorzugehen,408 wobei dieser Grundsatz gleichzeitig als Untermassverbot wirkt.409 403 Vgl. Egli, S. 299 m.w.H. und unter Heranziehung des polizeirechtlichen Gefährdungsbegriffs. Ausführlich zur Wahrscheinlichkeit und deren Beurteilung Schefer, Gefährdung, S. 477 ff. 404 Vgl. dazu auch Schefer, Gefährdung, S. 473, der darauf hinweist, dass einerseits die Schwere des potenziellen Übergriffs und andererseits dessen Wahrscheinlichkeit in Betracht gezogen werden müssen; die beiden Kriterien stünden in enger Wechselwirkung zueinander. Anschaulich BVerfGer, 2 BvR 2502/08 vom 18. Februar 2010: Mangels ausreichender Substanziierung unzulässige Verfassungsbeschwerde gegen eine Versuchsreihe der Europäischen Organisation für kernphysikalische Forschung («CERN»); die Beschwerdeführerin fürchtete ein «Schadenereignis apokalyptischen Ausmasses» durch schwarze Löcher. Gemäss den Verfassungsrichtern ist ein solches aber «nach dem Stand von Wissenschaft und Technik praktisch ausgeschlossen» (v.a. Rn. 12). 405 Müller J.P., Verwirklichung, S. 73; Mohler, Polizeirecht, Rn. 779 mit Fn. 1497; vgl. BGE 128 I 327 E. 4.2. Zu Recht betont Martin, S. 106, in diesem Zusammenhang die Bedeutung der Umstände des Einzelfalls. 406 Wiederkehr, AJP 2001, S. 217. 407 Isensee, Handbuch, Rn. 219 lit. a und f; Wiederkehr, AJP 2001, S. 217; Schefer, Gefährdung, S. 479 f. Vgl. auch Calliess, Gewährleistung, S. 1098; Müller L., Diss., S. 191. 408 Vgl. Besson, obligations positives, S. 77; Egli, S. 306. Zur Verhältnismässigkeit im Einzelnen statt vieler BSK BV-Epiney, Art. 36 N 53 ff. 409 Kiener/Kälin/Wyttenbach, § 9 N 149; Weber-Dürler, S. 154; ausführlich Calliess, Schutzpflicht, S. 329. Zum Begriff des Untermassverbots Canaris, S. 228. 54 Teil 2: Die drohnengestützte Datenbearbeitung im Lichte der Grundrechte Da bei der Wahrnehmung von Schutzpflichten kein «konkretisierbares Unterlassen»410 verlangt wird, kommt dem Staat bzw. der zuständigen Behörde ein grosser Gestaltungs- bzw. Ermessensspielraum bezüglich der zu treffenden Massnahme zu.411 In der Rechtsprechung des EGMR zeigen sich bei der Auslegung dieses Spielraums412 — je nach betroffener Garantie — Unterschiede: So hat der Gerichtshof etwa im Kontext von Art. 6 EMRK (Recht auf ein faires Verfahren) Schutzpflichten relativ strikt ausgelegt.413 In den meisten Fällen prüft er jedoch einzig, ob der Staat angemessene und geeignete Massnahmen getroffen hat, um die Wahrnehmung der Rechte des Einzelnen zu gewährleisten,414 und gesteht den Staaten somit einen grossen Ermessensspielraum zu.415 Neben gesetzgeberischen kommen auch organisatorische, verfahrensmässige und finanzielle Schutzvorkehrungen in Betracht.416 Vorausgesetzt ist selbstredend, dass die infrage kommenden Schutzmassnahmen selbst grundrechtskonform sind, mithin nicht unrechtmässig die Grundrechte der (potenziellen) Störer oder Dritter einschränken.417 Insgesamt müssen die in Realisierung der Schutzpflichten getroffenen Schutzmassnahmen so ausgestaltet sein, dass der bestmögliche Schutz grundrechtlicher Ansprüche resultiert. Ausschlaggebend ist die Effektivität des Schutzes.418 Dieser ist durch das faktisch und rechtlich Mögliche beschränkt, wobei auch die Begrenztheit der sachlichen und persönlichen 410 Unruh, S. 69; vgl. Martin, S. 107. 411 Vgl. Besson, obligations positives, S. 83 f.; Egli, S. 305; Unruh, S. 79. Anschaulich (zu Art. 8 EMRK) EGMR (GK), Von Hannover c. Deutschland (Nr. 2), NRn. 40660/08 und 60641/08 (2012) Ziff. 104. 412 Ausführlich dazu Arai-Takahashi, S. 85 f. und S. 217 ff.; Lavrysen, Restrictions, S. 327 ff. 413 Harris et al., S. 24 mit Hinweis auf EGMR, Zimmermann und Steiner c. Schweiz, Nr. 8737/79 (1983) Ziff. 29; vgl. auch Arai-Takahashi, S. 58 f. 414 So z.B. EGMR, Plattform «Ärzte für das Leben» c. Österreich, Nr. 10126/82 (1988) Ziff. 34. 415 Vgl. EGMR, Abdulaziz, Cabales und Balkandali c. Vereinigtes Königreich, NRn. 9214/80, 9473/81, 9474/81 (1985) Ziff. 67; Johnston u.a. c. Irland, Nr. 9697/82 (1986) Ziff. 55; EGMR (GK), A, B und C. Irland, Nr. 25579/05 (2010) Ziff. 247 ff. Vgl. auch Harris et al., S. 512 f. 416 Biaggini, Komm. BV, Art. 35 N 7. 417 Kiener, Grundrechtsschranken, Rn. 16; BSK BV-Waldmann, Art. 35 N 53; Calliess, Schutzpflicht, S. 326; Isensee, Handbuch, Rn. 220; Marsch, S. 263. Rupp, S. 37, spricht in diesem Zusammenhang von einem «Übermassverbot hinsichtlich der Beschränkung der Freiheit des Dritten». 418 BGE 138 I 475 E. 4.1; Besson, obligations positives, S. 85; Egli, S. 320 ff.; Martin, S. 136; Schefer, Kerngehalte, S. 253 f. Anschaulich BVerfGE 92, 26 (Rn. 74): «Das Bundesverfassungsgericht kann deswegen die Verletzung einer Schutzpflicht nur feststellen, wenn die öffentliche Gewalt Schutzvorkehrungen entweder überhaupt nicht getroffen hat oder die getroffenen Regelungen und Massnahmen gänzlich ungeeignet oder völlig unzulänglich sind, das gebotene Schutzziel zu erreichen […].» § 2 Grundrechtliche Schutzpflichten 55 Mittel des Staates zu beachten ist.419 Ein optimaler oder umfassender Schutz kann nicht beansprucht werden.420 Besteht die zu ergreifende Schutzmassnahme in einer gesetzlichen Regelung, so hat diese auch einen wirksamen Vollzugsmechanismus zu beinhalten. Nichts anderes ergibt sich auch aus dem Prinzip des effektiven Rechtsschutzes.421 Erweist sich eine in der Vergangenheit getroffene Massnahme aufgrund technologischer Fortschritte oder eines gesellschaftlichen Wandels als nicht mehr ausreichend, so sind neue Vorkehrungen zu treffen, um ein ausreichendes Schutzniveau aufrechtzuerhalten.422 IX. Schutzberechtigte Schutzpflichten aktualisieren sich unter der Voraussetzung, dass diejenige Person, deren Schutz infrage steht, vom Geltungsbereich eines Grundrechts erfasst ist.423 Somit sind primär natürliche Personen schutzberechtigt,424 juristische Personen des Privatrechts dagegen nur, wenn «die fragliche Garantie nicht an natürliche Qualitäten des Menschen anknüpft und von ihren Funktionen her auch auf juristische Personen passt.»425 X. Justiziabilität grundrechtlicher Schutzpflichten 1. Grundsätzliche Justiziabilität Anders als bei Unterlassungsansprüchen von Grundrechten wurde die Justi ziabilität von Schutzpflichten zunächst kontrovers diskutiert.426 Die h.L. geht aber heute davon aus, dass Schutzpflichten von betroffenen Personen gericht- 419 Unruh, S. 78; Müller L., Diss., S. 192; Leutert, S. 96; Besson, obligations positives, S. 95; Künzli, S. 240. 420 Martin, S. 136; Schefer, Kerngehalte, S. 271. 421 Vgl. Isensee, Sicherheit, S. 21; Stinner, Schutzpflicht, S. 59; Wiederkehr, Diss., S. 251 f.; Calliess, Schutzpflicht, S. 327; Müller L., Diss., S. 313. Es müssen Sach- und Verfahrensnormen geschaffen werden (Isensee, Handbuch, Rn. 267; Bacher, Rn. 201). Zum Anspruch auf effektiven Rechtsschutz statt vieler BSK BV-Schaub, Art. 146 N 2; Kiener/Kälin/Wyttenbach, § 42 N 4. 422 Calliess, Schutzpflicht, S. 328, spricht in diesem Zusammenhang vom «dynamischen Charakter der Schutzpflicht». Vgl. Rupp, S. 75 sowie vorne, S. 51. 423 Vorne, S. 52. 424 Statt aller Auer/Malinverni/Hottelier, Rn. 112 f. 425 Kiener/Kälin/Wyttenbach, § 5 N 20. Ausführlich und differenzierend Egli, S. 301 ff. (zu Minderjährigen, Ausländerinnen und Ausländern etc.). 426 Bacher, Rn. 203 mit zahlreichen Hinweisen in Fn. 563. 56 Teil 2: Die drohnengestützte Datenbearbeitung im Lichte der Grundrechte lich durchgesetzt werden können.427 Auch die Rechtsprechung in Deutschland428 sowie die Praxis menschenrechtlicher Überwachungsorgane zeigen auf, dass «Schutzpflichten an sich ohne weiteres justiziabel»429 sind. Das Bun desgericht hat sich bisher noch nie explizit zur Frage geäussert; implizit hat es sich aber bereits mehrfach mit (Verletzungen von) Schutzpflichten befasst.430 2. Durchsetzungsproblematik vor Bundesgericht Die Frage nach der Justiziabilität von Schutzpflichten stellt sich primär dort, wo der Gesetzgeber zur Wahrnehmung derselben berufen ist. Das Bundesgericht ist bislang mit der Feststellung, dass der Gesetzgeber die Rechtsunterworfenen ungenügend schützt bzw. den Grundrechtsschutz nur ungenügend verwirklicht, eher zurückhaltend.431 Des Weiteren steht fest, dass der Bundesgesetzgeber vom Bundesgericht nicht gezwungen werden kann, ein Gesetz zu erlassen bzw. zu revidieren oder neue Normen zu schaffen.432 Stellt das Bundesgericht eine Verletzung von grundrechtlichen Schutzpflichten fest, so kann dies aber durchaus eine Gesetzesrevision auslösen.433 In BGE 137 I 305 hat das Bundesgericht die Möglichkeit eines subjektiven Anspruchs auf ein Tätigwerden des Gesetzgebers grundsätzlich bejaht, wenn der grundrechtlich geschützte Anspruch hinsichtlich des «Ob» sowie auch des «Wie»434 hinreichend klar bestimmt ist.435 Im 427 Bacher, Rn. 205 f.; Besson, obligations positives, S. 63; Egli, S. 336; Martin, S. 120; Müller J.P., Verwirklichung, S. 72; Pulver, S. 423; Wiederkehr, AJP 2001, S. 218; vgl. auch Alexy, S. 427 f.; Gächter, § 30 Rn. 47 f.; Schefer, Kerngehalte, S. 269 ff.; Unruh, S. 64 f.; Kiener, Grundrechte in der BV, Rn. 30. 428 Vgl. dazu die zahlreichen Hinweise auf die Rechtsprechung des BVerfG bei Bacher, Rn. 204 mit Fn. 568. 429 Kälin/Künzli, Rn. 3.77. Zur EMRK vgl. Frowein/Peukert, Art. 8 EMRK N 11 ff.; Wyss, S. 67. 430 So etwa in BGE 118 V 206 E. 5a ff.; 119 Ia 28 E. 2 f.; 126 II 300 E. 5a; 140 II 315 E. 4.8. Vgl. auch Bacher, Rn. 204; Besson, obligations positives, S. 63; Gächter, § 30 Rn. 47 f. 431 BSK BV-Diggelmann, Art. 13 N 9. 432 Egli, S. 332 f. und S. 338; Martin, S. 121. Dies ergibt sich aus der Gewaltenteilung (auf Bundesebene), vgl. dazu Tschannen, § 27 Rn. 6 ff.; Häfelin/Haller/Keller/ Thurnherr, Rn. 1406 ff., v.a. Rn. 1416. Vgl. auch Art. 190 BV und dazu statt aller BSK BV-Epiney, Art. 190 N 35 ff. 433 Egli, S. 333; Martin, S. 121; vgl. auch BSK BV-Epiney, Art. 190 N 36 m.w.H. 434 Vgl. ausführlich zu diesen beiden Aspekten Martin, S. 117 f. 435 BGE 137 I 305 E. 2.4 f. Inhaltlich ging es um die Nichtfortführung der (zeitlich befristeten) Kommission für die Gleichstellung von Frau und Mann im Kanton Zug. Vgl. dazu auch BSK BV-Diggelmann, Art. 13 N 9 mit Fn. 24. In BGE 126 II 300 E. 2c erwähnte das Bundesgericht sodann die Möglichkeit, bei Vorliegen eines schutzwürdigen Interesses (in casu aus Art. 10 BV) eine «konkrete rechtsgestaltende Anordnungen zu bean tragen». Vgl. auch BGE 104 Ia 284 E. 2b. § 2 Grundrechtliche Schutzpflichten 57 konkreten Fall richtete sich der legislatorische Auftrag jedoch an einen kantonalen Gesetzgeber. Ein subjektives und gerichtlich durchsetzbares Recht auf Erlass bzw. Revision eines Bundesgesetzes ist vor diesem Hintergrund zu verneinen. 3. Praxis des EGMR Der EGMR auferlegt sich weniger Zurückhaltung. Anschaulich ist in diesem Kontext das Urteil in Fall Marckx c. Belgien436. Der Gerichtshof befasste sich darin mit der Ausgestaltung des belgischen Familienrechts mit Blick auf das Recht auf Achtung des Privat- und Familienlebens. Der Umstand, dass zwischen einer unverheirateten Mutter und deren leiblichen Tochter nicht ex lege durch die Geburt ein Abstammungsverhältnis entsteht, stellte gemäss Strassburger Richter eine Verletzung von Art. 8 EMRK dar.437 Als die nationalen Gerichte diesem Urteil nicht zum Durchbruch verhalfen, hielt der Gerichtshof in einem anderen Verfahren fest, er «ne discerne pas ce qui pouvait empêcher la cour d’appel de Bruxelles, puis la Cour de cassation de se conformer aux conclusions de l’arrêt Marckx.»438 XI. Zwischenfazit Seit ihrer Anerkennung in der schweizerischen Lehre und Rechtsprechung haben die Schutzpflichten stetig an Bedeutung gewonnen. Diese «Verwirklichungstendenz der Grundrechte»439 ist kaum überraschend, gehen Gefährdungen von grundrechtlich geschützten Rechtspositionen doch vermehrt auch von Privaten aus.440 Vereinfacht ausgedrückt, entsteht eine grundrechtliche Schutzpflicht dort, wo der Staat von einer aktuellen oder drohenden Gefährdung einer gewissen Schwere für die Grundrechte von betroffenen Privaten weiss oder hätte wissen müssen. In einer solchen Situation ist primär der Gesetzgeber 436 EGMR, Marckx c. Belgien, Nr. 6833/74 (1979), v.a. Ziff. 31. Vgl. aus der neueren Rechtsprechung des Gerichtshofs z.B. EGMR, Khadija Ismayilova c. Aserbaidschan, NRn. 65286/13 und 57270/14 (2019) Ziff. 112 ff. 437 Diese Missachtung des Prinzips mater semper certa est sei inkompatibel mit der staatlichen Schutzpflicht, Vorkehrungen zu treffen, welche die Integration eines Kindes in die Familie von Geburt an ermöglichen (EGMR, Marckx c. Belgien, Nr. 6833/74 (1979), Ziff. 31). Vgl. dazu und zur Weiterentwicklung dieser Rechtsprechung Arai-Takahashi, S. 66 ff. 438 EGMR, Vermeire c. Belgien, Nr. 12849/87 (1991), Ziff. 25. Inhaltlich ging es um die Erbschaft von Frau Vermeire, welche als uneheliche Tochter gegenüber den ehelichen Kindern der Erblasser benachteiligt war. 439 Unruh, S. 76. 440 Vgl. Besson, obligations positives, S. 94. 58 Teil 2: Die drohnengestützte Datenbearbeitung im Lichte der Grundrechte berufen, die grundrechtlichen Schutzpflichten wahrzunehmen. Der grundsätzlich justiziable, subjektive Anspruch auf Umsetzung scheitert auf Bundesebene indes daran, dass das Parlament vom Bundesgericht nicht zum Erlass von Gesetzen bzw. Einzelbestimmungen gezwungen werden kann. Bevor die in diesem Abschnitt diskutierten Tatbestandselemente und die daraus erwachsenden Rechtsfolgen der grundrechtlichen Schutzpflichten im hier interessierenden Kontext dargestellt und analysiert werden können,441 müssen zunächst die relevanten Grundrechtsgarantien diskutiert werden. § 3 Privatsphäre I. Vorbemerkungen 1. Die einzelnen Aspekte der Privatsphäre Jede Person hat Anspruch auf Achtung ihres Privat- und Familienlebens, ihrer Wohnung sowie ihres Brief-, Post- und Fernmeldeverkehrs (Art. 13 Abs. 1 BV); hinzu kommt der Anspruch auf Schutz vor Missbrauch ihrer persönlichen Daten (Art. 13 Abs. 2 BV). Nach der bundesgerichtlichen Rechtsprechung gewährleistet Abs. 1 allgemein das Recht auf eine Privat- und Geheimsphäre, wobei Abs. 2 im Besonderen das Recht auf informationelle Selbstbestimmung schützt.442 Inhaltlich weitgehend übereinstimmende Garantien finden sich in Art. 8 Ziff. 1 EMRK.443 Der grundrechtliche Anspruch auf Achtung des Familienlebens wird durch die drohnengestützte Datenbearbeitung nicht tangiert.444 Aus diesem Grund wird dieser Teilgehalt von Art. 13 Abs. 1 BV nicht weiter vertieft. 2. Persönlicher Schutzbereich Zum persönlichen Schutzbereich der nachfolgend behandelten Ansprüche gemäss Art. 13 Abs. 1 BV kann festgehalten werden, dass dieser alle natürlichen 441 Hinten, S. 69 ff. 442 BGE 142 II 340 E. 4.2; vgl. Biaggini, Komm. BV, Art. 13 N 11; Fasnacht, Rn. 135; BGE 128 II 259 E. 3.2; 137 I 167 E. 3.2. Ausführlich zur informationellen Selbstbestimmung hinten, S. 62 ff. 443 BGE 144 I 126 E. 4.1; 140 I 353 E. 8.3; 140 IV 181 E. 2.3; Kiener/Kälin/Wyttenbach, § 14 N 1; Haller W., § 209 N 34; Chadoian, S. 249. Gleiches gilt auch für Art. 17 UNO-Pakt II (vgl. Bygrave, S. 83 ff.; Schweizer/Rechsteiner, Rn. 2.47 f. und 2.55). 444 Vgl. ausführlich zum Schutzbereich des Anspruchs auf Achtung des Familienlebens Kiener/Kälin/Wyttenbach, § 14 N 22 ff. § 3 Privatsphäre 59 Personen erfasst. Juristische Personen können sich einzig auf jene Teilgehalte der Privatsphäre berufen, deren Schutzziel nicht untrennbar an die Existenz einer natürlichen Person gebunden ist.445 II. Achtung des Brief-, Post- und Fernmeldeverkehrs 1. Inhalt und sachlicher Schutzbereich Der Anspruch auf Achtung des Brief-, Post und Fernmeldeverkehrs schützt die Individualkommunikation als Ausprägung der Privatsphäre; auch moderne Kommunikationsformen wie z.B. Internettelefonate werden erfasst.446 Der Schutz erstreckt sich neben dem Inhalt der Kommunikation auch auf die Randdaten wie z.B. den Zeitpunkt, die Dauer und die Anzahl der Teilnehmer447 sowie sog. Standortdaten.448 2. Bei der drohnengestützten Datenbearbeitung durch Private insbesondere Das Fernmeldegeheimnis wird durch von Privatpersonen betriebene Drohnen und die damit einhergehende Datenbearbeitung kaum tangiert.449 Es wäre zwar vorstellbar, dass ein auf einer Drohne angebrachter Sensor Kommunikationsvorgänge abfangen kann (etwa ein IMSI-Catcher). Da die Verwendung von solcher Technik durch Private und auch deren Vertrieb nicht erlaubt sind,450 wird vorliegend auf eine ausführlichere Auseinandersetzung mit dem Fernmeldegeheimnis verzichtet. Im Übrigen hat das Bundesgericht unlängst festgehalten, dass der Einsatz eines IMSI-Catchers dann nicht das Fernmeldegeheimnis, sondern die informationelle Selbstbestimmung tangiert, wenn die Feststellung einer Geräte- oder Kartennummer eines Mobiltelefons unabhängig von einem Kommunikationsvorgang zwischen Menschen erfolgt.451 Bei so gearteten 445 Statt vieler BSK BV-Diggelmann, Art. 13 N 8; Dubey, Vol. II, Rn. 1766 ff.; Kiener/ Kälin/Wyttenbach, § 14 N 4 f. Die Garantien der Achtung der Wohnung und des Brief-, Post- und Fernmeldeverkehrs schützen folglich auch juristische Personen. 446 Rhinow/Schefer/Uebersax, Rn. 1402; Chadoian, S. 251; BGE 140 I 353 E. 8.3. 447 BGE 140 I 353 E. 8.3; 140 IV 181 E. 2.3 f.; Müller/Schefer, S. 203 mit zahlreichen Hinweisen. 448 Chadoian, S. 251 m.w.H. 449 Szenarien, in welchen durch eine drohnengestützte Datenbearbeitung das Recht auf Achtung des Brief- oder Postverkehrs verletzt wird, sind kaum erdenklich. Darauf wird deshalb nicht weiter eingegangen. 450 Vgl. zum Ganzen vorne, S. 42 f. 451 BGer, 1B_251/2017 vom 21. Februar 2018 E. 6.6. Vgl. dazu auch Sprecher et al., S. 666. 60 Teil 2: Die drohnengestützte Datenbearbeitung im Lichte der Grundrechte Einsätzen des IMSI-Catchers «‹kommunizierten› ausschliesslich technische Geräte miteinander».452 III. Achtung der Wohnung 1. Inhalt und sachlicher Schutzbereich Eine Wohnung im Sinne von Art. 13 Abs. 1 BV ist eine Räumlichkeit, «die eine gewisse Privatsphäre sichert und auf einen bestehenden oder künftigen, dauernden oder vorübergehenden Lebensmittelpunkt schliessen lässt.»453 Auch Neben- und Aussenräume wie Balkone, Innenhöfe sowie umfriedete Gärten fallen darunter.454 Die Garantie schützt primär vor unbefugtem (physischem) Eindringen in die Wohnung; doch auch das Aushorchen oder Ausspionieren von aussen (z.B. mit Kameras oder Richtmikrofonen) ist erfasst.455 Dabei ist unerheblich, ob die Sensoren an einer Drohne befestigt sind oder nicht. 2. Bei der drohnengestützten Datenbearbeitung durch Private insbesondere Im Bereich der drohnengestützten Datenbearbeitung tritt das Recht auf Achtung der Wohnung regelmässig hinter den Schutz des Privatlebens und der informationellen Selbstbestimmung zurück.456 Anders könnte es sich etwa verhalten, wenn ein Privater seine Drohne gezielt zum Ausspähen von Wohnräumen oder umfriedeten Gärten etc. eines Dritten einsetzt.457 Die damit einhergehende Datenbearbeitung eröffnet aber auch in diesen Fällen (zusätzlich) den Schutzbereich der informationellen Selbstbestimmung.458 452 BGer, 1B_251/2017 vom 21. Februar 2018 E. 6.6 mit Hinweis auf BVerfGer, 2 BvR 1345/03 vom 22. August 2006 (v.a. Rn. 57). 453 SGK BV-Breitenmoser, Art. 13 N 61 m.w.H. 454 SGK BV-Breitenmoser, Art. 13 N 61, erstes Lemma; BSK BV-Diggelmann, Art. 13 N 25; Rhinow/Schefer/Uebersax, Rn. 1396. 455 BSK BV-Diggelmann, Art. 13 N 26; Kälin/Künzli, Rn. 12.44; Kiener/Kälin/Wyttenbach, § 14 N 42. 456 Vgl. zur sog. unechten Grundrechtskonkurrenz BSK BV-Epiney, Art. 36 N 22; Kiener/ Kälin/Wyttenbach, § 7 N 7. 457 Vgl. BSK BV-Diggelmann, Art. 13 N 26; Kornmeier, S. 124. 458 Zur informationellen Selbstbestimmung ausführlich sogleich, S. 62 ff. § 3 Privatsphäre 61 IV. Achtung des Privatlebens 1. Inhalt und sachlicher Schutzbereich Eine präzise und umfassende Definition der in Art. 13 BV nebeneinanderstehenden Begriffe der Privatsphäre (Marginalie) und des Privatlebens (Abs. 1) ist unmöglich anzugeben.459 Mit Sicherheit umfasst der Schutz des Privatlebens die Gesamtheit der privaten Lebenssachverhalte, «die der Einzelne als Privatsache abgeschirmt haben möchte, unabhängig davon, ob diese Vorgänge als schutzwürdig erscheinen oder nicht.»460 Gemäss Bundesgericht schützt der Anspruch auf Achtung des Privatlebens somit «l’identité, les relations sociales et les comportements intimes de chaque personne physique, l’honneur et la réputation, ainsi que notamment toutes les informations se rapportant à une personne qui ne sont pas accessible au public.»461 Der Schutzpflichtengehalt des Grundrechts auf Achtung des Privatlebens ist mithin auf private Ausforschung gerichtet.462 2. Bei der drohnengestützten Datenbearbeitung durch Private insbesondere Setzen Private Drohnen ein, so geht es nicht «nur» um die Datenbearbeitung an sich, sondern — je nach den Umständen des Einzelfalls — auch um eine Einschränkung der Achtung des Privatlebens. Wird eine Drohne ausschliesslich zum Zwecke des Ausspionierens verwendet, ohne dass dabei Foto- oder Videoaufnahmen erstellet werden, so steht der Schutz des Privatlebens im Vordergrund.463 Das Gefühl des «Beobachtet-Werdens» durch Drohnen an sich kann — unabhängig von einer Datenaufzeichnung im konkreten Fall — bereits einen «Überwachungsdruck»464 bzw. einen «chilling effect»465 auslö459 BSK BV-Diggelmann, Art. 13 N 6; SGK BV-Breitenmoser, Art. 13 N 8. Vgl. auch BGE 144 I 266 E. 3.1, wo das BGer zum Begriff des Privatlebens gemäss Art. 8 Ziff. 1 EMRK festhält, dass dieser «sich einer abschliessenden Definition verschliesst». 460 Kiener/Kälin/Wyttenbach, § 14 N 11; vgl. Kälin/Künzli, Rn. 12.4; Tschentscher/ Lienhard/Sprecher, Rn. 265 . Für eine Aufzählung weiterer geschützter Aspekte vgl. Biaggini, Komm. BV, Art. 13 N 5 m.w.H. 461 BGE 135 I 198 E. 3.1; ähnlich auch BGE 140 I 381 E. 4.1; 137 II 371 E. 6.1. 462 Tschentscher/Lienhard/Sprecher, Rn. 265, die Stalking und Paparazzi als Beispiele nennen. Vgl. auch Biaggini, Komm. BV, Art. 13 N 16. 463 Bei diesem Vorgang liegt wohl regelmässig auch ein Beschaffen von Personendaten vor (vgl. zum Begriff vorne, S. 33). Dieser Umstand tritt jedoch, da er nicht primärer Zweck des Drohneneinsatzes ist, in den Hintergrund. 464 Zum Begriff Rose, S. 139 f.; vgl. Griebel, S. 112. 465 Dazu statt vieler Rhinow/Schefer/Uebersax, Rn. 1189 f. und Rn. 1564 f. Vgl. BGE 143 I 147 E. 3.3; 140 I 2 E. 10.6.3. 62 Teil 2: Die drohnengestützte Datenbearbeitung im Lichte der Grundrechte sen.466 Als «eyes in the sky»467 oder «verlängertes Auge»468 ermöglichen Drohnen eine Echtzeitbeobachtung, welche für sich allein genommen bereits eine Einschränkung des Rechts auf Achtung des Privatlebens darstellen kann.469 Dies gilt umso mehr, wenn aus voyeuristischen Beweggründen oder aus blosser Neugierde Drohnen zur Beobachtung eingesetzt werden.470 Da die allermeisten Drohnen jedoch mit audiovisuellen Sensoren ausgestattet sind, die gerade auf eine Datenbearbeitung abzielen,471 und die privatrechtlichen Schutzbehelfe in beiden Fällen parallel ausgestaltet sind,472 wird für die vorliegende Untersuchung das Recht auf informationelle Selbstbestimmung in den Fokus gerückt. Dieses soll sogleich vertieft werden. § 4 Informationelle Selbstbestimmung I. Entstehung und Einordnung Gemäss Art. 13 Abs. 2 BV hat jede Person Anspruch auf Schutz vor Missbrauch ihrer persönlichen Daten. Diese Bestimmung verankert einen grundrecht lichen Anspruch auf Datenschutz.473 Im Anschluss an das sog. «Volkszählungsurteil» des deutschen Bundesverfassungsgerichts474 sowie die Schweizer 466 Vgl. dazu vorne, S. 5 f., sowie Calo, S. 33; Cassart, RDTI, S. 87; Griebel, S. 112; Müller, Diss., S. 111 f.; Schmid, S. 83; Straub, S. 55; SGK BV-Schweizer, Art. 13 N 76. 467 Diesen Begriff findet man verschiedentlich in englischsprachigen Werken zu Drohnen, z.B. bei Farber. 468 Müller L., Diss., S. 23. Der Autor erwähnt auch das sog. Kamera-Monitor-Prinzip, wonach die aufgenommenen Bilder einzig auf einen Monitor (bei Drohnen regelmässig der Bildschirm eines Tablets oder Mobiltelefons) übertragen, aber nicht abgespeichert werden. Vgl. dazu auch Martini, S. 733. 469 Vgl. Ruegg et al., S. 53 f. Vgl. sodann aus deutschrechtlicher Optik Martini, S. 735 mit den Hinweisen in Fn. 37. 470 Vgl. Müller L., Diss., S. 113 f. 471 Vgl. dazu vorne, S. 40 (Sensoren) bzw. S. 33 ff. (drohnengestützte Datenbearbeitung). 472 Das Datenschutzgesetz verweist für die Durchsetzung von Ansprüchen bei Persönlichkeitsverletzungen auf die Regeln des ZGB (Art. 28 ff.). Diese sind ohne Weiteres auf Persönlichkeitsverletzungen anwendbar, die keine Datenbearbeitung beinhalten. Vgl. ausführlich zum Ganzen hinten, S. 106 ff. 473 BGE 128 II 259 E. 3.2; Botschaft BV, S. 153; Biaggini, Komm. BV, Art. 13 N 11. Vgl. Baeriswyl, Verselbständigung, S. 121, der das Grundrecht auf Datenschutz auch als Teil der persönlichen Freiheit (Art. 10 Abs. 2 BV) versteht. 474 BVerfGE 65, 1 (43 ff.) vom 15. Dezember 1983 (begriffsprägend); vgl. ausführlich dazu Flückiger, autodétermination, S. 849 ff. Vgl. sodann BGE 113 Ia 1 E. 4b/bb und BGE 129 I 232 E. 4.3.1. § 4 Informationelle Selbstbestimmung 63 Lehre475 hat das Bundesgericht entschieden, dass Art. 13 Abs. 2 BV ein Recht auf informationelle Selbstbestimmung garantiert.476 Es handelt sich um das spezifischste und zugleich zentralste Grundrecht im Kontext der Datenbearbeitung. Auch die EMRK garantiert als Teilaspekt von Art. 8 das Recht auf informationelle Selbstbestimmung.477 II. Schutzbereich 1. Persönlicher Schutzbereich Es können sich sowohl natürliche als auch juristische Personen auf die informationelle Selbstbestimmung berufen.478 Bei Letzteren werden Geschäftsge heimnisse jedoch primär von der Wirtschaftsfreiheit (Art. 27 BV) geschützt.479 2. Sachlicher Schutzbereich 2.1 Herrschende Lehre und Rechtsprechung Die h.L.480 geht davon aus, dass Art. 13 Abs. 2 BV — entgegen dem zu eng gera tenen Wortlaut der Norm — nicht nur vor Datenmissbrauch schützt, sondern jegliche Bearbeitung von Personendaten481 erfasst.482 Jede Person hat folglich 475 Der Begriff wurde damals bereits von Huber R., S. 40 und Tercier, S. 67 Rn. 460, verwendet. Der Schöpfer des Begriffs war Wilhelm Steinmüller (vgl. Bull, Selbstbestimmung, S. 25 mit Fn. 54). 476 Zum ersten Mal wird diese Formulierung in BGE 120 II 118 E. 3a, ohne Bezugnahme auf die BV, verwendet; vgl. ausführlich zu diesem Urteil Aebi-Müller, Rn. 593 mit Fn. 1555. Kurz darauf ebenso in BGE 122 I 143 E. 6b/aa. Seither gefestigte Rechtsprechung, vgl. u.a. BGE 144 II 91E. 4.4; 144 II 77 E. 5.2; 142 II 340 E. 4.2; 140 I 381 E. 4.1; 138 I 256 E. 5.5; 133 I 77 E. 5.3; 129 I 232 E. 4.3.1; 128 II 259 E. 3.2. Ausführlich zur «Entstehungsgeschichte» dieses Grundrechts Waldmeier, S. 6 ff. 477 Explizit EGMR (GK), Satakunnan Markkinapörssi OY und Satamedia OY c. Finnland, Nr. 931/13 (2017) Ziff. 137; seither etwa EGMR, M.L. und W.W. c. Deutschland, NRn. 60798/10 und 65599/10 (2018) Ziff. 87; Benedik c. Slowenien, Nr. 62357/14 (2018) Ziff. 103; vgl. Grabenwarter, S. 784, und bereits Breitenmoser, S. 246. Vgl. auch BGE 138 I 256 E. 5.5. Zurückhaltend dagegen Siemen, S. 132 f.; Lynskey, S. 178 f. 478 BGE 141 I 201 E. 4.1; Kiener/Kälin/Wyttenbach, § 14 N 5. Anders verhält es sich unter dem E-DSG; dieser ist auf juristische Personen nicht anwendbar (vgl. dazu hinten, S. 75). 479 Schweizer/Rechsteiner, Rn. 2.5; Müller/Schefer, S. 167. Vgl. zur Wirtschaftsfreiheit statt anderer BSK BV-Uhlmann, Art. 27 N 3 ff. 480 Trotz der in den letzten Jahren aufgekommenen kritischen Stimmen in der Lehre (vgl. dazu sogleich S. 64) rechtfertigt es sich, von der «herrschenden Lehre» zu sprechen. So auch Bieri, Rn. 625 mit Fn. 1783. 481 Vgl. zu den Begriffen Daten und Datenbearbeitung vorne, S. 29 ff. bzw. S. 33 ff. 482 Biaggini, Komm. BV, Art. 13 N 11; SGK BV-Schweizer, Art. 13 N 74; Müller L., Diss., S. 122; Tschentscher/Lienhard/Sprecher, Rn. 267; vgl. BGE 137 I 167 E. 3.2; 128 II 259 E. 3.2. 64 Teil 2: Die drohnengestützte Datenbearbeitung im Lichte der Grundrechte u.a. das Recht, selbst zu entscheiden, ob, wem und wann sie persönliche Lebenssachverhalte, Anschauungen, Empfindungen und Gedanken bekannt gibt.483 Gemäss Bundesgericht beinhaltet die informationelle Selbstbestimmung, «dass jede Person gegenüber fremder, staatlicher oder privater Bearbeitung und Speicherung von sie betreffenden Informationen bestimmen können muss, ob und zu welchem Zweck diese Informationen über sie bearbeitet und gespeichert werden.»484 2.2 Kritische Lehrstimmen In der Lehre stösst das Konzept der informationellen Selbstbestimmung teilweise auf Kritik.485 Gächter/Egli lehnen eine Übernahme eines informationellen Selbstbestimmungsrechts in das schweizerische Verfassungsrecht bereits deshalb ab, weil dieses in Deutschland aus der allgemeinen Handlungs-freiheit abgeleitet wurde,486 welche in der Schweiz nicht vorgesehen ist.487 Vielmehr sei diese Verfassungsbestimmung im Sinne von «Datenverkehrsrecht» zu verstehen, welches sich an den Gesetzgeber richte; ähnlich den Verfahrensgarantien würden dadurch Ansprüche an das Verfahrens- und Organisationsrecht gestellt.488 Belser vertritt ebenfalls die Ansicht, dass durch Art. 13 Abs. 2 BV kein umfassendes Selbstbestimmungsrecht gewährleistet werde. Der Schutzbereich umfasse vielmehr bloss den Schutz vor Missbrauch; die Verfassungsbestimmung sei wortgetreu zu verstehen.489 2.3 Verfassungsrevision? Im Zusammenhang mit der konkreten Tragweite der informationellen Selbstbestimmung waren unlängst auf politischer Ebene interessante Entwicklungen zu beobachten. Eine parlamentarische Initiative verlangte, den 483 Müller/Schefer, S. 167; Bieri, Rn. 625; Handkomm. DSG-Rosenthal, Art. 1 N 4; ähnlich Tschentscher/Lienhard/Sprecher, Rn. 266. 484 BGE 142 II 340 E. 4.2; ständige Rechtsprechung: BGE 140 I 2 E. 9.1; 140 I 381 E. 4.1; 138 II 346 E. 8.2. 485 Für eine ausführliche Darstellung der Kritik Flückiger, autodétermination, passim, v.a. S. 853 ff. 486 Ausführlich zur Konzeption und Herleitung der informationellen Selbstbestimmung gemäss BVerfGer Albers, S. 152 ff. 487 Gächter/Egli, Rn. 23; Müller J.P., Elemente, S. 18; Schrepfer, S. 178; Simon, S. 107 f.; vgl. auch Fasnacht, Rn. 124. 488 Gächter/Egli, Rn. 26; ebenso Gächter/Werder, S. 89 ff.; vgl. auch Belser, in: Belser/Epiney/Waldmann, Rn. 120; Fasnacht, Rn. 125. 489 Belser, in: Belser/Epiney/Waldmann, § 6 Rn. 118 ff.; Belser, Tragweite, S. 28 ff.; ihr folgend Fasnacht, Rn. 121 ff. i.V.m. Rn. 158; ähnlich auch Belser/Waldmann, S. 79 Rn. 87. § 4 Informationelle Selbstbestimmung 65 Wortlaut von Art. 13 Abs. 2 BV in ein Recht auf informationelle Selbstbestimmung eabzuändern.490 Die staatspolitischen Kommissionen beider Räte gaben der Initiative Folge; schliesslich wurde sie jedoch am 29. September 2017 vom Nationalrat abgeschrieben.491 Die Ratsmehrheit sprach sich gegen zusätzliche Bürokratie aus; ausserdem war sie gegen «l’introduction d’un article qui existe déjà dans la Constitution fédérale».492 2.4 Zwischenfazit Insgesamt ist vor dem Hintergrund dieser politischen Entwicklungen und mit der ständigen Rechtsprechung des Bundesgerichts493 sowie der h.L.494 festzuhalten, dass Art. 13 Abs. 2 BV ein umfassendes Selbstbestimmungsrecht über die eigenen Personendaten garantiert. III. Bei der drohnengestützten Datenbearbeitung durch Private insbesondere Die informationelle Selbstbestimmung wird durch die drohnengestützte Datenbearbeitung direkt tangiert.495 Jede (unerwünschte) Datenbearbeitung mittels Drohnen stellt grundsätzlich eine Einschränkung des in Art. 13 Abs. 2 BV garantierten Grundrechts dar. Da die allermeisten Drohnen Sensoren tragen, welche — wie Foto- und Videokameras — auf eine Datenbearbeitung ausgelegt sind,496 rückt die informationelle Selbstbestimmung für die vor liegende Auseinandersetzung klar ins Zentrum.497 Die Schutzpflichten dimension der informationellen Selbstbestimmung ist deshalb genauer zu analysieren.498 490 Parlamentarische Initiative Vischer (14.413). 491 Amtl. Bull. NR 2017 S. 1682. 492 Amtl. Bull. NR 2017 S. 1682 (Votum Nantermod). 493 Vgl. dazu die Hinweise vorne, Fn. 476. Für zahlreiche weitere Hinweise (auf die ältere Rechtsprechung) vgl. Müller L., Diss., S. 122 mit Fn. 769. 494 Vgl. dazu die Hinweise vorne, Fn. 482 f. 495 Ebenso aus deutschrechtlicher Optik Kornmeier, S. 119. 496 Vgl. dazu vorne, S. 40 ff. (Sensoren). 497 Vgl. auch vorne, S. 61 f. 498 Vgl. dazu hinten, S. 69 ff. 66 Teil 2: Die drohnengestützte Datenbearbeitung im Lichte der Grundrechte § 5 Weitere möglicherweise tangierte Grundrechte I. Menschenwürde 1. Inhalt und Schutzbereich Gemäss Art. 7 BV ist die Würde des Menschen zu achten und zu schützen. So verankert die BV ein fundamentales Prinzip der gesamten Verfassungsordnung.499 Gleichzeitig kommt der Menschenwürde ein Status als justiziables «Auffanggrundrecht»500 zu, dessen Anwendung erst dann in Betracht kommt, wenn die «spezialisierten Garantien nicht greifen».501 Eine Verletzung ist nur zurückhaltend anzunehmen.502 Die Menschenwürde entzieht sich gemäss bundesgerichtlicher Rechtsprechung einer Definition. Der Verfassungsgeber habe auch deshalb auf eine Definition verzichtet, «weil eine verfassungsrechtliche Bestimmung dessen, was die Würde und den Wert eines Menschen ausmacht, grundsätzlich problematisch wäre.»503 Die einzelnen Gehalte dieser Garantie lassen sich deshalb weder positiv noch negativ eindeutig definieren.504 2. Bei der drohnengestützten Datenbearbeitung durch Private insbesondere Die Menschenwürde bietet u.a. Schutz gegen ausgeprägte Formen von Persönlichkeitsverletzungen.505 Gemäss Belser wiegt die Gefahr für den Einzelnen beim Einsatz von zivilen Drohnen, die mit Infrarotkameras bestückt sind, so schwer, dass mit Verletzungen der Menschenwürde zu rechnen ist.506 Eine solche Verletzung durch Drohnen des Grenzwachtkorps, die tatsächlich mit Infrarotkameras bestückt sind,507 wurde vor Bundesverwaltungsgericht bereits einmal gerügt, von diesem aber klar verneint.508 499 Biaggini, Komm. BV, Art. 7 N 4; Mahon, Petit commentaire, Art. 7 Cst. N 4; vgl. Kley, S. 325. 500 BGE 132 I 49 E. 5.1; 143 IV 77 E. 4.1; Kiener/Kälin/Wyttenbach, § 10 N 14; Müller L., Diss., S. 99; Rhinow/Schefer/Uebersax, Rn. 174. 501 Biaggini, Komm. BV, Art. 7 N 4; Mahon, Petit commentaire, Art. 7 Cst. N 4. 502 Biaggini, Komm. BV, Art. 7 N 4; Rhinow/Schefer/Uebersax, N 174; Müller L., Diss., S. 100 m.w.H. 503 BGE 143 IV 77 E. 4.1; zustimmend Kiener/Kälin/Wyttenbach, § 10 N 11. 504 Vgl. Haller W., § 209 N 15; Mahon, Petit commentaire, Art. 7 Cst. N 5; SGK BVMastronardi, Art. 7 N 40 ff. 505 BVGer, A-2482/2007 vom 26. Juni 2007 E. 6.3; vgl. Müller L., Diss., S. 100. 506 Belser, Tragweite, S. 30. Vgl. zu Infrarot- und Wärmebildkameras vorne, S. 42. 507 Vgl. dazu und allgemein zum Einsatz von Drohnen durch das GWK vorne, S. 35 f . 508 BVGer, A-2482/2007 vom 26. Juni 2007 E. 6.1 und E. 6.3. § 5 Weitere möglicherweise tangierte Grundrechte 67 Mithin darf — a maiore minus — davon ausgegangen werden, dass die Menschenwürde bei drohnengestützten Datenbearbeitungen durch Private nicht eingeschränkt wird. Von den heute auf dem Markt angebotenen Drohnen für Privatpersonen und den daran angebrachten Sensoren509 geht keine Gefahr einer so schwerwiegenden Persönlichkeitsverletzung aus, dass Art. 7 BV herangezogen werden müsste.510 II. Glaubens- und Gewissensfreiheit 1. Inhalt und Schutzbereich Art. 15 BV gewährleistet eine sachlich umfassende Glaubens- und Gewissensfreiheit511 (oftmals auch Religionsfreiheit genannt512).513 Geschützt ist insbesondere das Recht «eine religiöse Überzeugung zu haben, zu äussern, zu verbreiten oder zu praktizieren oder gemäss einer religiösen Überzeugung zu handeln.»514 Gleichzeitig ist jeder Zwang zu entsprechendem Verhalten verboten.515 2. Bei der drohnengestützten Datenbearbeitung durch Private insbesondere Bei der drohnengestützten Datenbearbeitung durch Private ist nicht auszuschliessen, dass Einblicke in religiöse oder weltanschauliche Bereiche ermöglicht werden.516 Dieser Umstand ist insofern von Bedeutung, als dass bei der Wahrnehmung von Schutzpflichten die Bearbeitung von besonders schützenswerten Personendaten517 speziell zu berücksichtigen ist.518 Eine darüber hinausgehende Bedeutung kommt der Glaubens- und Gewissensfreiheit in diesem Kontext jedoch nicht zu. 509 Vgl. dazu ausführlich vorne, S. 40 ff. 510 Vgl. auch Kornmeier, S. 115 ff., wo bei einer grundrechtlichen Auseinandersetzung mit Drohneneinsätzen (aus deutschrechtlicher Optik) die Menschenwürde nicht erwähnt wird. 511 Biaggini, Komm. BV, Art. 15 N 3; vgl. Karlen, Religionsfreiheit, S. 266. 512 So z.B. BGE 129 I 12 E. 8.4; 135 I 79 E. 2.2; 144 I 281 E. 3.2. 513 Zur ähnlichen Garantie in Art. 9 EMRK statt aller Frowein/Peukert, Art. 9 EMRK N 1 ff. 514 BGE 125 I 347 E. 3a. Vgl. BGE 145 I 121 E. 5.1; 142 I 49 E. 3.4. Zu Schutzobjekt und Inhalt im Einzelnen BSK BV-Pahud de Mortanges, Art. 15 N 28 ff.; Biaggini, Komm. BV, Art. 15 N 4 ff. 515 Kiener/Kälin/Wyttenbach, § 29 N 1; BSK BV-Pahud de Mortanges, Art. 15 N 110. 516 Vgl. KGer LU, 7H 17 49 vom 18. April 2018 E. 3.5; Kornmeier, S. 119 f. In Betracht kommen etwa auch Aufnahmen von Besuchern einer Kirche oder Moschee. 517 Vgl. dazu vorne, S. 34. 518 Vgl. Belser, Tragweite, S. 37 mit Fn. 53. 68 Teil 2: Die drohnengestützte Datenbearbeitung im Lichte der Grundrechte III. Meinungsäusserungs- und Versammlungsfreiheit 1. Inhalt und Schutzbereich Des Weiteren seien hier kurz die durch Art. 16 bzw. Art. 22 BV garantierte Meinungsäusserungs- und Versammlungsfreiheit erwähnt.519 Werden Drohnen durch Behörden — wie z.B. durch die Polizei — eingesetzt, so besteht die Gefahr eines «chilling effect».520 Dadurch kann der Schutzbereich von Art. 16 und/oder Art. 22 BV tangiert sein.521 2. Bei der drohnengestützten Datenbearbeitung durch Private insbesondere Im Kontext privat betriebener Drohnen ist kaum eine Situation vorstellbar, in welcher der Schutzbereich der Meinungsäusserungs- und/oder Versammlungsfreiheit eröffnet wäre. Eine drohnengestützte Überwachung oder Beobachtung einer Demonstration durch einen Privaten dürfte kaum einmal einen psychologischen Druck verursachen, der eine abschreckende Wirkung auf die Grundrechtsausübung der Betroffenen mit sich bringt.522 In Übereinstimmung mit dem Fokus der vorliegenden Arbeit wird deshalb auf weitere Ausführungen dazu verzichtet. IV. Eigentumsgarantie 1. Inhalt und Schutzbereich Die Bundesverfassung gewährleistet in Art. 26 Abs. 1 das Eigentum.523 Der verfassungsrechtliche Eigentumsbegriff ist autonom, d.h. nicht zivilrechtsabhängig;524 er umfasst aber auch die zivilrechtlichen Institute des Fahrnisund Grundeigentums sowie des Besitzes.525 Insgesamt schützt Art. 26 BV 519 Ausführlich zum Gehalt dieser Grundrechte statt aller Biaggini, Komm. BV, Art. 16 N 4 und N 12 ff. und Art. 22 N 3 ff. 520 Zum Begriff: Müller/Schefer, S. 375 ff.; Rhinow/Schefer/Uebersax, Rn. 1189 f. und Rn. 1564 f. Vgl. auch BGE 143 I 147 E. 3.3; 140 I 2 E. 10.6.3; 136 I 167 E. 3.2.3. 521 Vgl. BSK BV-Hertig, Art. 16 N 40 und Art. 22 N 16; Clarke, S. 287; Martini, S. 738 ff. 522 Vgl. Kiener/Kälin/Wyttenbach, § 9 N 31. 523 Das in Art. 1 des 1. Zusatzprotokolls zur EMRK verankerte Recht auf Schutz des Eigentums wird nicht behandelt, da die Schweiz dieses nicht ratifiziert hat. Die Entschädigungsfunktion der Eigentumsgarantie (vgl. Art. 26 Abs. 2 BV) ist sodann für die vorliegende Untersuchung unerheblich. 524 Komm. BV-Biaggini, Art. 26 N 12; BSK BV-Waldmann, Art. 26 N 17; vgl. BGE 144 II 367 E. 3.2. 525 BGE 128 I 295 E. 6a; 120 Ia 120 E. 1b. § 6 Schutzpflichten im Kontext der informationellen Selbstbestimmung 69 die konkreten Eigentumsrechte wie etwa die Verfügungsmacht über das Eigentum und dessen Nutzung.526 2. Bei der drohnengestützten Datenbearbeitung durch Private insbesondere Art. 667 ZGB hält fest, dass sich das Eigentum an Grund und Boden nach oben und unten auf den Luftraum und das Erdreich erstreckt, soweit für die Ausübung des Eigentums ein Interesse besteht. Es lässt sich demnach nicht abstrakt bestimmen, bis zu welcher Höhe ein schutzwürdiges Interesse an der Ausübung des Eigentumsrechts innerhalb der geschützten «Luftsäule» über einem Grundstück gegeben ist.527 Überfliegt ein Privater ein Grundstück mit einer Drohne in einer Höhe von weniger als 120 m,528 so ist nicht vollständig auszuschliessen, dass die Eigentumsgarantie tangiert wird.529 Dies gilt umso mehr, wenn der Überflug in geringer Höhe (wenige Meter über dem Boden bzw. über den sich darauf befindlichen Gebäuden) erfolgt.530 Werden gleichzeitig Daten bearbeitet, so rückt die Eigentumsgarantie jedoch in den Hintergrund, weshalb sie nicht weiter vertieft wird. § 6 Schutzpflichten im Kontext der informationellen Selbstbestimmung I. Anerkannte Schutzpflichtendimension Es ist noch nicht abschliessend geklärt, welche Grundrechte in welchem Umfang Schutzpflichten auslösen.531 Dabei ist in Lehre und Rechtsprechung anerkannt, dass alle Grundrechte grundsätzlich auch eine Schutzdimension 526 Kiener/Kälin/Wyttenbach, § 30 N 10 f. mit zahlreichen Beispielen. Vgl. zu den drei justiziablen Teilgehalten der Eigentumsgarantie statt vieler BSK BV-Waldmann, Art. 26 N 33 ff. 527 Hrubesch-Millauer/Bruggisser, Rn. 7 ff., v.a. Rn. 20; Christen et al., S. 166 f. 528 Vgl. zu dieser Maximalflughöhe vorne, S. 25 529 Vgl. BGE 142 II 128 E. 2.2; 131 II 137 E. 3.1.2, je m.w.H.; KGer GR, ZK1 14 34 vom 9. Juli 2014 E. 4c. 530 Vgl. Hrubesch-Millauer/Bruggisser, Rn. 20. 531 Kälin/Künzli, Rn. 3.100 ff.; Kiener/Kälin/ yttenbach, § 4 N 18; vgl. BSK BV-Waldmann, Art. 35 N 49. Eine abschliessende Darstellung der Grundrechte, welchen Praxis und Lehre eine Schutzdimension zuerkennen, kann und soll die vorliegende Arbeit nicht leisten. Vgl. für eine Diskussion besonderer Schutzaufträge gemäss BV Egli, S. 160 ff. 70 Teil 2: Die drohnengestützte Datenbearbeitung im Lichte der Grundrechte aufweisen.532 «Innerhalb der einzelnen Grundrechte sind jedoch (…) einige Gehalte besser ‹geeignet›, Schutzpflichten aufleben zu lassen, als andere.»533 Einigkeit herrscht bezüglich Art. 13 Abs. 2 BV und der darin verankerten Schutzdimension: Diese Grundrechtsnorm umfasst klarerweise (auch) Schutzpflichten und kann mithin ein Tätigwerden des Gesetzgebers erforderlich machen.534 Sie enthält denn auch explizit einen «besonderen Schutzauftrag», namentlich den Anspruch auf Schutz vor Missbrauch persönlicher Daten.535 Gemäss dem EGMR begründet Art. 8 EMRK staatliche Schutzpflichten, die «the adoption of measures designed to secure respect for private life even in the sphere of the relations of individuals themselves» beinhalten können.536 Die dort verankerten Ansprüche umfassen auch Schutz durch den Staat,537 namentlich bei Gefährdungen der Privatsphäre und der informationellen Selbstbestimmung.538 Dementsprechend ist dieser gehalten, Verletzungen der Privatsphäre und des Datenschutzes zu verbieten und bei Widerhandlungen Sanktionen vorzusehen, die vor Gericht geltend gemacht werden können.539 Der Staat muss aktiv intervenieren,540 sofern die vorne dargestellten Voraussetzungen gegeben sind.541 532 Kiener/Kälin/Wyttenbach, § 4 N 18; Schefer, Kerngehalte, S. 248 m.w.H.; vgl. Rhinow/Schefer/Uebersax, Rn. 1173. 533 Schefer, Kerngehalte, S. 254; vgl. Auer/Malinverni/Hottelier, Rn. 131. Vgl. dazu den Wortlaut von Art. 35 Abs. 3 BV. 534 Biaggini, «Durchnummerieren», S. 82; Biaggini, Komm. BV, Art. 13 N 16; Egli, S. 164 ff.; Kiener/Kälin/Wyttenbach, § 4 N 18; Schwander, S. 119; Schweizer/ Rechsteiner, Rn. 2.32; Moeckli, Rn. 43. Vgl. auch Häfelin/Haller/Keller/ Thurnherr, Rn. 266. 535 Müller G., HdG VII/2, § 204 Rn. 8; Kiener/Kälin/Wyttenbach, § 4 N 18; Egli, S. 160 ff. 536 Erstmals in EGMR, X und Y c. Niederlande, Nr. 8978/80 (1985) Ziff. 23; seither ständige Rechtsprechung, vgl. z.B. EGMR, Jäggi c. Schweiz, Nr. 58757/00 (2006) Ziff. 33; EGMR (GK), Evans c. Vereinigtes Königreich, Nr. 6339/05 (2007) Ziff. 75; EGMR (GK), Von Han nover c. Deutschland (Nr. 2), Nr. 40660/08 und 60641/08 (2012) Ziff. 98; EGMR (GK), Bédat c. Schweiz, Nr. 56925/08 (2016) Ziff. 73; EGMR (GK), Nicolae Virgiliu Tănase c. Rumänien, Nr. 41720/13 (2019) Ziff. 125. 537 Lynskey, S. 114; Kälin/Künzli, Rn. 12.59, je m.w.H. Vgl. auch BGE 136 III 410 E. 6.2, wonach Art. 28 ff. ZGB die zivilrechtliche Konkretisierung von Art. 8 EMRK darstellen und somit den Auftrag für staatliche Schutzmassnahmen verwirklichen, indem sie der verletzten Person zivilrechtliche Rechtsbehelfe zur Verfügung stellen. 538 Vgl. Harris et al., S. 26. 539 Vgl. Nadakavukaren Schefer, S. 269; Kühling, S. 51 mit Hinweisen auf die Rechtsprechung des EGMR. 540 Vgl. Solove, Privacy, S. 34 («To preserve people’s ability to engage in self-definition, the state must actively intervene»); Auer/Malinverni/Hottelier, Rn. 132 («devoir d’intervention»). Vgl. auch Calliess, Schutzpflicht, S. 322. 541 Vorne, S. 51 f. § 6 Schutzpflichten im Kontext der informationellen Selbstbestimmung 71 In der Schweiz findet die Schutzpflichtendimension der informationellen Selbstbestimmung primär in der Datenschutzgesetzgebung sowie dem Persönlichkeitsschutz gemäss ZGB seinen Ausdruck.542 Das Bundesgericht hat in diesem Zusammenhang auch vom «privatrechtlichen, im DSG konkretisierten Recht auf informationelle Selbstbestimmung» gesprochen.543 Dieses Verständnis wird durch den Zweckartikel des E-DSG untermauert; bezweckt wird namentlich der «Schutz der Persönlichkeit und der Grundrechte von natürlichen Personen, über die Personendaten bearbeitet werden».544 Des Weiteren ist auch die Regelung in Art. 26 Abs. 2 lit. b E-DSG, wonach eine Datenbearbeitung entgegen der ausdrücklichen Willenserklärung der betroffenen Person eine Persönlichkeitsverletzung darstellt, als klarer Hinweis auf die Umsetzung der informationellen Selbstbestimmung im E-DSG zu verstehen.545 II. Verfassungsrechtliche Anforderungen an das Datenschutzrecht Durch den Erlass eines Datenschutzgesetzes nimmt der Staat grundsätzlich seine Schutzpflichten im Bereich der informationellen Selbstbestimmung wahr.546 Doch ist insbesondere dieses Gesetz einer ständigen Dynamik unterworfen: Vorkehrungen des Gesetzgebers können durch gesellschaftlichen oder technologischen Wandel unter Druck kommen oder sich als nicht (mehr) ausreichend erweisen,547 was Anpassungen des Gesetzes verlangen kann.548 Umgekehrt ist darauf zu achten, technische Entwicklungen nicht 542 Biaggini, Komm. BV, Art. 13 N 16; Kiener/Kälin/Wyttenbach, § 4 N 18; Moeckli, Rn. 43; Müller/Schefer, S. 166; Schweizer/Rechsteiner, Rn. 2.32. Vgl. Bacher, Rn. 510 mit Fn. 1290. 543 BGE 127 III 481 E. 3b; ähnlich BGE 143 I 253 E. 3.3; 120 II 118 E. 3a; Botschaft Totalrevision DSG, S. 7010. Vgl. BGE 138 II 346 E. 8; Botschaft DSG 1988, S. 417 ff.; Peter, S. 56 f.; SHK DSG-Fey, Art. 1 N 11 f.; Hausheer/Aebi-Müller, Rn. 13.24; Moeckli, Rn. 43; Amtl. Bull. NR 2019 S. 1778 (Votum Glättli). 544 Vgl. dazu BSK DSG-Mauer-Lambrou/Kunz, Art. 1 N 15 ff.; Müller L., Diss., S. 34 m.w.H. 545 Aebi-Müller, Rn. 592; Schweizer M., Rn. 351; Botschaft DSG 1988, S. 459 (je zu Art. 12 DSG). Vgl. auch Art. 26 Abs. 3 E-DSG. 546 Dies mindestens zu einem Teil. Die Wahrnehmung der Schutzpflichten in diesem Bereich finden auch in anderen Gesetzen und Normen ihren Ausdruck, so u.a. in Art. 28 ff. ZGB (vgl. BSK ZGB I-Meili, Art. 28 N 17) oder in den Art. 179 ff. StGB («Straf bare Handlungen gegen den Geheim- oder Privatbereich»; vgl. BSK StGB-Ramel/ Vogelsang, Art. 179 N 7). 547 Rupp, S. 74; vgl. Isensee, Handbuch, Rn. 219 lit. e. 548 Vgl. dazu auch vorne, S. 55. 72 Teil 2: Die drohnengestützte Datenbearbeitung im Lichte der Grundrechte zu verhindern oder zu bremsen. Dies nicht nur im Sinne der Verhältnismässigkeit,549 sondern auch mit Blick auf das Gebot der Technologieneutralität. Danach sollen alle Technologien — soweit wie möglich — gleichberechtigt behandelt werden; das Gesetz bleibt dadurch offen für technologische Entwicklungen und verhindert keine Innovationen.550 Das «programmatische Mitbedenken der Grundrechte [hier: der informationellen Selbstbestimmung] durch den Gesetzgeber» in Umsetzung der grundrechtlichen Schutzpflichten geniesst jedoch nicht immer Priorität. «Umso grössere Bedeutung für die Verwirklichung der Grundrechte gewinnt ihre verfahrensrechtliche Durchsetzung und damit der niederschwellige Zugang zur Justiz.»551 Insgesamt steht fest, dass ein — wenn nicht der zentrale — Aspekt der Schutzpflichtendimension der informationellen Selbstbestimmung in deren Durchsetzung zu sehen ist. Der Staat kann und darf nicht hinnehmen, dass der Grundrechtsschutz in diesem Bereich nicht effektiv wahrgenommen werden kann. Denn «Selbstbestimmung und Souveränität bedeuten, dass man weiss, wer wofür verantwortlich ist, oder jedenfalls, dass diese Informa tionen durch Fragen zu erlangen sind.»552 549 Vgl. vorne, S. 53. 550 Botschaft Totalrevision DSG, S. 6971; Mauchle, S. 817 m.w.H. Vgl. Harasgama, S. 38 f.; Burkert/Hettich/Thouvenin, S. 49 f.; kritisch Burkert, Rn. 99; Cottier, S. 35. 551 Kiener, Grundrechte in der BV, Rn. 33 in fine. 552 Stinner, Selbstbestimmung, S. 157 (Hervorhebung hinzugefügt); vgl. dazu auch Frei, Rn. 58 sowie vorne, S. 5 f. 73 Teil 3: Bestandesaufnahme (1): Der E-DSG § 1 Vorbemerkungen Nachfolgend wird der E-DSG analysiert und anhand desselben geprüft, ob der Gesetzgeber seinen grundrechtlichen Schutzpflichten im Kontext der informationellen Selbstbestimmung nachgekommen ist. Dabei liegt der Fokus auf der Frage, ob im Datenschutzrecht ein effektiver Schutz der informationellen Selbstbestimmung gewährleistet ist.553 Der Einfachheit halber soll nachfolgend primär von Foto- und Videoaufnahmen die Rede sein, da entsprechende Sensoren am weitesten verbreitet sind.554 Wie bereits ausgeführt, wird das DSG momentan totalrevidiert. Aufgrund der bestehenden Ähnlichkeiten zu den Regeln der EU und deren Bedeutung bei der Ausarbeitung der neuen Bestimmungen555 können bei der Auslegung derselben auch die europäischen Regelungen beigezogen werden.556 Bevor die zentralen Bestimmungen des E-DSG dargestellt und deren Geltung im hier interessierenden Kontext analysiert werden können, gilt es, die Anwendbarkeit des E-DSG auf die drohnengestützte Datenbearbeitung durch Private zu analysieren (S. 75 ff.). Sodann werden insbesondere die Bearbeitungsgrundsätze (S. 82 ff.), die Informationspflicht (S. 93 ff.), das Auskunftsrecht (S. 100 ff.) und die Rechte bei Persönlichkeitsverletzung (S. 106 ff.) detailliert untersucht. 553 Vgl. vorne, S. 54. 554 Vgl. zu den Sensoren vorne, S. 40 ff. Auf andere Arten der Datenbearbeitung sind die nachfolgenden Ausführungen ebenfalls anwendbar. 555 Vgl. dazu vorne, S. 11 ff. 556 Der Gesetzgeber weicht teilweise explizit von den Regelungen der EU ab (sog. «Swiss Finishes»); vgl. dazu z.B. Rosenthal, Entwurf, Rn. 99; Vasella/Sievers, passim; Amtl. Bull. NR 2019 S. 1781 (Voten BR Keller-Sutter, Jauslin). 74 Teil 3: Bestandesaufnahme (1): Der E-DSG § 2 Anwendbarkeit des Datenschutzgesetzes Um zu prüfen, ob die drohnengestützte Datenbearbeitung durch Private in den sachlichen Geltungsbereich557 des E-DSG fällt, müssen zunächst die für das Datenschutzrecht zentralen Termini des «Verantwortlichen» und der «betroffenen Person» definiert werden. Deren Verständnis ist für jede datenschutzrechtliche Auseinandersetzung unabdingbar. I. Verantwortlicher und betroffene Person 1. Verantwortlicher Gemäss Art. 4 lit. i E-DSG ist Verantwortlicher u.a. jede private Person, die allein oder zusammen mit anderen über den Zweck und die Mittel der Datenbearbeitung entscheidet. Der Begriff wurde im Zuge der DSG-Revision aus dem europäischen Recht übernommen, namentlich anstelle des bisherigen «Inhaber[s] der Datensammlung».558 Materiell ergaben sich daraus keine Änderungen.559 Werden mithilfe einer Drohne Daten bearbeitet, so ist der Drohnenbetreiber560 als Verantwortlicher zu qualifizieren. Daneben kommen auch weitere Personen infrage, welche «über den Zweck und die Mittel der Bearbeitung» mitentscheiden.561 Betraut ein Kunde einen Fotografen mit der Erstellung von Drohnenbildern, so sind beide als Verantwortliche im Sinne des E-DSG zu qualifizieren: Der Fotograf entscheidet (zusammen mit dem Kunden) über das Mittel der Datenbearbeitung; der Kunde wiederum entscheidet über den Zweck derselben.562 Im Rahmen der drohnengestützten Datenbearbeitung durch Private kommen somit jeweils der Drohnenbetreiber selbst sowie allfällige Drittpersonen, die mit diesem über den Drohneneinsatz und dessen Zweck entschieden haben, als Verantwortliche in Betracht. 557 Der räumliche Geltungsbereich des E-DSG muss nicht weiter behandelt werden; vgl. nur Art. 2a Abs. 1 E-DSG. 558 Botschaft Totalrevision DSG, S. 6971 und S. 7023. Dies geschah im Sinne einer «Modernisierung der Terminologie» (ebd., S. 6971). 559 Botschaft Totalrevision DSG, S. 7023; vgl. Botschaft DSG 1988, S. 448; Rosenthal, Entwurf, Rn. 18. 560 Vgl. zum Begriff und seiner Verwendung in der vorliegenden Untersuchung vorne, S. 27 f. 561 Ausführlich zu den Begriffen Zweck und Mittel der Bearbeitung Rosenthal, Controller, Rn. 16 ff.; vgl. auch Vasella, Auftragsbearbeitung, S. 110 ff. 562 Eigenes Beispiel nach Malgieri, S. 173. § 2 Anwendbarkeit des Datenschutzgesetzes 2. 75 Betroffene Person Betroffene Person ist die natürliche Person, über die Personendaten bearbeitet werden (Art. 4 lit. b E-DSG). Angesprochen ist damit jeweils diejenige natürliche Person, auf die sich die bearbeiteten Daten beziehen563 bzw. die Trägerin des Persönlichkeits- und Grundrechtsschutzes ist, den der E-DSG bezweckt.564 Anders als unter dem noch geltenden DSG können juristische Personen nicht mehr Betroffene im Sinne de E-DSG sein.565 II. Sachlicher Geltungsbereich Das Datenschutzgesetz gilt für die Bearbeitung von Personendaten natür licher Personen durch private Personen und Bundesorgane (Art. 2 Abs. 1 lit. a und b E-DSG). Wird eine Drohne von einer Privatperson zur Erstellung von Foto- und/oder Videoaufnahmen von anderen Personen, Grundstücken etc. verwendet, so fällt dies grundsätzlich in den Geltungsbereich des E-DSG, da regelmässig Personendaten bearbeitet werden.566 III. Ausnahme: Bearbeitung ausschliesslich zum persönlichen Gebrauch Der E-DSG sieht in Art. 2 Abs. 2 Ausnahmen von seinem Geltungsbereich vor.567 Für die vorliegende Untersuchung ist die Klärung der Frage, ob die drohnengestützte Datenbearbeitung durch Private unter die Ausnahmeregelung von Art. 2 Abs. 2 lit. a E-DSG fällt, von zentraler Bedeutung. Aus diesem Grund soll nachfolgend die gesetzliche Regelung im Detail besprochen und insbesondere anhand von deren normativer Grundlage sowie Beispielen analysiert werden. 563 Vgl. Ziebarth, in: Sydow, Art. 4 DSGVO N 9. 564 SHK DSG-Rudin, Art. 3 N 16; vgl. Art. 1 DSG. 565 Mit der Revision des DSG wurde der Schutz der Daten juristischer Personen aufgehoben, wobei ebenfalls eine Angleichung an die Regelung in der EU vorgenommen wurde. Vgl. dazu ausführlich Botschaft Totalrevision DSG, S. 7011 sowie bereits vorne, S. 29 mit den Hinweisen in Fn. 218. 566 Vgl. zu den Begriffen im Einzelnen vorne, S. 29 ff. bzw. S. 33. und zur drohnengestützten Datenbearbeitung S. 33 ff. Vgl. sodann Leissler, S. 535 m.w.H. 567 Die Ausnahmebestimmungen in Art. 2 lit. b und c E-DSG (parlamentarische Beratungen bzw. Immunität nach Gaststaatgesetz [GSG]) sind vorliegend klarerweise nicht einschlägig und werden deshalb nicht vertieft. 76 1. Teil 3: Bestandesaufnahme (1): Der E-DSG Die Regelung des E-DSG Der E-DSG ist nicht anwendbar auf «Personendaten, die von einer natürlichen Person ausschliesslich zum persönlichen Gebrauch bearbeitet werden» (Art. 2 Abs. 2 lit. a E-DSG).568 So sollen rein private Datenbearbeitungen bzw. «die Verwendung von Personendaten im engeren Privat- und Familienleben»569 vom Anwendungsbereich des Gesetzes ausgenommen werden. Die Ausnahmeregelung von Art. 2 Abs. 2 lit. a E-DSG soll dann nicht greifen, wenn bestimmte Datenbearbeitungen «hinsichtlich der Daten oder ihrer Zwecke den persönlichen oder familiären Bereich überschreiten».570 Im Unterschied zum noch geltenden DSG enthält die Ausnahmebestimmung keine Präzisierung mehr, dass für deren Anwendbarkeit die Daten auch nicht an Aussenstehende bekannt gegeben werden dürfen.571 Gemäss Botschaft beinhaltet diese redaktionelle Anpassung jedoch keine materiellen Änderungen der Bestimmung.572 Das europäische Datenschutzrecht enthält eine materiell gleichbedeutende Vorschrift573 in Art. 2 Abs. 2 lit. c DSGVO (sog. Haushaltsausnahme oder Haushaltsprivileg574). Danach findet die Verordnung «keine Anwendung auf die Verarbeitung personenbezogener Daten575 durch natürliche Personen zur Ausübung ausschliesslich persönlicher oder familiärer Tätigkeiten». Literatur und Rechtsprechung zu dieser Bestimmung können somit vorliegend zur Auslegung des E-DSG beigezogen werden. 568 Greift die Ausnahmebestimmung, so kann sich die betroffene Person immer noch auf die allgemeinen Regeln des Persönlichkeitsschutzes (Art. 28 ff. ZGB) berufen (Meier, Rn. 385; BSK DSG- Maurer-Lambrou/Kunz, Art. 2 N 22; Botschaft DSG 1988, S. 441; vgl. Gola, in: Gola, Art. 2 DSGVO N 19). 569 BSK DSG-Maurer-Lambrou/Kunz, Art. 2 N 21. 570 Vgl. Rossnagel, in: Simitis et al., Art. 2 DSGVO N 31; ähnlich Schmidt, in: Taeger/ Gabel, Art. 2 DSGVO N 21. 571 Art. 2 Abs. 2 lit. a DSG lautet: «[Das DSG] ist nicht anwendbar auf Personendaten, die eine natürliche Person ausschliesslich zum persönlichen Gebrauch bearbeitet und nicht an Aussenstehende bekannt gibt.» 572 Botschaft Totalrevision DSG, S. 7012. 573 So bereits zur Rechtslage vor Inkrafttreten der DSGVO Weber/Oertly, Rn. 22. 574 Gola, in: Gola, Art. 2 DSGVO N 19; Leissler, S. 535; Schmidt, in: Taeger/Gabel, Art. 2 DSGVO N 16; Voigt/von dem Bussche, S. 16. 575 «Personenbezogene Daten» ist die Bezeichnung der DSGVO (vgl. Art. 4 Ziff. 1) und weiterer europäischer Regelwerke für Personendaten (vgl. dazu vorne, S. 29 ff.). Die beiden Begriffe sind Synonyme. § 2 Anwendbarkeit des Datenschutzgesetzes 2. 77 Interessenausgleich als normative Grundlage Mit der Haushaltsausnahme soll ein Ausgleich zwischen den durch das Daten schutzgesetz zu schützenden Grundrechten und denjenigen des Datenbearbeiters geschaffen werden.576 Auch der Verantwortliche hat Anspruch auf Achtung seiner Privatsphäre sowie seines Privat- und Familienlebens; es gilt mithin, die sich gegenüberstehenden Interessen abzuwägen und einen Inter essenausgleich zu schaffen.577 Darin ist denn auch die normative Grundlage der Vorschrift zu sehen; es handelt sich nicht um eine «Bagatellklausel».578 Bei der Prüfung, ob die Haushaltsausnahme einschlägig ist, muss jeweils danach gefragt werden, ob das Verhalten des Verantwortlichen als schutzwürdig erscheint bzw. ob dessen Schutz im Einzelfall demjenigen der grundrechtlich geschützten Ansprüche der betroffenen Person vorgehen kann und soll.579 Aufgrund der Tatsache, dass die Ausnahme den Anwendungsbereich des Datenschutzgesetzes generell ausschliesst — und dies selbst dann, wenn besonders schützenswerte Personendaten bearbeitet werden580 —, muss sie eng und streng sowie grundrechtskonform ausgelegt werden.581 Nur so kann der Zweck des datenschutzrechtlichen Grundrechtsschutzes erfüllt werden.582 576 In Art. 1 E-DSG (und Art. 1 DSG) sind neben der informationellen Selbstbestimmung und der Privatsphäre (Art. 13 BV; Art. 8 EMRK) auch die persönliche Freiheit (Art. 10 Abs. 2 BV) sowie grundsätzlich alle übrigen durch die BV und die EMRK garantierten Grundrechte angesprochen (vgl. dazu BSK DSG-Mauer-Lambrou/Kunz, Art. 1 N 15 ff.; Müller L., Diss., S. 34 m.w.H.; Handkomm. DSG-Rosenthal, Art. 1 N 3). 577 SGK BV-Schweizer, Art. 13 N 74; de Terwangne, champ d’application, Rn. 18; Kühling/Raab, in: Kühling/Buchner, Art. 2 DSGVO N 10 und N 23 in fine; Rossnagel, in: Simitis et al., Art. 2 DSGVO N 23; Weichert, in: Däubler et al., Art. 2 DSGVO N 17. Vgl. Gola, in: Gola, Art. 2 DSGVO N 19; Ennöckl, in: Sydow, Art. 2 DSGVO N 10. 578 So aber Zerdick, in: Ehmann/Selmayr, Art. 2 DSGVO N 10. Wie hier Weichert, in: Däubler et al., Art. 2 DSGVO N 17. 579 Vgl. Botschaft DSG 1988, S. 438: «Der Zweckartikel [des DSG] soll als Leitlinie für die Auslegung der einzelnen Datenschutzbestimmungen den Bezug zum Persönlichkeitsschutz und zu den Grundrechten hervorheben.» 580 Rossnagel, in: Simitis et al., Art. 2 DSGVO N 23; BSK DSG-Maurer-Lambrou/Kunz, Art. 2 N 20. Vgl. auch die Legaldefinition in Art. 4 lit. c E-DSG sowie vorne, S. 31. 581 Weber/Oertly, Rn. 22; Gerschwiler, Rn. 3.36; Rossnagel, in: Simitis et al., Art. 2 DSGVO N 23; Kühling/Raab, in: Kühling/Buchner, Art. 2 DSGVO N 23; de Terwangne, RDTI, S. 47; Weichert, in: Däubler et al., Art. 2 DSGVO N 17; vgl. EuGH, Rs. C-212/13 vom 11. Dezember 2014 František Ryneš, Ziff. 28 f. 582 Vgl. Art. 1 E-DSG und vorne, S. 69 ff., v.a. S. 71 f. 78 3. Teil 3: Bestandesaufnahme (1): Der E-DSG Anwendungsbeispiele Nicht anwendbar ist das Datenschutzgesetz auf ein persönliches Notizbuch oder eine persönliche Agenda583, private Briefsammlungen584 oder Adressverzeichnisse,585 Gästelisten für ein privates Fest oder Daten des persönlichen Konsums (Rechnungen, Bestellungen, Lieferscheine etc.)586 sowie Urlaubsfotos und -filme.587 Es soll verhindert werden, dass der Verantwortliche über Daten Auskunft erteilen muss, die eng mit seiner Privatsphäre und/oder seinem Familienleben verbunden sind.588 Führt man sich die genannten Beispiele vor Augen, so wird klar, dass in diesen Fällen den schutzwürdigen Interessen des Verarbeiters Vorrang vor dem Anspruch auf Datenschutz der Betroffenen zukommen soll. 4. Bekanntgabe an Aussenstehende Die Haushaltsausnahme ist nicht einschlägig, wenn Personendaten nach der Beschaffung Aussenstehenden bekannt gegeben werden.589 Ebenso verhält es sich, wenn eine solche Bekanntgabe bei der Beschaffung bzw. Erstellung der Daten bereits fest beabsichtigt ist.590 Die Bekanntgabe ist als irgendwie geartetes Zugänglichmachen zu verstehen (namentlich etwa eine Publikation im Internet).591 Aussenstehende ist jede Person, die nicht dem engeren Privat- und Familienleben des Datenbearbeiters zugerechnet werden kann.592 Zwischen den Personen muss 583 SHK DSG-Rudin, Art. 2 N 21. 584 Botschaft DSG 1988, S. 441. 585 BSK DSG-Maurer-Lambrou/Kunz, Art. 2 N 20. 586 Rossnagel, in: Simitis et al., Art. 2 DSGVO N 27 mit zahlreichen Hinweisen. Vgl. auch EG 18 DSGVO. 587 Gola, in: Gola, Art. 2 DSGVO N 19. 588 Anschaulich Pedrazzini, Grundlagen, S. 24 («[W]ir haben in den Expertenkommissionen tagelang über diese Grenzziehung der privaten Sphäre diskutiert […]»); Botschaft DSG 1988, S. 441; vgl. Gnehm, S. 89. 589 Vgl. BSK DSG-Maurer-Lambrou/Kunz, Art. 2 N 21; Marzocchi, S. 23; Rossnagel, in: Simitis et al., Art. 2 DSGVO N 29; Schmidt, in: Taeger/Gabel, Art. 2 DSGVO N 18 m.w.H.; EuGH, Rs. C-101/01 vom 6. November 2003 Bodil Lindqvist, Ziff. 47. 590 Müller L., Sicherheit & Recht 2/2012, S. 63 m.w.H.; vgl. auch SHK DSG-Rudin, Art. 2 N 23; BGer, 6B_1188/2018 vom 26. September 2019 E. 3.1 ff. (zur Publikation vorgesehen). Wohl a.M. bezüglich privater Dashcam-Aufnahmen Berger, Rn. 19. 591 SHK DSG-Rudin, Art. 2 N 24. 592 Handkomm. DSG-Rosenthal/Jöhri, Art. 2 N 23. A.M. SHK DSG-Rudin, Art. 2 N 24, der «jede andere Person als der Inhaber der Daten» als Aussenstehenden qualifiziert. Unklar BSK DSG-Maurer-Lambrou/Kunz, Art. 2 N 21. Auf die genaue Anzahl Personen, denen die Daten bekannt gegeben werden, kommt es nicht an (vgl. Uriante, S. 69). § 2 Anwendbarkeit des Datenschutzgesetzes 79 aber nicht zwingend ein Verwandtschaftsverhältnis im Sinne des Zivilrechts593 vorliegen; eine persönliche Beziehung kann auch auf informeller Basis bestehen.594 Bei sozialen Netzwerken etwa ist eine differenzierte Betrachtung notwendig: Beschränkt sich die Zugriffsmöglichkeit auf einen eng begrenzten Personenkreis (z.B. Einzelnachricht), so ist die betreffende Datenbearbeitung grundsätzlich von der Ausnahme erfasst. Wird aber ein unbestimmter Personenkreis erreicht, so greift sie nicht.595 IV. Haushaltsausnahme bei der drohnengestützten Datenbearbeitung? 1. Ausgangsbeispiel: «Nachbarschafts-Drohne» Weber/Oertly hielten zum noch geltenden DSG fest, dass «[m]it Blick auf die ‹Nachbarschafts-Drohnen› davon auszugehen [ist] (…), dass diese regelmässig den Ausnahmetatbestand erfüllen, wenn die aufgenommenen Bilder nicht Drittpersonen zugänglich gemacht werden.»596 Die Autoren gehen dabei von folgendem Sachverhalt aus: «Beispielhaft sei an die Person X zu denken, welche eine Drohne, die geeignet ist, Aufnahmen zu tätigen, direkt über das Gebäude des Nachbargrundstücks kreisen lässt und dadurch Einblicke erhält, die ihr sonst verborgen blieben. Behält X die darauf entstandenen Aufnahmen für sich, ohne sie seinen Eltern, Freunden oder anderen Personen zugänglich zu machen, greift die Ausnahme von Art. 2 Abs. 2 lit. a DSG.»597 593 Vgl. zur Verwandtschaft Art. 20 ZGB (sowie zur Schwägerschaft Art. 21 ZGB); ausführlich dazu statt aller Steinauer/Fountoulakis, Rn. 311 ff. 594 Vgl. Handkomm. DSG-Rosenthal/Jöhri, Art. 2 N 23; Ernst, in: Paal/Pauly, Art. 2 DSGVO N 18; Grafenstein, in: Gierschmann et al., Art. 2 DSGVO N 35; vgl. Pabst, in: Schwartmann et al., Art. 2 DSGVO N 38. 595 Kühling/Raab, in: Kühling/Buchner, Art. 2 DSGVO N 25; Ennöckl, in: Sydow, Art. 2 DSGVO N 13. Dies gilt auch dann, wenn die Personen in diesem Umfeld «Freunde» oder «Follower» genannt werden, vgl. dazu Rossnagel, in: Simitis et al., Art. 2 DSGVO N 29. Vgl. sodann Schmidt, in: Taeger/Gabel, Art. 2 DSGVO N 18. Kritisch zu dieser «strengen Auffassung» Grafenstein, in: Gierschmann et al., Art. 2 DSGVO N 47. 596 Weber/Oertly, Rn. 22. 597 Weber/Oertly, Rn. 21. Ähnlich und die Haushaltsausnahme extensiver auslegend Pauner/Viguri, S. 97 f. (zur Rechtslage in der EU vor Inkrafttreten der DSGVO). Fraglich ist, ob Eltern und Freunde überhaupt als Aussenstehende im Sinne des DSG zu betrachten wären (vgl. dazu vorne, S. 78 f.). 80 2. Teil 3: Bestandesaufnahme (1): Der E-DSG Detailprüfung Diese Auffassung ist insbesondere mit Blick auf die Ziele der DSG-Revision genauer zu überprüfen.598 Der Schutzzweck des Datenschutzrechts und die dadurch gebotene enge Auslegung des Ausnahmetatbestands599 müssen zur Anwendbarkeit des Datenschutzgesetzes führen, wenn moderne Kamerasysteme in Autos600 oder an Drohnen601 mehr als rein private Bereiche überwachen.602 In diesen Fällen wird der persönliche oder familiäre Bereich, welcher das Datenschutzgesetz von seiner Anwendung ausnehmen will, überschritten. Der Ausnahmetatbestand sollte — wie aufgezeigt — nur greifen, wenn es sich mit Blick auf die schutzwürdigen Interessen des Verantwortlichen tatsächlich rechtfertigt, der Datenschutzgesetzgebung die Anwendung zu versagen.603 Wird eine Drohne im Rahmen der Freizeitgestaltung verwendet und entstehen dabei (auch) Foto- oder Videoaufnahmen von fremden Grund stücken und/oder Personen, so ist die Haushaltsausnahme grundsätzlich solange einschlägig, wie diese Daten keinem Aussenstehenden zugänglich gemacht werden.604 Wird ein fremdes Grundstück oder eine bestimmte Person hingegen über eine längere Dauer bzw. systematisch beobachtet, so ist von einer Überwachung auszugehen, was die Ausnahme entfallen lässt.605 Gleich verhält es sich, wenn die Aufnahme einer bestimmten Person bzw. von bestimmten fremden Grundstücken oder Gebäuden explizit beabsichtigt oder zumindest billigend in Kauf genommen wurde. In diesem Fall hat das schutz würdige Interesse des Verantwortlichen hinter dasjenige der betroffenen 598 Vgl. dazu vorne, S. 11 f. 599 Vgl. dazu vorne, S. 77. 600 Hier geht es um sog. Dashcams (d.h. in oder auf Motorfahrzeugen installierte Kameras). In den Erläuterungen des EDÖB dazu wird die Ausnahmeregelung von Art. 2 Abs. 2 lit. c DSG gar nicht thematisiert (vgl. EDÖB, Dashcams). Berger, Rn. 19, hält dafür, dass die private Verwendung von Dashcams grundsätzlich von der Haushaltsausnahme erfasst wird; a.M. Arnosti, Rn. 60 ff., v.a. Rn. 67. 601 Rossnagel, in: Simitis et al., Art. 2 DSGVO N 31; Schmidt, in: Taeger/Gabel, Art. 2 DSGVO N 21 in fine. 602 Gola, in: Gola, Art. 2 DSGVO N 19; Plath, in: Plath, Art. 2 DSGVO N 24. Vgl. de Terwangne, champ d’application, Rn. 19 m.w.H. 603 Vgl. unter Bezugnahme auf die Privatsphäre des Verarbeiters Müller L., Diss., S. 40. Ausführlich zur Interessenabwägung vorne, S. 77. 604 Vgl. Rossnagel, in: Simitis et al., Art. 2 DSGVO N 31; Bischof, S. 144; de Terwangne, champ d’application, Rn. 19; Kühling/Raab, in: Kühling/Buchner, Art. 2 DSGVO N 27; Unabhängiges Datenschutzzentrum Saarland, S. 111 f. 605 Gola, in: Gola, Art. 2 DSGVO N 19 in fine; ähnlich Griebel, S. 111; vgl. auch Rose, S. 140. § 3 Zentrale Bestimmungen des E-DSG 81 Person zurückzutreten. Werden drohnengestützt Daten im öffentlichen Raum beschafft, so entfällt die Haushaltsausnahme ebenfalls.606 3. Zusammenfassung und Fazit Insgesamt kann auch unter dem E-DSG festgehalten werden, dass gewisse drohnengestützte Datenbearbeitungen durch Private nicht in den Anwendungsbereich des E-DSG fallen. Die erwähnte «Nachbarschafts-Drohne» fällt jedoch nicht unter die Haushaltsausnahme: Der Umstand, dass die Person X gemäss Beispiel die Drohne «direkt über das Gebäude des Nachbargrundstücks kreisen lässt und dadurch Einblicke erhält, die ihr sonst verborgen blieben», spricht für eine bewusste und beabsichtigte Beobachtung. Im Rahmen der Interessenabwägung im Einzelfall erscheint dieses Verhalten als wenig schutzwürdig; die datenschutzrechtlichen Ansprüche der Betroffen müssen vorgehen. Somit wird die Mehrzahl der privat erstellten Aufnahmen mit Drohnen in den Anwendungsbereich des E-DSG fallen.607 § 3 Zentrale Bestimmungen des E-DSG Nachdem nun feststeht, dass der E-DSG ohne Weiteres auf die drohnen gestützte Datenbearbeitung durch Private anwendbar ist und die Ausnahme gemäss Art. 2 Abs. 2 lit. a E-DSG nur in Einzelfällen greift, gilt es, die zentralen Vorschriften dieses Gesetzes und deren Anwendung auf die drohnengestützte Datenbearbeitung zu erläutern.608 Aufgrund der Totalrevision des DSG609 soll eingangs kurz dargestellt werden, in welchen Bereichen das neue Datenschutzrecht Änderungen mit sich bringt und wo die bisher geltenden Regelungen übernommen wurden. Die in Art. 5 und 7 E-DSG festgehaltenen Grundsätze der Datenbearbeitung wurden praktisch unverändert aus dem alten Recht übernommen;610 deshalb sollen diese nachfolgend nur kurz dargestellt und auf ihre Bedeu606 Dieckert, in: Dieckert/Eich, S. 408. Vgl. Uschkereit/Zdanowiecki, S. 447; EuGH, Rs. C-212/13 vom 11. Dezember 2014 František Ryneš, Ziff. 31 ff. Wohl anders für das deutsche Recht vor Inkrafttreten der DSGVO Solmecke/Novak, S. 433. Kritisch Schmidt, in: Taeger/Gabel, Art. 2 DSGVO N 21. 607 Im Ergebnis gleich Griebel, S. 111 (zur DSGVO). 608 Nicht behandelt werden etwa folgende Bestimmungen des E-DSG: Art. 11: Verzeichnis der Bearbeitungstätigkeiten; Art. 12: Zertifizierung; Art. 13–15: Datenbekanntgabe ins Ausland; sowie Art. 22: Meldung von Verletzungen der Datensicherheit. Diese sind für die vorliegende Untersuchung nicht von Bedeutung oder gar nicht einschlägig. 609 Vgl. dazu vorne, S. 11 ff. 610 Vgl. Botschaft Totalrevision DSG, S. 7024 ff.; Rosenthal, Entwurf, Rn. 28. 82 Teil 3: Bestandesaufnahme (1): Der E-DSG tung im Zusammenhang mit der drohnengestützten Datenbearbeitung hin untersucht werden (dazu sogleich). Die Rechte der Betroffenen sind jedoch ausgebaut und verstärkt worden. Ein Ziel der Revision war es denn auch, die Transparenz von Datenbearbeitungen zu erhöhen; dies ging mit ausgebauten Informationspflichten einher (hinten, S. 93 ff.).611 Auch das Auskunftsrecht, welches die Informationspflicht des Verantwortlichen ergänzt, wurde ausgebaut (dazu hinten, S. 100 ff.).612 Keine materiellen Änderungen ergaben sich bei den Rechten der betroffenen Person bei Persönlichkeitsverletzungen; diese Normen wurden übernommen (S. 106 ff.). Neu eingeführt wurden dagegen die Vorgaben zu «Privacy by Design» (S. 89 ff.) sowie die Datenschutz-Folgenabschätzung (dazu S. 98 ff.). Der Aufbau der nachfolgenden Ausführungen orientiert sich an der Systematik des E-DSG. I. Bearbeitungsgrundsätze Die Grundsätze der Datenbearbeitung sind in den Art. 5 und 7 E-DSG geregelt. Werden diese nicht eingehalten, führt dies zu einer Persönlichkeitsverletzung, welche möglicherweise durch einen Rechtfertigungsgrund gedeckt ist (Art. 26 f. E-DSG).613 Nachfolgend ist im Einzelnen zu skizzieren, welche Bedeutung den datenschutzrechtlichen Bearbeitungsgrundsätzen allgemein und insbesondere im Zusammenhang mit der drohnengestützten Daten bearbeitung durch Private zukommt. 1. Rechtmässigkeit Gemäss Art. 5 Abs. 1 E-DSG müssen Personendaten rechtmässig bearbeitet werden. Rechtswidrig ist eine Datenbearbeitung insbesondere, wenn damit gegen eine Bestimmung der Schweizer Rechtsordnung verstossen wird, wobei zivilrechtliche oder strafrechtliche Normen im Vordergrund stehen.614 Vorausgesetzt ist zudem, dass die verletzte Bestimmung «zumindest auch, direkt oder indirekt, den Schutz der Persönlichkeit einer Person bezweckt».615 611 Botschaft Totalrevision DSG, S. 6972 f. und S. 7050 ff. Kritisch dazu Rosenthal, Entwurf, Rn. 93 ff. 612 Botschaft Totalrevision DSG, S. 7066 ff. 613 So verhält es sich auch unter dem noch geltenden DSG. Vgl. dazu statt anderer Rosenthal, Entwurf, Rn. 29; Handkomm. DSG-Rosenthal, Art. 4 N 2 (umstritten; vgl. ausführlich Epiney, in: Belser/Epiney/Waldmann, § 9 Rn. 5 ff.). Zu den Rechten bei Persönlichkeitsverletzungen und den Rechtfertigungsgründen hinten, S. 106 ff. 614 BSK DSG-Maurer-Lambrou/Steiner, Art. 4 N 6; SHK DSG-Baeriswyl, Art. 4 N 11 ff.; Meier, Rn. 640 f.; Handkomm. DSG-Rosenthal, Art. 4 N 7. 615 BVGer, A-3548/2018 vom 19. März 2019 E. 5.4.4. Zustimmend Vasella, Helsana+; kritisch Baeriswyl, Entwicklungen, S. 593. § 3 Zentrale Bestimmungen des E-DSG 83 Aus strafrechtlicher Sicht stehen die Bestimmungen von Art. 179 ff. StGB616 und insbesondere der Tatbestand der Verletzung des Geheim- oder Privatbereichs durch Aufnahmegeräte (Art. 179quater StGB) im Vordergrund. Es ist nicht auszuschliessen, dass sich ein Privater, der drohnengestützt Bilder von Grundstücken und den sich darauf befindlichen Personen erstellt, gemäss Art. 179quater StGB strafbar macht.617 Aus zivilrechtlicher Optik sind etwa die Täuschung (Art. 28 OR) und Drohung (Art. 29 f. OR) zu beachten.618 2. Treu und Glauben und Verhältnismässigkeit 2.1 Verhältnismässigkeit Die Bearbeitung muss nach Treu und Glauben erfolgen und verhältnismässig sein (Art. 5 Abs. 2 E-DSG). Verhältnismässigkeit bedeutet in diesem Zusammenhang, dass die Art und Weise sowie der Zweck der Bearbeitung geeignet und erforderlich sein müssen, um das angestrebte Ziel zu erreichen. Zudem hat ein angemessenes Verhältnis zwischen dem Zweck und dem verwendeten Mittel zu bestehen.619 Über diese drei klassischen Vorgaben der Verhältnismässigkeit hinaus sind die Rechte der betroffenen Personen soweit wie möglich zu wahren.620 Die Grundsätze der Datenvermeidung und der Datensparsamkeit ergeben sich aus dem datenschutzrechtlichen Verhältnismässigkeitsgrundsatz.621 Die in Art. 5 Abs. 4 E-DSG verankerte Regelung zur Dauer der Aufbewahrung von Daten ist als Regel der Datensparsamkeit bzw. -vermeidung und mithin als Ausfluss des Verhältnismässigkeitsprinzips zu verstehen.622 Namentlich sollen jeweils nur so viele Daten bearbeitet werden, wie zur Erreichung des 616 Epiney, in: Belser/Epiney/Waldmann, § 9 Rn. 13; SHK DSG-Baeriswyl, Art. 4 N 11. 617 Dazu aus Sicht der (statischen) Videoüberwachung Müller L., Sicherheit & Recht 2/2012, S. 67. Daneben könnte auch Art. 179 ter (Unbefugtes Aufnehmen von Gesprächen) einschlägig sein. Allgemein zu diesen Strafbestimmungen statt anderer BSK StGB-Ramel/Vogelsang, Art. 179quater N 5 ff. bzw. Art. 179 ter N 3 ff. 618 Handkomm. DSG-Rosenthal, Art. 4 N 7. Vgl. dazu statt aller BSK OR I-Schwenzer/ Fountoulakis, Art. 28 und Art. 29/30, passim. 619 BGE 138 II 346 E. 9.2; Botschaft DSG 1988, S. 450; Müller L., Sicherheit & Recht 2/2012, S. 70; Epiney, in: Belser/Epiney/Waldmann, § 9 Rn. 23 und ausführlich zu den einzelnen Teilaspekten Rn. 26 f. 620 Botschaft Totalrevision DSG, S. 7050; vgl. Handkomm. DSG-Rosenthal, Art. 4 N 20 f. 621 SHK DSG-Baeriswyl, Art. 4 N 23; Epiney, in: Belser/Epiney/Waldmann, § 9 Rn. 27 mit Fn. 83. Diese beiden Grundsätze werden im DSG nicht explizit genannt. Teilweise wird auch der Begriff der Datenminimierung verwendet (so Art. 5 Abs. 1 lit. c DSGVO); vgl. dazu Frenzel, in: Paal/Pauly, Art. 5 DSGVO N 34. 622 Botschaft Totalrevision DSG, S. 7026; Epiney, in: Belser/Epiney/Waldmann, § 9 Rn. 27, zweites Lemma in fine; Meier, Rn. 633. 84 Teil 3: Bestandesaufnahme (1): Der E-DSG Zwecks notwendig ist.623 Des Weiteren fordert diese Norm, dass Daten vernichtet oder anonymisiert werden, sobald sie zum Zweck der Bearbeitung nicht mehr erforderlich sind.624 Im Kontext der drohnengestützten Datenbearbeitung durch Private kommen verschiedene Massnahmen zur Datensparsamkeit in Betracht. So könnte etwa ein Drohnenbetreiber die Kamera nicht während des gesamten Fluges laufen lassen und/oder das Mikrofon nur aktivieren, wenn dies notwendig ist.625 2.2 Treu und Glauben Die bereits in der Bundesverfassung (Art. 5 Abs. 3 und Art. 9 BV) sowie im Zivilrecht (Art. 2 Abs. 1 ZGB) verankerte «Fundamentalnorm»626 von Treu und Glauben besagt allgemein, dass im Rechtsverkehr ein loyales und vertrauenswürdiges Verhalten geboten ist; widersprüchliches Verhalten läuft dem zuwider.627 Im Datenschutzrecht stellt dieser Grundsatz eine Generalklausel dar, welche dort zum Zuge kommt bzw. kommen kann, wo die anderen Grundsätze nicht greifen.628 Im Zusammenhang mit der drohnengestützten Datenbearbeitung durch Private kommt dem Grundsatz von Treu und Glauben insofern Bedeutung zu, als eine heimliche Datenbearbeitung oder eine solche, mit der die betroffene Person nicht rechnen muss, diesen Grundsatz verletzt.629 Die Datenbearbeitung ist mithin rechtswidrig, und dies selbst dann, wenn gegen keine (andere) Rechtsnorm verstossen wird.630 623 Dies bringt der E-DSG in Art. 6 Abs. 3 explizit zum Ausdruck. Vgl. dazu Botschaft Totalrevision DSG, S. 7030. 624 Die ausdrückliche Nennung im Gesetz sollte diesen Vorgaben «im Hinblick auf die technologische Entwicklung und die beinahe unbegrenzten Speichermöglichkeiten» Nachdruck verleihen; vgl. dazu Botschaft Totalrevision DSG, S. 7026. 625 Vgl. Harris/Given, S. 9. 626 BSK DSG-Maurer-Lambrou/Steiner, Art. 4 N 7 mit Hinweis auf BGE 128 III 201 E. 1c. 627 SHK DSG-Baeriswyl, Art. 4 N 17; Epiney, in: Belser/Epiney/Waldmann, § 9 Rn. 21; Kiener/Kälin/Wyttenbach, § 34 Rn. 5 f.; SGK BV-Schindler, Art. 5 N 55. 628 Epiney, in: Belser/Epiney/Waldmann, § 9 Rn. 22; SHK DSG-Baeriswyl, Art. 4 N 18; Handkomm. DSG-Rosenthal, Art. 4 N 14; vgl. BSK DSG-Maurer-Lambrou/Steiner, Art. 4 N 8. 629 Vgl. Botschaft DSG 1988, S. 449; SHK DSG-Baeriswyl, Art. 4 N 19. Die Abgrenzung zur Erkennbarkeit kann sich im konkreten Fall als schwierig erweisen; vgl. dazu auch sogleich, S. 85 f. 630 BVGer, A-3144/2008 vom 27. Mai 2009 E. 9.3.1; BSK DSG-Maurer-Lambrou/Steiner, Art. 4 N 8; SHK DSG-Baeriswyl, Art. 4 N 19. § 3 Zentrale Bestimmungen des E-DSG 3. 85 Zweckbindung und Erkennbarkeit (Transparenz) Art. 5 Abs. 3 E-DSG schreibt vor, dass Personendaten nur zu einem bestimmten und für die betroffene Person erkennbaren Zweck beschafft werden dürfen; sie dürfen nur so bearbeitet werden, dass es mit diesem Zweck vereinbar ist. Diese Bestimmung wurde im E-DSG neu gefasst und enthält nun die Grundsätze der Zweckbindung sowie der Transparenz; materiell ergaben sich daraus keine Änderungen im Verhältnis zum noch geltenden Recht.631 3.1 Zweckbindung Gemäss dem Grundsatz der Zweckbindung soll eine betroffene Person bereits zu Beginn wissen, wofür die über sie bearbeiteten Daten verwendet werden.632 Der Verantwortliche darf die Daten sodann nicht «zweckentfremden», d.h., er darf sie nicht ohne Weiteres für einen anderen als den ursprünglich vorgesehenen Zweck bearbeiten.633 Informiert ein Drohnenbetreiber die betroffene Person zwar, verwendet die entstandenen Aufnahmen anschliessend jedoch zu anderen Zwecken, so ist der Grundsatz der Zweckbindung verletzt. Im Kontext der drohnen gestützten Datenbearbeitung durch Private kommt der Zweckbindung aber keine besondere Bedeutung zu. Ist eine Datenbearbeitung an sich für die betroffene Person nicht erkennbar, so kann sie sich auch über den Zweck der Bearbeitung nicht im Klaren sein. 3.2 Erkennbarkeit Der Grundsatz der Erkennbarkeit (oder Transparenz) verlangt, dass die Beschaffung von Personendaten und insbesondere der Zweck ihrer Bearbeitung für die betroffene Person erkennbar sind.634 «Die Erkennbarkeit der Datenbearbeitung — das Prinzip der Transparenz — ist Grundlage des Rechts auf in formationelle Selbstbestimmung und Voraussetzung für die Wahrnehmung 631 Botschaft Totalrevision DSG, S. 7024 f. Die beiden Grundsätze stehen denn auch in einem engen Zusammenhang (Epiney, in: Belser/Epiney/Waldmann, § 9 Rn. 29). 632 Botschaft DSG 1988, S. 451; Epiney, in: Belser/Epiney/Waldmann, § 9 Rn. 29; BSK DSG-Maurer-Lambrou/Steiner, Art. 4 N 13; vgl. Peter, S. 126 f. 633 Epiney, in: Belser/Epiney/Waldmann, § 9 Rn. 29 und Rn. 35; vgl. BSK DSG-Maurer-Lambrou/Steiner, Art. 4 N 14b; BGE 138 II 346 E. 9.1. 634 Botschaft Revision DSG 2003, S. 2124; Epiney, in: Belser/Epiney/Waldmann, § 9 Rn. 37; BSK DSG-Maurer-Lambrou/Steiner, Art. 4 N 16a. Vgl. zum Zusammenspiel zwischen den Grundsätzen der Erkennbarkeit und von Treu und Glauben Gerschwiler, Rn. 3.89 f. 86 Teil 3: Bestandesaufnahme (1): Der E-DSG datenschutzrechtlicher Ansprüche.»635 Die Verbesserung dieser Transparenz war denn auch Teil verschiedener DSG-Revisionen.636 Sie kann als «Eckpfeiler des ganzen Datenschutzsystems» bezeichnet werden.637 Ob die Erkennbarkeit bejaht werden kann, ist nach den konkreten Umständen des Einzelfalls zu beurteilen.638 Ist eine Bearbeitung nicht oder nicht deutlich erkennbar, so hat der oder die Verantwortliche zusätzliche Informationen bereitzustellen. Gleiches gilt, wenn die Datenbeschaffung in einer konkreten Situation erkennbar, der Zweck derselben aber nicht ersichtlich ist.639 Nicht vorgegeben ist, in welcher Form die Erkennbarkeit sichergestellt werden soll.640 Auch diesbezüglich gilt, den Umständen des Einzelfalls Rechnung zu tragen sowie anhand der Vorgaben der Verhältnismässigkeit und von Treu und Glauben vorzugehen. Im Kontext der drohnengestützten Datenbearbeitung durch Private ist dem Transparenzgebot regelmässig nicht Genüge getan. Die betroffenen Personen sind sich über den Zweck einer drohnengestützten Datenbearbeitung meist nicht im Klaren. In den allermeisten Fällen kann eine allfällig betroffene Person nicht einmal erkennen, ob und welche Daten mit einer Drohne bearbeitet werden.641 4. Datenrichtigkeit Der Grundsatz der Datenrichtigkeit wurde aus Art. 5 DSG übernommen und mit den anderen zentralen Grundsätzen des E-DSG in einer einzigen Bestimmung zusammengeführt.642 Er besagt, dass, wer Personendaten bearbeitet, sich über deren Richtigkeit vergewissern muss. «Sie oder er muss alle angemessenen Massnahmen treffen, damit die Daten berichtigt, gelöscht oder vernichtet werden, die im Hinblick auf den Zweck ihrer Beschaffung oder Bearbeitung unrichtig oder unvollständig sind. Die Angemessenheit der Massnahmen hängt namentlich ab von der Art und dem Umfang der 635 SHK DSG-Baeriswyl, Art. 4 N 48. Vgl. auch Bull, Selbstbestimmung, S. 60. 636 Vgl. Botschaft Revision DSG 2003, S. 2124 ff. und S. 2130 ff.; Botschaft Totalrevision DSG, S. 7010 und S. 7050. 637 Botschaft Revision DSG 2003, S. 2126; SHK DSG-Baeriswyl, Art. 4 N 48; vgl. Rosenthal, Entwurf, Rn. 28. 638 Epiney, in: Belser/Epiney/Waldmann, § 9 Rn. 39; Meier, Rn. 707; SHK DSG-Baeriswyl, Art. 4 N 48. 639 SHK DSG-Baeriswyl, Art. 4 N 50; vgl. Epiney, in: Belser/Epiney/Waldmann, § 9 Rn. 40; Meier, Rn. 708 ff. 640 Meier, Rn. 711 ff. mit Beispielen. 641 Vgl. Masutti/Tomasello, S. 223; vgl. auch vorne, S. 5 f. 642 Botschaft Totalrevision DSG, S. 7026. § 3 Zentrale Bestimmungen des E-DSG 87 Datenbearbeitung sowie von den Risiken, welche die Bearbeitung für die Persönlichkeit und Grundrechte der betroffenen Personen mit sich bringt» (Art. 5 Abs. 5 E-DSG).643 Die Bestimmung ist vor allem im Bereich von wirtschaftlichen Transaktionen von Bedeutung. Wird z.B. bei der Beurteilung der Kreditwürdigkeit einer Person auf nicht korrekte Angaben zur finanziellen Situation oder auf eine falsche Wohnadresse derselben abgestellt, so sind diese Daten zu berichtigen.644 Bei der drohnengestützten Datenbearbeitung durch Private rückt die Datenrichtigkeit deshalb in den Hintergrund. 5. Einwilligung Art. 5 Abs. 6 E-DSG schreibt vor, dass die Einwilligung der betroffenen Person — falls sie erforderlich ist — nur gültig ist, wenn sie für eine oder mehrere bestimmte Bearbeitungen nach angemessener Information freiwillig erteilt wird. Die Einwilligung ist an keine Form gebunden; eine schriftliche Erklärung ist nicht erforderlich.645 Die Einwilligung kann somit auch durch eine stillschweigende bzw. konkludente Willenserklärung erfolgen (vgl. Art. 1 OR).646 Eine Information ist dann «angemessen» im Sinne der hier diskutierten Bestimmung, wenn die betroffene Person darauf gestützt die Datenbearbeitung in Bezug auf die Risiken für ihre Persönlichkeitsrechte einschätzen kann.647 Primär soll dazu über Umfang und Zweck der Datenbearbeitung informiert werden.648 Unfreiwillig erfolgt die Einwilligung, «wenn bei einer Verweigerung Nachteile drohen, die mit dem Bearbeitungszweck in keinem sachlichen Zusammenhang stehen oder aus anderen Gründen unverhältnismässig sind».649 643 Vgl. auch Handkomm. DSG-Rosenthal, Art. 5 N 1. Zu den gesetzlichen Ausnahmen der Berichtigungs-, Vernichtungs- und Löschungspflicht vgl. Botschaft Totalrevision DSG, S. 7026 f. 644 Ausführlich zum Ganzen Meier, Rn. 743 ff.; SHK DSG-Baeriswyl/Blonski, Art. 5 N 5 ff. 645 Botschaft Totalrevision DSG, S. 7027; Botschaft Revision DSG 2003, S. 2127; Meier, Rn. 874; Handkomm. DSG-Rosenthal, Art. 4 N 77; vgl. zur DSGVO Scheurer, S. 178 ff. 646 Haas, Rn. 229 ff.; Botschaft Totalrevision DSG, S. 7027; vgl. auch BGE 123 III 53 E. 5a; Hausheer/Aebi-Müller, Rn. 12.18. Zum Begriff der stillschweigenden Willenserklärung statt vieler BSK OR I-Zellweger-Gutknecht, Art. 1 N 17 m.w.H. 647 Vgl. SHK DSG-Baeriswyl, Art. 4 N 59; Meier, Rn. 826 f. 648 Botschaft Totalrevision DSG, S. 7027. Zu weiteren Aspekten SHK DSG-Baeriswyl, Art. 4 N 60 f.; Handkomm. DSG-Rosenthal, Art. 4 N 72 f. 649 Statt vieler Vasella, Jusletter, Rn. 14 mit zahlreichen Hinweisen. Vgl. auch BGE 138 II 331 E. 7.4.1. 88 Teil 3: Bestandesaufnahme (1): Der E-DSG Je sensibler die infrage stehenden Personendaten sind, desto höhere Anforderungen sind an die Eindeutigkeit zu stellen.650 Diesem Anspruch lebt Art. 5 Abs. 7 E-DSG denn auch nach, indem er für die Bearbeitung von besonders schützenswerten Personendaten eine ausdrückliche Einwilligung verlangt.651 «Eine Willenserklärung ist ‹ausdrücklich›, wenn sie durch geschriebene oder gesprochene Worte oder ein Zeichen erfolgt und der geäusserte Willen [recte: Wille] aus den verwendeten Worten oder dem Zeichen unmittelbar hervorgeht.»652 Eine ausdrückliche Willenserklärung kann mithin «auch etwa durch Kopfnicken und generell durch ein gesellschaftlich anerkanntes oder durch die Parteien im Einzelfall vereinbartes Kommunikationsmittel» erfolgen.653 Damit eine gültige Einwilligung vorliegen würde, müsste der Private, welcher mit seiner Drohne Daten bearbeiten will, die (möglicherweise) betroffenen Personen vorab über die Bearbeitung und deren Zweck informieren. Da für die Bearbeitung besonders schützenswerter Personendaten eine Einwilligung ausdrücklich zu erfolgen hat, müssten z.B. Grundstückeigentümer direkt kontaktiert werden.654 Diese Vorgaben werden in der Praxis wohl kaum einmal erfüllt werden (können).655 Der Einwilligung kommt im vor liegend interessierenden Bereich somit keine allzu grosse Bedeutung zu.656 6. Rechtsfolge bei Missachtung Werden die Bearbeitungsgrundsätze nicht eingehalten, liegt regelmässig eine Persönlichkeitsverletzung im Sinne von Art. 26 E-DSG vor. Dagegen kann sich die betroffene Person mit den Klagen nach Art. 28 E-DSG zur Wehr setzen.657 650 Vgl. Botschaft Totalrevision DSG, S. 7027. 651 Botschaft Totalrevision DSG, S. 7028; vgl. Botschaft Revision DSG 2003, S. 2127; Meier, Rn. 897. Eine detaillierte Diskussion der in der Schweiz vertretenen Auslegungen der «Ausdrücklichkeit» findet sich bei Vasella, Jusletter, Rn. 21 ff. mit zahlreichen Hinweisen. 652 Botschaft Totalrevision DSG, S. 7028 u.a. mit Hinweis auf BGE 121 III 31 E. 2c. Differenzierend zur Ausdrücklichkeit der Einwilligung Epiney, S. 103 mit Fn. 25. 653 Vasella, Jusletter, Rn. 26 m.w.H.; Handkomm. DSG-Rosenthal, Art. 4 N 78; vgl. Rosenthal, Entwurf, Rn. 39; Scheurer, S. 177 f. 654 Zur drohnengestützten Bearbeitung besonders schützenswerter Personendaten vgl. vorne, S. 34. 655 In Betracht käme etwa ein Hochzeitsfotograf, welcher die Anwesenden vor der Fotosession informiert. Diese Konstellation ist jedoch nicht Fokus der vorliegenden Untersuchung. Vgl. in diesem Kontext auch EASA, GM Implementing Regulation, S. 6 f. 656 Vgl. BSK ZGB II-Ernst, Art. 928 N 3a, zur Einwilligung des Besitzers (eines Grundstücks) in den Überflug mit einer Drohne. 657 Ausführlich dazu hinten, S. 106 ff. § 3 Zentrale Bestimmungen des E-DSG 89 Strafbewehrt ist eine Missachtung der Grundsätze von Art. 5 und 7 E-DSG dagegen nicht.658 Eine Bestrafung wäre denn auch unverhältnismässig; ein zivilrechtliches Vorgehen erscheint als geeignetere Massnahme, um Abhilfe zu schaffen. II. Privacy by Design Art. 6 E-DSG normiert «Datenschutz durch Technik und datenschutzfreundliche Voreinstellungen». Gemeint sind damit «Privacy by Design»659 bzw. «Data Protection by Design»660 und «Privacy by Default».661 1. Die Regelung im Einzelnen 1.1 Datenschutz durch Technik «Der Verantwortliche ist verpflichtet, die Datenbearbeitung technisch und organisatorisch so auszugestalten, dass die Datenschutzvorschriften eingehalten werden, insbesondere die Grundsätze nach Artikel 5 [E-DSG].662 Er berücksichtigt dies ab der Planung» (Art. 6 Abs. 1 E-DSG).663 «Datenschutzvorschriften» im Sinne dieser Bestimmung meint neben den exemplarisch erwähnten Datenbearbeitungsgrundsätzen auch die weiteren Anforderungen des E-DSG; diese sind einzuhalten und die Rechte der betroffenen Personen zu schützen.664 Ziel der Norm ist die Verwirklichung von datenschutzrechtlichen Anforderungen im System, und zwar dergestalt, dass die Gefahr von Verstössen gegen datenschutzrechtliche Vorgaben reduziert oder ausgeschlossen wird.665 658 Vgl. Art. 54 ff. E-DSG. 659 Botschaft Totalrevision DSG, S. 7029. Zum Ursprung dieses Begriffs Solove, Privacy by Design, S. 191 m.w.H. Kritisch zur Verwendung von «Privacy» im Zusammenhang mit der DSGVO, da dieser Begriff nicht mit «Datenschutz» gleichzusetzen sei, Hansen, in Simitis et al., Art. 25 DSGVO N 23; vgl. auch González Fuster, S. 254 ff., die von einem «Process of Legal Miscommunication» spricht. 660 Zu diesem Begriff bspw. Sydow, in: Sydow, Einleitung N 93. 661 Art. 6 Abs. 3 E-DSG. Im Kontext der drohnengestützten Datenbearbeitung durch Private kommt den datenschutzfreundlichen Voreinstellungen keine Bedeutung zu. Die Vorschrift zielt vielmehr auf ein Nutzer-Anwendungsanbieter-Verhältnis ab; vgl. Botschaft Totalrevision DSG, S. 7030; Hansen, in Simitis et al., Art. 25 DSGVO N 40. 662 Vgl. zu den Bearbeitungsgrundsätzen vorne, S. 82 ff. 663 Die Bestimmung ist inhaltlich an Art. 25 DSGVO angelehnt. Die Botschaft Totalrevision DSG, S. 7029, verweist denn auch explizit darauf. Literatur zur DSGVO kann somit auch hier zur Auslegung beigezogen werden. 664 Vgl. Mantz, in: Sydow, Art. 25 DSGVO N 19; Koops/Leenes, S. 163. 665 Botschaft Totalrevision DSG, S. 7029; vgl. Tamò-Larrieux, S. 86; Mantz, in: Sydow, Art. 25 DSGVO N 19. 90 Teil 3: Bestandesaufnahme (1): Der E-DSG Dabei müssen die Massnahmen des «Data Protection by Design» während des gesamten Lebenszyklus der Daten666 aufrechterhalten und ggf. angepasst bzw. verbessert werden.667 Als Umsetzungsbeispiele kommen die regelmässige Löschung von Daten oder deren standardmässige Anonymisierung in Betracht;668 kommt eine Kamera zum Einsatz, können z.B. Gesichter oder Personen durch Software unkenntlich gemacht werden.669 Aus organisatorischer Sicht sind sodann etwa die Regelung von Informationsflüssen, die Festlegung von Verhaltensregeln und das Abhalten von Schulungen denkbar.670 Hervorzuheben ist, dass Hersteller von Produkten und Applikationen nicht von Art. 6 E-DSG erfasst sind; Normadressat ist einzig der Verantwortliche.671 Letzterer ist mithin bei der Wahl der Massnahme auf diejenige Technik beschränkt, welche ihm angeboten wird.672 Dem Beispiel der DSGVO folgend, zielt auch der E-DSG darauf ab, Datenschutz durch Technik «übers Dreieck» zu erreichen, indem die Pflichten der Verantwortlichen die Nachfrage nach entsprechenden Produkten, Applikationen und Diensten bei den Herstellern erzeugt.673 1.2 Beurteilung der Angemessenheit Die technischen und organisatorischen Massnahmen müssen insbesondere dem Stand der Technik, der Art und dem Umfang der Datenbearbeitung sowie den Risiken, welche die Bearbeitung für die Persönlichkeit und Grundrechte der betroffenen Personen mit sich bringt, angemessen sein (Art. 6 Abs. 2 E-DSG).674 «Je höher das Risiko, je grösser die Eintrittswahrscheinlichkeit und je umfangreicher die Datenbearbeitung ist, umso höher 666 Ausführlich zum «life cycle of data» Tamò-Larrieux, S. 149 ff. 667 Tamò-Larrieux, S. 86; Mantz, in: Sydow, Art. 25 DSGVO N 19. 668 Botschaft Totalrevision DSG, S. 7029. Vgl. zur automatischen Anonymisierung BGE 138 II 436 E. 14.1. 669 Leenes et al., S. 28; vgl. auch BGE 138 II 436 E. 10.6.2; Vivet/Smith, S. 8 f. 670 Vgl. Mantz, in: Sydow, Art. 25 DSGVO N 60; Martini, in: Paal/Pauly, Art. 25 DSGVO N 28. 671 Vgl. Botschaft Totalrevision DSG, S. 7029; Hansen, in Simitis et al., Art. 25 DSGVO N 21. Wohl a.M. Masutti/Tomasello, S. 224, die auch Drohnenhersteller und -designer als Adressaten verstehen. 672 Martini, in: Paal/Pauly, Art. 25 DSGVO N 25; vgl. Klitou, S. 295 m.w.H. Rose, S. 140, fordert deshalb, dass sich entsprechende Vorschriften an Hersteller bzw. Anbieter der Technik richten sollen. 673 Martini, in: Paal/Pauly, Art. 25 DSGVO N 25; Tamò-Larrieux, S. 86 m.w.H.; vgl. EG 78 DSGVO. 674 Vgl. die ähnliche Regelung in Art. 8 Abs. 2 und 3 VDSG. § 3 Zentrale Bestimmungen des E-DSG 91 sind die Anforderungen an die technischen Vorkehren, damit sie (…) als angemessen gelten können.»675 Dem Verantwortlichen kommt bei der Wahl der Massnahmen ein relativ grosser Ermessensspielraum zu. Wird dieser überschritten, besteht die Gefahr der Verletzung von Datenbearbeitungsgrundsätzen und damit wiederum das Risiko von Persönlichkeitsverletzungen.676 Obwohl im E-DSG nicht explizit erwähnt, ist im Rahmen einer Verhältnismässigkeitsprüfung («Zweck-Mittel-Relation», Art. 5 Abs. 2 BV) auch eine wirtschaftliche Komponente zu berücksichtigen. So kann sichergestellt werden, dass der Verantwortliche die Implementierungskosten einer technischen oder organisatorischen Massnahme nicht in unbegrenztem Umfang zu tragen hat.677 1.3 Stand der Technik insbesondere Gemäss Art. 6 Abs. 2 E-DSG müssen die technischen und organisatorischen Massnahmen u.a. dem Stand der Technik angemessen sein. Die Botschaft zum E-DSG führt nicht aus, was damit gemeint ist.678 Auch neuere Urteile des Bundesgerichts zu datenschutzrechtlichen Fragen erwähnen den «Stand der Technik», ohne näher auf dessen Bedeutung und Inhalt einzugehen.679 Der im Umweltrecht oftmals verwendete Begriff680 beschreibt Massnahmen, die einem entwickelten Stadium von technischen Möglichkeiten zu einem bestimmten Zeitpunkt entsprechen und auf gesicherten Erkenntnissen der Wissenschaft, Technik und Erfahrung basieren. Ausserdem müssen diese Massnahmen in ausreichendem Mass verfügbar sein, um angemessen umgesetzt zu werden.681 675 Botschaft Totalrevision DSG, S. 7030. 676 Vgl. Mantz, in: Sydow, Art. 25 DSGVO N 36; Martini, in: Paal/Pauly, Art. 25 DSGVO N 37 ff.; Koops/Leenes, S. 163. 677 Zum DSG BSK DSG-Stamm-Pfister, Art. 7 N 9; Meier, Rn. 792. Vgl. den Wortlaut von Art. 25 Abs. 1 DSGVO und dazu Martini, in: Paal/Pauly, Art. 25 DSGVO N 41; Feiler/ Forgó/Weigl, Art. 25 GDPR N 3 sowie ausführlich Mantz, in: Sydow, Art. 25 DSGVO N 45 f. 678 Vgl. Botschaft Totalrevision DSG, S. 7029 f. 679 Vgl. etwa BGE 144 I 126 E. 8.3.5; 138 II 436 E. 10.6.5 und E. 14.1. 680 In der Schweizer Gesetzgebung ist er z.B. in Art. 32e Abs. 4 Umweltschutzgesetz (USG), Art. 4 Altlastenverordnung (AltlV) und Art. 26 Abfallverordnung (VVEA) zu finden. 681 Mantz, in: Sydow, Art. 25 DSGVO N 38; Martini, in: Paal/Pauly, Art. 25 DSGVO N 39; Hartung, in: Kühling/Buchner, Art. 25 DSGVO N 21. 92 2. Teil 3: Bestandesaufnahme (1): Der E-DSG Sanktionierung Werden die Vorschriften von Art. 6 E-DSG nicht eingehalten, so sieht das Gesetz keine strafrechtliche Sanktionierung vor (vgl. Art. 54 ff. E-DSG). Eine Nichteinhaltung kann dagegen (indirekt) zu einer Persönlichkeitsverletzung führen, namentlich wenn dadurch Bearbeitungsgrundsätze verletzt werden.682 3. Bedeutung bei der drohnengestützten Datenbearbeitung durch Private Privacy by Design hält in verschiedener Hinsicht Lösungsansätze für datenschutzrechtliche Probleme im Bereich der Drohnenfliegerei bereit. So kons� tatierte etwa Roma: «Privacy by design might prove an extremely useful tool to ensure effective enforcement of EU and national legislation, especially where Mini RPAS683 are concerned.»684 Mögliche Umsetzungsvarianten von Privacy by Design bei Drohnen sind z.B. eine Beschränkung von Dauer und Auflösung von Videoaufnahmen oder das Anbringen von Warnlichtern bzw. Warnsignalen, welche auf eine Datenbearbeitung hinweisen.685 Gesichter und/oder Nummern (z.B. auf Kontrollschildern von Motorfahrzeugen) könnten automatisch erkannt und unkenntlich gemacht werden.686 Doch auch das äussere Erscheinungsbild einer Drohne und die damit assoziierten Risiken für Sicherheit und Privatsphäre sollten im vorliegenden Zusammenhang mitbedacht werden.687 So kann etwa durch die Verwendung von helleren Farben und das Anbringen 682 Vgl. Husi-Stämpfli, S. 52; Botschaft Totalrevision DSG, S. 7029, wo explizit auf die Bearbeitungsgrundsätze Bezug genommen wird. Rosenthal, Entwurf, Rn. 41 mit Fn. 54, leitet dagegen aus der Tatsache, dass Art. 6 E-DSG in Art. 26 Abs. 2 lit. a E-DSG nicht aufgeführt wird, ab, dass eine Verletzung von ersterer Bestimmung keine Persönlichkeitsverletzung darstellt. Zu den Bearbeitungsgrundsätzen im Einzelnen vorne, S. 82 ff. 683 Remotely Piloted Aircraft System, vgl. dazu vorne, S. 17. 684 Roma, S. 30; ähnlich Cassart, RDTI, S. 88; Cavoukian, S. 16 ff.; Masutti/Tomasello, S. 231 f. Vgl. auch Boucher, S. 25; Dieckert, in: Dieckert/Eich, S. 415 f.; Harris/Given, S. 9; Klitou, S. 61 ff. 685 Cassart, RDTI, S. 88; Leenes et al., S. 29; Harris/Given, S. 9. Vgl. Froomkin/ Colangelo, S. 64; Groh, S. 167; Martini, S. 740; Rose, S. 139. 686 DroneRules, Guide, S. 18. Vgl. zu dieser Vorgehensweise auch BGE 138 II 346 E. 10.3 f. und 10.6.2. Vereinzelt wurde gar ein Verbot der drohnengestützten Bearbeitung von «personenbezogenen Bilddaten» propagiert (Leissler, S. 536 m.w.H.). 687 In der Studie von Chang/Chundury/Chetty, v.a. S. 6771, wurde festgestellt, dass die Probanden je nach Erscheinungsbild einer Drohne die damit einhergehenden Sicherheits- und Privatsphärerisiken anders beurteilten. § 3 Zentrale Bestimmungen des E-DSG 93 von Logos bzw. Kennzeichen eine Drohne weniger einschüchternd wirken und ggf. Transparenz geschaffen werden.688 Nicht alle der genannten Optionen sind jedoch ohne Weiteres umsetzbar. Denn die Programmierung von Datenschutzregeln in Software ist sehr komplex und teilweise gar nicht durchführbar. Zu Recht hielten Koops/ Leenes fest: «Some legal rules are intentionally open, vague, overinclusive or underinclusive, and often they are indeterminate and require interpretation.»689 Gerade die zentralen Bearbeitungsgrundsätze, insbesondere die Verhältnismässigkeit sowie Treu und Glauben,690 sind gewollt offen formuliert, was eine softwareseitige Umsetzung derselben erschwert, wenn nicht gar verunmöglicht.691 Die vorprogrammierten Datenschutzregeln würden ausserdem wohl oft (zu) rigide ausfallen (um Persönlichkeitsverletzungen mit Sicherheit zu vermeiden) und folglich keine Kontextsensitivität zulassen.692 Unter der Geltung des zukünftigen DSG wird sich erst zeigen müssen, welche Umsetzungsvarianten der Vorgabe von Datenschutz durch Technik die Drohnen- und Drohnenzubehörhersteller anbieten werden. Rechtsprechung zu Mindestanforderungen könnte «ein starker Anreiz [sein], erstens auf dem Markt ‹gerichtsfeste› Modelle anzubieten und zweitens auch nur solche Modelle zu verwenden».693 Entsprechende Gerichtspraxis fehlt indes bis anhin. III. Informationspflicht Die in Art. 17 E-DSG geregelte Informationspflicht, d.h. die Pflicht des Verantwortlichen, die betroffene Person angemessen über die Beschaffung von Personendaten zu informieren, ist primär darauf ausgerichtet, Transparenz bei der Datenbearbeitung herzustellen. Denn regelmässig kann die betroffene Person nicht erkennen, dass Daten über sie bearbeitet werden; folglich kann sie auch ihre Rechte gemäss E-DSG nicht wahrnehmen.694 Die Informationspflicht soll hier Abhilfe schaffen und dient somit auch einer effektiven 688 Ausführlich DroneRules, Guide, S. 10 ff.; vgl. auch Ruijten/Cuijpers, S. 68 ff.; Martini, S. 740 f.; Bajde et al., Public reactions, S. 5. 689 Koops/Leenes, S. 166; vgl. auch Leenes et al., S. 28. 690 Ausführlich zu den Bearbeitungsgrundsätzen vorne, S. 82 ff. 691 Leenes et al., S. 28; vgl. Koops/Leenes, passim, v.a. S. 166. 692 Leenes et al., S. 29; vgl. auch Boucher, S. 26. 693 Rose, S. 140, zum Einfluss von Rechtsprechung zur Verwertbarkeit von Dashcam-Aufnahmen auf die Gestaltung der Technik. Vgl. dazu auch BGer, 6B_1188/2018 vom 26. September 2019 E. 3.1 ff. (zur Publikation vorgesehen) 694 Botschaft Totalrevision DSG, S. 7050. 94 Teil 3: Bestandesaufnahme (1): Der E-DSG Rechtsdurchsetzung.695 Im Vergleich zum geltenden Recht wurde sie ausgeweitet; gemäss Art. 14 DSG trifft Private nur dann eine Informationspflicht, wenn sie besonders schützenswerte Personendaten beschaffen.696 1. Voraussetzungen Art. 17 Abs. 1 E-DSG schreibt vor, dass der Verantwortliche die betroffene Person angemessen über die Beschaffung von Personendaten informiert. Diese Informationspflicht gilt auch, wenn die Daten nicht bei der betroffenen Person beschafft werden; damit sind Fälle angesprochen, in denen die Daten bei Dritten beschafft werden.697 Als Dritter in diesem Sinn gilt jede vom Verantwortlichen und dem oder der Betroffenen verschiedene (natürliche oder juristische) Person.698 Wird auf öffentliche Quellen — wie etwa Medienberichte oder fremde Webseiten699 — zurückgegriffen, stellt dies ebenfalls eine Beschaffung bei Dritten dar.700 Die Pflicht zur Information wird durch die «Beschaffung» ausgelöst. Beim Beschaffen handelt es sich um einen Bearbeitungsvorgang im Sinne von Art. 4 lit. c E-DSG. «Beschaffen» beschreibt «die Aktivität […], wodurch [der Verantwortliche] gewollt Kenntnis von den Daten erhält oder die Verfügung darüber begründet».701 Es wird mithin ein aktives Tun vonseiten des Verantwortlichen vorausgesetzt.702 Die drohnengestützte Datenbearbeitung (unabhängig vom verwendeten Sensor) stellt ohne Weiteres eine «Beschaffung» im Sinne von Art. 17 E-DSG dar. Dieser Vorgang bezieht sich direkt auf die betroffene Person und die Daten werden somit direkt bei ihr beschafft. 2. Umfang und Modalitäten Inhaltlich schreibt Art. 17 Abs. 2 E-DSG vor, dass der betroffenen Person diejenigen Informationen mitgeteilt werden müssen, die erforderlich sind, damit 695 Vgl. Paal, in: Paal/Pauly, Art. 13 DSGVO N 4. 696 Daneben bestand die Informationspflicht auch dann, wenn «Persönlichkeitsprofile» beschafft wurden; dieser Begriff wurde mit dem E-DSG jedoch abgeschafft. Weder das europäische Recht noch andere ausländische Gesetzgebungen kennen diesen Begriff, er war eine Schweizer Eigenheit. An seiner Stelle wird im E-DSG der Begriff des «Profiling» verwendet (vgl. Art. 4 lit. f E-DSG sowie ausführlich Botschaft Totalrevision DSG, S. 7021). 697 Vgl. dazu den Wortlaut von Art. 14 Abs. 1 DSG. 698 BSK DSG-Rampini/Fuchs, Art. 14 N 6; vgl. Handkomm. DSG-Rosenthal, Art. 7a N 12. 699 Betreibt ein Unternehmen eine eigene Webseite, werden dort registrierte Daten regel mässig direkt bei der betroffenen Person beschafft. 700 Handkomm. DSG-Rosenthal, Art. 7a N 12. 701 SHK DSG-Rudin, Art. 3 N 34. 702 BSK DSG-Rampini/Fuchs, Art. 14 N 5; vgl. SHK DSG-Wermelinger, Art. 14 N 3. § 3 Zentrale Bestimmungen des E-DSG 95 sie ihre Rechte nach dem E-DSG geltend machen kann und eine transparente Datenbearbeitung gewährleistet ist. Im Sinne eines Mindestkatalogs führt das Gesetz folgende Informationen auf, die mitgeteilt werden müssen: die Identität und die Kontaktdaten des Verantwortlichen (lit. a); der Bearbeitungszweck (lit. b); und ggf. die Empfängerinnen und Empfänger oder die Kategorien von Empfängerinnen und Empfängern, denen Personendaten bekanntgegeben werden (lit. c).703 Der E-DSG legt nicht fest, auf welche Weise die Information erfolgen muss. Der Verantwortliche muss jedoch sicherstellen, dass die betroffene Person die Information tatsächlich zur Kenntnis nehmen kann.704 Sie muss demnach ausdrücklich und aktiv erfolgen sowie präzise, verständlich und in einer klaren und einfachen Sprache ausgedrückt werden.705 Der Verantwortliche hat sicherzustellen, dass die Möglichkeit besteht, sich in leicht zugänglicher Weise zu informieren, nicht aber, dass sich die betroffene Person im konkreten Fall tatsächlich informiert.706 Eine allgemeine Information kann genügen, wenn Personendaten bei der betroffenen Person selbst beschafft werden. In diesem Fall sind gemäss Botschaft «eine Datenschutzerklärung auf einer Website, aber gegebenenfalls auch Symbole oder Piktogramme, soweit sie die nötigen Informationen wiedergeben» denkbar.707 Es besteht keine Formvorschrift bezüglich der Information; sie kann also grundsätzlich auch mündlich erfolgen. Aus Beweisgründen sowie aus Transparenzüberlegungen ist aber eine schriftliche Information empfehlens wert.708 Insgesamt muss eine Form gewählt werden und zudem die Information so verständlich sein, dass sie dem Zweck einer transparenten Datenbearbeitung auch tatsächlich dient.709 Bei der drohnengestützten Datenbearbeitung durch Private kommt eine mündliche Information bezüglich Identität des Verantwortlichen und Zweck der Bearbeitung grundsätzlich in Betracht. 703 Vgl. zu den «Empfängerinnen und Empfänger[n]» und «Kategorien von Empfänger innen und Empfänger[n]» Handkomm. DSG-Rosenthal, Art. 7a N 17 und Art. 11a N 35; SHK DSG-Wermelinger, Art. 14 N 6. 704 Botschaft Totalrevision DSG, S. 7050. 705 Botschaft Revision DSG 2003, S. 2131; Epiney/Fasnacht, in: Belser/Epiney/Waldmann, § 11 Rn. 8 m.w.H.; Paal, in: Paal/Pauly, Art. 13 DSGVO N 5. 706 Botschaft Totalrevision DSG, S. 7050. 707 Botschaft Totalrevision DSG, S. 7050. 708 BSK DSG-Rampini/Fuchs, Art. 14 N 13; Handkomm. DSG-Rosenthal, Art. 7a N 12; Botschaft Totalrevision DSG, S. 7051; Botschaft Revision DSG 2003, S. 2131. 709 Botschaft Totalrevision DSG, S. 7051. 96 3. Teil 3: Bestandesaufnahme (1): Der E-DSG Zeitpunkt der Information Werden Daten direkt bei der betroffenen Person beschafft, so ist diese gemäss Art. 17 Abs. 2 E-DSG bei der Beschaffung zu informieren. Gemäss Botschaft bedeutet dies, dass der Verantwortliche die betroffene Person «in diesem Zeitpunkt» informieren muss.710 Daraus ergibt sich aber nicht zwingend eine Gleichzeitigkeit der Vorgänge; dies wäre auch nicht in jedem Fall praktikabel. Vielmehr hat die Information vor, während oder unmittelbar nach der (ersten) Datenbeschaffung zu erfolgen.711 Wie bei der eigentlichen Beschaffung ist auch hier ein aktives Tun des Verantwortlichen erforderlich.712 Für Daten, die nicht bei der betroffenen Person selbst beschafft werden, gibt Art. 17 Abs. 5 E-DSG eine Frist vor. Danach hat die Information spätestens einen Monat, nachdem der Verantwortliche die Daten erhalten hat, zu erfolgen. Eine kürzere Frist ist für den Fall vorgesehen, dass der Verantwortliche die Personendaten vor Ablauf der Monatsfrist (an einen oder mehrere) Empfänger bekannt gibt. In diesem Fall muss er die betroffene Person spätestens zum Zeitpunkt der Bekanntgabe informieren.713 4. Ausnahmen und Einschränkungen 4.1 Ausnahmen Das Gesetz sieht Ausnahmen zur und Einschränkungen der Informationspflicht vor. So entfällt diese gemäss Art. 18 Abs. 1 E-DSG, wenn eine der folgenden Voraussetzungen erfüllt ist: die betroffene Person verfügt bereits über die entsprechenden Informationen (lit. a); die Bearbeitung ist gesetzlich vorgesehen (lit. b); es handelt sich beim Verantwortlichen um eine private Person, die gesetzlich zur Geheimhaltung verpflichtet ist (lit. c); oder die Information erfordert einen unverhältnismässigen Aufwand (lit. e).714 Werden die Personendaten nicht bei der betroffenen Person beschafft, so entfällt die Informationspflicht zudem, solange die Person nicht mit verhältnismässigem Aufwand bestimmt werden kann (Art. 18 Abs. 2 E-DSG). Im Zusammenhang mit der drohnengestützten Datenbearbeitung durch Private besteht keine Rechtsgrundlage, welche eine entsprechende Daten710 Botschaft Totalrevision DSG, S. 7052. 711 Epiney/Fasnacht, in: Belser/Epiney/Waldmann, § 11 Rn. 8; BSK DSG-Rampini/ Fuchs, Art. 14 N 14; Handkomm. DSG-Rosenthal, Art. 7a N 23; vgl. SHK DSG-Wermelinger, Art. 14 N 8. Für eine Information jeweils vor der Datenbeschaffung plädiert Mester, in: Taeger/Gabel, Art. 13 DSGVO N 34 in fine. 712 Vgl. BSK DSG-Rampini/Fuchs, Art. 14 N 13. 713 Botschaft Totalrevision DSG, S. 7052. 714 Die in Art. 18 Abs. 1 lit. d E-DSG genannte Ausnahme bezieht sich auf Medien. Sie ist für die vorliegende Auseinandersetzung nicht beachtlich. § 3 Zentrale Bestimmungen des E-DSG 97 bearbeitung im Sinne von Art. 18 Abs. 1 lit. b E-DSG vorsehen würde. Ausserdem ist in diesem Kontext nicht ersichtlich, wie die betroffene Person bereits Kenntnis der entsprechenden Informationen haben oder dass ein Privater gesetzlich einer Geheimhaltungspflicht unterliegen könnte (lit. a und c). Des Weiteren werden die Daten nicht bei Dritten beschafft, womit die Ausnahmen gemäss Art. 18 Abs. 2 E-DSG nicht greifen.715 Insgesamt ist bei der drohnengestützten Datenbearbeitung durch Private keine der genannten Ausnahmen einschlägig. 4.2 Einschränkungen Des Weiteren sieht das Gesetz in Art. 18 Abs. 3 E-DSG vor, dass der Verantwortliche die Mitteilung der Informationen in gewissen Fällen einschränken, aufschieben oder darauf verzichten kann.716 Solche Massnahmen sind etwa möglich, wenn sie wegen überwiegender Interessen Dritter erforderlich sind (lit. a). Private Verantwortliche können zudem so vorgehen, wenn eigene überwiegende Interessen es erfordern (lit. c). Werden von einem Privaten mit einer Drohne Daten bearbeitet, so wird regelmässig keiner der im E-DSG vorgesehenen Tatbestände einschlägig sein. Denn es ist kaum eine Situation denkbar, in welcher überwiegende Inter essen des Drohnenbetreibers (als Verantwortlicher) oder von Dritten einer Information entgegenstehen könnten. Die Mitteilung der Informationen gemäss Art. 17 E-DSG kann somit nicht eingeschränkt, aufgeschoben oder auf sie verzichtet werden. 5. Sanktionierung Die Verletzung von Informationspflichten ist gemäss Art. 54 Abs. 1 lit. a und b E-DSG sanktionsbewehrt. So werden private Personen auf Antrag mit Busse bis zu 250’000 Franken bestraft, die ihre Pflichten nach den Art. 17, 19 und 23–25 E-DSG verletzen, indem sie vorsätzlich eine falsche oder unvollständige Auskunft erteilen (lit. a). Mit der gleichen Busse können private Personen belegt werden, die es vorsätzlich unterlassen, die betroffene Person nach Art. 17 Abs. 1 E-DSG zu informieren (lit. b Ziff. 1)717 oder ihr die Angaben nach Art. 17 Abs. 2 E-DSG zu liefern (lit. b Ziff. 2). 715 Vgl. dazu vorne, S. 94. 716 Die lit. b (Vereitelung des Zwecks der Datenbearbeitung) und d (Bearbeitung durch Bundesorgan) werden nicht weiter vertieft. Ausführlich zu den einzelnen Tatbeständen Botschaft Totalrevision DSG, S. 7054 ff. 717 Art. 54 Abs. 1 lit. b Ziff. 1 nimmt ausserdem Bezug auf Art. 19 Abs. 1 E-DSG. Dieser bezieht sich auf die Informationspflicht bei automatisierten Einzelentscheidungen und ist für die vorliegende Untersuchung nicht von Belang. 98 6. Teil 3: Bestandesaufnahme (1): Der E-DSG Bedeutung bei der drohnengestützten Datenbearbeitung durch Private Die Informationspflicht gemäss Art. 17 E-DSG ist ohne Weiteres auf Private anwendbar, die drohnengestützt Daten bearbeiten. Die gesetzlichen Ausnahmen der Informationspflicht sind nicht einschlägig. Es ist jedoch davon auszugehen, dass bei der drohnengestützten Datenbearbeitung eine Information der betroffenen Personen regelmässig unterbleibt. Dies dürfte sich primär daraus ergeben, dass sich Drohnenbetreiber einer entsprechenden Pflicht nicht bewusst sind oder aber diese nicht erfüllen. Eine Sanktionierung gemäss Art. 54 E-DSG kommt somit meist nicht in Betracht, da in so gearteten Fällen regelmässig keine Vorsätzlichkeit vorliegt.718 Die mit der Informationspflicht angestrebte Verbesserung der Transparenz wird bei der drohnengestützten Datenbearbeitung mithin nicht erreicht. Aus diesem Grund kommt dem datenschutzrechtlichen Auskunftsrecht (dazu hinten, S. 100 ff.) in diesem Kontext eine besondere Bedeutung zu. IV. Datenschutz-Folgenabschätzung 1. Voraussetzungen Wenn eine Bearbeitung ein hohes Risiko für die Persönlichkeit oder die Grundrechte der betroffenen Person mit sich bringen kann, erstellt der Verantwortliche vorgängig eine Datenschutz-Folgenabschätzung. Sind mehrere ähnliche Bearbeitungsvorgänge geplant, so kann eine gemeinsame Abschätzung erstellt werden (Art. 20 Abs. 1 E-DSG).719 Das «hohe Risiko» ergibt sich, insbesondere bei Verwendung neuer Technologien, aus der Art, dem Umfang, den Umständen und dem Zweck der Bearbeitung (Art. 20 Abs. 2 E-DSG).720 Ein hohes Risiko liegt namentlich bei einer umfangreichen Bearbeitung besonders schützenswerter Personendaten, oder wenn systematisch umfangreiche öffentliche Bereiche überwacht werden, vor (Art. 20 Abs. 2 lit. a und c E-DSG).721 Inhaltich verlangt Art. 20 Abs. 3 E-DSG «eine Beschreibung der geplanten Bearbeitung, eine Bewertung der Risiken für die Persönlichkeit oder die Grundrechte der betroffenen Person sowie die Massnahmen zum Schutz der 718 Gemäss dem klaren Wortlaut der Strafbestimmung reicht (Grob-)Fahrlässigkeit nicht aus (vgl. vorne, S. 97 und Botschaft Totalrevision DSG, S. 7101). 719 Eine ähnliche, aber ausführlichere Regelung findet sich auch in Art. 35 DSGVO. 720 Vgl. auch § 10 IDG/ZH und dazu PK IDG/ZH-Blattmann, § 10 N 10, sowie § 13 IDG/BS und dazu PK IDG/BS-Rudin, § 13 N 11. 721 Der vom Bundesrat vorgeschlagene lit. b wurde vom NR gestrichen (vgl. Amtl. Bull. NR 2019 S. 1810 f.). § 3 Zentrale Bestimmungen des E-DSG 99 Persönlichkeit und der Grundrechte». Datenbearbeitungen durch Private, die zur Erfüllung einer gesetzlichen Pflicht des Verantwortlichen erfolgen, sind gemäss Abs. 4 von der Erstellung einer Datenschutz-Folgenabschätzung ausgenommen.722 2. Sanktionierung Wird die Pflicht zur Erstellung einer Datenschutz-Folgenabschätzung nicht eingehalten, hat der untätig gebliebene Verantwortliche keine strafrechtlichen Folgen zu gewärtigen: Die Missachtung von Art. 20 E-DSG ist in den Strafbestimmungen von Art. 54 ff. E-DSG nicht enthalten. In diesem Fall ist auch keine Persönlichkeitsverletzung möglich, da die Folgenabschätzung vor der Datenbearbeitung ansetzt und somit zu diesem Zeitpunkt noch keine Personen betroffen sind.723 Wird eine Datenbearbeitung durchgeführt, welche einer Folgenabschätzung bedurft hätte, so ist jedoch die Einhaltung der Bearbeitungsg rundsätze gefährdet, was Persönlichkeitsverletzungen nach sich ziehen kann.724 3. Bedeutung bei der drohnengestützten Datenbearbeitung durch Private Bei der drohnengestützten Datenbearbeitung durch Private kommt der Datenschutz-Folgenabschätzung geringe Bedeutung zu. Zwar ist ein privater Verantwortlicher grundsätzlich der Pflicht zur Erstellung einer solchen unterworfen; jedoch ist nicht davon auszugehen, dass bei einer drohnengestützten Datenbearbeitung durch einen Privaten oft ein «hohes Risiko» im Sinne von Art. 20 Abs. 1 und 2 E-DSG besteht. Der Umfang, die Umstände und der Zweck einer solchen Datenbearbeitung erreichen diese Schwelle regelmässig nicht.725 Bei den in dieser Untersuchung interessierenden Bearbeitungsvorgängen liegt keine systematische Überwachung von umfangreichen öffentlichen Bereichen vor (vgl. Art. 20 Abs. 2 lit. c E-DSG).726 Dagegen ist eine umfangreiche 722 Die in Art. 20 Abs. 5 E-DSG vorgesehene Ausnahme von der Erstellung einer Datenschutz-Folgenabschätzung ist zwar auf private Verantwortliche anwendbar. Aufgrund von deren Anwendungsvoraussetzungen kann eine Vertiefung der Bestimmung vorliegend unterbleiben. Vgl. dazu Botschaft Totalrevision DSG, S. 7062. 723 Vgl. den Wortlaut von Art. 20 Abs. 1 E-DSG. 724 Vgl. zu den Bearbeitungsgrundsätzen vorne, S. 82 ff. 725 Eine Konsultation des EDÖB, wie dies in Art. 21 E-DSG vorgesehen ist, falls die Datenschutz-Folgenabschätzung ein hohes Risiko aufzeigt, ist deshalb bei der drohnengestützten Datenbearbeitung durch Private kaum nötig. Auf eine Vertiefung dieser Regelung kann deshalb verzichtet werden. 726 Gemäss Botschaft Totalrevision DSG, S. 7061, soll diese Bestimmung etwa «die Überwachung einer Bahnhofshalle» erfassen. 100 Teil 3: Bestandesaufnahme (1): Der E-DSG Bearbeitung von besonders schützenswerten Personendaten im Sinne von Art. 20 Abs. 2 lit. a E-DSG nicht auszuschliessen.727 Umfangreich ist eine Daten bearbeitung dann, wenn grosse Mengen an Daten bearbeitet werden und/oder eine grosse Zahl von Personen betroffen ist bzw. betroffen sein kann.728 Demnach könnte etwa eine (beabsichtigte) Aufnahme einer Demonstration729 mit einer Drohne durch einen Privaten die Pflicht zur Erstellung einer Datenschutz-Folgenabschätzung mit sich bringen.730 V. Auskunftsrecht 1. Zentrale Bedeutung des Anspruchs Das in Art. 23 E-DSG enthaltene Auskunftsrecht stellt eine Ergänzung der Informationspflicht des Verantwortlichen dar; es bildet die Grundlage dafür, dass die betroffene Person die Rechte aus dem Datenschutzgesetz überhaupt wahrnehmen kann.731 Das Auskunftsrecht ist mithin ein zentrales, wenn nicht das zentrale Institut der Durchsetzung des (datenschutzrechtlichen) Persönlichkeitsschutzes732 bzw. des Datenschutzrechts überhaupt.733 Es soll den betroffenen Personen ermöglichen, die über sie «bearbeiteten Daten zu kontrollieren mit dem Ziel, die Einhaltung der datenschutzrechtlichen Grund sätze und Bestimmungen zu überprüfen und gegebenenfalls durchzusetzen».734 So kann eine betroffene Person z.B. die Berichtigung unrichtiger Daten (vgl. Art. 5 Abs. 2 E-DSG), die Sperrung der Bekanntgabe gewisser Daten oder die 727 Die Botschaft Totalrevision DSG, S. 7060, enthält keine Präzisierungen des Begriffs «umfangreich», führt als Beispiel aber medizinische Forschungsprojekte an. 728 Gemäss Baumgartner, in: Ehmann/Selmayr, Art. 35 DSGVO N 38, kann «im Einzelfall auch bei deutlich weniger» als 5’000 Personen eine grosse Zahl gegeben sein. Vgl. Schwendemann, in: Sydow, Art. 35 DSGVO N 13 m.w.H.; Karg, in: Simitis, Art. 35 DSGVO N 33. 729 Religiöse, weltanschauliche oder politische Ansichten stellen besonders schützenswerte Personendaten dar; vgl. dazu vorne, S. 31 und S. 34. 730 Die diesbezüglichen luftfahrtrechtlichen Vorgaben sollen hier nicht vertieft werden. 731 BGE 144 I 126 E. 8.3.7; Botschaft Totalrevision DSG, S. 7066; Peter, S. 211 m.w.H.; vgl. BGE 140 V 464 E. 4.2; 139 V 492 E. 3.2; 125 II 473 E. 4b; anschaulich (zur DSGVO) Tombal, Rn. 39: «la porte d’entrée, voire même la condition préalable». 732 BGE 144 I 126 E. 8.3.7; 138 III 425 E. 5.3; 120 II 118 E. 3b; Botschaft DSG 1988, S. 433; BSK DSG-Gramigna/Maurer-Lambrou, Art. 8 N 1. Vgl. auch bereits Schucan, S. 82: «Kernstück der individuellen Gegenrechte gegen die datenverarbeitende Stelle». 733 SHK DSG-Rudin, Art. 8 N 1. Vgl. SGK BV-Schweizer, Art. 13 N 85; Biaggini, Komm. BV, Art. 13 N 14; Belser, in: Belser/Epiney/Waldmann, § 6 Rn. 99; Fasnacht, Rn. 155; Widmer, Rn. 5.2. 734 BGE 144 I 126 E. 8.3.7; EDÖB, Tätigkeitsbericht 2017/18, S. 17. Vgl. auch BGE 138 I 6 E. 7.5.2; Widmer, Rn. 5.2. § 3 Zentrale Bestimmungen des E-DSG 101 Löschung und Vernichtung derselben (Art. 28 Abs. 2 E-DSG735) verlangen.736 Dem Auskunftsrecht kommt folglich eine Kontrollfunktion zu.737 2. Auskunftsberechtigte und Auskunftsverpflichtete Jede Person kann gemäss Art. 23 Abs. 1 E-DSG vom Verantwortlichen kostenlos738 Auskunft darüber verlangen, ob Personendaten über sie bearbeitet werden. Als subjektives, höchstpersönliches Recht kann es auch von urteilsfähigen handlungsunfähigen Personen selbständig, ohne Zustimmung ihres gesetzlichen Vertreters, geltend gemacht werden (vgl. Art. 19c ZGB).739 Aufgrund des klaren Wortlauts von Art. 23 Abs. 1 E-DSG kann Auskunft darüber verlangt werden, ob Personendaten bearbeitet werden (und nicht etwa nur darüber, welche Personendaten bearbeitet werden).740 Auskunftsverpflichtet ist derjenige, der für eine Datenbearbeitung verantwortlich ist bzw. vermutungsweise dafür verantwortlich zeichnet.741 3. Voraussetzungen Das Auskunftsrecht besteht voraussetzungslos.742 Der Auskunftsberechtigte hat weder ein schutzwürdiges Interesse noch eine besondere Motivation nachzuweisen;743 auch blosse Neugier reicht aus.744 Grenze bildet einzig der Rechts- 735 Vgl. dazu detailliert hinten, S. 112 ff. 736 Vgl. BGE 125 II 473 E. 4b; 126 II 126 E. 5c/bb. 737 Vgl. BGE 138 III 425 E. 5.3; 120 II 118 E. 3b. 738 Vgl. aber Art. 23 Abs. 6 E-DSG, wonach der Bundesrat Ausnahmen von der Kostenlosigkeit vorsehen kann, namentlich wenn der Aufwand unverhältnismässig gross ist. 739 Botschaft Totalrevision DSG, S. 7066; Peter, S. 212. Ausführlich zum Begriff des höchstpersönlichen Rechts und dessen Bedeutung statt vieler KuKo ZGB-Hotz, Art. 19c N 1 ff. Ausführlich zu den Begriffen der Urteilsfähigkeit und der Handlungsfähigkeit (bzw. -unf ähigkeit) statt vieler Steinauer/Fountoulakis, Rn. 75 ff. und Rn. 163 ff. 740 Vgl. Epiney/Fasnacht, in: Belser/Epiney/Waldmann, § 11 Rn. 24; BSK DSG-Gramigna/ Maurer-Lambrou, Art. 8 N 24. Selbstredend können vom Verantwortlichen keine Angaben gefordert werden, über die er gar nicht verfügt (vgl. BGE 136 II 508 E. 3.7). 741 «Lässt der Verantwortliche Personendaten von einem Auftragsbearbeiter bearbeiten, so bleibt er auskunftspflichtig» (Art. 23 Abs. 4 E-DSG). Auftragsdatenbearbeiter ist gemäss Legaldefinition in Art. 4 lit. j E-DSG die «private Person oder [das] Bundes organ, die oder das im Auftrag des Verantwortlichen Personendaten bearbeitet». In Übereinstimmung mit dem Fokus der vorliegenden Untersuchung wird nicht weiter auf die Auftragsdatenbearbeitung eingegangen. 742 Handkomm. DSG-Rosenthal, Art. 8 N 5; Epiney/Fasnacht, in: Belser/Epiney/Waldmann, § 11 Rn. 21. 743 BGE 144 III 119 E. 7.1.1; 127 V 219 E. 1a/aa; Handkomm. DSG-Rosenthal, Art. 8 N 12. 744 Botschaft Totalrevision DSG, S. 7069. 102 Teil 3: Bestandesaufnahme (1): Der E-DSG missbrauch (vgl. Art. 2 Abs. 2 ZGB).745 Besteht der Verdacht einer rechtsmissbräuchlichen oder querulatorischen Geltendmachung des Auskunftsrechts, so darf der Verantwortliche vom Auskunftsberechtigten eine Begründung des Auskunftsbegehrens verlangen;746 ansonsten ist grundsätzlich keine solche zu liefern. 4. Ausübung des Auskunftsrechts 4.1 Auskunftsbegehren Das Auskunftsbegehren ist prinzipiell schriftlich einzureichen,747 es kommen aber auch mündliche (z.B. telefonische) oder elektronische Gesuche in Betracht.748 Inhaltlich sind keine hohen Anforderungen an das Begehren zu stellen.749 Es kann jedoch vom Auskunftsberechtigten verlangt werden, wenigstens die «ihm möglichen und zumutbaren zweckdienlichen Angaben zur Erleichterung des Auffindens der Daten zu machen».750 Im Rahmen der drohnengestützten Datenbearbeitung liegt hier der Knackpunkt des Auskunftsrechts. Der Drohnenbetreiber, welcher als (allfälliger) Verantwortlicher die Auskunft zu erteilen hätte, ist nicht ohne Weiteres zu identifizieren. Somit kann die betroffene Person ihr Auskunftsbegehren gar nicht erst stellen. 4.2 Identifikation des Auskunftsberechtigten Der Verantwortliche hat die Identität des Gesuchstellers zu überprüfen; würde er dies nicht tun, bestünde die Gefahr von Persönlichkeitsverletzungen, indem Daten Dritter eingesehen werden könnten.751 Je schutzwürdiger die Daten, umso höhere Anforderungen sind an die Qualität des Identitätsnachweises zu stellen.752 Regelmässig geschieht die Überprüfung anhand einer Kopie eines amtlichen Identitätsausweises, der dem Gesuch beigelegt wird.753 745 Ausführlich zum Rechtsmissbrauch bei Ausübung des Auskunftsrechts BGE 138 III 432 E. 5.5 f. m.w.H. 746 BGE 138 III 425 E. 5.4 f.; 123 II 534 E. 2e; vgl. BSK DSG-Gramigna/Maurer-Lambrou, Art. 8 N 39. 747 Vgl. Art. 1 Abs. 1 VDSG. 748 Art. 1 Abs. 2 VDSG; SHK DSG-Rudin, Art. 8 N 20; Widmer, Rn. 5.18. 749 Epiney/Fasnacht, in: Belser/Epiney/Waldmann, § 11 Rn. 32; SHK DSG-Rudin, Art. 8 N 21; vgl. Peter, S. 219. 750 BSK DSG-Gramigna/Maurer-Lambrou, Art. 8 N 39; vgl. Belser U., S. 58. 751 BSK DSG-Gramigna/Maurer-Lambrou, Art. 8 N 40; SHK DSG-Rudin, Art. 8 N 22. 752 Epiney/Fasnacht, in: Belser/Epiney/Waldmann, § 11 Rn. 31; BSK DSG-Gramigna/ Maurer-Lambrou, Art. 8 N 40. 753 Die nicht notwendigen Informationen auf dem Identitätsausweis dürfen geschwärzt oder anders unleserlich gemacht werden; SHK DSG-Rudin, Art. 8 N 21. § 3 Zentrale Bestimmungen des E-DSG 5. 103 Erteilung der Auskunft 5.1 Form und Inhalt Die Auskunft ist grundsätzlich schriftlich, in Form eines Ausdrucks oder einer Fotokopie, zu erteilen.754 Auf jeden Fall müssen die Daten in lesbarer bzw. verständlicher und — falls sie in irgendeiner Weise verschlüsselt sind — entschlüsselter Form übermittelt werden.755 Je nach Art der bearbeiteten Daten ist dies jedoch kein gangbarer Weg: Geht es um Video- oder Tonaufnahmen, so ist der Auskunftsverpflichtete prinzipiell gehalten, dem Berechtigten eine Kopie der Daten auf einem geeigneten Speichermedium zuzustellen.756 Inhaltlich bestimmt Art. 23 Abs. 2 E-DSG, welche Angaben die betroffene Person erhält. Namentlich soll sie diejenigen Informationen erhalten, die erforderlich sind, damit sie ihre Rechte nach dem Datenschutzgesetz geltend machen kann. Gemäss dem Wortlaut der Bestimmung werden der betroffenen Person ausschliesslich die darin genannten Informationen mitgeteilt. Dabei handelt es sich u.a. um die Identität und die Kontaktdaten des Verantwortlichen (lit. a), die bearbeiteten Personendaten als solche (lit. b)757 sowie den Bearbeitungszweck (lit. c).758 Der Inhalt der erteilten Auskunft muss wahrheitsgetreu und vollständig sein.759 Vollständigkeit bedeutet in diesem Zusammenhang, dass der Gehalt der Auskunft dem Gegenstand des Gesuchs entspricht.760 Es gilt des Weiteren allfällige Einschränkungen zu beachten, was eine sorgfältige Prüfung im Einzelfall verlangt.761 754 De lege lata explizit Art. 8 Abs. 5 DSG. 755 BSK DSG-Gramigna/Maurer-Lambrou, Art. 8 N 49; SHK DSG-Rudin, Art. 8 N 35. 756 BSK DSG-Gramigna/Maurer-Lambrou, Art. 8 N 49; SHK DSG-Rudin, Art. 8 N 50. Vgl. in diesem Zusammenhang Art. 25a f. E-DSG, wo das «Recht auf Datenherausgabe und -übertragung» (sog. Datenportabilität) geregelt ist. Darauf soll nicht weiter eingegangen werden; vgl. dazu Amtl. Bull. NR 2019 S. 1813 (Votum Glättli) und S. 1816 (Votum BR Keller-Sutter). 757 Der Zusatz «als solche» wurde vom NR eingefügt und sollte gemäss Kommissionsmehrheit die Verständlichkeit der Bestimmung verbessern; dieses Ziel wurde damit aber nicht erreicht (ebenso Amtl. Bull. NR 2019 S. 1813 f. [Votum Flach]). 758 Die weiteren, in lit. d bis g genannten Informationen sind für die vorliegende Aus einandersetzung kaum von Bedeutung, weshalb hier auf eine Vertiefung derselben verzichtet wird. Weiterführend Botschaft Totalrevision DSG, S. 7067 f. 759 Botschaft DSG 1988, S. 453; Epiney/Fasnacht, in: Belser/Epiney/Waldmann, § 11 Rn. 36; BSK DSG-Gramigna/Maurer-Lambrou, Art. 8 N 51. 760 Epiney/Fasnacht, in: Belser/Epiney/Waldmann, § 11 Rn. 36. 761 Vgl. BSK DSG-Gramigna/Maurer-Lambrou, Art. 8 N 51. 104 Teil 3: Bestandesaufnahme (1): Der E-DSG 5.2 Einschränkungen Art. 24 Abs. 1 E-DSG sieht vor, dass der Verantwortliche die Auskunft verweigern, einschränken oder aufschieben kann, wenn ein Gesetz im formellen Sinn dies vorsieht, namentlich um ein Berufsgeheimnis zu schützen (lit. a), dies aufgrund überwiegender Interessen Dritter erforderlich ist (lit. b) oder das Auskunftsgesuch offensichtlich unbegründet ist, namentlich wenn es einen datenschutzwidrigen Zweck verfolgt oder offensichtlich querulatorisch ist (lit. c). Darüber hinaus kann ein privater Verantwortlicher gemäss Art. 24 Abs. 2 lit. a E-DSG die Auskunft verweigern, einschränken oder aufschieben, wenn «überwiegende Interessen des Verantwortlichen die Massnahme [erfordern]». Werden von einem Privaten mit einer Drohne Daten bearbeitet, so ist regelmässig keiner der vorgenannten Tatbestände erfüllt.762 Zunächst ist kaum eine Situation denkbar, in welcher überwiegende Interessen des Drohnenbetreibers als Verantwortlicher oder von Dritten der Auskunftserteilung entgegenstehen würden. Ausserdem ist kein Gesetz im formellen Sinn ersichtlich, das einschlägig wäre. Der Tatbestand des offensichtlich unbegründeten Auskunftsgesuchs ist sodann eng auszulegen und nicht leichthin anzunehmen.763 Aufgrund der Tatsache, dass das Auskunftsrecht bereits bei blosser Neugier und ohne spezifischen Interessennachweis zu erteilen ist,764 kommt eine Verweigerung oder eine Einschränkung der Auskunft wegen offensichtlicher Unbegründetheit grundsätzlich nur in Ausnahmefällen infrage, etwa bei unzulässiger Beweisausforschung im Vorfeld eines Prozesses.765 Querulatorische Auskunftsersuchen liegen dann vor, wenn diese z.B. häufig wiederholt werden, ohne dass dies schlüssig begründet würde, oder wenn sich diese an einen Verantwortlichen richten, von dem die gesuchstellende Person bereits weiss, dass er keine Daten über sie bearbeitet.766 762 Auch die in Art. 25 E-DSG vorgesehenen Einschränkungen des Auskunftsrechts für Medien sind für die vorliegende Auseinandersetzung nicht von Bedeutung. Aus diesem Grund wird nicht weiter darauf eingegangen. Vgl. dazu Botschaft Totalrevision DSG, S. 7070 sowie (zum materiell übernommenen alten Recht) Meier, Rn. 1164 ff. 763 Botschaft Totalrevision DSG, S. 7069. Dieser Umstand wird durch die Verwendung des Begriffs «offensichtlich» im Gesetzestext unterstrichen. 764 Vgl. dazu vorne, S. 101 f. 765 Botschaft Totalrevision DSG, S. 7069. Die Beweisausforschung lässt sich nicht klar von einer rechtsmissbräuchlichen Verwendung des Auskunftsrechts trennen. 766 Botschaft Totalrevision DSG, S. 7069. § 3 Zentrale Bestimmungen des E-DSG 6. 105 Zeitpunkt der Auskunft Das Auskunftsrecht kann ohne zeitliche Beschränkung, d.h. jederzeit, ausgeübt werden.767 Es ist unverjährbar.768 Die Auskunft ist in der Regel innert 30 Tagen seit Eingang des Auskunftsbegehrens zu erteilen (vgl. Art. 23 Abs. 7 E-DSG).769 Das Auskunftsrecht ist zudem unverzichtbar; das Gesetz hält dies in Art. 23 Abs. 5 E-DSG explizit fest, indem es bestimmt, dass niemand im Voraus auf das Auskunftsrecht verzichten kann.770 Es ist aber zulässig, dass eine betroffene Person gegenüber einem Verantwortlichen auf die (erneute) Ausübung des Auskunftsrechts verzichtet oder aber ein Gesuch wieder zurückzieht.771 7. Sanktionierung Die Verletzung von Auskunftspflichten ist gemäss Art. 54 Abs. 2 lit. a E-DSG sank tionsbewehrt. So werden private Personen auf Antrag mit Busse bis zu 250’000 Franken bestraft, die ihre Pflichten nach den Art. 17, 19 und 23–25 E-DSG verletzen, indem sie vorsätzlich eine falsche oder unvollständige Auskunft erteilen. 8. Bedeutung bei der drohnengestützten Datenbearbeitung durch Private Wie im Rahmen der Untersuchung der Informationspflicht festgestellt, ist es bei der drohnengestützten Datenbearbeitung unwahrscheinlich, dass die Verantwortlichen derselben nachkommen.772 Das Auskunftsrecht, als Ergänzung der Informationspflicht und als Kontrollrecht der betroffenen Person, ist somit von umso grösserer Bedeutung. Aus den vorangehenden Ausführungen erhellt jedoch, dass auch das Auskunftsrecht in diesem Kontext die ihm zugedachte Funktion nicht erfüllen kann. Denn ist der oder die Verantwortliche nicht zu identifizieren, so ist bereits das Stellen eines Auskunftsbegehrens ausgeschlossen. Dieser Umstand fällt besonders ins Gewicht, da bei der drohnengestützten Datenbearbeitung nachweislich ein Transparenzbedürfnis zu Art und Zweck derselben ausgelöst wird.773 767 Epiney/Fasnacht, in: Belser/Epiney/Waldmann, § 11 Rn. 21 m.w.H. 768 SHK DSG-Rudin, Art. 8 N 30 m.w.H.; vgl. auch BSK DSG-Gramigna/Maurer-Lambrou, Art. 8 N 61. 769 Soll die Auskunft beschränkt werden, so sollte der begründete Entscheid über die Beschränkung desselben innert gleicher Frist ergehen (vgl. Art. 8 Abs. 4 VDSG). 770 Diese Bestimmung wurde aus dem DSG, namentlich dessen Art. 8 Abs. 6, übernommen. 771 Weiterführend dazu Epiney/Fasnacht, in: Belser/Epiney/Waldmann, § 11 Rn. 42; BSK DSG-Gramigna/Maurer-Lambrou, Art. 8 N 61, je m.w.H. 772 Vgl. vorne, S. 98. 773 Vgl. zu den diesbezüglichen Studien vorne, S. 5 mit den Hinweisen in Fn. 34. 106 Teil 3: Bestandesaufnahme (1): Der E-DSG VI. Rechte bei Persönlichkeitsverletzungen Die in Art. 26 bis 28 E-DSG enthaltenen Vorschriften entsprechen weitgehend, mit einigen redaktionellen Änderungen, den heute geltenden Art. 12, 13 und 15 DSG.774 Inhaltlich konkretisieren diese Normen die in Art. 28 ff. ZGB geregelten Ansprüche bei Verletzungen der Persönlichkeit und allfällige Rechtfertigungsgründe im Kontext des Datenschutzrechts.775 1. Persönlichkeitsverletzung Wer Personendaten bearbeitet, darf die Persönlichkeit der betroffenen Personen nicht widerrechtlich verletzen (Art. 26 Abs. 1 E-DSG). Eine Persönlichkeitsverletzung liegt gemäss der Auflistung in Abs. 2 insbesondere vor, wenn die Datenbearbeitungsgrundsätze776 nicht eingehalten werden (lit. a).777 Daneben sind auch die Bearbeitung entgegen der ausdrücklichen Willens erklärung778 der betroffenen Person sowie die Bekanntgabe an Dritte von besonders schützenswerten Personendaten explizit erwähnt (lit. b und c). Die Liste ist — wie sich bereits aus der Verwendung des Begriffs «insbesondere» ergibt — als nicht abschliessend zu verstehen.779 Bei der drohnengestützten Datenbearbeitung durch Private werden regelmässig Datenbearbeitungsgrundsätze nicht eingehalten, wobei der Grundsatz der Erkennbarkeit im Vordergrund steht.780 In diesen Fällen ist gemäss Art. 26 Abs. 2 lit. a E-DSG grundsätzlich eine Persönlichkeitsverletzung gegeben; diese kann jedoch möglicherweise gerechtfertigt sein.781 Die zu Art. 12 Abs. 2 lit. a DSG geführte Diskussion, ob eine Rechtfertigung einer Persönlichkeitsverletzung aufgrund der Verletzung eines Datenbearbeitungsgrundsatzes überhaupt möglich ist,782 dürfte mit dem E-DSG erledigt 774 Botschaft Totalrevision DSG, S. 7070 f.; vgl. auch Rosenthal, Entwurf, Rn. 33 f. und 117. 775 BGE 136 II 508 E. 6.3.2; 127 III 481 E. 3 a/bb; Aebi-Müller, Rn. 545; Meier, Rn. 1517 ff.; Botschaft Totalrevision DSG, S. 7071; vgl. auch Schweizer M. Rn. 354 ff. 776 Ausführlich zu den Bearbeitungsgrundsätzen vorne, S. 82 ff. 777 Vgl. dazu anschaulich BSK DSG-Rampini, Art. 12 N 9. 778 Vgl. zur Bedeutung dieses Widerspruchsrechts im Kontext der informationellen Selbstbestimmung Handkomm. DSG-Rosenthal, Art. 4 N 66. 779 Botschaft Totalrevision DSG, S. 7072; vgl. SHK DSG-Wermelinger, Art. 12 N 4; BSK DSG-Rampini, Art. 12 N 7; Aebi-Müller, Rn. 574. 780 Ausführlich dazu vorne, S. 85 f. 781 Zu den Rechtfertigungsgründen sogleich, S. 108 f. 782 Vgl. dazu BGE 136 II 508 E. 5.3.1 ff.; BSK DSG-Rampini, Art. 12 N 9b. § 3 Zentrale Bestimmungen des E-DSG 107 sein.783 Eine Rechtfertigung der Bearbeitung von Personendaten entgegen den allgemeinen Bearbeitungsgrundsätzen ist demnach nicht generell ausgeschlossen; die Rechtfertigungsgründe dürfen aber im konkreten Fall nur mit grosser Zurückhaltung bejaht werden.784 Da die Aufnahmen von Personen und Grundstücken mithilfe von Drohnen besonders schützenswerte Personendaten darstellen können,785 kommt — bei deren Bekanntgabe — auch der Regelung von Art. 26 Abs. 2 lit. c E-DSG Gewicht zu. Eine solche Bekanntgabe, etwa eine Weitergabe an Dritte, bedarf somit in jedem Fall eines Rechtfertigungsgrundes,786 ansonsten sie eine widerrecht liche Persönlichkeitsverletzung darstellt. Von geringerer Bedeutung ist jedoch Art. 26 Abs. 2 lit. b E-DSG. Gemäss dieser Bestimmung liegt eine Persönlichkeitsverletzung vor, wenn Personendaten entgegen der ausdrücklichen Willenserklärung der betroffenen Person bearbeitet werden. Ein solches Verbot kann jedoch nicht pauschal ausgesprochen werden; es muss vielmehr gegenüber bestimmten Verantwortlichen und bezogen auf bestimmte Daten und Bearbeitungen verhängt werden.787 Bei der drohnengestützten Datenbearbeitung durch Private dürfte eine entsprechende «ausdrückliche» Willenserklärung der betroffenen Personen kaum einmal vorliegen bzw. kaum vom allfälligen Bearbeiter zur Kenntnis genommen werden.788 Anders würde es sich nur verhalten, wenn etwa die betroffene Person die Drohne sowie deren Betreiber identifizieren, diesen direkt ansprechen und eine allfällige Datenbearbeitung explizit verbieten kann. Keine Persönlichkeitsverletzung liegt in der Regel vor, wenn die betroffene Person die Personendaten allgemein zugänglich gemacht und eine Bearbeitung nicht ausdrücklich untersagt hat (Art. 26 Abs. 3 E-DSG). Ein «allgemeines Zugänglichmachen» von Daten setzt voraus, dass dies in den Medien 783 Vgl. Botschaft Totalrevision DSG, S. 7072: «In Buchstaben b und c [von Art. 26 Abs. 2 E-DSG] wurde die Bezugnahme auf den Rechtfertigungsgrund entfernt, wie dies bei der Revision im Jahre 2003 bereits für Buchstabe a [von Art. 12 Abs. 2 DSG] erfolgte. Auch dies dient lediglich der Klarheit und entspricht Artikel 28 ZGB, in dem die Verletzung der Persönlichkeit und die Rechtfertigungsgründe ebenfalls in zwei Teilbestimmungen behandelt werden.» 784 BGE 136 II 508 E. 5.2.4; 138 II 346 E. 7.2; vgl. BSK DSG-Rampini, Art. 12 N 9b in fine; ausführlich zur Kontroverse Handkomm. DSG-Rosenthal, Art. 12 N 16 ff. 785 Vgl. dazu vorne, S. 33 ff. 786 Vgl. BSK DSG-Rampini, Art. 12 N 14; Handkomm. DSG-Rosenthal, Art. 12 N 44. Zu den Rechtfertigungsgründen sogleich, Rn. 298 ff. 787 Botschaft Totalrevision DSG, S. 7071 f.; BSK DSG-Rampini, Art. 12 N 11; Handkomm. DSG-Rosenthal, Art. 12 N 31. 788 Vgl. Botschaft Totalrevision DSG, S. 7071 f.; BSK DSG-Rampini, Art. 12 N 11; SHK DSG-Wermelinger, Art. 12 N 6 f., der vom «Empfang des Verbots» spricht. Vgl. sodann ausführlich Meier, Rn. 1553 ff. 108 Teil 3: Bestandesaufnahme (1): Der E-DSG oder im Internet geschieht;789 so sind sie für «eine unbestimmte Zahl von Personen […] ohne wesentliche Hindernisse» zugänglich.790 Für die vorliegende Auseinandersetzung ist dieser Ausschluss unbeachtlich. 2. Rechtfertigungsgründe Gemäss Art. 27 Abs. 1 E-DSG ist eine Persönlichkeitsverletzung widerrechtlich, wenn sie nicht durch Einwilligung der betroffenen Person, durch ein überwiegendes privates oder öffentliches Interesse oder durch Gesetz gerechtfertigt ist. Diese Bestimmung entspricht der Regelung in Art. 28 Abs. 2 ZGB. Die Rechtfertigungsgründe des Gesetzes sowie der Einwilligung der betroffenen Person finden im Zusammenhang mit der drohnengestützten Datenbearbeitung durch Private kaum einmal Anwendung.791 Zunächst ist kein Gesetz ersichtlich, welches in diesem Kontext eine Datenbearbeitung rechtfertigen würde. Vorausgesetzt wäre dafür, dass der Gesetzgeber eine Datenbearbeitung «ausdrücklich fordert, erlaubt oder zumindest stillschweigend voraussetzt».792 Des Weiteren darf auch das Vorliegen einer wirksamen Einwilligung regelmässig verneint werden.793 Eine solche ist gemäss Art. 5 Abs. 6 E-DSG nur gültig, wenn sie für eine oder mehrere bestimmte Bearbeitungen nach angemessener Information freiwillig erteilt wird. Bei drohnengestützten Datenbearbeitungen durch Private sind nur wenige Situationen vorstellbar, in welcher diese Voraussetzungen erfüllt sind.794 Art. 27 Abs. 2 E-DSG enthält eine nicht abschliessende Liste von Fällen, in denen ein überwiegendes Interesse des Verantwortlichen als Rechtfertigungsgrund in Betracht fällt.795 So werden u.a. der Abschluss und die Durchführung eines Vertrags (lit. a), die Überprüfung der Kreditwürdigkeit (lit. c), 789 Meier, Rn. 1573; BSK DSG-Rampini, Art. 12 N 16, erwähnt zusätzlich öffentliche Veranstaltungen; vgl. SHK DSG-Wermelinger, Art. 12 N 10. 790 Handkomm. DSG-Rosenthal, Art. 12 N 54. Des Weiteren ist vorausgesetzt, dass dies mit Wissen und Willen der betroffenen Person geschieht (Handkomm. DSG-Rosen thal, Art. 12 N 55 f.). 791 Vgl. zur grundsätzlichen Bedeutung der Einwilligung als Rechtsfertigungsgrund SHK DSG-Baeriswyl, Art. 4 N 54; BSK DSG-Rampini, Art. 13 N 3. 792 SHK DSG-Wermelinger, Art. 13 N 15; vgl. BSK DSG-Rampini, Art. 13 N 15 und N 18 (mit Beispielen); Handkomm. DSG-Rosenthal, Art. 13 N 24. 793 Vgl. allgemein zur Einwilligung vorne, S. 87 f. 794 In Betracht kommt z.B. ein Hochzeitsfotograf, der die gesamte Hochzeitsgesellschaft vorab informiert (bzw. informieren lässt) und anschliessend mit einer Drohne Fotos schiesst. Die diesbezüglichen luftfahrtrechtlichen Vorgaben sollen hier ausser Acht gelassen werden. 795 Ausführlich zu den Einzelnen Tatbeständen Botschaft Totalrevision DSG, S. 7073 ff.; Meier, Rn. 1631 ff. § 3 Zentrale Bestimmungen des E-DSG 109 die Veröffentlichung im redaktionellen Teil eines periodisch erscheinenden Mediums (lit. d) oder die Verwendung zu Zwecken der Forschung, Planung oder Statistik (lit. e) genannt. Die aufgeführten Tatbestände sind im Zusammenhang mit der drohnengestützten Datenbearbeitung durch Private kaum einschlägig. Persönlichkeitsverletzungen können damit nicht gerechtfertigt werden. Handelt es sich bei der betroffenen Person um «eine Person des öffentlichen Lebens», d.h. eine (absolute oder relative) Person der Zeitgeschichte wie z.B. Politiker, Profisportler, Musiker, Schauspieler etc.,796 könnte u.U. Art. 27 Abs. 2 lit. f E-DSG zur Anwendung kommen. Gemäss dieser Bestimmung fällt ein überwiegendes Interesse des Verantwortlichen in Betracht, wenn dieser «Personendaten über eine Person des öffentlichen Lebens [sammelt], die sich auf das Wirken dieser Person in der Öffentlichkeit beziehen». Für den hier interessierenden Kontext ist ein so geartetes überwiegendes Interesse wohl in den allermeisten Fällen zu verneinen, da sich die drohnengestützte Datenbearbeitung auf das «Wirken der Person in der Öffentlichkeit» beziehen müsste.797 Ein überwiegendes privates Interesse des Verantwortlichen, das nicht im Gesetz erwähnt ist, kommt im Grundsatz ebenfalls als Rechtfertigungsgrund in Betracht. Ein solches setzt jedoch ein schützenswertes Interesse voraus, welches dasjenige der betroffenen Person überwiegt.798 Im hier interessierenden Kontext dürfte somit regelmässig keine so geartete Rechtfertigung in Betracht kommen.799 Insgesamt ist im Bereich der drohnengestützten Datenbearbeitung durch Private in den allermeisten Fällen kein Rechtfertigungsgrund gegeben. Die nicht gerechtfertigten Persönlichkeitsverletzungen bringen somit grundsätzlich die Anwendbarkeit der Ansprüche gemäss Art. 28 E-DSG mit sich. 796 Ausführlich zu den Begriffen der absoluten und relativen Person der Zeitgeschichte BGE 127 III 481 E. 2c/aa. Vgl. auch SHK DSG-Wermelinger, Art. 13 N 29; Meier, Rn. 1694. 797 Die Bestimmung wurde denn auch im Interesse von Medienschaffenden und Historikern ins DSG eingefügt (vgl. Handkomm. DSG-Rosenthal, Art. 13 N 67 m.w.H.). Sie wurde unverändert aus dem DSG in den E-DSG übernommen. 798 Ausführlich zur Beurteilung dieser Interessen Handkomm. DSG-Rosenthal, Art. 13 N 7 ff. 799 Vgl. dazu die Ausführungen zur Interessenabwägung im Zusammenhang mit der Haushaltsausnahme (vorne, S. 77). 110 3. Teil 3: Bestandesaufnahme (1): Der E-DSG Rechtsansprüche 3.1 Übersicht Art. 28 E-DSG enthält die Ansprüche, welche eine betroffene Person gegenüber privaten Verantwortlichen vor dem Zivilgericht geltend machen kann.800 Gemäss Art. 28 Abs. 2 E-DSG richten sich die Klagen zum Schutz der Persönlichkeit nach den Art. 28, 28a sowie 28g–28l ZGB.801 Darauf soll sogleich vertieft eingegangen werden. Daneben gewährt Art. 28 Abs. 1 E-DSG der betroffenen Person das Recht zu verlangen, dass unrichtige Personendaten berichtigt werden.802 Kann weder die Richtigkeit noch die Unrichtigkeit der betreffenden Personen daten festgestellt werden, so kann die klagende Partei verlangen, dass ein Bestreitungsvermerk angebracht wird (Art. 28 Abs. 3 E-DSG). Dem Berichtigungsanspruch kommt für die vorliegende Untersuchung untergeordnete Bedeutung zu, da die Richtigkeit der drohnengestützt bearbeiteten Daten kaum umstritten sein dürfte.803 Deshalb wird dieser Anspruch nicht weiter behandelt. Die betroffene Person kann zudem verlangen, dass die Berichtigung, die Löschung oder die Vernichtung, das Verbot der Bearbeitung oder der Bekanntgabe an Dritte, der Bestreitungsvermerk oder das Urteil Dritten mitgeteilt oder veröffentlicht wird (Art. 28 Abs. 4 E-DSG). 3.2 Klagen zum Schutz der Persönlichkeit Die datenschutzrechtlichen Klagen «richten sich nach» den Bestimmungen des ZGB (Art. 28 Abs. 2 E-DSG). Letztere sind folglich nicht unbesehen auf das Datenschutzgesetz zu übertragen; vielmehr ist kurz auf das Zusammenspiel zwischen den beiden Gesetzen einzugehen. Zunächst enthält Art. 28 Abs. 1 ZGB den Grundsatz, dass, wer in seiner Persönlichkeit widerrechtlich verletzt wird, zu seinem Schutz gegen jeden, der an der Verletzung mitwirkt, das Gericht anrufen kann. Abs. 2 dieser Be- 800 Die Ansprüche, welche gegenüber einem verantwortlichen Bundesorgan geltend gemacht werden können, finden sich in Art. 37 E-DSG. Vgl. dazu Botschaft Totalrevision DSG, S. 7084 ff. 801 Auf das bestehende Kosten- und Prozessrisiko kann vorliegend nicht weiter eingegangen werden; vgl. zu den diesbezüglichen Revisionsvorschlägen Botschaft Totalrevision DSG, S. 7123 f.; vgl. auch Handkomm. DSG-Rosenthal, Art. 15 N 2, und im Allgemeinen Kiener, Zugang, S. 54 f. 802 Es sei denn, eine gesetzliche Vorschrift verbietet die Änderung (lit. a) oder die Personen daten werden zu Archivzwecken im öffentlichen Interesse bearbeitet (lit. b). 803 Vgl. zur Datenrichtigkeit vorne, S. 86 f. § 3 Zentrale Bestimmungen des E-DSG 111 stimmung nennt Rechtfertigungsgründe; diese enthält der E-DSG selbst und fast gleichlautend bereits in Art. 27 Abs. 1. Als lex specialis geht die Bestimmung des E-DSG vor.804 Die Art. 28g–28l ZGB behandeln das Recht auf Gegendarstellung; sie sind für die vorliegende Untersuchung unbeachtlich.805 Art. 28a ZGB regelt die einzelnen Ansprüche, welche der betroffenen Person zur Verfügung stehen: Unterlassung (Abs. 1 Ziff. 1), Beseitigung (Abs. 1 Ziff. 2); Feststellung (Abs. 1 Ziff. 3); Schadenersatz (Abs. 3 i.V.m. Art. 41 ff. OR806); Genugtuung (Abs. 3 i.V.m. Art. 49 OR) und Gewinnherausgabe (Abs. 3 i.V.m. Art. 423 OR).807 Art. 28 Abs. 2 E-DSG seinerseits verdeutlicht die Unterlassungs- und Beseitigungsansprüche im datenschutzrechtlichen Kontext, indem er namentlich das Verbot einer bestimmten Datenbearbeitung (lit. a) und die Unter sagung einer bestimmten Bekanntgabe von Personendaten an Dritte (lit. b) explizit erwähnt.808 Daneben nennt die Bestimmung auch den Anspruch auf Löschung oder Vernichtung von Personendaten (lit. c).809 Aus Art. 26 Abs. 2 lit. a E-DSG ergibt sich — zusammen mit dem Löschungs- und Vernichtungsanspruch — des Weiteren ein einklagbares «Recht auf Vergessenwerden».810 Im Zusammenhang mit der drohnengestützten Datenbearbeitung durch Private sind dem Widerspruchs- bzw. Beseitigungsrecht sowie dem Unterlassungsanspruch zentrale Bedeutung zuzumessen. Dagegen kommt 804 Vgl. SHK DSG-Wermelinger, Art. 15 N 7. 805 Gemäss Art. 28g Abs. 1 ZGB hat, wer durch Tatsachendarstellungen in periodisch erscheinenden Medien, insbesondere Presse, Radio und Fernsehen, in seiner Persönlichkeit unmittelbar betroffen ist, Anspruch auf Gegendarstellung. Vgl. ausführlich zum Gegendarstellungsrecht statt vieler BSK ZGB I-Schwaibold, Art. 28g ff., passim. 806 Besteht zwischen dem Verantwortlichen und der betroffenen Person ein Vertrag, so können sich auch daraus Ansprüche aus Persönlichkeitsverletzung ergeben. Vom Bestehen eines entsprechenden Vertrags ist in den hier interessierenden Konstellationen nicht auszugehen. Vgl. dazu statt aller Handkomm. DSG-Rosenthal, Art. 15 N 9 f. 807 Die in Art. 28a Abs. 2 ZGB enthaltenen Ansprüche auf Mitteilung und Veröffentlichung werden in Art. 28 Abs. 4 E-DSG konkretisiert (Botschaft Totalrevision DSG, S. 7078; vgl. Poledna/Codoni, S. 274). 808 Vgl. Botschaft Totalrevision DSG, S. 7077. 809 «Der Begriff ‹Vernichten› ist stärker als der Begriff ‹Löschen› und impliziert, dass die Daten unwiederbringlich zerstört werden. Wenn die Daten auf Papier vorhanden sind, ist dieses zu verbrennen oder zu schreddern» (Botschaft Totalrevision DSG, S. 7021). Vgl. ausführlich zur Vernichtung von elektronischen Daten und deren Abgrenzung zur Löschung BVGE 2015/13 E. 3.3.4 sowie BSK DSG-Bühler, Art. 21 N 14 f. 810 Vgl. Rosenthal, Entwurf, Rn. 32; Botschaft Totalrevision DSG, S. 7077. Dieses ist in der Rechtsprechung zum zivilrechtlichen Persönlichkeitsschutz bereits seit längerer Zeit anerkannt; vgl. BGE 111 II 209 E. 3c; 122 III 449 E. 3b. 112 Teil 3: Bestandesaufnahme (1): Der E-DSG den Ansprüchen auf Schadenersatz811 und Gewinnherausgabe812 nur untergeordnete Bedeutung zu.813 Eine Genugtuungsklage käme jedoch prinzipiell in Betracht; gleiches gilt für die Feststellungsklage. 3.3 Die Ansprüche im Einzelnen a Beseitigung Gestützt auf Art. 28a Abs. 1 Ziff. 2 ZGB kann die betroffene Person verlangen, dass eine bestehende Persönlichkeitsverletzung beseitigt wird. Die Geltendmachung eines Beseitigungsanspruchs setzt voraus, dass die betroffene Person nachweist, dass die beklagte Partei ihre Daten in persönlichkeitsverletzender Weise (vgl. Art. 26 E-DSG) bearbeitet und diese Bearbeitung andauert.814 Im Rahmen des Datenschutzrechts kommen verschiedenste Arten der Beseitigung in Betracht. So kann neben der Löschung und Vernichtung (Art. 28 Abs. 2 lit. c E-DSG) auch ein Verbot der (weiteren) Bearbeitung und Bekanntgabe (vgl. Art. 28 Abs. 2 lit. a und b E-DSG) verlangt werden.815 Des Weiteren kann die betroffene Person etwa die Herausgabe, das Unbrauchbarmachen der Daten oder die Entfernung derselben von sozialen Netzwerken anbegehren.816 Unter dem Titel der Beseitigung kann zudem das Recht auf Auskunft gemäss Art. 23 E-DSG gerichtlich durchgesetzt werden.817 Wird ein Herausgabeanspruch geltend gemacht, so kann — im Rahmen der Verhältnismässigkeit — vom Verantwortlichen u.U. verlangt werden, dass er Datenträger wie USB-Sticks oder externe Speicherkarten wie z.B. SDKarten, 811 Eine drohnengestützte Datenbearbeitung durch Private wird regelmässig keinen Vermögensschaden im Sinne des Obligationenrechts mit sich bringen (zum Schadens begriff statt aller BSK OR I-Kessler, Art. 41 N 3 ff.). Aus diesem Grund wird auf eine weitere Vertiefung des Schadenersatzanspruchs verzichtet. Vgl. ausführlich und mit datenschutzrechtlichen Anwendungsbeispielen BSK DSG-Rampini, Art. 15 N 20 f.; Meier, Rn. 1782 ff. 812 Für den hier interessierenden Zusammenhang ist kaum ein Beispiel erdenklich, in welchem der Verantwortliche einen Gewinn erzielen würde. Die praktische Bedeutung dieses Anspruchs ist im Datenschutzrecht und allgemein im Kontext des Persönlichkeitsschutzes beschränkt (Poledna/Codoni, S. 276; Meier, Rn. 1788). Vgl. aber die Anwendungsbeispiele in BGE 133 III 153 und BGE 143 III 297. 813 Detailliert zu diesen Ansprüchen und deren Geltendmachung Meier, Rn. 1774 ff. mit zahlreichen Hinweisen. 814 BSK DSG-Rampini, Art. 15 N 9. Ausführlich zu den einzelnen Voraussetzungen Meier, Rn. 1731 ff. 815 Vgl. ausführlich dazu Handkomm. DSG-Rosenthal, Art. 15 N 28 ff. 816 Vgl. BSK DSG-Rampini, Art. 15 N 7 f.; SHK DSG-Wermelinger, Art. 15 N 9 ff.; Meier, Rn. 1744. 817 SHK DSG-Wermelinger, Art. 15 N 7 und N 11; Poledna/Codoni, S. 277. Vgl. Art. 243 Abs. 2 lit. d ZPO (Geltung des vereinfachten Verfahrens für diese Klagen). § 3 Zentrale Bestimmungen des E-DSG 113 auf welchen die strittigen Daten gespeichert sind, herausgibt.818 Trägt eine Drohne eine Kamera, so kommt daher grundsätzlich auch in Betracht, dass die Speicherkarte derselben her ausgefordert wird. Dabei gilt zu beachten, dass — wenn Daten verschiedener Personen darauf gespeichert sind — nicht durch die Herausgabe neuerliche Persönlichkeitsverletzungen begangen werden. Bezieht sich ein Beseitigungsanspruch auf die Löschung bzw. Vernichtung von elektronisch gespeicherten Personendaten, so geht es um einen technisch komplexen Vorgang.819 Vorausgesetzt ist, dass allfällige Kopien der Dateien in Clouds820 oder auf externen Festplatten erfasst werden (können).821 Die physische Zerstörung von Datenträgern soll vom Gericht nur als ultima ratio angeordnet werden.822 Sind Personendaten sodann im Internet, z.B. auf sozialen Netzwerken, gespeichert, führt dies oftmals dazu, dass diese auch nach einer Löschung auf der «Ursprungsseite» anderswo noch auffindbar bleiben.823 b Unterlassung Eine drohende oder die Wiederholung einer bereits begangenen Verletzung kann mit dem Unterlassungsanspruch unterbunden werden (vgl. Art. 28a Abs. 1 Ziff. 1 ZGB und Art. 28 Abs. 2 lit. a und b E-DSG). Unter diesem Titel kann etwa verlangt werden, dass eine unmittelbar bevorstehende bzw. beabsichtigte Bekanntgabe von Daten an Dritte oder Veröffentlichung von Daten (z.B. in sozialen Medien) untersagt wird.824 Die Geltendmachung des Unterlassungsanspruchs verlangt, dass die betroffene Person die Gefahr einer drohenden Persönlichkeitsverletzung nachweist. Ein Unterlassungsbegehren setzt mithin «ein hinreichendes Rechtsschutzinteresse voraus, das besteht, wenn die widerrechtliche Handlung unmittelbar droht, d.h., wenn das Verhalten des Beklagten die künftige Rechtsverletzung ernsthaft befürchten lässt.»825 Aus datenschutzrechtlicher 818 BSK DSG-Rampini, Art. 15 N 7 in fine; zurückhaltend Handkomm. DSG-Rosenthal, Art. 15 N 33; vgl. BSK ZGB I-Meili, Art. 28a N 5. 819 SHK DSG-Wermelinger, Art. 15 N 10 mit Hinweis auf PK IDG/BS-Rudin, § 16 N 13 ff.; vgl. auch BSK DSG-Bühler, Art. 21 N 14, und zu technischen Aspekten Burris, passim. 820 Cloud-Computing «ist eine weitgefasste Bezeichnung für eine IT-Architektur zur verteilten Bereitstellung von IT-basierten Diensten» (ausführlich dazu Weiss, S. 1 ff.). 821 SHK DSG-Wermelinger, Art. 15 N 10; vgl. Meier, Rn. 1746. 822 Meier, Rn. 1746. 823 SHK DSG-Wermelinger, Art. 15 N 10, nennt in diesem Zusammenhang «Archiv webseiten». 824 Vgl. BSK DSG-Rampini, Art. 15 N 10. 825 BGE 124 III 72 E. 2a (zu Art. 9 Abs. 1 lit. a UWG). Ähnlich BGE 128 III 96 E. 2e (zum Markenschutzrecht). Vgl. auch Meier, Rn. 1735; SHK DSG-Wermelinger, Art. 15 N 12. 114 Teil 3: Bestandesaufnahme (1): Der E-DSG Perspektive ist hier z.B. an Fälle zu denken, in denen der Verantwortliche in der Vergangenheit bereits eine ähnlich gelagerte Persönlichkeitsverletzung begangen hat und/oder eine Verwarnung durch die betroffene Person wirkungslos blieb. Ebenso wäre ein hinreichendes Rechtsschutzinteresse zu bejahen, wenn der Verantwortliche die Unrechtmässigkeit einer Bearbeitung bestreitet, sodass sich eine Wiederholung derselben abzeichnet.826 Da Datenbearbeitungen oftmals auf Dauer angelegt sind und die ernsthafte Gefahr einer Wiederholung der Persönlichkeitsverletzung bestehen kann, lassen sich Beseitigungs- und Unterlassungsansprüche nicht immer sauber trennen bzw. drängt sich eine Kombination derselben auf.827 Das Gericht kann dem Beklagten, ggf. unter Androhung der Bestrafung nach Art. 292 StGB,828 verbieten, die fragliche Datenbearbeitung vorzunehmen.829 Da damit vorwiegend präventive Zwecke verfolgt werden, ist einem solchen Begehren nur mit grosser Zurückhaltung und unter Wahrung des Grundsatzes der Verhältnismässigkeit stattzugeben.830 Im Kontext der drohnengestützten Datenbearbeitung durch Private könnte ein Unterlassungsbegehren darauf abzielen, eine beabsichtigte Veröffentlichung von Bildern oder Videos, z.B. auf sozialen Medien, gerichtlich verbieten zu lassen. c Genugtuung Gestützt auf Art. 28a Abs. 3 ZGB i.V.m. Art. 49 Abs. 1 OR hat eine Person, die in ihrer Persönlichkeit widerrechtlich verletzt wurde, Anspruch auf Leistung einer Geldsumme als Genugtuung, sofern die Schwere der Verletzung es rechtfertigt und diese nicht anders wiedergutgemacht worden ist.831 Vorausgesetzt ist mithin eine objektiv schwere Persönlichkeitsverletzung und dass diese auch subjektiv hinreichend schwer wiegt (d.h. als «seelischer Schmerz» 826 BSK DSG-Rampini, Art. 15 N 11. 827 Meier, Rn. 1733; BSK DSG-Rampini, Art. 15 N 12; Handkomm. DSG-Rosenthal, Art. 15 N 35. 828 «Ungehorsam gegen amtliche Verfügungen»; ausführlich dazu statt aller BSK StGB-Riedo/Boner, Art. 292 N 9 ff. mit zahlreichen Hinweisen. 829 Poledna/Codoni, S. 272; SHK DSG-Wermelinger, Art. 15 N 13. 830 BSK ZGB I-Meili, Art. 28a N 2; Meier, Rn. 1741. Vgl. auch BGE 135 III 145 E. 5 ff. 831 Als Wiedergutmachung kommen z.B. die Gutheissung eines Feststellungsbegehrens und die Urteilspublikation in Betracht (so BGE 133 III 153 E. 4). «Eine Geldsumme als Genugtuung soll nur gewährt werden, wenn der Betroffene Beeinträchtigungen erlitten hat, die wegen ihrer Intensität das Mass überschreiten, was eine Person nach der heute geltenden Auffassung ohne besonderen Rechtsschutz zu erdulden hat» (Botschaft Persönlichkeitsschutz, S. 681) § 3 Zentrale Bestimmungen des E-DSG 115 empfunden wird).832 Diese Voraussetzungen sind (auch) im datenschutzrechtlichen Kontext vom Kläger nachzuweisen.833 Neben der widerrechtlichen Persönlichkeitsverletzung muss für einen Genugtuungsanspruch ein Verschulden des Verletzers834 sowie ein adäquater Kausalzusammenhang835 zwischen der persönlichkeitsverletzenden Datenbearbeitung und der erlittenen seelischen Unbill vorliegen.836 Eine Genugtuung kommt u.a. in Betracht, wenn besonders schützenswerte Personendaten unrechtmässig bearbeitet werden.837 Diese Voraussetzungen können grundsätzlich bei einer drohnengestützten Datenbearbeitung durch Private vorliegen.838 Regelmässig werden aber Daten, welche Private mithilfe einer Drohne bearbeiten (z.B. in der Form der Bekanntgabe an Dritte), keine seelische Unbill von der erforderlichen Schwere verursachen.839 d Feststellung Gestützt auf Art. 28a Abs. 1 Ziff. 3 ZGB hat eine in ihrer Persönlichkeit verletzte Person Anspruch, die Widerrechtlichkeit einer Verletzung feststellen zu lassen, wenn sich diese weiterhin störend auswirkt. Vorausgesetzt ist, dass die Verletzung bereits eingetreten ist, diese aber nicht mehr andauert.840 In diesem Fall würden sowohl der Unterlassungs- sowie der Beseitigungsanspruch ins Leere laufen. Die Feststellungsklage ist denn auch subsidiär zu diesen Ansprüchen.841 832 BSK DSG-Rampini, Art. 15 N 22; BSK ZGB I-Meili, Art. 28a N 17; Handkomm. DSG-Rosenthal, Art. 15 N 49; Hausheer/Aebi-Müller, Rn. 14.63. Vgl. BGE 143 III 297 E. 9; 129 III 715 E. 4.4; 120 II 97 E. 2. 833 Art. 8 ZGB; BGE 120 II 97 E. 2b; BGer, 4A_465/2012 vom 10. Dezember 2012 E. 3.2; SHK DSG-Wermelinger, Art. 15 N 18; BSK DSG-Rampini, Art. 15 N 22. 834 Ständige Rechtsprechung seit BGE 126 III 161 E. 5b/aa; vgl. BGE 133 III 26 E. 12.1; BSK OR IKessler, Art. 49 N 14; anders dagegen BGE 120 II 97 E. 2c (obiter). Vgl. auch BSK ZGB IMeili, Art. 28a N 17 m.w.H. 835 Handkomm. DSG-Rosenthal, Art. 15 N 49. Zum Begriff des adäquaten Kausalzusammenhangs statt aller BSK OR I-Kessler, Art. 41 N 16 ff. mit zahlreichen Hinweisen. 836 Zur Bedeutung des Verschuldens bei der Bemessung einer allfälligen Genugtuung in Form einer Geldsumme BSK ZGB I-Meili, Art. 28a N 17; BSK DSG-Rampini, Art. 15 N 24. 837 Dazu und mit weiteren Anwendungsbeispielen BSK DSG-Rampini, Art. 15 N 22; SHK DSG-Wermelinger, Art. 15 N 19. Zurückhaltend Handkomm. DSG-Rosenthal, Art. 15 N 50. 838 Zur Bearbeitung von besonders schützenswerten Personendaten durch Drohnen vgl. vorne, S. 34. 839 Vgl. die Beispiele bei SHK DSG-Wermelinger, Art. 15 N 19; BSK DSG-Rampini, Art. 15 N 22. 840 Anschaulich BSK ZGB I-Meili, Art. 28a N 6; vgl. Handkomm. DSG-Rosenthal, Art. 15 N 36. 841 Anders verhält es sich im Verhältnis zu den anderen Klagen gemäss Art. 28a Abs. 2 und 3 ZGB (BSK DSG-Rampini, Art. 15 N 15 m.w.H.). 116 Teil 3: Bestandesaufnahme (1): Der E-DSG Zur erfolgreichen Geltendmachung des Feststellungsanspruchs muss sich die (abgeschlossene) Persönlichkeitsverletzung weiterhin oder erneut störend auswirken.842 «Der Feststellungsklage kommt […] die Funktion zu, eine eingetretene Persönlichkeitsverletzung zu beseitigen. Besteht ein durch eine Verletzung in den persönlichen Verhältnissen hervorgerufener Störungszustand, nimmt das Begehren um gerichtliche Feststellung einer widerrechtlichen Persönlichkeitsverletzung eine dem Verletzten dienende Beseitigungsfunktion wahr.»843 Die Feststellungsklage steht im Vordergrund, wenn für den Kläger ideelle Überlegungen ausschlaggebend sind; diese können sodann mit dem Rechtsbegehren der Mitteilung oder der Veröffentlichung verknüpft werden.844 Ein solches Vorgehen kommt z.B. in Betracht, wenn durch die persönlichkeitsverletzende Datenbearbeitung wie eine Publikation im Internet ein «nachteiliges Vorstellungsbild in der Öffentlichkeit oder bei Dritten» entstanden ist.845 An das Feststellungsinteresse sind keine hohen Anforderungen zu stellen.846 Die infrage stehende Persönlichkeitsverletzung muss somit nicht schwer wiegen.847 Im Zusammenhang mit der drohnengestützten Datenbearbeitung durch Private käme eine Feststellungsklage demnach in Betracht, wenn etwa durch ungewollte, auf sozialen Netzwerken veröffentlichte Foto- oder Videoaufnahmen ein nachteiliges Bild der betroffenen Person bei Dritten entstanden ist. 4. Bedeutung bei der drohnengestützten Datenbearbeitung durch Private Die in diesem Abschnitt gemachten Ausführungen zeigen klar auf, dass bei der drohnengestützten Datenbearbeitung regelmässig widerrechtliche Persönlichkeitsverletzungen begangen werden, wogegen die in Art. 28 E-DSG enthaltenen Ansprüche geltend gemacht werden können. Die zur Verfügung stehenden Klagevarianten sind denn auch grundsätzlich geeignet, um bei persönlichkeitsverletzenden Datenbearbeitungen mittels Drohnen Abhilfe 842 Vgl. den Wortlaut von Art. 28a Abs. 1 lit. c; BGE 127 III 481 E. 1c/aa; SHK DSG-Wermelinger, Art. 15 N 16; Handkomm. DSG-Rosenthal, Art. 15 N 36. 843 BGer, 5A_286/2012 vom 29. Oktober 2012 E. 2.1. Vgl. auch BGE 127 III 481 E. 1c/aa und grundlegend bereits BGE 95 II 481 E. 9. 844 Vgl. SHK DSG-Wermelinger, Art. 15 N 17; Handkomm. DSG-Rosenthal, Art. 15 N 37. 845 BSK DSG-Rampini, Art. 15 N 15; Meier, Rn. 1752; vgl. BGE 122 III 449 E. 2b. 846 BSK DSG-Rampini, Art. 15 N 15; Meier, Rn. 1753; Pedrazzini, Rechtsschutz, S. 82 f.; Handkomm. DSG-Rosenthal, Art. 15 N 37. 847 BGE 127 III 481 E. 1c/bb; Meier, Rn. 1753. Vgl. auch BSK ZGB I-Meili, Art. 28a N 8 mit Hinweisen auf die uneinheitliche Praxis zu dieser Frage vor BGE 127 III 481. § 4 Gesamtbetrachtung und Überleitung 117 zu schaffen. Die betroffenen Personen werden jedoch kaum einmal wirksam eine Klage zum Schutz der Persönlichkeit einreichen können, da ihnen die Kenntnis über die Identität des Drohnenbetreibers fehlt. § 4 Gesamtbetrachtung und Überleitung Der dritte Teil der vorliegenden Untersuchung hat die drohnengestützte Datenbearbeitung im Lichte des zukünftigen Schweizer Datenschutzgesetzes beleuchtet. Wie sich gezeigt hat, kommt die Haushaltsausnahme auf die drohnengestützte Datenbearbeitung durch Private nur in Ausnahmefällen zur Anwendung. Grundsätzlich greift mithin der E-DSG und die darin vorgesehenen Rechte und Pflichten sind anwendbar.848 Der Informationspflicht bei der Beschaffung von Personendaten, welche auch bei der drohnengestützten Datenbearbeitung einzuhalten wäre, kommen die Verantwortlichen wohl in den allermeisten Fällen nicht nach.849 Doch auch die Ausübung des Auskunftsrechts ist den betroffenen Personen kaum einmal möglich.850 Es muss deshalb konstatiert werden, dass die von einer drohnengestützten Datenbearbeitung betroffenen Personen sich nicht über Identität des Bearbeiters und Zweck der Bearbeitung informieren können. Die Rechte bei Persönlichkeitsverletzungen würden es prinzipiell erlauben, gegen Drohnenbetreiber vorzugehen, welche unter Verletzung von Persönlichkeitsrechten Daten bearbeiten.851 Jedoch scheitert dieses Vorgehen an der Anonymität der oder des Verantwortlichen. Ein effektiver Rechtsschutz fehlt somit. Insgesamt steht fest, dass das vom Gesetzgeber angestrebte Ziel der einfachgesetzlichen Konkretisierung und Verwirklichung der informationellen Selbstbestimmung im E-DSG nur teilweise erreicht wird. Die vorangehend analysierten Bestimmungen weisen klare Mängel bei der Rechtsdurchsetzung auf. Es gilt deshalb nun, die Bestandesaufnahme zur drohnengestützten Datenbearbeitung und der Durchsetzung von datenschutzrechtlichen Ansprüchen mit Bezug auf das Luftfahrtrecht fortzuführen. 848 Ausführlich zum Ganzen vorne, S. 75 ff., v.a. S. 81. 849 Vgl. dazu vorne, S. 93 ff. 850 Vgl. vorne, S. 100 ff. 851 Vgl. dazu vorne, S. 106 ff. 119 Teil 4: Bestandesaufnahme (2): Die «Drohnenverordnungen» § 1 Vorbemerkungen Das Schweizer Luftfahrtrecht kennt nur wenige Vorschriften zum Datenschutz. Art. 107a und 107b LFG852 beziehen sich zum einen auf die Datenbearbeitung durch das BAZL und weitere im Bereich des Luftfahrtrechts zuständige Behörden und Organisationen.853 Zum anderen sind dort die Zugriffsrechte zum Luftfahrzeugregister geregelt; Drohnen werden darin nicht eingetragen.854 Aus diesem Grund stehen die Bestimmungen der drei Drohnenverordnungen mit ihren Bezügen zum Datenschutz im Vordergrund.855 Nachfolgend werden deshalb die Vorschriften zur Identifizierung von Drohnenbetreibern, namentlich die Registrierung (sogleich, S. 120 ff.), die Kennzeichnung (S. 135 ff.), die Fernidentifizierung (S. 138 ff.) sowie der sog. U-Space (S. 142 ff.), behandelt. Ausserdem gilt es, auf den Spezialfall der selbst gebauten Drohnen einzugehen (S. 144 ff.) sowie auf gewisse Schwierigkeiten bei der Umsetzung der Drohnenverordnungen hinzuweisen (S. 146 ff.). In einem anschliessenden Abschnitt werden einige präventive und flankierende Massnahmen in den europäischen Regelwerken in ihren Bezügen zum Datenschutz untersucht. Neben den Aufklärung- und Informationsmassnahmen (S. 148 ff.) werden unter diesem Titel auch die Themen GeoAwareness (S. 151 ff.) und Positionslichter (S. 153 ff.) analysiert.856 852 Die im Rahmen der Totalrevision des DSG vorgeschlagene Änderung des Art. 107a LFG wirkt sich inhaltlich nicht auf die vorliegende Untersuchung aus (vgl. Botschaft Totalrevision DSG, S. 7142; vgl. auch hinten, S. 172). 853 Vgl. dazu etwa BVGer, A2 401/2011 vom 6. Januar 2012 E. 4.1 ff. 854 Vgl. dazu bereits vorne, S. 3 f., sowie insbesondere die Ausführungen hinten, S. 131 ff. 855 Vgl. zu den drei Verordnungen vorne, S. 13 ff. 856 Auf eine Analyse von weiteren Aspekten der Drohnenverordnungen wird verzichtet. 120 Teil 4: Bestandesaufnahme (2): Die «Drohnenverordnungen» Zusammen mit den Resultaten der Bestandesaufnahme zum E-DSG857 ergeben die hier gemachten Ausführungen ein Gesamtbild, welches in einem nachfolgenden Schritt anhand der in Teil 2 herausgeschälten grundrecht lichen Vorgaben beurteilt werden kann.858 § 2 Identifizierung von Drohnenbetreibern I. Registrierung 1. Zentrale Bedeutung der Registrierung Die Registrierung von Drohnen und deren Betreibern ist in den allermeisten Fällen859 ein zentraler und notwendiger Schritt zur Identifizierung des jeweiligen Drohnenbetreibers860 oder piloten.861 Gleichzeitig handelt es sich jedoch «nur» um den ersten Schritt; zusätzlich muss eine Identifikation der Drohne aus der Ferne möglich sein.862 Die Registrierung ist mithin für die Rechtsdurchsetzung — gerade auch im Bereich des Datenschutzrechts — von zentraler Bedeutung.863 2. Registrierungspflicht für Drohnen und Betreiber Die Grundlage der Registrierungspflicht findet sich in Art. 57 lit. d und in Anhang IX Ziff. 4 EASA-Grundverordnung sowie den darauf basierenden Regeln in der Durchführungsverordnung. In Anhang IX Ziff. 4.1 schreibt die EASA- Grundverordnung vor, dass «unbemannte Luftfahrzeuge, für deren Konstruktion eine Zertifizierung gemäss Artikel 56 Absatz 1 erforderlich ist, im Einklang mit den in Artikel 57 genannten Durchführungsrechtsakten registriert [werden].»864 Die Betreiber von Drohnen werden registriert, wenn (a) das Luftfahrzeug bei einem Zusammenstoss kinetische Energie von mehr als 80 Joule auf 857 Vgl. vorne, S. 73 ff., v.a. S. 117. 858 Hinten, S. 157 ff. 859 Je nach Entfernung zur Drohne und der Einsehbarkeit des Geländes kann der Drohnenbetreiber ggf. visuell und durch direkte (persönliche) Kontaktaufnahme identifiziert werden. Vgl. dazu auch hinten, S. 160. 860 Vgl. zu den Begriffen «Pilot» und «Betreiber» ausführlich vorne, S. 27 f. 861 Grest, S. 120 f.; Delise, S. 25; Hänsenberger, AJP 2017, S. 164; Hänsenberger, Diss., S. 207 und S. 231 f.; Krumm, Anpassungsbedarf, S. 120; Schubert, S. 232. 862 Dazu ausführlich hinten, S. 138 ff. 863 Burger/Scuderi, S. 89 in fine. Vgl. auch EASA, Opinion, S. 27; Leissler, S. 536. 864 Es geht mithin um die Registrierung der Drohne an sich; vgl. zur Definition des unbemannten Luftfahrzeugs vorne, S. 20 f. § 2 Identifizierung von Drohnenbetreibern 121 einen Menschen übertragen kann,865 wenn (b) sein Betrieb mit Risiken für die Privatsphäre, den Schutz von personenbezogenen Daten,866 die Sicherheit oder die Umwelt verbunden ist oder wenn (c) für dessen Konstruktion eine Zertifizierung gemäss Art. 56 Abs. 1 EASA-Grundverordnung erforderlich ist.867 3. Die Kriterien im Einzelnen 3.1 Zertifizierung der Konstruktion Die Formulierung von Art. 56 Abs. 1 EASA-Grundverordnung weicht vom Wortlaut der Bestimmungen in Anhang IX Ziff. 4.1 und Ziff. 4.2 lit. c EASA- Grundverordnung ab. Erstere Vorschrift erwähnt nämlich keine «Zertifizierung», sondern «eine Zulassung/ein Zeugnis». Diese terminologische Besonderheit in Art. 56 Abs. 1 EASA-Grundverordnung lässt sich dadurch erklären, dass die darin aufgelisteten Sachverhalte («Konstruktion», «Herstellung», «Propeller», «Personal, einschliesslich Fernpilot» etc.) nicht alle (sinnvoll) vom Begriff der «Zertifizierung» erfasst werden können.868 Die Formulierung «Zulassung/Zeugnis» bezweckt somit die Erfassung aller aufgelisteten Sachverhalte, so auch die Zertifizierung der Konstruktion gemäss Anhang IX Ziff. 4.1 EASA-Grundverordnung.869 Die Zertifizierung der Konstruktion steht denn auch im Zentrum der Registrierungspflicht.870 Angesprochen sind damit Drohnen der zulassungspflichtigen Kategorie.871 Das Risiko, das von diesen Drohnen und deren Betrieb ausgeht (etwa, weil sie für die Beförderung von Menschen 865 Joule ist die SI-Einheit für Energie. Vgl. dazu ausführlich sogleich, S. 122 f. 866 Vgl. zum Begriff die Hinweise vorne, Fn. 575. 867 Anhang IX Ziff. 4.2 EASA-Grundverordnung; vgl. Josipovic, S. 440. 868 Es wäre unüblich, von einer «Zertifizierung» als Pilot zu sprechen; vielmehr wird in diesem Zusammenhang von einer «Zulassung» oder einer «Lizenz» gesprochen (vgl. z.B. Art. 3 lit. c Durchführungsverordnung). 869 Vgl. zur Änderung des Wortlauts von Art. 56 Abs. 1 EASA-Grundverordnung während des Gesetzgebungsverfahrens auch Rat der Europäischen Union, Vorschlag Dezember 2015, S. 48 sowie Rat der Europäischen Union, Vorschlag Dezember 2016, S. 97 (jeweils zu Art. 46 des ursprünglichen Vorschlags). 870 EASA, Opinion, S. 16, verweist denn auch auf das sog. Lufttüchtigkeitszeugnis (Certi�ficate of Airworthiness, CofA). 871 Vgl. die Marginalie zu Art. 14 Durchführungsverordnung «Registrierung […] zulassungspflichtiger UAS»; vgl. sodann zum Begriff vorne, S. 23 f. Unter gewissen Umständen können auch Drohnen der «speziellen» Kategorie eine solche Zulassung benötigen (vgl. EASA, Opinion, S. 30); auf weitere technische Ausführungen dazu wird verzichtet. 122 Teil 4: Bestandesaufnahme (2): Die «Drohnenverordnungen» konstruiert sind872), erfordert eine eingehende Prüfung derselben durch die zuständige Behörde.873 Die unter dem Aspekt der Zertifizierung der Konstruktion erfassten Drohnen und deren Betreiber sind für die vorliegende Auseinandersetzung vernachlässigbar. Wie bereits aufgezeigt, sind die von Privaten betriebenen Drohnen regelmässig der offenen Kategorie zuzuordnen; deren Konstruktion bedarf keiner Zertifizierung.874 3.2 Kinetische Energie a Begriff und Bedeutung Die Formel für kinetische Energie lautet «halbe Masse mal Geschwindigkeit im Quadrat» (½ m × v2). Der Geschwindigkeit kommt folglich eine viel grössere Bedeutung zu als der Masse.875 Das Abstellen auf die kinetische Energie, welche im Fall einer Kollision beim Aufprall von einer Drohne übertragen werden kann, leuchtet ohne Weiteres ein. Im Strassenverkehrsrecht wird die Betriebsgefahr ebenfalls anhand der kinetischen Energie beurteilt.876 Die Grenze von 80 Joule kinetischer Energie als Anknüpfungspunkt für die Registrierung soll anhand der folgenden Beispiele illustriert werden: Eine 1 kg schwere Drohne, die mit einer Geschwindigkeit von 45 km/h aufschlägt, überträgt eine kinetische Energie von 78,125 Joule.877 Die gleiche kinetische Energie überträgt eine Drohne mit einer Masse von 250 g, die mit einer Geschwindigkeit von 90 km/h aufschlägt.878 Ziemlich genau 80 Joule an kinetischer Energie überträgt eine Drohne mit einer Masse von 423 g, die mit einer Geschwindigkeit von 70 km/h aufschlägt. Diese Beispiele zeigen auf, dass die Schwelle von 80 Joule kinetischer Energie relativ schnell erreicht wird. Die Verletzungsgefahr von Personen am Boden879 rückt dadurch bei der Beurteilung der Betriebsgefahr in den 872 Art. 40 Abs. 1 lit. b Delegierte Verordnung. 873 Vgl. zu den einzelnen erfassten Drohnen Art. 40 Delegierte Verordnung. Vgl. sodann EASA, Opinion, S. 16. Eine tiefergehende Auseinandersetzung mit den einzelnen erfassten Betriebsarten kann die vorliegende Untersuchung nicht leisten. 874 Vgl. dazu vorne, S. 22. 875 Rothen, S. 52; Hänsenberger, Diss., S. 14; Schmid, S. 75. 876 OFK SVG-Giger, Art. 58 N 53a; Schaffhauser/Peter, S. 38. Vgl. auch BGer, 1C_273/2016 vom 5. Dezember 2016 E. 5.5. 877 Vgl. Josipovic, S. 440 mit Fn. 24. Vgl. zur Berechnung der kinetischen Energie beim Aufprall einer Drohne ausführlich Dalamagkidis/Valavanis/Piegl, S. 101 ff.; vgl. sodann die tabellarische Übersicht bei Hänsenberger, Diss., S. 15 f. 878 Vgl. Masutti/Tomasello, S. 75 f. 879 Die Beförderung von Menschen mit Drohnen wird in Zukunft möglich sein; vgl. vorne, S. 121, sowie Art. 6 Abs. 1 lit. b Durchführungsverordnung; Preznuk, S. 99 ff.; zurück- § 2 Identifizierung von Drohnenbetreibern 123 Vordergrund.880 Bei einer Einwirkung von kinetischer Energie im Umfang von 80 Joule auf den (ungeschützten) Kopf eines Menschen beträgt die Wahrscheinlichkeit der Todesfolge des Aufpralls ungefähr 30%.881 Umstände des Einzelfalls wie z.B. das Alter oder die Gesundheit des betroffenen Menschen bleiben unberücksichtigt; Gleiches gilt für die Orientierung der Drohne beim Aufprall (z.B. mit Propellern oder mit der Ladung voran etc.).882 Zu Recht weist Hänsenberger in diesem Zusammenhang darauf hin, dass «der Rückgriff auf objektivierte Werte für die Bestimmung des allgemeinen Betriebs risikos (…) unumgänglich» ist.883 Neben der Masse und der Geschwindigkeit wirken sich selbstverständlich auch Faktoren wie die Konstruktionsweise884 und ggf. angehängte Sensoren885 auf die konkrete Betriebsgefahr aus.886 Aufgrund mangelnder Objektivierbarkeit bleiben sie im Rahmen der Registrierungsvorschriften aber unberücksichtigt. Vielmehr sind diese Aspekte bei der Herstellung und während des Betriebs zu beachten.887 b Regelung in der Durchführungsverordnung Der Grenzwert von 80 Joule wurde mit Blick auf den Übergang von Klasse C1 zu C2888 festgelegt.889 Drohnenbetreiber müssen sich registrieren, wenn sie eine haltend Ebinger, S. 141 f. 880 Personen am Boden sind denn auch von Drohnenflügen am meisten gefährdet, da sich heute (noch) keine Personen an Bord der Drohne befinden (vgl. EASA, Explanatory Note, S. 15). 881 Aviation Rulemaking Committee, S. 8; Masutti/Tomasello, S. 75. Diese Werte variieren je nach Aufprallstudie und deren genauen Ausgestaltung; vgl. dazu auch EASA, Explanatory Note, S. 15 f.; EASA, Opinion, S. 16; Arterburn et al., S. 10 ff. und dies., Annex C S. 27 ff. 882 Vgl. Schmid, S. 75; zur Auswirkung der Orientierung der Drohne anschaulich Tegler, passim. 883 Hänsenberger, Diss., S. 16. 884 Die Frage, ob Propeller (und ggf. wie viele) vorhanden sind, kann hier von Bedeutung sein. Vgl. dazu auch die Ausführungen zur Konstruktionsweise von Drohnen vorne, S. 18 ff. 885 Sensoren, wie z.B. eine Kamera, können in der Drohne verbaut oder daran angehängt werden. Dies beeinflusst deren Einwirkung bei einem Aufprall. Auch die Dichte der Objekte wirkt sich aus. Vgl. dazu ausführlich EASA, Explanatory Note, S. 15 f. 886 EASA, Explanatory Note, S. 15 f.; daran anschliessend Hänsenberger, Diss., S. 16 f. und S. 19 ff. 887 Vgl. Teil 1 Ziff. 5, Teil 2 Ziff. 6, Teil 3 Ziff. 6 Anhang Delegierte Verordnung (u.a. soll gemäss den dortigen Vorgaben die Verletzung von Menschen während des Betriebs minimiert und sollen scharfe Kanten vermieden werden). 888 Vgl. dazu vorne, S. 24 f. 889 EASA, Opinion, S. 16 und S. 21. Vgl. auch die Berechnungsbeispiele vorne, S. 122. 124 Teil 4: Bestandesaufnahme (2): Die «Drohnenverordnungen» Drohne mit einer Startmasse890 von 250 g oder mehr betreiben, die bei einem Aufprall auf einen Menschen eine kinetische Energie von über 80 Joule891 übertragen kann.892 Innerhalb der offenen Kategorie sind demnach Betreiber von Drohnen der Klassen C2 und C3 gehalten, sich zu registrieren.893 Betreiber von Drohnen mit einer Startmasse von unter 250 g bzw. von solchen, die nur weniger als 80 Joule kinetische Energie übertragen können (d.h. solche der Produkteklasse C0 und C1), müssen sich nicht registrieren.894 Die Bestimmung ist aus Verhältnismässigkeitsüberlegungen eingefügt worden und soll Betreiber von «harmlosen» Drohnen von der Registrierungspflicht ausnehmen.895 Es mag zutreffen, dass eine Drohne mit einer Startmasse von weniger als 250 g nur eine geringe Verletzungsgefahr birgt. Die von Drohnen ausgehenden Risiken für den Datenschutz bzw. die informationelle Selbstbestimmung bestehen jedoch auch bei sehr leichten Drohnen. Diese wurden bei der Regelung der Registrierungspflicht denn auch explizit berücksichtigt. 3.3 Risiken für Privatsphäre und den Schutz von Personendaten Wenn der Betrieb einer Drohne mit Risiken für die Privatsphäre oder den Schutz von personenbezogenen Daten verbunden ist,896 so führt dies zu einer Registrierungspflicht des Betreibers.897 Prima facie stellt dies ein relativ 890 Vgl. zum Begriff «Startmasse» vorne, S. 25 mit den Hinweisen in Fn. 187. 891 Vgl. dazu vorne, S. 122 f. 892 Art. 14 Abs. 5 lit. a Ziff. i Durchführungsverordnung. 893 Art. 14 Abs. 5 lit. a Ziff. i Durchführungsverordnung. Drohnen, die 80 Joule oder mehr an kinetischer Energie übertragen können, fallen in Klasse C2 (vgl. dazu vorne, S. 25). 894 Gemäss Formulierung der Bestimmung kann auch der Betreiber einer Drohne mit einer Startmasse von mehr als 250 g auf eine Registrierung verzichten, wenn die Drohne keine kinetische Energie von 80 Joule übertragen kann. Dies hängt — wie dargelegt (vorne, S. 122 f.) — von der möglichen Geschwindigkeit der Drohne ab. Auf weitere Ausführungen hierzu wird verzichtet. 895 EASA, Opinion, S. 17; Masutti/Tomasello, S. 75. Letztere Autoren äussern sich jedoch kritisch zum festgesetzten Schwellenwert (Masutti/Tomasello, S. 78 f. mit Fn. 35). Vgl. auch EG 15 Durchführungsverordnung; Schmid, S. 77. 896 In der englischen Sprachfassung der Durchführungsverordnung ist von «privacy, and protection of personal data» die Rede. Es ist umstritten, ob «privacy» mit «Privatsphäre» treffend übersetzt wird (vgl. nur die Zusammenstellung verschiedenster Begriffsannäherungen und Definitionsversuchen zu «privacy» bei Solove/ Schwartz, S. 42 ff.). In der vorliegenden Untersuchung soll der Begriffsverwendung der Durchführungsverordnung gefolgt werden. 897 Anhang IX Ziff. 4.2 lit. b EASA-Grundverordnung; Art. 14 Abs. 1 Durchführungsverordnung. Erfasst werden sollen auch Drohnen, die ein Risiko für die Sicherheit und die Gefahrenabwehr und/oder für die Umwelt darstellen können. Darauf soll in Übereinstimmung mit dem Fokus der Untersuchung nicht weiter eingegangen werden. § 2 Identifizierung von Drohnenbetreibern 125 unklares Anknüpfungskriterium dar, denn es wird nicht angegeben, anhand welcher Faktoren solche Risiken beurteilt werden sollen. In der Durchführungsverordnung wird diesbezüglich jedoch Klarheit geschaffen. Dort heisst es, dass «Betreiber solcher unbemannte[r] Luftfahrzeuge registriert werden [sollten], die mit einem Sensor zur Erfassung personenbezogener Daten ausgestattet sind.»898 Dies bedeutet, dass bei der Beurteilung der Datenschutzrisiken (und folglich der Risiken für die informationelle Selbstbestimmung899) kein strenger Massstab anzulegen ist. Sofern eine Drohne die Fähigkeit zur Datenbearbeitung besitzt, ist deren Betrieb mit entsprechenden Risiken verbunden. Grundsätzlich müssen sich somit die Betreiber aller Drohnen, die zur Datenbearbeitung in der Lage sind,900 registrieren. 3.4 Ausnahme für «Spielzeugdrohnen» a Begriff Die Registrierungspflicht für Betreiber von Drohnen der offenen Kategorie,901 die mit einem «Sensor, der personenbezogene Daten erfassen kann», ausgerüstet sind, soll nicht gelten, wenn es sich um ein «Spielzeug» handelt.902 Diese Ausnahme muss vertieft analysiert werden, da eine Legaldefinition von Spielzeugdrohnen nicht existiert.903 Ausgangspunkt ist aufgrund der klaren Verweisung in Art. 14 Abs. 5 lit. a Ziff. ii Durchführungsverordnung das europäische Spielzeugrecht. Gemäss Art. 2 Abs. 1 Spielzeugrichtlinie sind Spielzeuge «Produkte, die — ausschliesslich oder nicht ausschliesslich — dazu bestimmt oder gestaltet sind, von Kindern unter 14 Jahren zum Spielen verwendet zu werden».904 Nach dieser Spielzeugdefinition richten sich auch die Standardised European Rules of the Air (SERA);905 danach ist ein Spielzeugluftfahrzeug «ein unbemanntes Luft898 EG 16 Durchführungsverordnung (Hervorhebung hinzugefügt); vgl. EASA, Opinion, S. 17. «Erfassen» fällt ohne Weiteres unter den weiten Begriff der «Bearbeitung»; vgl. dazu vorne, S. 33. 899 Vgl. dazu eingehend vorne, S. 63 ff. 900 Vgl. zu den Sensoren im Einzelnen vorne, S. 40 ff., zum (weiten) Begriff der Daten bearbeitung vorne, S. 33 ff. 901 Zur offenen Kategorie vorne, S. 22. 902 Art. 14 Abs. 5 lit. a Ziff. ii Durchführungsverordnung mit Verweis auf Richtlinie 2009/48/EG des Europäischen Parlaments und des Rates über die Sicherheit von Spielzeug (Spielzeugrichtlinie); vgl. auch EG 16 Durchführungsverordnung; Krumm, Rechtsrahmen, S. 484. 903 Hänsenberger, Diss., S. 136. 904 Vgl. die inhaltlich identische Legaldefinition in Art. 65 Lebensmittel- und Gebrauchsgegenständeverordnung (LGV); vgl. auch Art. 1bis Abs. 2 Spielzeugverordnung (VSS); BLV, Erläuterungen LGV, S. 20 f. 905 Diese gelten auch in der Schweiz; vgl. Anhang Ziff. 5 LVA CH–EU. 126 Teil 4: Bestandesaufnahme (2): Die «Drohnenverordnungen» fahrzeug, das ausschliesslich oder nicht ausschliesslich für den Spielgebrauch durch Kinder unter 14 Jahren konzipiert oder bestimmt ist».906 Es wird mithin auf den sog. «Spielwert»907 abgestellt, was eine Grauzone entstehen lässt. Denn «[f]ür ein Kind hat zwar praktisch alles einen Spielwert, doch fällt deshalb nicht jeder Gegenstand unter die Definition von Spielzeug.»908 In diesem Graubereich ist anhand verschiedener Kriterien im Einzelfall zu entscheiden, ob ein Spielzeug im Rechtssinn vorliegt oder nicht.909 Geht es um Drohnen, können u.a. folgende Indikatoren für die Beur teilung der Spielzeugqualität herangezogen werden:910 — der Vertrieb als Spielzeug, ausgerichtet auf Kinder (Ort, Zielgruppe der Verpackung);911 — einfache Bedienungs- und Flugfunktionen (links/rechts und auf/ab); — fehlende Fähigkeit zu komplexen Flugmanövern; — einfache Montage, wenige Komponenten; — nur kurze Maximalflugzeit (einige wenige Minuten); — Grösse (Aussendurchmesser bis ca. 50 cm). Neben den vorgenannten Kriterien wird auch regelmässig das Gewicht in die Beurteilung miteinbezogen. So wird vereinzelt bei Drohnen mit einem Gewicht von weniger als 250 g von einem Spielzeug gesprochen.912 Andere Autoren gehen bis zu einem Gewicht von «ca. 200 g» von einem Spielzeug aus.913 Auf jeden Fall sollte die Schwelle mit Blick auf das Gefährdungspotenzial auch sehr leichter Drohnen nicht allzu hoch angesetzt werden. b Fähigkeit zur Datenbearbeitung als Ausschlusskriterium? Teilweise wird bei der Beurteilung der Spielzeugqualität das Vorhandensein einer «Bild-/Video-/Ton-Aufnahme-Übertragungs-Funktion» als Kriterium 906 Art. 2 Ziff. 129a SERA. 907 Vgl. Europäische Kommission, Leitlinien, S. 17. Eine Legaldefinition des Begriffs existiert nicht. Im Schweizer Recht wird er einzig in Anhang I Ziff. 14 Spielzeugverordnung (VSS) verwendet. 908 Europäische Kommission, Leitlinien, S. 17. Vgl. auch Graumann, S. 63. 909 Vgl. Langner/Klindt/Schucht, Rn. 149 m.w.H. sowie aus luftfahrtrechtlicher Sicht Schwenk/Giemulla, Kap. 7 Rn. 12. 910 Zum Ganzen Arbeitskreis ZLS, S. 13; Schucht, S. 143; Hänsenberger, Diss., S. 137; vgl. auch Langner/Klindt/Schucht, Rn. 149 in fine. 911 Wobei — in Übereinstimmung mit der Spielzeugdefinition der Spielzeugrichtlinie — von Kindern bis 14 Jahren ausgegangen wird. 912 Voland/Qui, S. 346 mit Blick auf das deutsche Recht (vor Inkrafttreten der Drohnenverordnungen); vgl. EASA, Opinion, S. 17. 913 Hänsenberger, Diss., S. 137; Arbeitskreis ZLS, S. 13 (unter Bezugnahme auf «fliegende Spielzeuge mit Rotorblättern […] für den Aussenbereich, die für Kinder über 12 Jahren bestimmt sind»). § 2 Identifizierung von Drohnenbetreibern 127 verwendet.914 Inhaltlich ist bei dieser etwas umständlichen Formulierung die Fähigkeit gemeint, Daten zu bearbeiten. Ist eine Drohne dazu in der Lage, kann gemäss Beschluss eines deutschen Expertengremiums nicht mehr von einem Spielzeug ausgegangen werden.915 Diese Ansicht steht im Widerspruch zur Durchführungsverordnung. Letztere schliesst nicht aus, dass eine Drohne einen «Sensor, der personenbezogene Daten erfassen kann», trägt und gleichzeitig als Spielzeug im Sinne der Spielzeugrichtlinie qualifiziert werden kann.916 Betrachtet man die Ausnahmebestimmung von Art. 14 Abs. 5 lit. a Ziff. ii Durchführungsverordnung im gesamten Normgefüge, steht fest, dass eine «Bild-/Video-/Ton-Aufnahme- Übertragungs-Funktion» die Spielzeugeigenschaft nicht gänzlich auszuschliessen vermag.917 Ansonsten würde die Ausnahme ihres Anwendungsbereichs beraubt.918 Es drängt sich deshalb eine differenzierte Betrachtung des in der Ausnahmebestimmung erwähnten Sensors auf. Bei einfachen Foto- und Videokameras mit geringer Auflösung kann ein Spielzeug vorliegen. Trägt eine Drohne jedoch hochtechnische Sensoren, die etwa hochauflösende Aufnahmen erlauben,919 liegt kein Spielzeug im Rechtssinn mehr vor, da solche Technik regelmässig nicht für Kinder konzipiert ist.920 Auf jeden Fall steht fest, dass sich im Zusammenhang mit der Registrierung von «Spielzeugdrohnen» schwierige Abgrenzungsfragen stellen werden. Nach hier vertretener Ansicht sollten sich die Betreiber von allen Drohnen, die Daten bearbeiten können, in jeden Fall registrieren müssen. «Spielzeugdrohnen» könnten ansonsten den Datenschutz gefährden.921 Auch die 914 Arbeitskreis ZLS, S. 13. Eine vergleichbare Stellungnahme von Schweizer Experten oder des zuständigen Bundesamts für Lebensmittelsicherheit und Veterinärwesen existiert nicht. 915 Arbeitskreis ZLS, S. 13; wohl gl.M. Hänsenberger, Diss., S. 137; vgl. Schucht, S. 143. 916 Vgl. den Wortlaut von Art. 14 Abs. 5 lit. a Ziff. ii Durchführungsverordnung; Krumm, Rechtsrahmen, S. 484. 917 Die Maxime «lex superior derogat legi inferiori» muss umso mehr gelten, wenn es sich bei den niederrangigen Regelungen nicht um Gesetze, sondern — wie hier — «nur» um einen Beschluss eines Expertengremiums handelt. Vgl. BGE 142 V 547 E. 3.3; BGer, 9C_921/2008 vom 23. April 2009 E. 3.3; Kramer, S. 131 f. 918 Eine Auslegung ist zu vermeiden, wenn dadurch eine Regelung unanwendbar oder sonst zweck- und funktionslos wird (Bydlinski, S. 444; Kramer, S. 123; vgl. BGE 112 II 167 E. 2b). 919 Vgl. EASA, Opinion, S. 17. 920 Vgl. zu diesem Kriterium vorne, S. 126. 921 Verschiedene Autoren kritisieren denn auch Ausnahmen für Spielzeuge ganz generell, vgl. nur Huttunen, Unmanned, S. 353; vgl. Masutti/Tomasello, S. 56 und S. 78 f. Vgl. sodann die Motion Reynard (18.420), wo gefordert wird, dass der Datenschutz bei der Sicherheit von Spielzeug ebenfalls mitberücksichtigt werden soll. 128 Teil 4: Bestandesaufnahme (2): Die «Drohnenverordnungen» EASA wies im Gesetzgebungsprozess auf das durch die Ausnahmeregelung für Spielzeug geschaffene Risiko diesbezüglich hin.922 Aus diesem Grund sollte die Ausnahme für Spielzeugdrohnen mit der Fähigkeit zur Datenbearbeitung im Rahmen einer Revision der Durchführungsverordnung gestrichen werden. 4. Zwischenfazit Die Registrierungspflicht für Drohnen und vor allem deren Betreiber ist von eminenter Bedeutung für die Sicherheit des Luftverkehrs insgesamt, aber ebenfalls für den Datenschutz. In der offenen Kategorie, welche für die vorliegende Untersuchung zentral ist, müssen sich grundsätzlich die Betreiber aller Drohnen registrieren, welche zur Datenbearbeitung in der Lage sind. Ausgenommen davon sind einzig die Betreiber von Spielzeugdrohnen. 5. Registrierungsverfahren Drohnenbetreiber registrieren sich selbst in dem Staat, in welchem sie ihren Wohnsitz923 bzw. — bei juristischen Personen — ihren Hauptgeschäftssitz haben; sie stellen sicher, dass die von ihnen registrierten Informationen korrekt sind. Ein Betreiber kann zu keinem Zeitpunkt in mehr als einem Staat registriert sein.924 In der Schweiz ist das BAZL — als zuständige Fachbehörde des Bundes im Bereich der Luftfahrt925 — grundsätzlich für alle Aspekte des Registrierungsverfahrens zuständig. Es kann diese Aufgabe auch an einen externen Anbieter vergeben.926 Da noch unklar ist, ob und in welcher Form eine solche Vergabe in Betracht kommt, soll nachfolgend von der Zuständigkeit des BAZL selbst ausgegangen werden.927 Die Erfassung der Drohnenbetreiber erfolgt als Selbstregistrierung. Das Einreichen von Belegen ist nicht notwendig bzw. sollte durch das Hochladen von Scans (z.B. der Identitätskarte) erledigt werden können. Es drängt sich deshalb 922 EASA, Opinion, S. 17; vgl. auch EG 31 EASA-Grundverordnung. 923 Dieser beurteilt sich nach Art. 23 ZGB. Vgl. dazu statt aller Steinauer/Fountoulakis, Rn. 347 ff. 924 Art. 14 Abs. 6 Durchführungsverordnung. 925 Art. 7 Abs. 1 OV-UVEK; vgl. Art. 3 Abs. 2 LFG. Zum Aufgabenbereich des BAZL sowie dessen Stellung innerhalb der Bundesverwaltung Henseler, Digitalisierung, S. 188 f. m.w.H. 926 Ausführlich zur Vergabe von Dienstleistungsaufträgen Galli et al., Rn. 225 ff. 927 Aus diesem Grund soll nachfolgend nur vereinzelt auf die mögliche Zuständigkeit eines externen Dienstleisters verwiesen werden. § 2 Identifizierung von Drohnenbetreibern 129 die Verwendung eines Onlineformulars auf.928 Dies ermöglicht nicht nur eine einfache und unkomplizierte Registrierung für die einzelnen Drohnenbetreiber, sondern hält auch den administrativen Aufwand seitens des Bundes tief.929 Da Drohnen auch von Minderjährigen betrieben werden können, stellt sich die Frage, wer (Eltern und/oder Kind) und ab welchem Alter ein Kind der Registrierungspflicht unterliegt. Dazu bietet sich ein Rückgriff auf das vorgesehene Mindestalter für den Betrieb von Drohnen der offenen Kategorie an; dieses liegt grundsätzlich bei 16 Jahren.930 Für Jugendliche ab diesem Alter ist deshalb im Prinzip eine selbständige Registrierung zu verlangen. Fliegt dagegen ein Kind unter 12 Jahren eine Drohne in der offenen Kategorie,931 so sollte sich ausschliesslich der bzw. ein gesetzlicher Vertreter des Kindes932 regis trieren.933 Ab 12 und bis 16 Jahre kann, je nach den Umständen des Einzelfalls, eine Selbstregistrierung des Kindes in Betracht kommen.934 Findet keine solche statt, so ist eine Registrierung des gesetzlichen Vertreters zu verlangen. Um die Korrektheit der im Register gespeicherten Informationen sicherzustellen, sollten die registrierten Drohnenbetreiber in regelmässigen Abständen (z.B. alle zwei Jahre935) aufgefordert werden, ihre Angaben zu prüfen und ggf. zu korrigieren bzw. zu ergänzen. Diese Aufforderung kann automatisch erfolgen, wobei eine E-Mail-Erinnerung die effizienteste Lösung sein dürfte.936 Durch dieses Vorgehen wird zugleich dem datenschutzrechtlichen Grundsatz der Datenrichtigkeit nachgelebt.937 928 Vgl. Art. 14 Abs. 2 Durchführungsverordnung («Felder für die Eingabe […] [von] Informationen»); Groh, S. 165. Das BAZL spricht denn auch von der «Registrierungsplattform» (BAZL, Übernahme). 929 Vgl. JARUS, Categorization, S. 25. 930 Vgl. Art. 9 Abs. 1 Durchführungsverordnung. 931 Ein Kind unter 12 Jahren darf etwa mit einer selbst gebauten Drohne (vgl. dazu hinten, S. 144 ff.) mit einer Startmasse von < 250 g oder einer Spielzeugdrohne (vgl. vorne, S. 125 ff.) fliegen; ansonsten ist eine Beaufsichtigung notwendig (vgl. zu den Voraussetzungen solcher Flüge im Einzelnen Art. 9 Abs. 2 Durchführungsverordnung). 932 Dabei handelt es sich primär um die Eltern bzw. den Inhaber der elterlichen Sorge und/oder einen Beistand (vgl. zur gesetzlichen Vertretung statt vieler Hausheer/ Aebi-Müller, Rn. 07.50 ff.). 933 Art. 9 Abs. 1 i.V.m. Art. 9 Abs. 3 lit. a Durchführungsverordnung sieht vor, dass das Mindestalter in der offenen Kategorie von 16 auf 12 Jahre heruntergesetzt werden kann. Dies liegt in der Kompetenz der einzelnen Staaten. 934 Vgl. zur Rechtsstellung von urteilsfähigen handlungsunfähigen Personen Art. 19 ff. ZGB und dazu statt aller Hausheer/Aebi-Müller, Rn. 07.08 ff.; vgl. sodann ausführlich zur Beurteilung der Urteilsfähigkeit im Kindesalter dies., Rn. 06.42 f. 935 Dieser Zeitraum entspricht in etwa der «Lebensdauer» einer Drohne der offenen Kategorie (EASA, Opinion, S. 17). 936 Vgl. zu den registrierten Daten sogleich, S. 130 f. 937 Ausführlich zu diesem Grundsatz vorne, S. 86 f. 130 Teil 4: Bestandesaufnahme (2): Die «Drohnenverordnungen» Das BAZL stellt eine «eindeutige digitale Registrierungsnummer» für Drohnenbetreiber sowie für registrierungspflichtige Drohnen aus, sodass sie «individuell identifiziert werden können».938 Dabei ist nach Standards vorzugehen, welche die Interoperabilität zwischen den Registrierungssystemen der einzelnen EU-Staaten und der Schweiz sicherstellen.939 Die zusätzlichen Ressourcen, welche dafür beim BAZL notwendig werden, können durch eine Registrierungsgebühr gedeckt werden.940 6. Anzugebende Informationen Im Registrierungssystem für Drohnenbetreiber müssen u.a. folgende Informationen angegeben werden:941 — bei natürlichen Personen: vollständiger Name und Geburtsdatum; — bei juristischen Personen: Name oder Firma und UnternehmensIdentifikationsnummer (UID942); — Adresse des Betreibers;943 — E-Mail-Adresse und Telefonnummer des Betreibers; — Nummer der Versicherungspolice für die Drohne, sofern eine solche erforderlich ist.944 Juristische Personen haben entweder eine Firma oder einen Namen.945 Art. 14 Abs. 2 lit. a Durchführungsverordnung, der nur «Namen und Identifizierungsnummer» der juristischen Person auflistet, ist somit zu einschränkend formuliert. Eine UID-Nummer wird ihnen unabhängig von der Rechtsform zugewiesen.946 Es erscheint nicht zwingend notwendig, dass ein Drohnenbetreiber eine EMail-Adresse und eine Telefonnummer hinterlegt. Verfügt eine natürliche 938 Vgl. dazu weiterführend hinten, S. 136 f. 939 Vgl. Art. 14 Abs. 6 Durchführungsverordnung; EASA, Explanatory Note, S. 7. Vgl. allgemein zur Bedeutung von Standards im Bereich der Drohnenfliegerei in Europa Cuccuru, S. 352 ff. 940 Vgl. EASA, Explanatory Note, S. 7. 941 Vgl. Art. 14 Abs. 2 lit. a bis d Durchführungsverordnung. Die weiteren geforderten Angaben sind für die vorliegende Auseinandersetzung vernachlässigbar. 942 Die UID ist im Bundesgesetz über die Unternehmens-Identifikationsnummer (UIDG) sowie der zugehörigen Verordnung (UIDV) geregelt. Mit dieser Nummer können Unternehmen eindeutig identifiziert werden (Art. 1 UIDG); sie besteht aus dem Präfix «CHE», einer nach dem Zufallsprinzip zugeteilten achtstelligen Zahl und einer Prüfziffer (Art. 5 lit. a bis c UIDV). 943 In Art. 14 Abs. 2 lit. b Durchführungsverordnung ist von «Anschrift» die Rede. 944 Die Versicherungspflicht für Drohnen ist in Art. 20 VLK geregelt. Vgl. dazu de lege ferenda Hänsenberger, Diss., S. 224 ff. 945 Vgl. Art. 950 Abs. 1 OR; Hausheer/Aebi-Müller, Rn. 16.07 ff. 946 Vgl. Art. 3 Abs. 1 lit. c und Art. 4 Abs. 1 UIDG. § 2 Identifizierung von Drohnenbetreibern 131 Person über keine E-Mail-Adresse und/oder (persönliche) Telefonnummer, so sollte dies eine Registrierung nicht ausschliessen. Die Hinterlegung einer EMail-Adresse ermöglicht eine einfache Kontaktaufnahme, was zu begrüssen ist.947 Die weiteren Angaben sind jedoch zwingend zu verlangen, da ansonsten der Zweck der Registrierungspflicht gefährdet werden könnte.948 Natürliche Personen geben ihre Wohnsitzadresse, juristische Personen die Adresse des Sitzes949 an, da die Selbstregistrierung daran anknüpft und jeweils nur in einem Staat eine Registrierung möglich ist.950 Des Weiteren sollten juristische Personen dazu angehalten werden, Name und Direktwahl der bzw. einer für die Drohnenflüge verantwortlichen natürlichen Person anzugeben. Dadurch ist gewährleistet, dass die Kommunikation zwischen BAZL und Betreiber (sowie ggf. Dritten) effizient abgewickelt werden kann. 7. «Drohnenbetreiberregister» 7.1 Ausgestaltung des Registers Wenn die Informationen der Drohnenbetreiber registriert wurden und ihnen eine Betreibernummer zugeteilt ist, stellt sich die Frage nach der Ausgestaltung des Registers und insbesondere den diesbezüglichen Zugriffsberechtigungen. Für Drohnen der zulassungspflichtigen Kategorie951 drängt sich eine Ergänzung der Bestimmungen zum Luftfahrzeugregister (Art. 52 ff. LFG, Art. 3 ff. LFV) auf.952 Für Betreiber von Drohnen der offenen und speziellen Kategorie953 müssen dagegen neue Normen geschaffen werden. Da der Fokus eines solchen Registers auf den Betreibern und nicht auf den Drohnen an sich liegt, handelt es sich dabei vielmehr um ein «Drohnenbetreiberregister» als um ein «Drohnenregister».954 947 So können die registrierten Drohnenbetreiber etwa zur Überprüfung ihrer Angaben im Register angehalten werden (vgl. dazu vorne, S. 129). 948 Vgl. dazu vorne, S. 120. 949 Mit Sitz wird grundsätzlich der Ort bezeichnet, wo die Verwaltung geführt wird (vgl. Art. 56 ZGB; BSK ZGB I-Huguenin/Reitze, Art. 56 N 1 ff.; BSK OR II-Schenker, Art. 626 N 5). Eine juristische Person kann neben dem Sitz auch über Zweig- und Geschäfts niederlassungen verfügen (vgl. dazu BSK ZGB I-Huguenin/Reitze, Art. 56 N 2; ausführlich Meier-Hayoz/Forstmoser/Sethe, § 16 Rn. 178, § 24 Rn. 14 ff.). 950 Art. 14 Abs. 6 Durchführungsverordnung. 951 Vgl. zur zulassungspflichtigen Kategorie vorne, S. 23 f. 952 Aufgrund der Ähnlichkeit zur bemannten Luftfahrt sollten die diesbezüglichen Vorschriften weiterentwickelt werden. Vgl. Art. 14 Abs. 3 und Abs. 7 Durchführungsverordnung; EASA, Explanatory Note, S. 5; Hänsenberger, Diss., S. 232. 953 Betreiber von Drohnen der speziellen Kategorie registrieren sich selber, unabhängig von der Masse der Drohne (Art. 14 Abs. 5 lit. b Durchführungsverordnung). 954 So aber etwa Fumagalli, S. 31; Pugin, S. 11. Vgl. auch vorne, S. 128. 132 Teil 4: Bestandesaufnahme (2): Die «Drohnenverordnungen» Die EASA-Grundverordnung und die Durchführungsverordnung enthalten zur konkreten Ausgestaltung eines solchen Registers nur wenige Vorgaben. Die einzelnen Staaten sind gehalten «die Informationen über die Registrierung von unbemannten Luftfahrzeugen und über die Betreiber (…) in digitalen, harmonisierten und interoperablen nationalen Registrierungssystemen» zu speichern.955 Des Weiteren müssen die Systeme «es ermöglichen, über die in [Art. 74 EASA-Grundverordnung956] genannte Datenbank gegenseitig auf die Daten zuzugreifen und diese auszutauschen».957 Es ist somit die Verwendung eines europaweiten Standards bei der Ausstellung der Registrierungsnummern notwendig.958 In den Drohnenverordnungen wird jedoch kein solcher vorgegeben.959 Gemäss Auskunft der EASA vom September/Oktober 2019 wurde zwischen Vertretern der EU-Mitgliedstaaten und der Industrie ein entsprechender Standard vereinbart. Dieser soll jedoch erst im zweiten oder dritten Quartal 2020 der Öffentlichkeit zugänglich gemacht werden.960 Daneben sind bei der Ausgestaltung des Registers die datenschutzrechtlichen Vorschriften für Bundesbehörden einzuhalten.961 7.2 Vergleichbare Register Als Vorlage für das «Drohnenbetreiberregister» und insbesondere den für die Rechtsdurchsetzung zentralen Aspekt der Zugriffsberechtigungen kommt neben dem Luftfahrzeugregister auch das «Informationssystem Verkehrszulassung» in Betracht.962 Bei Letzterem handelt es sich um die Be955 Art. 56 Abs. 7 EASA-Grundverordnung; vgl. Krumm, Anpassungsbedarf, S. 116. 956 Der dort geregelte «Informationsspeicher» soll «eine wirksame Zusammenarbeit zwischen der [EASA] und den zuständigen nationalen Behörden bei der Wahrnehmung ihrer Zertifizierungs-, Aufsichts- und Durchsetzungsaufgaben im Rahmen der vorliegenden Verordnung» gewährleisten (Art. 74 Abs. 1 EASA-Grundverordnung) 957 Art. 14 Abs. 4 Durchführungsverordnung. Dies beinhaltet «a requirement for real- time accessibility» (EASA, Opinion, S. 17). 958 EASA, Opinion, S. 17. Vgl. dazu bereits vorne, S. 130. 959 Anders verhält es sich bei der «eindeutigen physischen Seriennummer» von Drohnen der Produkteklassen C1 bis C3; vgl. Teil 2 Ziff. 11, Teil 3 Ziff. 13 und Teil 4 Ziff. 8 Anhang Delegierte Verordnung. 960 EASA Communication Department, E-Mails vom 4. und 6. September sowie vom 22. Oktober 2019 (beim Autor einsehbar). Vgl. auch EASA, Opinion, S. 17. 961 Vgl. Art. 132 Abs. 1 EASA-Grundverordnung sowie die diesbezügliche Präzisierung für die Schweiz im Beschluss Nr. 1/2019, AS 2019 S. 2985; vgl. sodann Art. 2 Abs. 1 lit. b und Art. 29 ff. E-DSG. 962 Weitere Register wie z.B. das Schiffsregister gemäss Bundesgesetz über das Schiffsregister (SR 747.11) oder das Eisenbahnfahrzeugregister (Art. 17a EBG) werden nicht behandelt. Die dort erfassten Sachverhalte unterscheiden sich in wesentlicher Weise vom hier interessierenden Register für Drohnenbetreiber. § 2 Identifizierung von Drohnenbetreibern 133 zeichnung für das seit 1. Januar 2019 vom Bundesamt für Strassen (ASTRA) geführte System,963 welches u.a. das bis dahin existierende «Fahrzeug- und Fahrzeughalterregister» abgelöst hat.964 Die beiden Register sollen nachfolgend kurz diskutiert und darauf geprüft werden, ob sie als Vorlage für das Drohnenbetreiberregister dienen könnten. a Luftfahrzeugregister Das Luftfahrzeugregister dient primär «der öffentlichen Kontrolle und Sicherheit».965 Zudem erlaubt es, Art und Umfang von Rechtsbeziehungen zwischen (natürlichen und juristischen) Personen und eingetragenen Luftfahrzeugen abzubilden oder diese zu bestimmen.966 So werden u.a. der Name und die Adresse des Eigentümers und u.U. des Halters registriert.967 Gemäss Art. 107b Abs. 1 LFG sind «[d]ie im schweizerischen Luftfahrzeugregister enthaltenen Personendaten öffentlich. Sie können mittels Abrufverfahren zugänglich gemacht werden.» Ein solches Verfahren liegt vor, «wenn mehrere Verwaltungsstellen dasselbe Informationssystem betreiben oder wenn Dritte nach dem Prinzip der Selbstbedienung Zugriff auf die Daten des Systems haben.»968 Der Bundesrat hielt unlängst fest, dass «die Angaben innerhalb [des Luftfahrzeugregisters] auch in der Schweiz seit jeher öffentlich zugänglich [sind].»969 Auf der entsprechenden Webseite des BAZL können jederzeit und ohne Interessennachweis Angaben wie Name und Adresse des Eigentümers von registrierten Luftfahrzeugen eingesehen werden. b Informationssystem Verkehrszulassung Das Informationssystem Verkehrszulassung dient u.a. der Identifikation von Fahrzeughaltern und der Fahrzeugfahndung sowie der Erteilung, Kontrolle 963 Vgl. Art. 89a ff. SVG. 964 Daneben existierten drei weitere Register, namentlich das Fahrberechtigungsregister, das Administrativmassnahmen-Register sowie das Fahrzeugtypenregister. Vgl. ausführlich dazu Botschaft Via sicura, S. 8480 ff. 965 So bereits Botschaft LFG, S. 359; vgl. sodann McNally, S. 24 f.; Abeyratne, S. 262; Dempsey, S. 301. 966 Vgl. McNally, S. 23 ff.; Schwenk/Giemulla, Kap. 2 Rn. 130. Dieser Aspekt ist gemäss Botschaft LFG, S. 359, jedoch von untergeordneter Bedeutung. 967 Art. 8 Abs. 1 lit. f und Abs. 2 LFV. Vgl. zum Begriff des Halters vorne, S. 28. 968 BJ, Leitfaden, Rn. 838. Vgl. Baeriswyl, Fahndungsmittel, S. 13 mit Fn. 24; Wermelinger, S. 187; Handkomm. DSG-Jöhri, Art. 19 N 74. 969 So der Bundesrat 2009 in der Botschaft Teilrevision 1 LFG, S. 4969 (Hervorhebung hinzugefügt). Art. 107b LFG wurde (erst) per 1. April 2011 in Kraft gesetzt, um eine für die Datenbearbeitung (d.h. die öffentliche Bekanntgabe von Personendaten aus dem Luftfahrzeugregister) ausreichende Gesetzesgrundlage zu schaffen (vgl. dazu Botschaft Teilrevision 1 LFG, S. 4943). 134 Teil 4: Bestandesaufnahme (2): Die «Drohnenverordnungen» und dem Entzug von Ausweisen für die Zulassung von Personen und Fahrzeugen zum Strassenverkehr sowie von Bewilligungen.970 Im Informationssystem sind deshalb z.B. die Personalien von Fahrzeughaltern und Ausweisinhabern gemäss SVG gespeichert.971 Im Unterschied zum Luftfahrzeugregister «[sind] [d]ie Daten der Verkehrszulassung nicht öffentlich» (Art. 89g Abs. 1 SVG). Dies wurde «als wichtiger Grundsatz zur Gewährleistung des Datenschutzes» auf Gesetzesstufe geregelt.972 Gemäss Art. 89g Abs. 3 SVG dürfen die kantonalen Verkehrs zulassungsbehörden die Fahrzeughalter- und Versicherungsdaten Personen bekannt geben, die von einem Verkehrsunfall betroffen sind oder die im Hinblick auf ein Verfahren ein hinreichendes Interesse schriftlich geltend machen.973 «Die bisherige Praxis der voraussetzungslosen Halterauskünfte an jedermann (z.B. per Internet, SMS etc.)» ist mit dem Grundsatz der Nicht öffentlichkeit nicht vereinbar und somit unzulässig.974 Für verschiedene Behörden (Polizei, Strafverfolger, Bundesämter etc.) ist eine Einsichtnahme in bestimmte Daten des Informationssystems vorgesehen (vgl. Art. 89e SVG). Dem Datenschutz kommt beim Informationssystem Verkehrszulassung somit eine zentrale Rolle zu.975 Die Zugriffs- und Einsichtsrechte sind explizit auf Gesetzesstufe verankert und dem «Schutz der Persönlichkeitsrechte» der betroffenen Personen wird explizit Rechnung getragen.976 7.3 Zwischenfazit Bei der Ausgestaltung des Registers und der Regelung der Zugriffsrechte sollten neben den Anforderungen an die Datenbearbeitung durch Bundesorgane977 auch mögliche Ansprüche Privater aus dem Datenschutzrecht und dem Persönlichkeitsschutz mitberücksichtigt werden. Das Modell des Informations systems Verkehrszulassung, welches vom Grundsatz der Nichtöffentlichkeit ausgeht, scheint deshalb als Vorlage für das «Drohnenbetreiberregister» gut 970 Art. 89b lit. a und e SVG. 971 Art. 89c SVG. 972 Davor war der Grundsatz der Nichtöffentlichkeit auf Verordnungsstufe festgeschrieben; vgl. Botschaft Via sicura, S. 8509; OFK SVG-Giger, Art. 89g N 2. 973 Daneben dürfen die Daten auch Personen bekannt gegeben werden, die an einem Zulassungsverfahren beteiligt sind. Anderweitige Bekanntgaben sind nicht zulässig; es handelt sich in Art. 89g Abs. 3 SVG um eine abschliessende Liste (Komm. SVGWeissenberger, Art. 89g N 1; OFK SVG-Giger, Art. 89g N 4). 974 Botschaft Via sicura, S. 8508; OFK SVG-Giger, Art. 89g N 4. 975 Botschaft Via sicura, S. 8481. 976 Botschaft Via sicura, S. 8480. 977 Vgl. Art. 4 lit. h und Art. 29 ff. E-DSG sowie ausführlich hinten, S. 165 f. § 2 Identifizierung von Drohnenbetreibern 135 geeignet. Es erlaubt, einen Ausgleich zwischen den Interessen der Betreiber, Behörden und (betroffenen) Dritten zu schaffen.978 8. Datenschutzrechtliche Beurteilung und Überleitung Grundsätzlich müssen sich alle Betreiber von Drohnen, die Daten bearbeiten können, registrieren.979 Durch die bei der Registrierung anzugebenden Informationen, welche anschliessend mit einer eindeutigen Betreibernummer verknüpft werden, ist ein wichtiger Schritt zur Durchsetzung datenschutzrechtlicher Ansprüche getan. Die Pflicht zur Registrierung für sich allein genommen reicht jedoch noch nicht aus, um in einem konkreten Fall einen Drohnenbetreiber identifizieren zu können. Dazu sind weitere Vorgaben in den Drohnenverordnungen enthalten, namentlich in den Vorschriften zur Kennzeichnung. Darauf soll sogleich eingegangen werden. II. Kennzeichnung 1. Arten der Kennzeichnung Drohnen, welche registriert werden bzw. deren Betreiber sich registrieren müssen, sind einzeln zu markieren und zu kennzeichnen.980 Die Delegierte Verordnung kennt verschiedene Arten der Kennzeichnung,981 u.a. die «CEKennzeichnung»,982 die «Kennzeichnung des Schallleistungspegels»983 sowie die «Identifizierungskennzeichen der UA-Klasse»984. Letztere sollen auf einen Blick die Anforderungen an den Fernpiloten und den Betrieb der Drohne 978 Vgl. weiterführend hinten, S. 162 f. 979 Vgl. vorne, S. 128. 980 Vgl. Anhang IX Ziff. 4.3 EASA-Grundverordnung. Die deutsche Fassung dieser Bestimmung lässt Klarheit vermissen; besser geglückt ist die Formulierung in der englischen Sprachfassung. 981 Vgl. Art. 16 Delegierte Verordnung. 982 Die «CE-Kennzeichnung» (CE = Conformité Européenne) ist gemäss der Definition in Art. 3 Ziff. 12 Delegierte Verordnung «eine Kennzeichnung, durch die der Hersteller erklärt, dass das Erzeugnis den geltenden Anforderungen genügt, die in den Harmonisierungsrechtsvorschriften der Union, nach denen die Anbringung der Kennzeichnung vorgeschrieben ist, festgelegt sind». Vgl. dazu auch EG 32 und Art. 15 Delegierte Verordnung; vgl. sodann Verordnung (EG) Nr. 765/2008, welche die allgemeinen Grundsätze der CE-Kennzeichnung festlegt. 983 Vgl. dazu Teile 14 und 15 Anhang Delegierte Verordnung. 984 Die Abkürzung «UA» steht hier für «unbemanntes Luftfahrzeug» (engl. unmanned aircraft; vgl. dazu bereits vorne, S. 16). Gemeint sind damit die (Produkte-)Klassen C0 bis C3, vgl. dazu vorne, S. 24 ff. 136 Teil 4: Bestandesaufnahme (2): Die «Drohnenverordnungen» erkenn- und überprüfbar machen.985 Vorliegend interessiert jedoch primär die Kennzeichnung der Drohnen mit der «eindeutigen digitalen Registriernummer für UAS-Betreiber».986 Die weiteren Kennzeichnungen sollen nicht vertieft werden. 2. Kennzeichnung mit Betreibernummer 2.1 Begriff Die für die Registrierung zuständige Behörde987 stellt eine eindeutige digitale Registriernummer aus, sodass die erfassten Drohnen sowie deren Betreiber «individuell identifiziert werden können».988 Betreiber, welche sich registrieren müssen, bringen ihre Registriernummer auf jeder Drohne an.989 Die Terminologie der Delegierten Verordnung ist nicht einheitlich; sie verwendet neben dem Begriff «UAS-Betreibernummer» auch verschiedentlich die Bezeichnung «eindeutige digitale Registriernummer für UAS-Betreiber».990 Damit ist dasselbe gemeint, nämlich die eindeutige Nummer, welche jedem registrierten Drohnenbetreiber zugeteilt wird. Der Einfachheit halber soll nachfolgend einzig von der Betreibernummer gesprochen werden. 2.2 Praktische Umsetzung Die Kennzeichnung knüpft an die Selbstregistrierung durch den Betreiber an; somit sind auch bei Inkrafttreten der entsprechenden Bestimmungen bereits im Umlauf befindliche Drohnen zu kennzeichnen. In welcher Form die Betreibernummer auf der Drohne angebracht werden soll, wird nicht spezifiziert. Die Verwendung einer Plakette mit starkem Klebstoff drängt sich auf, da der Gebrauch von Schrauben, Nägeln etc. die Flugeigenschaften der Drohne beeinträchtigen oder diese gar schädigen könnte.991 Es ist zudem notwendig, dass die Registrierungsnummer «gut sichtbar, leserlich und dauerhaft» angebracht wird.992 Befindet sich die Drohne am Boden, so sollten die Angaben auf der Plakette ohne Hilfsmittel gelesen wer985 Groh, S. 166. Vgl. zu den Anforderungen im Einzelnen Anhang Delegierte Verordnung. 986 Die Kennzeichnung mit der «eindeutigen digitalen Registriernummer für registrierungspflichtige UAS» wird nicht weiter behandelt, da Drohnen der offenen Kategorie selbst nicht registriert werden müssen; vgl. dazu vorne S. 120 ff. und S. 128. 987 Vgl. zum Verfahren vorne, S. 128 ff. 988 Art. 14 Abs. 6 Durchführungsverordnung. 989 Art. 14 Abs. 8 Durchführungsverordnung. 990 Vgl. Teil 2 Ziff. 12, Teil 3 Ziff. 14 und Teil 4 Ziff. 9 Anhang Delegierte Verordnung. 991 Vgl. zur Flugmechanik von Drohnen Büchi, Buch der Drohnen, S. 122 ff. 992 Vgl. zu diesen Anforderungen für die CE- bzw. die Kennzeichnung der Produkteklasse Art. 16 Abs. 1 und 2 Delegierte Verordnung. Vgl. auch Hänsenberger, Diss., S. 232. § 2 Identifizierung von Drohnenbetreibern 137 den können.993 Mit dauerhaft ist in diesem Zusammenhang zumindest Feuerund Wasserfestigkeit gemeint.994 Auch Wetter- und Kältebeständigkeit der Plakette sollten gewährleistet sein. Die Grösse derselben sollte selbstredend derjenigen der Drohne angepasst werden. Ist etwa eine Kennzeichnung auf dem Rumpf nicht leserlich anzubringen, so kann diese auch in der Batteriekammer angebracht werden.995 Dem Drohnenbetreiber steht es selbstverständlich frei, weitere Informationen auf der Plakette zu vermerken (etwa Adresse oder Telefonnummer, was bei Verlust der Drohne hilfreich sein könnte). Auch die (zusätzliche) Verwendung eines QR-Codes ist zulässig.996 Um eine effiziente und möglichst flächendeckende Umsetzung der Kennzeichnungsvorschrift zu erreichen, sollten die Plaketten direkt bei den einzelnen Drohnenherstellern und/oder händlern997 bezogen werden können. Das BAZL verfügt nicht über die notwendigen personellen und technischen Ressourcen zur Herstellung derselben.998 Zentral ist, dass unleserliche oder nicht dauerhafte Kennzeichnungen (die z.B. mit nicht wasserbeständiger Farbe angebracht sind) vermieden werden. 3. Datenschutzrechtliche Beurteilung Aufgrund der Tatsache, dass die Kennzeichnungspflicht an der Registrierungspflicht der Betreiber anknüpft, sind grundsätzlich alle Drohnen, welche Personendaten bearbeiten können, eindeutig zu kennzeichnen.999 Insgesamt ist die so ausgestaltete Kennzeichnungspflicht von Drohnen durchaus zu begrüssen, da damit eine Möglichkeit der Identifizierung des Betreibers geschaffen wird. Es ist jedoch zweifelhaft, ob die Betreibernummer im Flug tatsächlich gelesen werden kann. In Anbetracht der Grösse, der möglichen Geschwindigkeiten und Flughöhen von Drohnen in der offenen Kategorie1000 dürfte es nur ausnahmsweise gelingen, die Betreibernummer (vollständig) zu 993 Brillen oder Kontaktlinsen werden in diesem Kontext nicht als Hilfsmittel verstanden (vgl. EASA, GM Implementing Regulation, S. 124). 994 Vgl. EASA, Opinion, S. 17. 995 EASA, GM Implementing Regulation, S. 124 f. 996 EASA, GM Implementing Regulation, S. 124. 997 Vgl. die Begriffsdefinition in Art. 3 Ziff. 16 Delegierte Verordnung. 998 Vgl. dazu auch vorne, S. 6. 999 Vgl. zur Registrierungspflicht vorne, S. 135. 1000 Vgl. dazu die Tabelle vorne, S. 25, sowie die Ausführungen zu den in der Schweiz verbreiteten Drohnen vorne, S. 39. 138 Teil 4: Bestandesaufnahme (2): Die «Drohnenverordnungen» erkennen.1001 Mit der Kennzeichnungspflicht allein können deshalb nicht alle datenschutzrechtlichen Bedenken ausgeräumt werden. III. Fernidentifizierung 1. Bedeutung und Begriff Die Identifizierung einer Drohne aus der Ferne ist neben der Registrierung der zentrale Aspekt für die Identifizierung des Betreibers.1002 Wie gerade aufgezeigt, ist dies mithilfe der auf der Drohne anzubringenden Betreibernummer, durch visuelle (Fern-)Identifizierung, wohl nur in Ausnahmefällen möglich. Es bedarf deshalb einer Möglichkeit der elektronischen Fernidentifizierung. Die Grundlagen dafür sind in der Durchführungsverordnung zu finden. Die «direkte» Fernidentifizierung1003 wird in Art. 2 Ziff. 13 Durchführungsverordnung definiert als «ein System, das die lokale Übertragung von Informationen über ein im Betrieb befindliches unbemanntes Luftfahrzeug gewährleistet und auch die Kennzeichnung des unbemannten Luftfahrzeugs umfasst, sodass diese Informationen ohne physischen Zugang zum unbemannten Luftfahrzeug abgerufen werden können.»1004 2. Erfasste Drohnen Innerhalb der offenen Kategorie ist die direkte Fernidentifizierung für Drohnen der Klassen C1 bis C31005 vorgeschrieben.1006 Ausnahmen sind für die Klassen C2 und C3 vorgesehen, sollten sie gefesselt (engl. tethered) betrieben werden. Dies bedeutet, dass die Drohne an einer Leine und/oder einem Kabel hängt, wodurch der Operationsradius eingeschränkt wird.1007 Insgesamt werden die allermeisten Drohnen, welche von Privaten eingesetzt werden, 1001 Vgl. dazu auch Groh, S. 166; JARUS, Categorization, S. 16. 1002 JARUS, Categorization, S. 25 und S. 29; vgl. BRTF, S. 8. 1003 Aus dem EU-Recht wird nicht klar, ob die direkte explizit von der «indirekten» Fern- identifizierung abgegrenzt werden sollte; Letztere wird in der Durchführungsverordnung denn auch nicht erwähnt. Als «indirekte» Variante käme z.B. in Betracht, dass eine Drohne keine spezifischen Signale zur Fernidentifizierung aussendet und somit mithilfe anderer Parameter identifiziert werden müsste. 1004 Vereinzelt ist in der Delegierten Verordnung von «Fernidentifikation» die Rede, ohne dass ein Grund für diese Abweichung ersichtlich wäre (vgl. Art. 1 Abs. 1 und 2 Delegierte Verordnung; vgl. auch EG 26 Durchführungsverordnung). In Übereinstimmung mit der Begriffsdefinition wird nachfolgend von der «Fernidentifizierung» gesprochen. 1005 Vgl. zu den (Produkte-)Klassen C0 bis C3 vorne, S. 24 ff. 1006 Teil 2 Ziff. 12, Teil 3 Ziff. 14 und Teil 4 Ziff. 9 Anhang Delegierte Verordnung. 1007 Teil 3 Ziff. 5 und Teil 4 Ziff. 4 Anhang Delegierte Verordnung. Vgl. dazu Schwenk/ Giemulla, Kap. 7 Rn. 24 in fine. § 2 Identifizierung von Drohnenbetreibern 139 von den Vorschriften zur Fernidentifizierung erfasst. Einzig Drohnen der Klasse C0 müssen keine Informationen zur Fernidentifizierung übertragen. Diese Ausnahme steht in Einklang mit derjenigen zur Registrierungspflicht für Drohnen mit einer Startmasse von weniger als 250 g.1008 3. Zusatzgerät für die direkte Fernidentifizierung 3.1 Allgemeine Anforderungen Die Delegierte Verordnung geht vom Verständnis aus, dass «die lokale Übertragung von Informationen über ein im Betrieb befindliches unbemanntes Luftfahrzeug» nicht durch die Drohne selber, sondern durch ein Zusatzgerät erfolgt. Aus diesem Grund stellt die Verordnung verschiedene Vorgaben dafür auf. So muss das Zusatzgerät für die direkte Fernidentifizierung gewährleisten, dass «während der gesamten Flugdauer (…) in Echtzeit direkt und regelmässig» bestimmte Informationen übermittelt werden. Diese müssen «innerhalb des Sendebereichs von vorhandenen Mobilfunkgeräten direkt empfangen werden können».1009 Das zentrale Element des Zusatzgeräts wird somit ein Chip sein, der die Übermittlung gewährleistet.1010 Die zu übermittelnden Informationen umfassen u.a.:1011 — die Drohnenbetreibernummer;1012 — die geografische Position der Drohne und ihre Höhe über der Oberfläche oder dem Startpunkt; — den Streckenverlauf, gemessen im Uhrzeigersinn vom geografischen Norden; — die Geschwindigkeit über Grund; — die geografische Position des Piloten oder, falls nicht verfügbar, des Startpunkts. Das Zusatzgerät für die direkte Fernidentifizierung muss ausserdem gewährleisten, dass der Nutzer diese Angaben nicht verändern kann.1013 Dadurch 1008 Vgl. dazu vorne, S. 123 f. 1009 Teil 6 Ziff. 3 Anhang Delegierte Verordnung. 1010 Vgl. EASA, A-NPA, S. 18; Hänsenberger, AJP 2017, S. 164; Hänsenberger, Diss., S. 231 f. 1011 Teil 6 Ziff. 3 Anhang Delegierte Verordnung. Das Zusatzgerät muss auch seine eigene eindeutige physische Seriennummer übermitteln (vgl. dazu Anhang Teil 6 Ziff. 2 Delegierte Verordnung). Diese Information ist für die vorliegende Auseinandersetzung jedoch nicht von Bedeutung. 1012 Vgl. dazu vorne, S. 136 f. 1013 Teil 6 Ziff. 4 Anhang Delegierte Verordnung. Die Betreibernummer ist in der Liste nicht enthalten. Aus den Gesetzesmaterialien wird nicht klar, weshalb dies so geregelt wurde. Die diesbezügliche Ziff. 4 ist in der finalen Fassung der Delegierten Verordnung erstmals enthalten (vgl. zur Entstehungsgeschichte der Delegierten Verordnung die zugehörige Webseite). 140 Teil 4: Bestandesaufnahme (2): Die «Drohnenverordnungen» wird zum Ausdruck gebracht, dass nicht nur der Drohnenbetreiber, sondern auch Dritte und ggf. Behörden keine Möglichkeit haben dürfen, Änderungen an den Informationen der Fernidentifizierung vorzunehmen. 3.2 Empfang durch Mobilfunkgeräte insbesondere Die vom Zusatzgerät ausgesendeten Informationen müssen «innerhalb des Sendebereichs von vorhandenen Mobilfunkgeräten direkt empfangen werden können.»1014 Es handelt sich demnach um eine «lokale Übertragung»1015 der Angaben «over a short range».1016 Die Bezugnahme auf den Empfang mit Mobilfunkgeräten spricht für eine applikationsbasierte Lösung, wobei auch eine Webseite in Betracht kommt.1017 Wer die entsprechende App auf seinem Mobiltelefon installiert oder Zugriff auf die entsprechende Webseite hat, kann damit die ausgesendeten Informationen direkt empfangen.1018 Technisch erfolgt die lokale Übertragung entweder über Bluetooth oder Wi-Fi.1019 Ein am 16. September 2019 vom BAZL, von Skyguide1020 und weiteren beteiligten Unternehmen durchgeführter Test zeigte auf, dass ein Benutzer einer entsprechenden App damit Drohnen identifizieren konnte, die vor bis zu 60 Sekunden im näheren Umkreis betrieben wurden. Dies ermöglicht eine Fernidentifizierung auch dann, wenn sich eine schnell bewegende Drohne in kurzer Zeit von einer betroffenen Person entfernt.1021 Diese zeitliche Beschränkung wird durch eine geografische ergänzt, welche Abfragen auf ein Gebiet mit einem Durchmesser von 3,6 km beschränkt.1022 Der Test zeigte jedoch ebenfalls auf, dass das System der lokalen Übertragung Nachteile mit sich bringt. So sind aufgrund der zeitlichen und geo1014 Teil 6 Ziff. 3 Anhang Delegierte Verordnung. So auch bereits EASA, A-NPA, S. 18. 1015 So die Definition in Art. 2 Ziff. 13 Durchführungsverordnung. 1016 EASA, Opinion, S. 26. 1017 Vgl. BAZL, Demo Report, S. 2. 1018 Vgl. EASA, A-NPA, S. 18. Die EASA zog ursprünglich auch die Datenübermittlung per Radio-Frequency Identification (RFID) in Betracht. 1019 BAZL, Demo Report, S. 1. Bluetooth ist ein Industriestandard für die Datenübertra- gung zwischen Geräten über kurze Distanz per Funktechnik; Wi-Fi bezeichnet die Zertifizierung eines Funknetzwerks wie etwa eines «Wireless Local Area Network» (WLAN). Vgl. ausführlich dazu Labiod/Afifi/De Santis, S. 75 ff. bzw. S. 5 ff. 1020 Skyguide ist die Abkürzung von «Skyguide Schweizerische Aktiengesellschaft für zivile und militärische Flugsicherung» (vgl. Art. 2 Abs. 1 VFSD). Dabei handelt es sich um eine gemischtwirtschaftliche Gesellschaft, welche gestützt auf Art. 40a LGF geschaffen wurde (Kunz, S. 158; Moor/Bellanger/Tanquerel, S. 250; a.M. Freudiger, S. 40 f. mit Fn. 181; vgl. auch Art. 762 OR und ausführlich dazu BSK OR II-Wernli/ Rizzi, Art. 762 N 5 ff.). 1021 Unmanned Airspace, passim; vgl. BAZL, Fernidentifizierung. 1022 BAZL, Demo Report, S. 3. § 2 Identifizierung von Drohnenbetreibern 141 grafischen Beschränkung auch die Polizei und andere Behörden grundsätzlich gehalten, sich in der Nähe einer zu identifizierenden Drohne aufzuhalten. Ansonsten ist ein Abrufen der entsprechenden Informationen nicht oder nicht mehr möglich. Dies kann nicht Zweck der Bestimmungen zur Fernidentifizierung sein.1023 Für Strafverfolgungs- und Aufsichtsbehörden drängt sich deshalb eine andere Lösung auf, namentlich die Übertragung von Informationen über das Internet (mittels Mobilfunknetz). Dies erlaubt einen zeitlich weiter zurückreichenden und geografisch unbeschränkten Zugriff, da die Informationen nicht nur lokal übertragen werden.1024 Private Betroffene sollten dagegen auf die zeitlich und geografisch limitierte Übertragung beschränkt bleiben; sie könnten sich — sollten eine direkte Fernidentifizierung so nicht gelingen — an die Behörden wenden. Eine Umsetzung dieser sog. Netzwerklösung setzt eine Revision der entsprechenden Verordnungsbestimmungen voraus.1025 Dabei sind strikte Vorgaben für den Zugriff auf die Informationen aufzustellen; eine vollständige und flächendeckende Überwachung der Drohnen und somit ihrer Betreiber wäre unverhältnismässig.1026 4. Datenschutzrechtliche Beurteilung Die Ausnahme von den Vorschriften zur Fernidentifizierung für Drohnen der Klasse C0 — unabhängig davon, ob sie Personendaten bearbeiten können oder nicht — ist aus datenschutzrechtlicher Sicht bedauerlich. Die Möglichkeit der Fernidentifizierung wäre auch für diese Klasse wünschenswert.1027 Die gefesselt betriebenen Drohnen stellen dagegen aus datenschutzrechtlicher Sicht kaum ein Risiko dar; die Ausnahme für diese Betriebsart ist nachvollziehbar. Durch die Vorgaben für die Klassen C1 bis C3 können grundsätzlich die Betreiber einer Vielzahl von Drohnen fernidentifiziert werden. Der lokale Charakter der Ferni dentifizierung weist Parallelen zur Identifizierung von Fahrzeughaltern anhand ihrer Kontrollschildnummern auf.1028 Bei «Kleinst1023 Vgl. Anhang IX Ziff. 1.3 EASA-Grundverordnung. 1024 Vgl. BAZL, Demo Report, S. 3 f. Vorausgesetzt ist einzig eine funktionierende Internet verbindung. 1025 Vgl. Art. 2 Ziff. 13 Durchführungsverordnung und Teil 6 Ziff. 3 Anhang Delegierte Ver- ordnung, wo nur die «lokale Übertragung von Daten» erwähnt wird. 1026 Vgl. zu den datenschutzrechtlichen Ansprüchen der Drohnenbetreiber hinten, S. 162 f. 1027 Gegen Ausnahmen bei der Fernidentifizierung — jedoch gestützt auf Luftsicherheits- überlegungen — spricht sich auch BRTF, S. 8, aus. 1028 Singh/Carthew/Xiong, S. 4 f. Vgl. Art. 2 Abs. 1 lit. a und Anhang 1 Ziff. 2 Verordnung über das Informationssystem Verkehrszulassung [IVZV]. 142 Teil 4: Bestandesaufnahme (2): Die «Drohnenverordnungen» drohnen» der Klasse C0, welche innert Sicht und somit nahe beim Betreiber fliegen müssen,1029 besteht sodann die Möglichkeit, diesen dadurch zu identifizieren.1030 Gemeinsam mit den Registrierungsvorschriften ermöglicht die Fernidentifizierung somit, Transparenz herzustellen bzw. diese stark zu verbessern.1031 IV. Flugverkehrsmanagement U-Space (UTM) 1. Begriff Ganz allgemein bezweckt das Flugverkehrsmanagement (Air Traffic Management, ATM1032) die Gewährleistung eines sicheren, geordneten und raschen Verkehrsflusses von Luftfahrzeugen im kontrollierten Luftraum.1033 In der Schweiz ist Skyguide1034 mit dem Flugverkehrsmanagement für den bemannten Luftverkehr betraut.1035 Die Integration von Drohnen in den kontrollierten Luftraum setzt die Schaffung einer Flugverkehrskontrolle spezifisch für diese Luftfahrzeuge voraus.1036 Nur so kann die Sicherheit und der ungestörte Betrieb der (bemannten) Luftfahrt gewährleistet werden. In diesem Zusammenhang wird der Begriff «Unmanned Aircraft System Traffic Management»1037 oder abgekürzt UTM verwendet.1038 Auf europäischer Ebene und auch in der Schweiz hat sich dafür der Begriff «Urban-Space» bzw. «U-Space» durchgesetzt.1039 Inhaltlich wird damit dasselbe, nämlich ein Flugverkehrsmanagementsystem 1029 Je kleiner die Drohe, desto näher am Betreiber muss diese fliegen, damit die grund- sätzlich geltende Vorgabe des direkten Sichtkontakts eingehalten werden kann (vgl. dazu Art. 2 Ziff. 7 und Art. 4 Abs. 1 lit. d Durchführungsverordnung). 1030 So EASA, Opinion, S. 17. 1031 DroneRules, Guide, S. 17. Zur Bedeutung dieses Aspekts, v.a. im Bereich des Daten� - schutzes, detailliert hinten, S. 157. 1032 Vgl. Art. 1 lit. a Verordnung über den Flugsicherungsdienst (VFSD). Flugverkehrs management umfasst als Überbegriff das Luftraum-Management (lit. b), das Verkehrsfluss- und Verkehrskapazitäts-Management (lit. c) und die Flugverkehrsdienste (lit. d). 1033 Hänsenberger, Diss., S. 70. Vgl. Art. 6 Abs. 1 sowie Anhang 1 VFSD. 1034 Vgl. zu Skyguide die Hinweise vorne, Fn. 1020. 1035 Art. 6 VFSD; vgl. auch Riedi, Rn. 648; Dettling-Ott, Flugsicherung, Rn. 583. 1036 BAZL, ConOps, S. 6; Hänsenberger, Diss., S. 70; Giemulla/Freiherr von Bothmer, S. 21. 1037 Vgl. zum Begriff Unmanned Aircraft System (UAS) vorne, S. 17 sowie S. 21. 1038 Vgl. Delise, S. 25; Masutti/Tomasello, S. 2; Schubert, S. 230. 1039 Vgl. SESAR, Master Plan, S. 4; BAZL, U-Space; BAZL, ConOps, passim; Stellungnahme des BR vom 29. August 2018 zur Interpellation Jositsch (18.3397); SUST, Ber icht Grenchen, S. 3. § 2 Identifizierung von Drohnenbetreibern 143 für Drohnen, beschrieben. Dieses soll den Zugang zum unteren Luftraum, insbesondere in urbanen Gebieten, ermöglichen.1040 Aufgrund des engen Zusammenhangs zwischen dem U-Space und den vorangehend dargestellten Vorschriften für Drohnen soll auch dieser kurz betrachtet werden.1041 2. Inhalt und Zweck Inhaltlich geht es um die Schaffung eines Systems, «mit dem eine automatisierte Verkehrsleitung für Drohnen eingeführt werden kann. Werden Drohnen dieser automatisierten Leitung unterstellt, sind ihre Identifizierung, die Überwachung im Luftraum, die Koordination mit anderen Luftverkehrsteilnehmenden sowie der Schutz von besonders sensiblen Gebieten sichergestellt.»1042 Als «all-encompassing framework»1043 soll der (Schweizer) U-Space zahlreiche Drohnenoperationen gleichzeitig und vor allem auf sichere und geordnete Weise ermöglichen.1044 Mit der zunehmenden Zahl an Drohnenflügen insgesamt und den komplexer werdenden Anwendungsfeldern ist dies für den bemannten Luftverkehr sowie für Dritte von zentraler Bedeutung.1045 Skyguide spielt bei der Einführung des U-Space in der Schweiz eine zentrale Rolle. Sie soll die anwendbaren Normen implementieren und die notwendigen Systeme entwickeln.1046 Die diesbezüglichen rechtlichen Vorgaben, welche ebenfalls auf Ebene der EU ausgearbeitet werden,1047 liegen jedoch noch nicht vor.1048 3. Datenschutzrechtliche Beurteilung Der U-Space als Flugverkehrsmanagementsystem ist nicht auf den Datenschutz und dessen Durchsetzung beim privaten Drohnenbetrieb ausgerichtet. 1040 Huttunen, U-space, S. 71 m.w.H. 1041 Registrierung und Fernidentifizierung sind (neben der Geo-Awareness; vgl. dazu hinten, S. 151 ff.) notwendige Voraussetzungen für die Einführung des U-Space (vgl. EASA, NPA, S. 12; Schubert, S. 230 ff.). 1042 Stellungnahme des BR vom 29. August 2018 zur Interpellation Jositsch (18.3397). 1043 BAZL, ConOps, S. 6. 1044 Vgl. zu den einzelnen Funktionen von U-Space und den damit verbundenen Umset- zungsschwierigkeiten Huttunen, U-Space, S. 80 ff. 1045 Vgl. nur SUST, Bericht Grenchen, S. 3. 1046 BAZL, Memorandum, S. 5. 1047 Vgl. zur Bedeutung europäischer Vorschriften in diesem Zusammenhang vorne, S. 13 ff. 1048 Eine «Opinion» der EASA zum U-Space soll im Februar 2020 publiziert werden. Diese hat selbst keine rechtliche Wirkung, dient aber der EU-Kommission als Hilfestellung beim Erlass entsprechender Normen (vgl. EASA, Opinions Information). 144 Teil 4: Bestandesaufnahme (2): Die «Drohnenverordnungen» Jedoch hielt der Bundesrat unlängst fest, dass der «U-Space sämtliche notwendigen Elemente für die Rechtsdurchsetzung enthalten [wird].»1049 Die entsprechende Interpellation bezog sich explizit auf Art. 179quater StGB und Art. 28 ZGB.1050 Da bisher noch keine gesetzlichen Grundlagen für den U-Space bestehen, konnte sich der Bundesrat nicht konkreter zu den angesprochenen Elementen der Rechtsdurchsetzung äussern. Die Identifizierung privater Drohnenbetreiber, welche in die Privatsphäre Dritter eingreifen, soll gemäss Bundesrat durch den U-Space aber klarerweise ermöglicht werden. Darauf zielte die vorerwähnte Interpellation denn auch ab; Ständerat Jositsch bezog sich in der Begründung auf die Schwierigkeit der «Rückverfolgung zum Besitzer» in «der konkreten Situation».1051 Aufgrund der engen Verknüpfung des Privat sphärenschutzes gemäss ZGB und den datenschutzrechtlichen Ansprüchen1052 müssen trotz ihrer fehlenden Nennung in der Interpellation auch Letztere erfasst sein. V. Spezialfall: Selbst gebaute Drohnen 1. Definition Drohnen, auch solche mit der Fähigkeit zur Datenbearbeitung, können mit einfachen Materialien und mit beschränktem finanziellem Aufwand selbst gebaut werden.1053 Aus diesem Grund hat der europäische Gesetzgeber solche Luftfahrzeuge explizit geregelt.1054 Eine selbst gebaute bzw. — in der Terminologie der Drohnenverordnungen — «privat hergestellte» Drohne ist in Art. 2 Ziff. 16 Durchführungsverordnung definiert als «ein UAS,1055 das vom Erbauer für seine eigenen Zwecke zusammengebaut oder hergestellt wurde, mit Ausnahme von UAS, die aus Bauteilen zusammengesetzt werden, die vom Hersteller als Fertigbausatz in Verkehr gebracht werden».1056 1049 Stellungnahme des BR vom 29. August 2018 zur Interpellation Jositsch (18.3397). 1050 Interpellation Jositsch (18.3397), Ziff. 1 und 2. Vgl. in diesem Zusammenhang die Ausführungen vorne, S. 106 ff. (ad Art. 28 ZGB) und BSK StGB-Ramel/Vogelsang, Art. 179quater N 5 ff. 1051 Interpellation Jositsch (18.3397). 1052 Vgl. dazu vorne, S. 106. 1053 Thomasen, Beyond Airspace Safety, S. 2; Juniper, S. 112; Jackman, S. 8 ff. Vgl. zum Eigenbau ausführlich Büchi, Buch der Drohnen, S. 188 ff. 1054 Vgl. EG 4 EASA-Grundverordnung. 1055 Vgl. zum Begriff Unmanned Aircraft System (UAS) vorne, S. 17 sowie S. 21. 1056 Diese Definition findet sich auch in Art. 3 Ziff. 22 Delegierte Verordnung. Nachfolgend soll von «selbst gebaut» die Rede sein, da «privat hergestellt» eher sperrig klingt und den geregelten Sachverhalt m.E. weniger gut beschreibt. § 2 Identifizierung von Drohnenbetreibern 2. 145 Anwendbare Vorschriften 2.1 Grundsätzliche Zugehörigkeit zur offenen Kategorie Um die auf selbst gebaute Drohnen anwendbaren Regeln darzustellen, ist nach deren Drohnenkategorie und ggf. -klasse zu fragen. Gemäss Art. 4 Abs. 1 lit. a Durchführungsverordnung werden selbst gebaute Drohnen der offenen Kategorie zugeordnet. Die Vorgaben der Durchführungsverordnung, welche für Operationen der offenen Kategorie gelten, müssen folglich auch von selbst gebauten Drohnen eingehalten werden. Die Startmasse derselben muss somit < 25 kg betragen und die Drohne darf nicht höher als 120 m fliegen.1057 2.2 Registrierung und Kennzeichnung Bei der Registrierungspflicht sind keine Ausnahmen für selbst gebaute Drohnen vorgesehen.1058 Da die Kennzeichnungspflicht an die Selbstregistrierung des Betreibers anknüpft, sind auch selbst gebaute Drohnen mit der eindeutigen Betreibernummer zu markieren. Auch hier hat diese Kennzeichnung «gut sichtbar, leserlich und dauerhaft» zu sein, was beim Bau der Drohne ggf. bereits beachtet werden muss.1059 2.3 Fernidentifizierung Gemäss Art. 2 Abs. 1 lit. a Delegierte Verordnung i.V.m. Art. 4 Abs. 1 lit. a Durchführungsverordnung sind Vorschriften und Bedingungen der Delegierten Verordnung auf selbst gebaute Drohnen nicht anwendbar. Sie werden keiner Produkteklasse zugeordnet und müssen somit u.a. die Vorgaben zur Fernidentifizierung nicht erfüllen. 3. Datenschutzrechtliche Beurteilung Mit Blick auf die Verhältnismässigkeit kann gegen die Registrierungs- und Kennzeichnungspflicht für selbst gebaute Drohnen eingewendet werden, dass bei diesen regelmässig das Interesse am Handwerklichen im Vordergrund steht. Aus Sicht des Datenschutzes ist jedoch zu begrüssen, dass diese Pflichten einzuhalten sind. Diese greifen etwa, wenn eine selbst gebaute Drohne mit einem Sensor ausgestattet ist, der Personendaten bearbeiten kann.1060 1057 Vgl. auch die weiteren Vorgaben in Art. 4 Abs. 1 lit. b bis f Durchführungsverordnung. 1058 Vgl. ausführlich zu den einzelnen Kriterien, die eine Registrierungspflicht des Drohnenbetreibers auslösen, vorne, S. 121 ff. 1059 Ausführlich zur Kennzeichnung vorne, S. 136 ff. 1060 Ausführlich dazu vorne, S. 124 f. 146 Teil 4: Bestandesaufnahme (2): Die «Drohnenverordnungen» Bedauerlich ist hingegen aus datenschutzrechtlicher Sicht, dass selbst gebaute Drohnen keine Informationen zur direkten Fernidentifizierung übertragen müssen. Die Identifizierung von deren Betreibern wird sich somit regelmässig schwierig gestalten. Es ist jedoch davon auszugehen, dass selbst gebaute Drohnen nicht sehr weit verbreitet sind und deren datenschutzrechtliches Gefährdungspotenzial deshalb beschränkt ist. VI. Herausforderungen bei der Umsetzung 1. Notwendige Mitwirkung der Drohnenbetreiber In der offenen Kategorie verlangen die gesetzlichen Regelungen die Mitwirkung der einzelnen Drohnenbetreiber. So ist eine Selbstregistrierung vorgesehen und auch die Kennzeichnung ist jedem Einzelnen übertragen.1061 Diese wenig restriktiven Vorgaben sind im Grundsatz zu begrüssen; strengere Regeln und höhere Anforderungen würden wohl von vielen privaten Drohnen betreibern als unverhältnismässig empfunden, was sodann zu illegalen Drohnenoperationen führen würde.1062 Bereits die vorangehend dargestellten Vorschriften zur Registrierung und Kennzeichnung werden wohl zunächst als übermässige administrative Bürde wahrgenommen werden (insbesondere im Unterschied zum bisherigen Schweizer Recht ohne vergleichbare Vorgaben). Dies ist jedoch bei einer so grundlegenden Neuregelung der Drohnenfliegerei unvermeidbar.1063 Die notwendige Mitwirkung ist primär durch Ausbildung und Aufklärung zu fördern.1064 Es gilt, gezielt auf die gewollten Wirkungen der (neuen) Vorschriften hinzuarbeiten.1065 Denn die Schweizer Rechtsordnung basiert auf der Annahme, dass sich die Rechtsunterworfenen grundsätzlich (freiwillig) normgemäss verhalten.1066 Die grundlegenden Änderungen der Rechtslage im Bereich der Drohnen verlangen folglich eingehende Informationsund Aufklärungsmassnahmen seitens der Behörden und insbesondere des BAZL.1067 Damit wird nicht nur der Vollzug des neuen Rechts erleichtert, 1061 Vgl. dazu vorne, S. 128 ff. (Registrierungsverfahren) bzw. S. 136 ff. (Kennzeichnung). 1062 Zu Recht weist JARUS, Categorization, S. 19, auf diese Problematik hin. Vgl. zur Bedeutung der Akzeptanz einer Regelung auch Rehbinder, Rn. 116. 1063 Vgl. dazu vorne, S. 13 ff. 1064 Noll, S. 150; Müller G., Rechtskenntnis, S. 557; Leissler, S. 536 m.w.H. 1065 Zur «Information über das Recht als Aufgabe des Staates» eingehend Müller/ Uhlmann, Rn. 327 m.w.H. Ausführlich zur Information und Aufklärung von Drohnenbetreibern hinten, S. 148 ff. 1066 Müller/Uhlmann, Rn. 312; ausführlich Rehbinder, Rn. 115 ff.; vgl. auch Noll, S. 150. 1067 Vgl. Müller/Uhlmann, Rn. 323. § 2 Identifizierung von Drohnenbetreibern 147 sondern gleichzeitig den Ängsten und Befürchtungen in der Bevölkerung entgegengewirkt.1068 Die Einhaltung der Vorschriften kann sodann durch die Androhung von Sanktionen «erzwungen» werden.1069 Die Schweiz ist gemäss Art. 131 EASA- Grundverordnung gehalten, «Vorschriften [zu erlassen] über Sanktionen, die bei Verstössen gegen diese Verordnung und die auf ihrer Grundlage erlassenen Delegierten Rechtsakte und Durchführungsrechtsakte zu verhängen sind (…). Die vorgesehenen Sanktionen müssen wirksam, verhältnismässig und abschreckend sein.» Die Androhung von Bussgeldern könnte als Sank tion etwa in Betracht kommen, wenn es um die Missachtung der Pflicht zur Selbstregistrierung oder um die Nicht- oder Falschkennzeichnung einer Drohne geht. Solche Bussen kennt das LFG bereits für Übertretungen verschiedener Vorschriften des Luftfahrtrechts.1070 2. Übergangsbestimmungen Im Kontext der Umsetzung sei zudem auf die vorgesehenen Übergangbestimmungen verwiesen.1071 Diese sehen verschiedene Ausnahmen zu den Vor gaben der Delegierten Verordnung (etwa Kennzeichnung der Produkteklasse, Fähigkeiten zur direkten Fernidentifizierung etc.) vor. Heute bereits verkaufte Drohnen können daher unter bestimmten Bedingungen weiterbetrieben werden;1072 eine «Nachrüstung» wird nicht verlangt. Die Übergangsfristen beschlagen jedoch nicht die Registrierung- und Kennzeichnungspflicht; dieser müssen Drohnenbetreiber ab dem 1. Januar 2021 nachkommen.1073 In Anbetracht der tiefgreifenden Änderungen, welche durch die Droh1068 Zur eher kritischen Haltung der Bevölkerung vorne, S. 5 f. 1069 Ob sich solche Sanktionen als wirksam erweisen werden, kann vorliegend nicht be- antwortet werden. Vgl. dazu Rehbinder, Rn. 115. 1070 Vgl. z.B. Art. 91 LFG. Für die Missachtung von Vorschriften für Drohnen der speziellen und v.a. der zulassungspflichtigen Kategorie sollten schärfere Sanktionen vorgesehen werden. Ausgehend vom Risiko, das die Missachtung der Regeln der jeweiligen Kategorie mit sich bringt, wäre auch die Androhung einer Freiheitsstrafe durchaus vertretbar (vgl. etwa Art. 89 und 90 LFG). 1071 Vgl. Art. 22 Durchführungsverordnung sowie die «besonderen Bestimmungen» in Art. 20 Durchführungsverordnung. Die Einzelheiten dieser Regelungen sollen nicht weiter vertieft werden. 1072 Art. 20 Durchführungsverordnung etwa erlaubt den Betrieb in der Kategorie A1 für Drohnen mit einer Startmasse von < 250 g bzw. in der Kategorie A3 für solche mit einer Startmasse < 25 kg (jeweils einschliesslich Nutzlast). Vgl. zu den Einsatzkategorien vorne, S. 22 mit den dortigen Hinweisen. 1073 Die Übergangsbestimmungen verweisen einzig auf die Vorgaben der Delegierten Verordnung (vgl. dazu im Einzelnen Anhang Delegierte Verordnung). Zum Geltungsbeginn der Durchführungsverordnung vorne, S. 14 mit Fn. 89. 148 Teil 4: Bestandesaufnahme (2): Die «Drohnenverordnungen» nenverordnungen veranlasst wurden, erscheinen solche Übergangsbestimmungen — für Hersteller wie Betreiber von Drohnen — unumgänglich.1074 Aus Sicht der Drohnenbetreiber wurde dadurch eine verhältnismässige Regelung geschaffen; sie dürfen ihre bereits gekauften Drohnen unter gewissen Voraussetzungen weiterbetreiben. Die damit einhergehenden Verzögerungen bis zur vollständigen Umsetzung des neuen Rechtsrahmens sind hinzunehmen. § 3 Präventive und flankierende Massnahmen Unter diesem Titel sollen weitere Vorschriften der Drohnenverordnungen analysiert werden, welche nicht auf die Identifizierung von Drohnenbetreibern ausgerichtet sind, sich jedoch positiv auf die Einhaltung von Datenschutzbestimmungen auswirken (können). Dabei werden zunächst die Information und Ausbildung von Drohnenbetreibern analysiert (sogleich). Danach soll kurz auf die Vorgaben zur Geo-Awareness (hinten, S. 151 ff.) sowie zu den Positionslichtern (hinten, S. 153 ff.) eingegangen werden. I. Information und Ausbildung Viele datenschutzrechtliche Vorschriften und deren Bedeutung sind einem Grossteil aller Privatpersonen wohl gar nicht bekannt.1075 Eine Sensibilisierung bezüglich dieser Thematik drängt sich deshalb auf; Betreibern und Pilo ten soll ab dem Zeitpunkt des Kaufs der Drohne bewusst gemacht werden, dass für deren Betrieb bestimmte Anforderungen und Beschränkungen gelten.1076 Der EU-Gesetzgeber schreibt in diesem Kontext Informationsbei blätter und eine Theorie-Prüfung für Drohnenpiloten vor. 1. Informationsbeiblatt Hersteller, Einführer1077 und Händler1078 von Drohnen müssen sicherstellen, 1074 Vgl. zu Zweck und Bedeutung von Übergangsregelungen Müller/Uhlmann, Rn. 345. 1075 Vgl. etwa EDÖB, Drohnen: «In einigen Fällen sind sich die [Drohnen-]Operateure nicht bewusst, dass sie etwas Unrechtmässiges (evtl. sogar Strafbares) machen.» Ähnlich Stöcker et al., S. 18. 1076 Vgl. EG 20 Durchführungsverordnung. 1077 Einführer (engl. importer) ist «jede in der Union niedergelassene natürliche oder juristische Person, die ein Erzeugnis aus einem Drittland auf dem Unionsmarkt in Verkehr bringt» (Art. 3 Ziff.15 Delegierte Verordnung). 1078 Händler (engl. distributor) ist «jede natürliche oder juristische Person in der Liefer- kette, die ein Erzeugnis auf dem Markt bereitstellt, mit Ausnahme des Herstellers oder des Einführers» (Art. 3 Ziff. 16 Delegierte Verordnung). § 3 Präventive und flankierende Massnahmen 149 dass diesen ein Informationsblatt beiliegt, welches klar, verständlich und lesbar sein muss.1079 Anders als die deutschen Sprachfassungen dieser Bestimmungen vermuten lassen, sind damit auch elektronische Instrumente wie QR-Codes erfasst.1080 Der Inhalt der Information ist von der EASA vorgegeben und klärt über die geltenden Beschränkungen und Auflagen für die jeweilige Produkteklasse1081 auf.1082 Inhaltlich liegt der Fokus auf der Verhinderung von Betriebsgefahren und der Gewährleistung der Luftsicherheit.1083 Die Informationsblätter der EASA1084 enthalten aber auch jeweils den folgenden Hinweis: «Do not take photographs, videos or sound recordings of people without their permission. Respect people’s privacy.»1085 Diese allgemein formulierten Verhaltensanweisungen vermögen für sich allein genommen noch keine ausreichende Sensibilisierung zu bewirken. Trotzdem sind sie zu begrüssen, da sie die Bedeutung des Datenschutzes im Kontext der Drohnenfliegerei unterstreichen.1086 2. Online-Theorieprüfung Für Flüge mit Drohnen der offenen Kategorie wird vom Drohnenpiloten1087 die Absolvierung eines Online-Lehrgangs, der erfolgreich mit einer OnlineTheorieprüfung abgeschlossen wird, verlangt.1088 Eine solche Prüfung ist für Drohnen der Klasse C0 nicht vorgeschrieben; die Kenntnisnahme des 1079 Vgl. Art. 6 Abs. 7, Art. 8 Abs. 4 und Art. 9 Abs. 2 Delegierte Verordnung. Vgl. JARUS, Categorization, S. 22. 1080 In der englischen Fassung ist denn auch von «information notice» und nicht mehr von «information leaflet» die Rede. Dies wurde mit Blick auf die Erweiterung der möglichen Instrumente angepasst. Vgl. dazu EASA, Opinion, S. 13; JARUS, Operations, S. 20 und S. 28 ff. 1081 Vgl. zu den (Produkte-)Klassen C0 bis C3 vorne,S. 24 ff. 1082 Vgl. Teil 1 Ziff. 9, Teil 2 Ziff. 19, Teil 3 Ziff. 20, Teil 4 Ziff. 16 und Teil 5 Ziff. 5 Anhang Delegierte Verordnung. Vgl. auch Krumm, Anpassungsbedarf, S. 118. 1083 EASA, Opinion, S. 12 f.; EASA, A-NPA, S. 20. 1084 Die EASA hat für jede Produkteklasse (C0 bis C4) ein eigenes Dokument ausgearbei- tet. Diese enthalten die jeweils geltenden Vorschriften; im Bereich des Datenschutzes und der Privatsphäre gilt für alle Produkteklassen dasselbe. 1085 Vgl. EASA, awareness leaflets, passim. In ähnlicher Form war dieser Hinweis auch bereits in EASA, NPA, S. 119 ff. enthalten. 1086 Vgl. dazu auch JARUS, Categorization, S. 29. 1087 Die Vorschriften zur Online-Theorieprüfung beziehen sich explizit auf die Drohnen- piloten, da sie die Drohnen fliegen. Dieser Terminologie soll in diesem Abschnitt gefolgt werden; vgl. grundsätzlich zur Begriffsverwendung vorne, S. 27 f. 1088 UAS.OPEN.020 Abs. 4 lit. b, UAS.OPEN.030 Abs. 2 lit. a, UAS.OPEN.040 Abs. 3 Anhang Teil A Durchführungsverordnung. Vgl. JARUS, Categorization, S. 22. 150 Teil 4: Bestandesaufnahme (2): Die «Drohnenverordnungen» Informationsbeiblatts reicht aus.1089 Der Zeitpunkt der Online-Theorieprüfung könnte mit der Selbstregistrierung der Betreiber verknüpft werden.1090 Die Prüfung umfasst 40 Multiple-Choice-Fragen, die sich «angemessen» auf verschiedene Themen wie Flugsicherheit, Luftraumbeschränkungen, Luftsicherheit und Luftrecht verteilen sollen.1091 Ebenfalls aufgeführt ist das Thema «Schutz der Privatsphäre und der Daten».1092 Die Piloten müssen über theoretische Kenntnisse bezüglich der in diesem Kontext bestehenden Risiken sowie der grundlegenden Prinzipien des Datenschutzes gemäss anwendbarem Datenschutzgesetz verfügen.1093 Das BAZL ist für die Ausstellung von Nachweisen über das erfolgreiche Bestehen der Online-Theorieprüfung zuständig.1094 Es ist jedoch nicht davon auszugehen, dass das Bundesamt den Online-Lehrgang selbst ausarbeitet und/oder die Online-Theorieprüfung selbst durchführt.1095 Vielmehr werden diese Aufgaben wohl von einem externen Dienstleister erbracht.1096 Das BAZL hat sicherzustellen, dass die Dienstleistungen den Vorgaben der Durch führungsverordnung entsprechen. Mithin hat es in einer allfälligen Aus schreibung entsprechende Fachkompetenzen des Dienstleisters (etwa im Datenschutzrecht) als anbieterbezogenes Eignungskriterium1097 zu defi nieren.1098 Bezüglich Fragen zum Datenschutz kommt zudem ein (infor meller) Austausch mit dem EDÖB in Betracht.1099 1089 Vgl. Josipovic, S. 441; EASA, Opinion, S. 21. Dazu vorne, S. 148 f. 1090 Vgl. zur Selbstregistrierung per Onlineformular vorne, S. 128 ff. 1091 Vgl. die Aufzählung in UAS.OPEN.020 Abs. 4 lit. b. Die weiteren geprüften Themen sind menschliches Leistungsvermögen und dessen Grenzen, Betriebsverfahren, allgemeine Kenntnisse zu UAS und Versicherung. 1092 UAS.OPEN.020 Abs. 4 lit. b Ziff. vii. Vgl. zur Verwendung des Begriffs «Privatsphäre» die Hinweise vorne, Fn. 896. 1093 Vgl. EASA, GM Part-UAS, S. 11. 1094 Art. 18 lit. c Durchführungsverordnung. Diese können auch elektronisch ausgestellt werden (EASA, GM Part-UAS, S. 12). 1095 Vgl. zu den beschränkten personellen Ressourcen beim BAZL Henseler, Digitalisie- rung, S. 184 f. und S. 197 f. m.w.H. 1096 Vgl. UAS.OPEN.020 Abs. 4 lit. b spricht in diesem Zusammenhang «von einer Stelle […], die von der zuständigen Behörde des Mitgliedstaats, in dem der UAS-Betreiber registriert ist, anerkannt wurde». Vgl. zur öffentlichen Vergabe von Dienstleistungsaufträgen Galli et al., Rn. 225 ff.; Poltier, Rn. 213. 1097 Zum Begriff Poltier, Rn. 321 ff. 1098 Vgl. Hänni/Stöckli, Rn. 762; Karlen, Verwaltungsrecht, S. 372 f.; Trüeb, Art. 9 BöB N 3 f. 1099 Vgl. Leissler, S. 536; Roma, S. 30. Zu datenschutzrechtlichen Themen erteilt das BAZL denn auch keine Auskunft, sondern verweist auf die Webseite des EDÖB. § 3 Präventive und flankierende Massnahmen 151 Der Datenschutz und die Privatsphäre sind «angemessen» bei den 40 Multiple- Choice-Fragen zu berücksichtigen. In Anbetracht der nicht zu unterschätzenden Bedeutung der weiteren Themen, primär mit Blick auf die Sicherheit von Drohnenflügen, werden wohl keine vertieften Kenntnisse im Bereich des Datenschutzes verlangt werden können. Trotzdem wird den Prüflingen eine Auseinandersetzung mit den wichtigsten Aspekten des Datenschutzrechts und des Rechts auf Schutz der Privatsphäre abverlangt. Dadurch kann eine gewisse Sensibilisierung erreicht werden, was zu begrüssen ist. 3. Staatliche Informationsmassnahmen In Ergänzung zu den dargestellten Informations- und Aufklärungsmassnahmen sollte das BAZL bei der Bekanntmachung der neuen Vorschriften für Drohnen proaktiv handeln;1100 dies hat es auch bis anhin auf innovative Weise getan.1101 Die gesellschaftliche Akzeptanz der Drohnentechnologie1102 würde durch eine Sensibilisierung der Gesamtbevölkerung auf datenschutzrechtliche Themen und die diesbezüglichen Vorschriften in den Drohnenverordnungen sicherlich zunehmen. Des Weiteren ist in diesem Zusammenhang eine enge Zusammenarbeit mit dem EDÖB und ggf. kantonalen Stellen wünschenswert, damit die neuen (datenschutzrechtlichen) Vorgaben in der breiten Öffentlichkeit bekannt gemacht werden können.1103 II. Geo-Awareness (Geofencing) 1. Begriff und Abgrenzung Geo-Awareness beschreibt eine Funktion, die eine potenzielle Verletzung von Luftraumgrenzen erkennt und die Piloten warnt, sodass diese sofortige und wirksame Massnahmen zur Vermeidung dieser Verletzung ergreifen können.1104 Ziel eines Geo-Awareness-Systems ist demnach, den Piloten in die Lage zu versetzen, den Eintritt einer drohenden Rechtsverletzung zu verhindern, indem es ihn mit Informationen über den Standort der Drohne in der Luft und ggf. von weiteren Luftfahrzeugen versorgt.1105 1100 Vgl. JARUS, Categorization, S. 29. Vgl. allgemein zur Information der Rechtsunter worfenen über Inhalt und Wirkung (neuer) Gesetze vorne, S. 146 m.w.H. 1101 So hat das BAZL etwa mehrere Videos zum Thema sowie ein «Drohnenquiz» erstellt (alles auf der Webseite des Bundesamts abrufbar). 1102 Vgl. vorne, S. 5 f. 1103 Vgl. Roma, S. 30; Stöcker et al., S. 18. 1104 Vgl. die Definition in Art. 2 Ziff. 15 Durchführungsverordnung. 1105 Krumm, Anpassungsbedarf, S. 118. 152 Teil 4: Bestandesaufnahme (2): Die «Drohnenverordnungen» Im Gegensatz dazu zielt Geofencing1106 darauf ab, Drohnen softwaregestützt daran zu hindern, bestimmte einprogrammierte Lufträume anzufliegen oder zu verlassen. Zu denken ist etwa an Flugverbotszonen rund um Flughäfen; es können aber auch Innenstädte oder kritische Infrastrukturen «abgezäunt» werden (sog. no-drone zones).1107 Des Weiteren sind Beschränkungen der Ge schwindigkeit und/oder der Flughöhe in gewissen Zonen möglich.1108 Die hier beschriebene Abgrenzung wird in der Literatur nicht immer vorgenommen. Der Begriff «Geo-Awareness» ist neuer und deshalb noch weniger verbreitet. Deshalb ist regelmässig von Geofencing die Rede, wenn eigentlich Geo-Awareness gemeint ist. Aufgrund der sehr ähnlichen Zielrichtung der beiden Funktionen ist eine klare Abgrenzung derselben regelmässig nicht zwingend notwendig. Die deutsche Wortschöpfung «Geo-Sensibilisierung», als Übersetzung von Geo-Awareness1109 ist kaum verbreitet und sollte deshalb nur verwendet werden, wenn dies unumgänglich ist.1110 2. Inhalt und Grenzen Geo-Awareness sollte bereits durch die Hersteller von Drohnen (vor-)installiert werden.1111 Die Delegierte Verordnung fordert ein «Geo-Awareness-System» für Drohnen der Klassen C1 bis C3.1112 Die Ausstattung damit erlaubt den zuständigen Behörden die Schaffung von spezifisch umgrenzten Gebieten, welche «aus Gründen der Sicherheit und Gefahrenabwehr, des Schutzes der Privatsphäre oder der Umwelt» ausgeschieden werden. Darin können bestimmte Drohnenoperationen verboten bzw. erlaubt und gleichzeitig Auflagen gemacht werden.1113 Wichtig ist, dass der Zugang zum Luftraum durch diese technische Beschränkung nicht zu stark behindert wird. Grundsätzlich sollte der niedrige Luftraum allen Drohnenbetreibern offenstehen, solange sie sich an die 1106 Auch Bezeichnungen wie «Electronic Fencing» oder «GPS-Käfig» sind vereinzelt anzutreffen, vgl. Landrock/Baumgärtel, S. 13. 1107 Schubert, S. 225; vgl. EASA, A-NPA, S. 18. 1108 Zum Ganzen Hänsenberger, Diss., S. 216 f.; Landrock/Baumgärtel, S. 13 f.; Huttunen, U-space, S. 83 f. Vgl. auch JARUS, Categorization, S. 21. 1109 Vgl. Art. 2 Ziff. 15 Durchführungsverordnung. 1110 Die Legaldefinitionen in Art. 2 Durchführungsverordnung enthalten denn auch an verschiedenen Stellen den englischen «Originalbegriff». 1111 BRTF, S. 20. Es bestehen (noch) keine international anerkannten Standards dazu (Masutti/Tomasello, S. 2). 1112 Teil 2 Ziff. 13, Teil 3 Ziff. 15 und Teil 4 Ziff. 10 Anhang Delegierte Verordnung. Vgl. zu den (Produkte-)Klassen C0 bis C3 vorne, S. 24 ff. 1113 Vgl. Art. 15 Abs. 1 Durchführungsverordnung; Groh, S. 182 f. § 3 Präventive und flankierende Massnahmen 153 geltenden Vorschriften halten.1114 Unverhältnismässig wäre es jedenfalls, wenn grossflächig Städte und Dörfer insgesamt als «no-drone zones» ausgeschieden und dadurch Drohnenflüge gänzlich unterbunden würden.1115 3. Datenschutzrechtliche Beurteilung Diese hier diskutierten Luftraumbeschränkungen sind vor allem aus Sicht der bemannten Luftfahrt zentral.1116 Doch auch aus datenschutzrechtlicher Optik, namentlich im Rahmen der Privacy by Design,1117 kann Geo-Awareness nützlich sein, indem z.B. in besonders sensiblen Gebieten Drohnenflüge verboten oder stark beschränkt werden können (z.B. im Umkreis von Spitälern oder Gotteshäusern).1118 Umgekehrt können auch die Betreiber die Geo-Awareness-Funktion ihrer Drohne nutzen, um Flugpläne zu erstellen und die Drohne so vom Fliegen ausserhalb des vorgesehenen Bereichs abzuhalten, wodurch das Risiko der Bearbeitung von Daten, die der Betreiber möglicherweise nicht bearbeiten darf oder will, verringert wird.1119 Selbstverständlich ist nicht jeder Drohnenbetreiber bereit, seinen Drohnenflug einzig auf einer vor definierten Route zu fliegen. Denkbar wäre aber jedenfalls, dass im Vorfeld bestimmte Gebiete, wie etwa Wohnsiedlungen, davon ausgenommen würden und so das Risiko von ungewollten Datenbearbeitungen minimiert würde. Dies setzt jedoch voraus, dass sich Drohnenbetreiber der datenschutz rechtlichen Risiken ihrer Aktivitäten bewusst sind.1120 III. Positionslichter 1. Erfasste Drohnen Als letzte präventive Massnahme seien hier die Positionslichter erwähnt. Für Drohnen der Klassen C1 bis C31121 fordert die Delegierte Verordnung die Ausrüstung mit Lichtern für die Steuerbarkeit1122 sowie die Unterscheidbarkeit 1114 Vgl. Huttunen, U-space, S. 84. 1115 Vgl. zur Verhältnismässigkeit diesbezüglicher Vorgaben in der offenen Kategorie JARUS, Categorization, S. 21. 1116 Vgl. Masutti/Tomasello, S. 78; BRTF, S. 20. 1117 Dazu ausführlich vorne, S. 92 f. 1118 Vgl. Koelle et al., S. 158; Euler/Cobus/Koelle, S. 150. 1119 Vgl. Vivet/Smith, S. 7. 1120 Vgl. zu Information und Ausbildung vorne, S. 148 ff. 1121 Vgl. zu den (Produkte-)Klassen C0 bis C3 vorne, S. 24 ff. 1122 Die Kenntnis der genauen Lage der Drohne in der Luft ist unerlässlich, um die Drohne steuern zu können. 154 Teil 4: Bestandesaufnahme (2): Die «Drohnenverordnungen» der Drohne bei Nacht. Die Anordnung der Lichter soll es einer Person am Boden ermöglichen, die Drohne von einem bemannten Luftfahrzeug unterscheiden zu können.1123 Nicht vorgeschrieben sind die Lichter für Drohnen der Klassen C0. Diese dürften aufgrund ihrer beschränkten Grösse nicht in der Lage sein, die notwendigen Lichter zu tragen, um diese Vorgaben zu erfüllen.1124 2. Datenschutzrechtliche Beurteilung Unter dem Titel der Privacy by Design wurden «Warnlichter» bereits angesprochen.1125 Die von der Delegierten Verordnung geforderten Lichter sind gemäss dem klaren Wortlaut der Bestimmungen aber auf die Steuerbarkeit der Drohnen ausgerichtet.1126 Werden Drohnen in der Nacht eingesetzt, so müssen sie anhand der Positionslichter von bemannten Luftfahrzeugen unterschieden werden können. Dies geht zumindest teilweise in Richtung des Datenschutzes durch Technik:1127 Eine möglicherweise betroffene Person kann eine Drohne anhand von deren Lichtern orten, als solche erkennen und ihr ggf. ausweichen. Ob überhaupt eine Datenbearbeitung durch die Drohne stattfindet, lässt sich zwar nicht erkennen. Es ist aber ohne Weiteres möglich, dass eine Drohne, welche in der Nacht eingesetzt wird, über eine Infrarot- oder Thermalkamera verfügt oder mithilfe des Steuerlichts und einer «normalen» Kamera Daten bearbeiten kann.1128 Insgesamt erscheinen die Vorschriften zu «Drohnenlichtern» dem Datenschutz zuträglich zu sein. Die bessere Sichtbarkeit der Drohne am Himmel und deren Identifizierbarkeit anhand der Positionslichter sind klar zu begrüssen. Denkt man den Datenschutz durch Lichtsignale weiter, so kämen zudem farbige Leuchtsignale oder Kennzeichen in Betracht, welche Aufschluss über die Fähigkeiten der Drohne sowie deren aktuellen Einsatz geben 1123 Teil 2 Ziff. 16, Teil 3 Ziff. 18 und Teil 4 Ziff. 14 Anhang Delegierte Verordnung. 1124 Die maximale Startmasse für eine Drohne der Klasse C0 beträgt < 250 g. Vgl. auch vorne, S. 24 f. 1125 Vorne, S. 92 f. 1126 Teil 2 Ziff. 16, Teil 3 Ziff. 18 und Teil 4 Ziff. 14 Anhang Delegierte Verordnung. Vgl. EASA, Opinion, S. 14. 1127 Vgl. Anhang IX Ziff. 1.3 EASA-Grundverordnung, wonach beim Betrieb von Drohnen auch «den Grundsätzen des Schutzes der Privatsphäre und personenbezogener Daten durch Technik und Voreinstellungen Rechnung [zu] tragen» ist. 1128 Vgl. ausführlich zu den Sensoren vorne, S. 40 ff., und spezifisch zu Infrarot- und Wärme bildkameras S. 42. § 4 Gesamtbetrachtung 155 würden (z.B. ob gefilmt wird).1129 Blinklichter, die in einer gewissen Abfolge ausgesendet werden, könnten zudem die Identifizierung der Drohne und/ oder des Betreibers ermöglichen. Solche Zeichen können von jeder handelsüblichen Kamera oder einem Smartphone erfasst und mit entsprechender Software verarbeitet werden.1130 Diesbezügliche Vorgaben müssten auf Ebene der EU (bzw. den gesamten europäischen Luftraum umfassend) beschlossen werden, damit sich die Farbcodes bzw. Lichtsequenzen durchsetzen und flächendeckend richtig interpretiert werden könnten.1131 Ein Vorteil der Nutzung von Lichtsignalen wäre, dass diese auch funktionieren, wenn keine (verlässliche) Funk- oder Satellitenabdeckung besteht.1132 De lege lata sind die Vorschriften zu den Positionslichtern aber «nur» auf die Steuerbarkeit und Unterscheidbarkeit der Drohne beschränkt. § 4 Gesamtbetrachtung Teil 4 der vorliegenden Untersuchung hat die drohnengestützte Datenbearbeitung vor dem Hintergrund der Drohnenverordnungen der EU und insbesondere der darin enthaltenen Bestimmungen mit Bezügen zum Datenschutz beleuchtet. Diese Bestandesaufnahme hat gezeigt, dass in verschiedenen Bereichen wichtige Bestimmungen eingeführt wurden, die dem Datenschutz zuträglich sind. Die Registrierungsvorschriften und die damit verbundene Kennzeichnungspflicht erfassen die allermeisten Drohnen der offenen Kategorie. Dadurch wird die Grundlage für eine Identifizierung der Drohnenbetreiber und somit die Durchsetzung verschiedener Rechtsansprüche diesen gegenüber geschaffen. Die Vorschriften zur Fernidentifizierung stellen sodann einen weiteren wesentlichen Schritt in dieselbe Richtung dar. Die präventiven und flankierenden Massnahmen wie Geo-Awareness und Positionslichter können ebenfalls einen Beitrag zur Einhaltung datenschutzrechtlicher Vorgaben leisten. Auch die für die allermeisten Drohnenpiloten vorgeschriebene Online-Theorieprüfung sowie Informationsmassnahmen seitens der Händler und Behörden spielen eine nicht zu unterschätzende Rolle. Das Bewusstsein für neue Regeln und die damit einhergehenden 1129 Froomkin/Colangelo, S. 61 ff.; Thomasen, Beyond Airspace Safety, S. 20; Koelle et al., S. 158. 1130 Vgl. Singh/Carthew/Xiong, S. 4. 1131 Zur Bedeutung von (internationalen) Standards in diesem Kontext Froomkin/ Colangelo, S. 63. 1132 Singh/Carthew/Xiong, S. 4. 156 Teil 4: Bestandesaufnahme (2): Die «Drohnenverordnungen» Pflichten von Drohnenbetreibern wird dadurch geschärft. Die gezielte Ausbildung und Information von Drohnenbetreibern bezüglich datenschutzrechtlicher Themen sind deshalb klar zu begrüssen. Insgesamt ist in den Drohnenverordnungen aber keine wirksame Möglichkeit zur Durchsetzung von datenschutzrechtlichen Ansprüchen vorgesehen. Sie enthalten zwar mit der Registrierungs- und Kennzeichnungspflicht sowie der Fernidentifizierung die Grundlagen dafür, doch fehlt eine Verbindung derselben mit dem Datenschutzrecht und den darin enthaltenen Ansprüchen. Es besteht somit noch immer eine Lücke im Rechtsschutz. 157 Teil 5: Die Durchsetzung von daten schutzrechtlichen Ansprüchen § 1 Schutzpflicht bei der drohnengestützten Datenbearbeitung durch Private I. Informations- und Machtungleichgewicht als Ausgangspunkt Das einleitend festgestellte Informations- und Machtungleichgewicht1133 zwischen Drohnenbetreiber und betroffener Person ist im Bereich des Datenschutzrechts besonders ausgeprägt.1134 Letztere weiss nicht, ob und welche Daten über sie bearbeitet werden. Hinzu kommt, dass sie die im E-DSG verankerten Rechte kaum einmal wirksam geltend machen kann, da es an Transparenz mangelt: Die Informationspflicht läuft ohne die Mitwirkung des Drohnenbetreibers ins Leere; das Auskunftsrecht kann in den allermeisten Fällen aufgrund fehlender Informationen über den Verantwortlichen nicht ausgeübt werden. Daraus folgt, dass auch die weiteren Rechtsschutzmöglichkeiten gemäss E-DSG und ZGB1135 nicht geltend gemacht werden können.1136 Es überrascht deshalb nicht, dass gemäss Studien Transparenz «eine der zentralen Forderungen» von betroffenen Personen ist.1137 Insgesamt zeigt die drohnengestützte Datenbearbeitung durch Private exemplarisch die erheblichen Vollzugsdefizite im Datenschutzrecht auf.1138 1133 Vgl. vorne, S. 6, mit den dortigen Hinweisen. 1134 Thomasen, Beyond Airspace Safety, S. 19 f.; Froomkin/Colangelo, S. 49. Vgl. zum «Machtgefälle zwischen Datenbearbeiter und betroffener Person» auch bereits Aebi-Müller, Rn. 590, fünftes Lemma. Vgl. sodann Müller L., Diss., S. 133 und S. 310, jeweils mit Blick auf die (statische) Videoüberwachung. 1135 Vgl. dazu vorne, S. 110 ff. 1136 Vgl. Roma, S. 30; DroneRules, Handbook, S. 7. Vgl. zur gleich gelagerten Problematik im Bereich des Besitzschutzes nach ZGB BSK ZGB II-Ernst, Art. 926 N 5a und Art. 928 N 3a; Hrubesch-Millauer/Bruggisser, Rn. 38 ff. 1137 Euler/Cobus/Koelle, S. 150. Die Studie befasste sich mit dem Thema «Smart Cams», worunter neben Drohnen auch Dashcams und Datenbrillen subsumiert wurden. 1138 Vgl. dazu nur Bericht Evaluation DSG, S. 342 f.; Vasella/Sievers, S. 44 und S. 48; sowie den «Schreckensbericht» von Kühling et al., passim (zum deutschen Recht). 158 Teil 5: Die Durchsetzung von datenschutzrechtlichen Ansprüchen Die Bestimmungen zur Identifizierung und Information von Drohnenbetreibern gemäss den im vorangehenden Teil dieser Untersuchung dargestellten Regelwerken vermögen keine hinreichende Abhilfe zu schaffen. Es liegt daher eine Situation vor, in welcher die grundrechtlich verankerten und im E-DSG konkretisierten Ansprüche der betroffenen Person nicht mehr effektiv zur Geltung kommen. Es gilt deshalb zu prüfen, ob sich daraus eine Schutzpflicht ergibt und wie diese ggf. wahrzunehmen ist. II. Tatbestand 1. Vorliegen eines Störerdreiecks Im hier interessierenden Bereich der drohnengestützten Datenbearbeitung durch Private ist das vorerwähnte Dreiecksverhältnis1139 zwischen «beeinträchtigtem Grundrechtsträger» — «Verursacher eines Übergriffs» — Staat offensichtlich. Die Drohnenbetreiber verursachen als Verantwortliche1140 regelmässig einen Übergriff in eine grundrechtlich geschützte Position. Die beeinträchtigten Grundrechtsträger sind andere, davon betroffene Private; deren Grundrechtsträgerschaft ist vorliegend klar zu bejahen.1141 2. Kenntnis der konkreten Gefährdung Des Weiteren steht fest, dass der Staat Kenntnis von der Problematik hat: So wiesen die Geschäftsprüfungskommissionen und die Geschäftsprüfungsdelegation der eidgenössischen Räte bereits im Jahr 2008 darauf hin, dass «mit Kameras ausgestattete Mini-Drohnen weit grössere Einblicke in die Privatsphäre des Bürgers ermöglichen», als dies bei Aufklärungsdrohnen der Armee der Fall sei.1142 Zahlreiche politische Vorstösse zum Thema unterstreichen diese Feststellung.1143 Ferner zeigt die laufende Revision des DSG und die damit angestrebte Stärkung der Rechte der betroffenen Personen, dass die verschiedenartigen Grundrechtsgefährdungen im Bereich der informationellen Selbstbestimmung bekannt waren und sind.1144 Diese aktualisieren sich bei drohnen 1139 Vgl. dazu vorne, S. 47. 1140 Vgl. zum Begriff vorne, S. 74. 1141 Vgl. dazu vorne, S. 63 und S. 65. 1142 Jahresbericht GPK/GPDel 2007, S. 5155. Zum Einsatz von Aufklärungsdrohnen der Ar- mee vgl. vorne, S. 35 f. 1143 Vgl. dazu die Hinweise vorne, in Fn. 6. 1144 Vgl. dazu ausführlich vorne, S. 11 f. § 1 Schutzpflicht bei der drohnengestützten Datenbearbeitung durch Private 159 gestützten Datenbearbeitungen durch Private in erheblichem Masse; die erforderliche Schwere der Gefährdung ist somit ebenfalls zu bejahen.1145 Hinzu kommt, dass der Bundesrat unlängst explizit Lücken bei der Rechtsdurchsetzung im Zusammenhang mit Drohnen eingeräumt hat.1146 Das Bewusstsein für die bestehenden Mängel im Grundrechtsschutz sind Bundesrat und Parlament demnach bekannt. 3. Kompetenz des Bundes Die Gesetzgebung im Bereich des Datenschutzes sowie der Luftfahrt ist Sache des Bundes. Für privatrechtliche Datenschutzbestimmungen kann sich der Bundesgesetzgeber auf die Rechtsetzungskompetenzen in den Bereichen Zivilrecht (Art. 122 BV), privatwirtschaftliche Erwerbstätigkeit (Art. 95 BV) und Verbraucherschutz (Art. 97 Abs. 1 BV) berufen.1147 Datenschutzvorschriften für Behörden und Verwaltungsstellen stützt der Bundesgesetz geber auf die organisatorische Zuständigkeit nach Art. 173 Abs. 2 BV.1148 Die Gesetzgebung über die Luftfahrt ist gemäss Art. 87 Abs. 1 BV dem Bund vorbehalten.1149 Dieser ist folglich rechtlich in der Lage, in den hier interessierenden Rechtsgebieten Massnahmen zu ergreifen. III. Adressat der Schutzpflicht Die Exekutive (unter Einschluss der Vollzugsbehörden), die Legislative und die Judikative können zur Wahrnehmung von Schutzpflichten berufen sein.1150 Gerichten kommt dabei im hier interessierenden Kontext kaum Bedeutung zu. Aus diesem Grund werden nachfolgend nur die Rolle der Vollzugsbehörden sowie diejenige des Gesetzgebers diskutiert. 1145 Vgl. dazu vorne, S. 52 f. 1146 Stellungnahme des BR vom 29. August 2018 zur Interpellation Jositsch (18.3397); vgl. dazu auch die Ausführungen vorne, S. 143 f. 1147 Botschaft Totalrevision DSG, S. 7185, sowie Botschaft DSG 1988, S. 424 ff. Ausführlich zur Zuständigkeit des Bundes Belser, in: Belser/Epiney/Waldmann, § 5 Rn. 7 ff.; vgl. auch SHK DSG-Fey, Art. 1 N 5 f. Die im E-DSG erstmals erwähnte Kompetenzgrundlage in Art. 97 Abs. 1 BV wurde in der Lehre teilweise kritisiert (vgl. z.B. Vasella, Helsana+). 1148 So explizit Botschaft Totalrevision DSG, S. 7186; vgl. auch Botschaft DSG 1988, S. 425. Gemäss Biaggini, Komm. BV, Art. 173 N 33, begründet diese Bestimmung aber keine Bundeskompetenzen; vielmehr handle es sich um «einen ‹Platzhalter› für eine inhärente Kompetenz des Bundes» (ebd., Vorbem. Art. 42–135 N 19); vgl. Belser, in: Belser/ Epiney/Waldmann, § 5 Rn. 33 f. 1149 Ausführlich dazu statt anderer BSK BV-Kern, Art. 87 N 24 ff.; Biaggini, Komm. BV, Art. 87 N 14 ff. 1150 Ausführlich dazu vorne, S. 51 f. 160 1. Teil 5: Die Durchsetzung von datenschutzrechtlichen Ansprüchen Vollzugsbehörden Als Vollzugsbehörde ist primär die Polizei zu nennen. Entsprechend ihrem Auftrag greift sie ein zur Gefahrenabwehr sowie zum Schutz der öffentlichen Sicherheit und Ordnung.1151 Dazu zählen auch die Verhinderung und die Ahndung von Straftaten.1152 Dabei kommen vorliegend neben verschiedenen kernstrafrechtlichen Bestimmungen1153 auch Delikte gemäss LFG in Be tracht.1154 Sodann ist auch an kantonale oder kommunale Bestimmungen1155 zu denken, welche Drohnenflüge verbieten bzw. einer Bewilligungspflicht unterstellen.1156 Nicht nur Private, sondern auch die Polizei sieht sich indes mit der Schwierigkeit konfrontiert, dass eine wirksame Verfolgung des «Störers» die Identifizierung desselben voraussetzt. Bisher gelang dies in einzelnen Fällen, indem die Ordnungshüter den Standort des Piloten aus demjenigen der Drohne am Himmel eruieren konnten.1157 In vielen Fällen bleibt ein rechts widriger Drohnenflug heute jedoch ungeahndet, da keine Verfolgung statt finden kann. Ein effektives Vorgehen gegen fehlbare Drohnenpiloten setzt demnach legislatorische Massnahmen voraus. 2. Gesetzgeber Wie aufgezeigt, ergibt sich eine Pflicht zu gesetzgeberischen Massnahmen dort, wo «Straf- oder Privatrecht Lücken aufweisen, welche voraussehbare private Übergriffe in menschenrechtlich geschützte Bereiche sanktionslos 1151 Vgl. dazu statt anderer Mohler, Polizeirecht, Rn. 83 ff. 1152 Auf die strafprozessuale Polizeitätigkeit soll vorliegend nicht weiter eingegangen werden; vgl. dazu z.B. Mohler, Polizeirecht, Rn. 141. 1153 Im StGB werden durch Art. 179 bis, 179 ter und 179 quater das Abhören und Aufnehmen fremder Gespräche, das unbefugte Aufnehmen von Gesprächen bzw. die Verletzung des Geheim- oder Privatbereichs durch Aufnahmegeräte unter Strafe gestellt. Diese Bestimmungen sind ohne Weiteres auf die drohnengestützte Datenbearbeitung durch Private anwendbar, wobei Art. 179quater StGB im Vordergrund steht (vgl. Schneider- Marfels/Kaufmann, Rn. 10; Meili A., S. 46; Interpellation Jositsch [18.3397]). Vgl. zu den Bestimmungen im Einzelnen BSK StGB-Ramel/Vogelsang, Art. 179 bis N 2 ff., Art. 179 ter N 3 ff. und Art. 179quater N 5 ff. 1154 Hier steht Art. 91 LFG i.V.m. Art. 14b und 17 f. VLK im Vordergrund. 1155 Vgl. zur (kantonalen) Kompetenz zum Erlass solcher Bestimmungen Art. 51 Abs. 2 LGF und Art. 2a Abs. 2 LFV. Auf die teilweise umstrittene Kompetenzverteilung zwischen Bund und Kantonen (bzw. Gemeinden) soll nicht weiter eingegangen werden. 1156 Vgl. z.B. § 7 Abs. 1 PolR Oberes Fricktal (Bewilligungspflicht für «Modellfliegen inkl. Drohnen»); § 18 Abs. 3 SchifffahrtsVO LU («Modellflugzeuge dürfen weder auf noch über einem Gewässer eingesetzt werden»); Art. 10 Abs. 1 RaLA (Flugverbot in einem Umkreis von 300 m um öffentliche Gebäude). 1157 Vgl. z.B. die Berichte von Fluck und Huber M. § 1 Schutzpflicht bei der drohnengestützten Datenbearbeitung durch Private 161 lassen».1158 Um gegen die vorliegend interessierenden Übergriffe in das Grundrecht der informationellen Selbstbestimmung vorzugehen, muss mit gesetzgeberischen Massnahmen ein privatrechtliches Vorgehen überhaupt erst ermöglicht werden. Es geht folglich darum, zivilrechtliche Rechtsbehelfe zu schaffen, welche ausreichend Schutz bieten;1159 (zusätzliche) strafrechtliche Bestimmungen erscheinen nicht erforderlich.1160 IV. Zwischenfazit Zusammenfassend steht fest, dass bei der drohnengestützten Datenbearbeitung durch Private eine Gefährdung der informationellen Selbstbestimmung von hinreichender Schwere besteht, dass sich eine Schutzpflicht aktualisiert. Die Normen des Datenschutz- und des Luftfahrtrechts vermögen die Rechtsdurchsetzung nicht hinreichend zu garantieren; in diesem Bereich ist der Gesetzgeber zur Wahrnehmung seiner Schutzpflicht berufen. Er ist gehalten, die tatsächlichen Hindernisse im Bereich des grundsätzlich zur Verfügung stehenden Rechtsschutzes legislatorisch zu adressieren und so kurativ einzugreifen.1161 Dazu ist er faktisch und rechtlich in der Lage. Solange der Staat keine Schritte in Richtung eines effektiven Rechtsschutzes unternimmt, besteht ausserdem die Gefahr, dass sich Private der Selbsthilfe bzw. der Notwehr bedienen und eine Drohne über ihrem Grundstück «abschiessen».1162 Die Lehrmeinungen in der Schweiz zur Zulässigkeit eines solchen Vorgehens sind nicht einhellig, wobei jedoch ein Teil «insbesondere angesichts der regelmässig anonymen zivilen Drohnennutzung, wie sie heute an der Tagesordnung ist (…)», für zulässig erachtet, «bestimmt gegen einzelne Störer vorzugehen und die entsprechenden Schranken (…) nicht zu hoch anzusetzen.»1163 Wäre ein auf Selbsthilfe basierender Abschuss einer Drohne durch Private ohne Weiteres akzeptiert, würde dies dem staatlichen 1158 Kälin/Künzli, Rn. 3.113. Vgl. dazu vorne, S. 52 f. 1159 Vgl. EGMR (GK), Söderman c. Schweden, Nr. 5786/08 (2013) Ziff. 85; BGE 136 III 410 E. 6.2; Müller L., Diss., S. 317 f. 1160 Zu den bestehenden Strafbestimmungen vgl. die Hinweise in Fn. 1153. Vgl. sodann EGMR (GK), Söderman c. Schweden, Nr. 5786/08 (2013) Ziff. 85; EGMR, K.U. c. Finnland, Nr. 2872/02 (2008) Ziff. 47. 1161 Vgl. dazu vorne, S. 52 ff. und v.a. S. 50. 1162 Vgl. illustrativ Schneider-Marfels/Kaufmann, Rn. 32 in fine, wo diese Autoren die Formulierung «Feuer frei» verwenden. BSK ZGB II-Ernst, Art. 926 N 5a spricht vom «gewaltsamen Herunterholen». 1163 Christen et al., S. 168; ähnlich auch Schneider-Marfels/Kaufmann, passim, v.a. Rn. 29 und 32. Für Zurückhaltung plädierend dagegen Hrubesch-Millauer/ Bruggisser, Rn. 35 ff. Vgl. sodann BSK ZGB II-Ernst, Art. 926 N 5a und Art. 928 N 3a. 162 Teil 5: Die Durchsetzung von datenschutzrechtlichen Ansprüchen Gewaltmonopol zuwiderlaufen. Dieses umfasst die Pflicht Privater, sich grundsätzlich jedweder Anwendung physischer Gewalt zu enthalten.1164 Stellt der Staat alternative Handlungsoptionen im Sinne eines effektiven Rechtsschutzes bereit, so ist dies ebenfalls Ausfluss seines Gewaltmonopols.1165 § 2 Wahrnehmung der Schutzpflicht de lege ferenda Vor diesem Hintergrund soll nun ein Vorschlag präsentiert werden, der die Durchsetzung von datenschutzrechtlichen Ansprüchen bei der drohnengestützten Datenbearbeitung durch Private ermöglicht. Dafür sind zwei Zwischenschritte notwendig: Zunächst ist die Perspektive der Drohnenbetreiber einzunehmen und danach zu fragen, wie deren Interessen im Rahmen der Wahrnehmung grundrechtlicher Schutzpflichten berücksichtigt werden müssen (sogleich). Des Weiteren ist der Frage nachzugehen, auf welcher Gesetzesstufe und in welcher Gesetzesmaterie ein legislatorisches Vorgehen korrekterweise angezeigt wäre (S. 164 ff.). I. Achtung der Ansprüche der Drohnenbetreiber Bei der Wahrnehmung von Schutzpflichten ist darauf zu achten, dass die infrage kommenden Schutzmassnahmen grundrechtskonform sind und die Schutzund Freiheitsbedürfnisse aller Betroffenen aufeinander abgestimmt werden.1166 Die Privatsphäre und die informationelle Selbstbestimmung der Drohnen betreiber ist mithin bei der Ausgestaltung und Anwendung der Registrierungs-, Kennzeichnungs-, und Fernidentifizierungsregelungen ebenfalls zu respektieren.1167 Der Fokus der vorliegenden Untersuchung, namentlich die Durchsetzung von datenschutzrechtlichen Ansprüchen gegenüber Drohnenbetreibern, darf nicht darüber hinwegtäuschen, dass auch deren Grundrechte zu schützen sind. Dieser Gedanke fand auch in EG 31 EASA-Grundverordnung Eingang. Dort heisst es, dass die «nationalen Registrierungssysteme (…) dem geltenden natio nalen Recht über den Schutz der Privatsphäre und die Verarbeitung personenbezogener Daten entsprechen [sollten]».1168 1164 Zum staatlichen Gewaltmonopol statt vieler Mohler, Gewaltmonopol, S. 157 ff. 1165 Mohler, Gewaltmonopol, S. 159; Stinner, S. 62; Rupp, S. 56. Vgl. auch Geiser/ Uttinger, S. 9. 1166 Kiener, Grundrechte in der BV, Rn. 30; Martin, S. 91. Vgl. dazu auch vorne,S. 54. 1167 Vgl. allgemein zur Schlichtung dieser «Grundrechtskollisionen» Martin, S. 91 m.w.H. 1168 So auch EG 19 Durchführungsverordnung. § 2 Wahrnehmung der Schutzpflicht de lege ferenda 163 Die Vorgaben zur Registrierung und Kennzeichnung1169 von Drohnen der offenen Kategorie sind diesbezüglich kaum risikobehaftet. Sie sind zumutbar, für die Identifizierung von Drohnenbetreibern erforderlich und somit ohne Weiteres als verhältnismässig zu qualifizieren. Anders verhält es sich bei der Fernidentifizierung, sofern diese eine dauerhafte und nicht anlassbezogene Überwachung der Drohnen und deren Betreiber ermöglicht.1170 Eine aktive Kontrolle über GPS-Tracker (oder alternative Systeme wie Galileo oder GLONASS1171) hätte zwar den Vorteil, dass den zuständigen Behörden zu jeder Zeit die Daten zu den Flugbewegungen von Drohnen zur Verfügung stünden.1172 Dies erscheint jedoch nicht erforderlich und folglich unverhältnismässig. Aus diesem Grund ist die in der Durchführungsverordnung getroffene Wertung zur Fernidentifizierung, namentlich deren Beschränkung auf eine «lokale Übertragung», zu begrüssen. Denn dies verunmöglicht die jederzeitige und anlasslose Überwachung von Drohnenoperationen.1173 Diese Überlegungen müssen umso mehr gelten, wenn es um die Implementierung des U-Space geht. Dieses System setzt grundsätzlich voraus, dass der exakte Standort von sich in der Luft befindlichen Drohnen jederzeit bekannt ist. Unklar ist derzeit, wer und in welcher Form Zugriff auf diese Positionsdaten hat.1174 Denn daraus liesse sich bei kommerziellen Anwendungen ggf. auf Geschäftspraktiken oder Geschäftsgeheimnisse schliessen.1175 Im Rahmen der nicht kommerziellen Nutzung kann vom Standort der Drohne auf denjenigen des Piloten geschlossen werden. Dies dürfte dazu führen, dass diese Positionsdaten regelmässig (auch) Personendaten darstellen.1176 Des Weiteren sind auch die Betreibernummern als Personendaten zu qualifizieren.1177 Diese Umstände sind von datenschutzrechtlicher Relevanz und mithin bei Überlegungen de lege ferenda unbedingt zu beachten.1178 1169 Vgl. dazu vorne, S. 120 ff. bzw. S. 135 ff. 1170 Vgl. nur BGer, 6B_908/2018 vom 7. Oktober 2019 E. 3.2 (zur Publikation vorgesehen) m.w.H. Zum «chilling effect» vgl. vorne, S. 61 mit den Hinweisen in Fn. 465. 1171 Dabei handelt es sich ebenfalls um globale Navigationssatellitensysteme (GNSS), die jedoch von Europa (Galileo) bzw. Russland (GLONASS) betrieben werden. Vgl. Büchi, Buch der Drohnen, S. 37, und ausführlich zu diesen Systemen Falcone/Hahn/Burger, passim (Galileo) bzw. Revnivykh et al., passim (GLONASS). 1172 Vgl. Grest, S. 121. 1173 Singh/Carthew/Xiong, S. 4 f. 1174 Vgl. dazu vorne, S. 142 ff. 1175 Vgl. Malgieri, S. 193. 1176 Dazu unter Geltung der DSGVO Malgieri, S. 194 f. Vgl. zum weit gefassten Begriff des Personendatums vorne, S. 29 ff. 1177 Mutatis mutandis BGE 138 II 346 E. 6.2 f. 1178 Vgl. zu diesem Aspekt mit Blick auf das Luftfahrzeugregister Botschaft Teilrevision 1 LFG, S. 4943; vgl. auch EG 19 Durchführungsverordnung. 164 Teil 5: Die Durchsetzung von datenschutzrechtlichen Ansprüchen II. Bestimmung des passenden Gesetzes Da es inhaltlich um die Durchsetzung von datenschutzrechtlichen Ansprüchen geht, liegt die Vermutung nahe, dass entsprechende Bestimmungen im DSG aufzunehmen wären.1179 Eine drohnenspezifische Regelung im Datenschutzgesetz ist mit Blick auf die Technologieneutralität jedoch nicht erstrebenswert.1180 Ausserdem soll es nicht darum gehen, die Technologie an sich zu regulieren, sondern die negativen Auswirkungen derselben.1181 Aus diesem Grund bietet sich eine sektorspezifische Regulierung an.1182 Namentlich bei Datenbearbeitungen, «die im anonymen Massenverkehr auftreten und sich damit der Beeinflussung durch die betroffenen Personen entziehen»,1183 ist ein solches Vorgehen einer Anpassung des Datenschutzgesetzes vorzuziehen. Es sollten folglich die Datenschutzbestimmungen des Luftfahrtrechts revidiert und ergänzt werden. Da es inhaltlich um eine Datenbearbeitung und bekanntgabe durch Bundesbehörden und namentlich das BAZL geht, sind die entsprechenden Normen in einen bundesrechtlichen Erlass aufzunehmen.1184 Eine Anpassung der VLK oder die Ausarbeitung einer neuen Verordnung (durch das UVEK1185) wäre zwar innerhalb kürzerer Frist möglich.1186 Der zeitlich aufwendigere Weg über die Bundesversammlung (und ggf. das Volk) drängt sich jedoch aufgrund der Regelungsmaterie auf.1187 Insgesamt sind deshalb neue Regelungen ins LFG aufzunehmen. 1179 Vgl. zu dieser Forderung in Deutschland Uschkereit/Zdanowiecki, S. 449. Stöcker et al., S. 18, halten dafür, dass alle Gefahren der drohnengestützten Datenbearbeitung beurteilt und angemessen in die nationalen Datenschutzgesetze eingearbeitet werden sollten. 1180 Vgl. zur Technologieneutralität vorne, S. 71 f. 1181 Vgl. Leenes et al., S. 7 m.w.H. 1182 So auch Davola, S. 160. Vgl. allgemein zur Bedeutung sektorspezifischer Regulierungen im Datenschutz Sprecher, S. 544 m.w.H. 1183 Schantz, in: Simitis et al., Art. 6 Abs. 1 DSGVO N 86. 1184 Vgl. Art. 30, Art. 32 E-DSG und v.a. Art. 30 Abs. 2 lit. c E-DSG. Zur Normstufe ausführlich Waldmann/Bickel, in: Belser/Epiney/Waldmann, § 12 N 44 ff. 1185 Vgl. zur diesbezüglichen Kompetenz des UVEK Art. 51 Abs. 1 und 2 LFG; Art. 2a Abs. 3, Art. 21 und Art. 125 Abs. 2 LFV; vgl. auch Dettling-Ott, Organisation, Rn. 9. 1186 Vgl. dazu Cottier, S. 37, der im Kontext der Regulierung der Digitalisierung eine «refonte des lents et dispersés processus traditionnels d’adoption des normes» fordert. 1187 Vgl. zur Aufnahme von Datenschutzbestimmungen im LFG auch Botschaft Teil revision 1 LFG, S. 4943, und zu parallelen Überlegungen im Strassenverkehrsrecht Botschaft Via sicura, S. 8480 f. und S. 8521. § 3 Vorschläge für eine Gesetzesbestimmung 165 Wie aufgezeigt, ist der Bund für die Schaffung entsprechender Bestimmungen zuständig.1188 Der Anstoss zu einer neuen gesetzlichen Regelung kann von jedem Mitglied des National- und Ständerats, von jeder Fraktion, von jeder parlamentarischen Kommission und von jedem Kanton ausgehen (Art. 160 Abs. 1 BV).1189 Dem Bundesrat als Kollegium steht dieses Recht ebenfalls zu (Art. 181 und Art. 177 Abs. 1 BV);1190 er kann die Ausarbeitung einer entsprechenden Vorlage der Verwaltung übertragen (Art. 177 Abs. 2 BV).1191 Vorliegend würde die Federführung dem UVEK und innerhalb desselben dem BAZL zufallen.1192 Für Beratung und Beschluss einer neuen oder revidierten Norm ist sodann die Bundesversammlung zuständig.1193 § 3 Vorschläge für eine Gesetzesbestimmung I. Zu regelnde Aspekte Vor diesem Hintergrund soll nun ein Formulierungsvorschlag für eine Bestimmung im LFG präsentiert werden. Dieser soll — im Einklang mit der Forschungsfrage — primär die Bekanntgabe von Daten aus dem «Drohnenbetreiberregister» zum Zweck der Durchsetzung von datenschutzrechtlichen Ansprüchen behandeln. Es könnte sich dabei um einen nArt. 107c LFG handeln. Dieser würde systematisch unter dem Titel «Datenschutz» stehen und thematisch an die Zugriffsrechte zum bereits bestehenden Luftfahrzeug register anknüpfen. Neben dem Zugriff sind weitere Aspekte wie Zweck und Inhalt des Registers zu regeln, denn die neue Bestimmung muss eine ausreichende Bestimmtheit aufweisen.1194 Ausserdem sollte die Kompetenz zur Führung des 1188 Vgl. vorne, S. 159. 1189 Zum Initiativ- und Antragsrecht ausführlich statt vieler Biaggini, Komm. BV, Art. 160 N 2 ff.; BSK BV-Thurnherr, Art. 160 N 4 ff. 1190 Vgl. zu dieser Befugnis Biaggini, Komm. BV, Art. 181 N 2 f. und N 5. 1191 Biaggini, Komm. BV, Art. 181 N 7 und Art. 177 N 11 ff. 1192 Art. 2 Abs. 2 und Art. 43 Abs. 1 RVOG; Art. 3 Abs. 1 lit. a und Art. 7, v.a. Art. 7 Abs. 3 lit. a OV-UVEK; Art. 3 Abs. 2 LFG; vgl. Dettling-Ott, Betrieb, Rn. 5; Dettling-Ott, Organi sation, Rn. 10. 1193 Art. 163 BV und dazu Sägesser, in: Sägesser, Art. 163 BV N 404 ff. Zum Ablauf des Ver- fahrens im Einzelnen vgl. auch Art. 71 ff. ParlG; Komm. ParlG-von Wyss, Art. 71 N 8; Komm. ParlG-Theler, Art. 73 N 8. 1194 Vgl. zu diesem Kriterium und den einzelnen davon erfassten Aspekten BSK DSG-Jöhri/ Studer, Art. 17 N 11; SHK DSG-Mund, Art. 17 N 6 ff.; Waldmann/Bickel, in: Belser/ Epiney/Waldmann, § 12 N 45 f. 166 Teil 5: Die Durchsetzung von datenschutzrechtlichen Ansprüchen Registers festgelegt werden.1195 Dadurch werden die Vorgaben des E-DSG und der EASA-Grundverordnung für die Datenbearbeitung durch das BAZL verwirklicht.1196 II. Kommentierter Formulierungsvorschlag 1. Kompetenz (Abs. 1) 1.1 Formulierungsvorschlag 1 Das BAZL führt das schweizerische Register der Betreiber von unbemann- ten Luftfahrzeugsystemen im Sinne von Art. 2 Ziff. 2 Verordnung (EU) Nr. 2019/947. 1.2 Bemerkungen Das BAZL hat die Plattform für die Selbstregistrierung der Drohnenbetreiber bereitzustellen.1197 Deshalb soll auch bezüglich der Führung des Betreiberregisters von der Kompetenz des Bundesamts ausgegangen werden.1198 Der Verweis auf das grundlegende europäische Regelwerk ist ohne Weiteres vertretbar;1199 solche sind im Schweizer Luftfahrtrecht denn auch nicht unüblich.1200 Aus Gründen der Klarheit und der Rechtssicherheit sowie mit Blick auf die Legaldefinition in der Durchführungsverordnung sollte zudem der Begriff «unbemanntes Luftfahrzeugsystem» (anstelle von Drohne) verwendet werden.1201 Das Betreiberregister ist im Gesetz in einem separaten Artikel zu behandeln; damit wird die Unterscheidung vom Luftfahrzeugregister verdeutlicht.1202 Die Bestimmung spricht vom «schweizerischen Register», da es sich gemäss Vorgaben der EASA-Grundverordnung um ein nationales (jedoch zugleich harmonisiertes und interoperables) System handeln soll.1203 1195 Vgl. zum Ganzen BJ, Leitfaden, Rn. 833. 1196 Vgl. Art. 5–7, Art. 30 und Art. 32 E-DSG; Art. 132 Abs. 1 EASA-Grundverordnung. Vgl. auch BJ, Leitfaden, Rn. 829 ff. 1197 Vgl. vorne, S. 128. 1198 Die Ausarbeitung und Führung des Registers könnte auch an eine dritte Institution vergeben werden (vgl. vorne, S. 128 mit den dortigen Hinweisen). 1199 Es handelt sich dabei um einen sog. «dynamisch-direkte Verweisung»; vgl. nur BJ, Leitfaden, Rn. 758. 1200 Zu finden sind solche etwa in Art. 14a VLK, Art. 2b Abs. 1, Art. 26 und Art. 77 LFV. 1201 Vgl. dazu vorne, S. 26 f. 1202 Vgl. zur Aufnahme von Drohnen der zulassungspflichtigen Kategorie ins Luftfahrzeug register vorne, S. 131. 1203 Vgl. vorne, S. 132. Auch die Pflicht zur Registrierung besteht im Staat des Wohnsitzes bzw. des Hauptgeschäftssitzes (vgl. dazu vorne, S. 128). § 3 Vorschläge für eine Gesetzesbestimmung 2. 167 Zweck (Abs. 2) 2.1 Formulierungsvorschlag 2 Das Register dient der Erfüllung folgender Aufgaben: a. Identifizierung von Betreibern von unbemannten Luftfahrzeugsystemen; b. Durchführung von Administrativ- und Strafverfahren gegen Betreiber und Fernpiloten von unbemannten Luftfahrzeugsystemen; c. Unterstützung von in- und ausländischen Behörden sowie internationalen Organisationen bei der Erfüllung ihrer Aufgaben gemäss der Verordnung (EU) Nr. 2018/1139 und den darauf gestützt erlassenen Rechtsakten; d. (…).1204 2.2 Bemerkungen Der primäre Zweck ist die Identifizierung der Betreiber. Sind Betreiber und Fernpilot nicht ein und dieselbe Person, so kann Letzterer nicht direkt durch Rückgriff auf das Register identifiziert werden, da sich Fernpiloten selbst nicht registrieren müssen. Die notwendigen Angaben können in diesem Fall beim Betreiber in Erfahrung gebracht werden.1205 Das Register soll zudem der Durchführung von Administrativ- und Strafverfahren dienen. Entsprechende Verfahren sind zur Durchsetzung der Sanktionen, welche die Schweiz gemäss Art. 131 EASA-Grundordnung für Verstösse gegen die Grundverordnung sowie die Durchführungs- und die Delegierte Verordnung vorzusehen hat, notwendig.1206 Neben Strafverfolgungsbehörden wird z.B. auch die SUST Zugriff auf Angaben von Drohnenbetreibern benötigen.1207 Des Weiteren soll das Register der Zusammenarbeit mit der EASA, der EU-Kommission und den zuständigen ausländischen Behörden dienen. Letzteres wird von der EASA-Grundverordnung explizit verlangt.1208 1204 Weitere Zwecke wie z.B. die Erstellung von Statistiken kämen ebenfalls in Betracht. In Übereinstimmung mit dem Fokus der vorliegenden Untersuchung werden diese aber nicht weiter vertieft. 1205 Der Betreiber ist gehalten, «seine» Fernpiloten zu überwachen; er sollte diese mithin auch identifizieren können (vgl. etwa Art. 14 Abs. 2 lit. e Durchführungsverordnung; UAS.SPEC.050 Abs. 1 lit. b und d Anhang Teil B Durchführungsverordnung; EASA, GM Part-UAS, S. 20). 1206 Vgl. dazu vorne, S. 147. 1207 Die SUST ist eine ausserparlamentarische Kommission, welche u.a. mit der Untersu- chung von Zwischenfällen in der Luftfahrt betraut ist (Art. 1 und Art. 6 Verordnung über die Sicherheitsuntersuchung von Zwischenfällen im Verkehrswesen [VSZV]). 1208 Art. 74 EASA-Grundverordnung, v.a. Abs. 1; vgl. auch EG 35, 37 und 48 EASA-Grund- verordnung. Vgl. sodann vorne, S. 132. 168 3. Teil 5: Die Durchsetzung von datenschutzrechtlichen Ansprüchen Inhalt (Abs. 3) 3.1 Formulierungsvorschlag 3 Das Register enthält folgende Daten: a. bei natürlichen Personen: vollständiger Name und Geburtsdatum; b. bei juristischen Personen: Name oder Firma und UnternehmensIdentifikationsnummer (UID); c. Adresse des Betreibers; d. E-Mail-Adresse und Telefonnummer des Betreibers; e. Nummer der Versicherungspolice jedes unbemannten Luftfahrzeug systems des jeweiligen Betreibers, sofern dafür eine Versicherung erforderlich ist. 3.2 Bemerkungen Der Formulierungsvorschlag zum Inhalt des Registers ist stark an Art. 14 Abs. 2 Durchführungsverordnung angelehnt; die dort aufgelisteten Informationen sind bei der Selbstregistrierung anzugeben und somit selbstredend auch im Register zu speichern.1209 Bei den registrierten Informationen handelt es sich um Personendaten bzw. um Daten,1210 weshalb dieser Begriff im Formulierungsvorschlag zu Abs. 3 sowie den nachfolgenden Absätzen verwendet wird. Kann eine natürliche Person bei der Registrierung keine E-Mail-Adresse und/oder keine (persönliche) Telefonnummer angeben, so wäre diese Angabe auch nicht im Register enthalten. Solange der Zweck der Identifizierung des Betreibers dennoch erreicht werden kann, ist nicht zwingend auf die Vollständigkeit der Angaben gemäss obigem Formulierungsvorschlag zu beharren.1211 1209 Vgl. zu den anzugebenden Daten vorne, S. 130 f. Die in Art. 14 Abs. 2 lit. e und f Durch- führungsverordnung aufgeführten Erklärungen und Dokumente werden im Formulierungsvorschlag nicht aufgeführt; sie sind denn auch für Betreiber von Drohnen der offenen Kategorie nicht von Bedeutung. 1210 Vgl. dazu eingehend vorne, S. 29 ff. 1211 Vgl. dazu auch bereits vorne, S. 130 f. § 3 Vorschläge für eine Gesetzesbestimmung 4. 169 Datenbekanntgabe (Abs. 4 und 5) 4.1 Formulierungsvorschlag 4 Das Register ist nicht öffentlich. Das BAZL darf folgende Daten gemäss Abs. 3 Personen bekannt geben: a. die im Hinblick auf die Geltendmachung von Ansprüchen gemäss dem Bundesgesetz über den Datenschutz und/oder dem Schweizerischen Zivilgesetzbuch ein hinreichendes Interesse geltend machen: Name und Adresse des Betreibers; b. (…).1212 5 Das BAZL stellt für Gesuche nach Abs. 4 ein Onlineformular zur Verfügung.» 4.2 Bemerkungen Das Register sollte vom Grundsatz der Nichtöffentlichkeit ausgehen und dieser explizit im Gesetz verankert werden.1213 Des Weiteren erfolgt die Identifizierung eines Drohnenbetreibers nicht direkt, d.h. gestützt auf die durch die Fernidentifizierung ausgesendeten Informationen. Vielmehr muss sich eine betroffene Person dafür an das BAZL wenden.1214 Von diesem Verständnis geht auch die Bestimmung zur direkten Fernidentifizierung aus; die Personalien des Betreibers werden nicht direkt übermittelt.1215 Betreiber von Drohnen, welche keine Informationen zur Fernidentifizierung übertragen müssen (d.h. solchen der Klasse C01216 sowie selbst gebauten Drohnen1217), können nur visuell identifiziert werden, was mit erheblichen Schwierigkeiten verbunden ist.1218 Somit verbleibt in diesem Bereich eine Lücke im Rechtsschutz. Relativiert wird die Problematik durch die beschränkte Verbreitung selbst gebauter Drohnen sowie den Umstand, dass (Kleinst-)Drohnen der Klasse C0 nahe dem Betreiber fliegen müssen, damit die relevanten Betriebsvorschriften eingehalten werden können.1219. 1212 Weitere Gründe für eine Datenbekanntgabe kommen zwar ohne Weiteres in Betracht (z.B. Personen, die von einem Unfall mit einer Drohne betroffen sind und haftungsrechtliche Ansprüche geltend machen wollen). Es kann jedoch im Rahmen der vorliegenden Untersuchung keine abschliessende Liste präsentiert werden. 1213 Zur Begründung vorne, S. 134 f. Dafür spricht ausserdem, dass u.U. Minderjährige im Register verzeichnet sind (vgl. dazu vorne, S. 129 sowie Spies, S. 102). 1214 Vgl. zur lokalen Übertragung von Angaben der Fernidentifizierung vorne, S. 140 f. 1215 Vgl. dazu vorne, S. 139 ff. 1216 Vgl. vorne, S. 138 f. 1217 Vgl. vorne, S. 145. 1218 Vgl. vorne, S. 137 f. 1219 Vgl. vorne, S. 141 f. 170 Teil 5: Die Durchsetzung von datenschutzrechtlichen Ansprüchen Bringt eine Person schlüssig vor, dass sie von einer (unerwünschten) drohnengestützten Datenbearbeitung betroffen war, so hat das BAZL die nachgesuchten Daten bekannt zu geben. Es ist somit kein strenger Massstab anzuwenden; Glaubhaftmachung genügt.1220 Im Sinne der Verhältnismässigkeit sind selbstredend jeweils nur diejenigen Daten bekannt zu geben, welche für den konkreten Zweck notwendig sind.1221 Zur Geltendmachung des datenschutzrechtlichen Auskunftsrechts etwa sind einzig Name und Adresse des (vermeintlich) Verantwortlichen notwendig.1222 Das Geburtsdatum einer natürlichen Person oder Angaben zur Versicherung der Drohne sind hingegen für die Durchsetzung von datenschutzrechtlichen Ansprüchen unerheblich. Aus diesem Grund sollte das Gesetz festlegen, dass «nur» Name und Adresse des Betreibers bekannt gegeben werden dürfen.1223 Trotz der offenen Formulierung («darf») hat bei Nachweis eines hinreichenden Interesses grundsätzlich eine Bekanntgabe zu erfolgen.1224 Dabei stellt bereits die Angabe der Betreibernummer im Hinblick auf die Geltendmachung von datenschutzrechtlichen Ansprüchen ein solches Interesse dar.1225 Gleich sollte es sich auch verhalten, wenn eine Person glaubhaft den Bestand von Ansprüchen gestützt auf das ZGB geltend macht. Dies kommt etwa in Betracht, wenn das Datenschutzrecht gar nicht anwendbar ist1226 bzw. ein Vorgehen über den Besitzschutz infrage kommt.1227 Damit stünden der betroffenen Person die für die Wahrnehmung ihrer Rechte notwendigen Angaben zur Verfügung. Die bestehende Schutzpflicht im Rahmen der informationellen Selbstbestimmung wäre dadurch wahrgenommen.1228 Das Onlineformular soll eine möglichst effiziente und einfache Art der Betreiberabfrage ermöglichen. Gleichzeitig ist das BAZL damit in der Lage, eine Übersicht über die eingegangenen Anfragen zu erhalten sowie ggf. bei rechtsmissbräuchlichen Anfragen (z.B. Massen- oder querulatorischen Anfragen) einzuschreiten.1229 1220 Zur Glaubhaftmachung statt aller Kiener/Rütsche/Kuhn, Rn. 729 m.w.H. 1221 Zur Verhältnismässigkeit als Datenbearbeitungsgrundsatz vgl. vorne, S. 83 f. 1222 Vgl. vorne, S. 102. 1223 Vgl. BJ, Leitfaden, Rn. 833. 1224 Es besteht gleichzeitig aber keine Pflicht dazu; vgl. zur Verhinderung von Rechtsmiss- brauch sogleich. 1225 Vgl. vorne, S. 134 f. sowie Botschaft Via sicura, S. 8509. 1226 Vgl. dazu vorne, S. 75 ff., v.a. S. 76 mit den Hinweisen in Fn. 568. 1227 Vgl. vorne, S. 157 mit den Hinweisen in Fn. 1136. Vgl. sodann BSK ZGB II-Ernst, Art. 926 N 5a und Art. 928 N 3a. 1228 Vgl. vorne, S. 69 ff. und v.a. S. 72 sowie S. 50. 1229 Vgl. dazu nur Art. 2 Abs. 2 ZGB und zu dessen Geltung in der gesamten Schweizer Rechtsordnung BGE 143 III 666 E. 4.2; 128 III 201 E. 1c; 122 II 193 E. 2c/ee. § 3 Vorschläge für eine Gesetzesbestimmung 171 Ein Miteinbezug des EDÖB bei der Datenbekanntgabe erscheint nicht sinnvoll. Dieser ist zwar für die Überwachung der Einhaltung von datenschutzrechtlichen Vorschriften zuständig, ist aber am Registrierungsverfahren nicht beteiligt und hat deshalb auch keinen (direkten) Zugang zum Register. Hinzu kommt, dass er de lege lata nur ausnahmsweise dazu berufen ist, bei Auseinandersetzungen zwischen Privaten einzugreifen.1230 Gemäss Art. 43 Abs. 1 E-DSG kann der EDÖB zwar grundsätzlich gegen jede «private Person» vorgehen;1231 aufgrund fehlender (personeller) Mittel wird dies aber realistischerweise weiterhin nur bei gravierenden und/oder systematischen Datenschutzverletzungen in Betracht kommen.1232 5. Zugriff im Abrufverfahren (Abs. 6) 5.1 Formulierungsvorschlag 6 Folgende Behörden können im Abrufverfahren Einsicht in Daten des Registers nehmen: a. die Polizei in Daten, welche die Identifizierung des Betreibers ermöglichen; b. die Strafverfolgungs- und Gerichtsbehörden im Rahmen von Verfahren zur Beurteilung von Widerhandlungen gegen Bestimmungen des Schweizer Rechts sowie Vorschriften der Verordnung (EU) Nr. 2018/1139 und der darauf gestützt erlassenen Rechtsakte; c. die Untersuchungsstelle im Rahmen von Untersuchungen von Zwischen fällen im Luftverkehr; d. ausländische Behörden und internationale Organisationen im Rahmen der Erfüllung ihrer Aufgaben gemäss der Verordnung (EU) Nr. 2018/1139 und den darauf gestützt erlassenen Rechtsakten; e. (…).1233 5.2 Bemerkungen Ein Einsichtsrecht im Abrufverfahren1234 von Polizei sowie Strafverfolgungsund Gerichtsbehörden sollte unbedingt vorgesehen werden. Im Unterschied zur Datenbekanntgabe an Private sind diese Behörden dadurch nicht auf 1230 Botschaft DSG 1988, S. 479; Waldmann/Oeschger, in: Belser/Epiney/Waldmann, § 15 Rn. 15; vgl. BGE 136 II 508 E. 6.3.2; Art. 29 DSG. 1231 Botschaft Totalrevision DSG, S. 7090; Rosenthal, Entwurf, Rn. 124. 1232 Vgl. Rosenthal, Entwurf, Rn. 125; Jöhri, Rn. 8.97 ff.; vgl. auch Art. 43 Abs. 2 E-DSG. 1233 Wie bereits zu Zweck und Datenbekanntgabe festgehalten, wäre auch hier eine Erweiterung der Liste möglich. Es kann jedoch im Rahmen der vorliegenden Untersuchung keine abschliessende Aufzählung präsentiert werden. 1234 Zum Begriff vorne, S. 133. 172 Teil 5: Die Durchsetzung von datenschutzrechtlichen Ansprüchen eine Mitwirkung des BAZL angewiesen, um den Inhalt des Registers zu konsultieren. Durch ihr Einsichtsrecht werden sie in die Lage versetzt, gegenüber fehlbaren Drohnenbetreibern das (Straf-)Recht durchzusetzen.1235 Die SUST wird im LFG als «Untersuchungsstelle» bezeichnet, weshalb dieser Begriff ebenso im Formulierungsvorschlag verwendet wird.1236 Die Datenbekanntgabe an ausländische Behörden sowie internationale Organisationen ist in Art. 107a Abs. 5 E-LFG bereits geregelt.1237 Die Bestimmung ist jedoch allgemein formuliert und deckt die Einspeisung von Daten in den Informationsspeicher gemäss Art. 74 Abs. 1 EASA-Grundverordnung durch das BAZL nicht hinreichend ab.1238 Da die Daten im Informationsspeicher jederzeit und ohne Mitwirkung und Kontrolle des BAZL abgerufen werden können, gehen damit erhöhte Risiken für die Persönlichkeitsrechte der Drohnenbetreiber einher.1239 Aus diesem Grund ist auch Datenbekanntgabe an Luftfahrtbehörden ausländischer Staaten sowie die europaweit wirkenden Luftfahrtorganisationen (primär EASA) zu normieren.1240 Durch die explizite Regelung dieser Form des Datenaustauschs wird sichergestellt, dass eine genügend spezifische Grundlage für den regelmässigen bzw. jederzeitigen Zugriff auf Personendaten durch ausländische Behörden sowie die EASA besteht.1241 III. Unkommentierter Formulierungsvorschlag Der Übersichtlichkeit halber wird der Formulierungsvorschlag nachstehend in seiner gesamten Länge und ohne Kommentierungen und Fussnoten wiedergegeben. 1 Das BAZL führt das schweizerische Register der Betreiber von unbemannten Luftfahrzeugsystemen im Sinne von Art. 2 Ziff. 2 Verordnung (EU) Nr. 2019/947. 1235 Vgl. zur Notwendigkeit gesetzgeberischer Massnahmen in diesem Kontext vorne, S. 160 f. 1236 Vgl. zu den Aufgaben der SUST vorne, S. 167 mit den Hinweisen in Fn. 1207. 1237 Vgl. zur geplanten Revision dieser Bestimmung im Rahmen der Totalrevision des DSG die Hinweise vorne, Fn. 852. 1238 Vgl. zum Informationsspeicher vorne, S. 132; zur notwendigen gesetzlichen Grund- lage für eine Datenbearbeitung durch das BAZL vorne, S. 164. 1239 Vgl. Waldmann/Bickel, in: Belser/Epiney/Waldmann, § 12 N 95; BSK DSG-Jöhri/ Studer, Art. 19 N 65; Wermelinger, S. 187. Vgl. sodann Art. 30 Abs. 2 lit. c und Art. 32 E-DSG; Botschaft Totalrevision DSG, S. 7083. 1240 Vgl. Botschaft Teilrevision 1 LFG, S. 4968 f. 1241 Vgl. BJ, Leitfaden, Rn. 838; Handkomm. DSG-Jöhri, Art. 19 N 74. § 3 Vorschläge für eine Gesetzesbestimmung 2 Das Register dient der Erfüllung folgender Aufgaben: 173 a. Identifizierung von Betreibern von unbemannten Luftfahrzeugsystemen; b. Durchführung von Administrativ- und Strafverfahren gegen Betreiber und Fernpiloten von unbemannten Luftfahrzeugsystemen; c. Unterstützung von in- und ausländischen Behörden sowie internationalen Organisationen bei der Erfüllung ihrer Aufgaben gemäss der Verordnung (EU) Nr. 2018/1139 und den darauf gestützt erlassenen Rechtsakten; d. (…). 3 Das Register enthält folgende Daten: a. bei natürlichen Personen: vollständiger Name und Geburtsdatum; b. bei juristischen Personen: Name oder Firma und UnternehmensIdentifikationsnummer (UID); c. Adresse des Betreibers; d. E-Mail-Adresse und Telefonnummer des Betreibers; e. Nummer der Versicherungspolice jedes unbemannten Luftfahrzeug systems des jeweiligen Betreibers, sofern dafür eine Versicherung erforderlich ist. 4 Das Register ist nicht öffentlich. Das BAZL darf folgende Daten gemäss Abs. 3 Personen bekannt geben: a. die im Hinblick auf die Geltendmachung von Ansprüchen gemäss dem Bundesgesetz über den Datenschutz und/oder dem Schweizerischen Zivilgesetzbuch ein hinreichendes Interesse geltend machen: Name und Adresse des Betreibers; b. (…). 5 Das BAZL stellt für Gesuche nach Abs. 4 ein Onlineformular zur Verfügung. 6 Folgende Behörden können im Abrufverfahren Einsicht in Daten des Registers nehmen: a. die Polizei in Daten, welche die Identifizierung des Betreibers ermög lichen; b. die Strafverfolgungs- und Gerichtsbehörden im Rahmen von Verfahren zur Beurteilung von Widerhandlungen gegen Bestimmungen des Schweizer Rechts sowie Vorschriften der Verordnung (EU) Nr. 2018/1139 und der darauf gestützt erlassenen Rechtsakte; c. die Untersuchungsstelle im Rahmen von Untersuchungen von Zwischen fällen im Luftverkehr; d. ausländische Behörden und internationale Organisationen im Rahmen der Erfüllung ihrer Aufgaben gemäss der Verordnung (EU) Nr. 2018/1139 und den darauf gestützt erlassenen Rechtsakten; e. (…). 175 Zusammenfassung und Schlussbemerkungen § 1 Zusammenfassung Die immer häufiger werdende Verwendung von Drohnen durch Private bedeutet eine Gefährdung der informationellen Selbstbestimmung. Nachweislich fühlen sich grosse Teile der Bevölkerung durch anonym operierende Drohnen verunsichert, da sie nicht identifizieren können, wer für die Drohne verantwortlich ist und was Letztere überhaupt tut. Gerade im Bereich des Datenschutzes stellt dies die Rechtsdurchsetzung vor grosse Herausforderungen. Die vorliegende Arbeit verfolgte deshalb das Ziel, eine Möglichkeit aufzuzeigen, wie die Identifizierung der für eine drohnengestützte Datenbearbeitung verantwortlichen Person gewährleistet werden kann. Vor diesem Hintergrund hat sie die (zukünftigen) Regeln des Datenschutzrechts sowie des Luftfahrtrechts analysiert und an den Vorgaben der grundrechtlichen Schutzpflichten im Kontext der informationellen Selbstbestimmung gemessen. Durch die drohnengestützte Datenbearbeitung können verschiedene Grundrechte tangiert werden, wobei die informationelle Selbstbestimmung klar im Vordergrund steht. In Lehre und Rechtsprechung besteht Einigkeit darüber, dass in Art. 13 Abs. 2 BV eine Schutzdimension verankert ist. Sie findet im Schweizer Recht primär in der Datenschutzgesetzgebung sowie dem Persönlichkeitsschutz gemäss ZGB ihren Ausdruck. Ein — wenn nicht der zentrale — Aspekt der grundrechtlichen Schutzpflichten im Kontext der informationellen Selbstbestimmung ist in deren Durchsetzung zu erblicken. Im Bereich des Datenschutzes verfolgt die noch laufende Revision des DSG insbesondere das Ziel, mittels Verbesserung der Transparenz der Datenbearbeitung sowie der Kontrollmöglichkeiten der betroffenen Personen, die staatlichen Schutzpflichten im Kontext der informationellen Selbstbestimmung effektiver wahrzunehmen. Die in dieser Untersuchung vorgenom mene Analyse zeigte auf, dass der E-DSG zwar auf die drohnengestützte Da tenbearbeitung durch Private anwendbar ist, die darin verankerten Rechte jedoch oftmals nicht durchgesetzt werden können. Die Anonymität der Droh nenbetreiber verunmöglicht die angestrebte Transparenz und erlaubt regelmässig keine effektive Kontrolle durch die betroffene Person. 176 Zusammenfassung und Schlussbemerkungen Die neuen luftfahrtrechtlichen Vorschriften zu Drohnen werden in vielerlei Hinsicht zu einer Verbesserung führen. Die Bestimmungen zur Registrierung von Drohnenbetreibern und zur Kennzeichnung von Drohnen sowie die Vorgaben zur Fernidentifizierung stellen die Grundlagen dar, um einen Drohnenbetreiber erfolgreich identifizieren zu können. Die präventiven und flankierenden Massnahmen, welche in den Drohnenverordnungen vorgesehen sind, ermöglichen, in gewissen Bereichen unterstützend auf die Einhaltung des Datenschutzes hinzuwirken. Insgesamt erweisen sich die Regeln aber bezüglich der Durchsetzung des Datenschutzrechts nicht als ausreichend. Unter Bezugnahme auf die grundrechtlichen Ausführungen wird deshalb festgestellt, dass im Bereich der informationellen Selbstbestimmung bei der drohnengestützten Datenbearbeitung durch Private eine staatliche Schutzpflicht besteht. Diese bezieht sich auf die Durchsetzung datenschutzrechtlicher Ansprüche und folglich auf die Identifizierung der verantwortlichen Drohnenbetreiber. Der Bund, namentlich der Bundesgesetzgeber, ist gehalten, diese wahrzunehmen. Dazu drängt sich eine Revision des LFG auf, und zwar in dem Sinne, dass eine neue Bestimmung zum Drohnenbetreiberregister geschaffen wird. Die Regelung zur Datenbekanntgabe an Private würde als zentraler Aspekt des Registers das Eingangstor zur Durchsetzung datenschutzrechtlicher Ansprüche und primär des Auskunftsrechts jedes Betroffenen darstellen. Gleichzeitig würden so die Kontrollrechte jedes Einzelnen gestärkt und die angestrebte Transparenz bei drohnengestützten Datenbearbeitungen verbessert. Die gleiche Funktion käme der Bestimmung für die im ZGB verankerten Rechte zu. Insgesamt würde so die staatliche Schutzpflicht im Kontext der drohnengestützten Datenbearbeitung durch Private wahrgenommen. Damit die vorgeschlagene Norm ihren Zweck erfüllen und eine wirksame Durchsetzung von datenschutzrechtlichen Ansprüchen ermöglichen kann, ist bei der Prüfung von entsprechenden Gesuchen kein strenger Massstab anzulegen. Wird bei der Ausgestaltung des Drohnenbetreiberregisters im Rahmen der Umsetzung der Drohnenverordnungen im Schweizer Recht auch der Datenschutz und dessen Durchsetzung explizit mitbedacht, so würde dies gleichzeitig die Schaffung eines möglichst umfassenden rechtlichen Rahmens für die technologische Realität von heute bedeuten. § 2 Schlussbemerkungen 177 § 2 Schlussbemerkungen Die laufenden Revisions- und Umsetzungsprozesse in den für diese Dissertation zentralen Rechtsgebieten waren äusserst spannend und zugleich sehr herausfordernd. Wie bei den Bemerkungen zu den zentralen Rechtsgrundlagen ausgeführt, sollen einzelne Aspekte der Drohnenverordnungen, wie etwa die Vorschriften zur Registrierungs- und Kennzeichnungspflicht, bereits ab Januar 2021 praktisch umgesetzt werden.1242 Schon bald sind deshalb Neuerungen zu erwarten, die sich inhaltlich auf die hier präsentierten Resultate auswirken könnten. Dafür spricht auch, dass das BAZL bei gewissen Aspekten der Drohnenregulierung europa- und sogar weltweit bisher eine führende Rolle eingenommen hat. So war etwa der Test der Fernidentifizierung von Drohnen, welcher im September 2019 in Bern durchgeführt wurde,1243 der erste und einzige in ganz Europa. Gemäss Auskünften des BAZL anlässlich von Gesprächen im Oktober 2019 ist der Zeitplan für die Einführung und Umsetzung der Vorgaben der Drohnenverordnungen von Arbeitshilfen der EU bzw. der EASA abhängig. Aufgrund der in verschiedenen Bereichen angestrebten Harmonisierung und Standardisierung ist dies nicht erstaunlich. Es ist jedoch zu hoffen, dass das BAZL und der Bund insgesamt mutig voranschreiten und mit Weitsicht ausgewogene Regeln für die immer stärker aufkommende Drohnentechnologie schaffen werden. 1242 Vgl. vorne, S. 13 ff. 1243 Vorne, S. 140 f. Über den Autor: David Henseler studierte Rechtswissenschaften an der Universität Freiburg i.Üe. und am Center for Transnational Legal Studies (CTLS) in London. Nach Praktika in einer Zürcher Wirtschaftskanzlei und am Kantonsgericht Luzern erhielt er im November 2016 das Anwaltspatent des Kantons Luzern. An der Universität Zürich arbeitete er sodann am Lehrstuhl für Staats-, Verwaltungs- und Verfahrensrecht von Prof. Regina Kiener. Seine Dissertation wurde am 1. April 2020 von der Rechtswissenschaftlichen Fakultät der Universität Zürich abgenommen. Heute ist David Henseler als Gerichtsschreiber am Verwaltungsgericht des Kantons Zürich tätig. sui generis ist ein Verein, der sich der Förderung des freien Zugangs zu juristischer Literatur, Gerichtsurteilen, Behördenentscheidungen und Gesetzesmaterialien verschrieben hat. Unter dem Label sui generis erscheint seit 2014 eine juristische Open-Access-Fachzeitschrift. 2019 erfolgte die Gründung des sui generis Verlags. sui generis Buchreihe herausgegeben von Daniel Hürlimann und Marc Thommen In dieser Reihe werden juristische Dissertationen und Habilitationen sowie Lehrbücher und Fachpublikationen einem breiten Publikum zugänglich gemacht. Die Bücher dieser Reihe erscheinen als gedruckte Werke und online. Die digitale Version ist weltweit kostenlos zugänglich (Open Access). Die Urheberrechte verbleiben bei den AutorInnen; die Werke werden unter einer Creative-Commons-Lizenz veröffentlicht. Bisher in der sui generis Reihe erschienen: 001 — Monika Simmler: Normstabilisierung und Schuldvorwurf 002 — Marc Thommen: Introduction to Swiss Law 003 — Silvio Hänsenberger: Die zivilrechtliche Haftung für autonome Drohnen unter Einbezug von Zulassungs- und Betriebsvorschriften 004 — Mais A.M. Qandeel: Enforcing Human Rights of Palestinians in the Occupied Territory 005 — Moritz Oehen: Der Strafkläger im Strafbefehls- und im abgekürzten Verfahren 006 — Jens Lehne: Crisis at the WTO: Is the Blocking of Appointments to the WTO Appellate Body by the United States Legally Justified? 007 — Lorenz Garland: Waffengleichheit im Vorverfahren 008 — Christoph Urwyler: Die Praxis der bedingten Entlassung aus dem Strafvollzug 009 — Dominik Elser: Die privatisierte Erfüllung staatlicher Aufgaben 010 — David Henseler: Datenschutz bei drohnengestützter Datenbearbeitung durch Private 011— Lorenz Raess: Court Assistance in the Taking of Evidence in International Arbitration Dieses Werk ist erschienen in der Reihe sui generis, herausgegeben von Daniel Hürlimann und Marc Thommen. 1. Auflage 20. Juli 2020 © 2020 David Henseler Dieses Werk wurde unter einer Creative Commons Lizenz als Open Access veröffentlicht, die bei Weiterverwendung nur die Nennung des Urhebers erfordert (CC BY 4.0 — https://creativecommons.org/licenses/by/4.0). Die vorliegende Dissertation wurde durch den Forschungskredit der Universität Zürich, Verfügung Nr. FK-18-011, unterstützt. Die Druckvorstufe dieser Publikation wurde vom Schweizerischen Nationalfonds zur Förderung der wissenschaftlichen Forschung unterstützt. ISBN: 978–3–907297–10–0 DOI: 10.38107/010 Korrektorat: Christoph Meyer Gestaltung: Müller+Hess, Basel Druck: Ebner & Spiegel, Ulm www.suigeneris-verlag.ch 010 DATENSCHU DROHNENGE DATENBEAR DURCH PRIV Der Einsatz von Drohnen nimmt stetig zu, auch unter Privaten. Wer eine Drohne hört oder sieht, weiss in der Regel nicht, ob er gerade beobachtet oder gefilmt wird und ob das Aufgenommene live übertragen oder aufgezeichnet wird. Ebenso bleibt regelmässig unklar, wer für die Drohne verantwortlich ist. Vor diesem Hintergrund nimmt sich dieses Buch der folgenden Frage an: Wie kann das Recht auf informationelle Selbstbestimmung — konkretisiert im Datenschutzrecht — bei der drohnengestützten Datenbearbeitung durch Private durchgesetzt werden? Der Autor analysiert dazu sowohl das zukünftige Datenschutzgesetz als auch die neuen Vorschriften zur Drohnenfliegerei und präsentiert gestützt darauf konkrete Vorschläge für eine Gesetzesanpassung. sui generis ISBN 978–3–907297–10–0 DOI 10.38107/010
https://openalex.org/W4387056232	https://www.qeios.com/read/49HAI8/pdf	English	null	Review of: "Modified Hawking radiation of Schwarzschild-like black hole in bumblebee gravity model"	null	2,023	cc-by	542	Qeios, CC-BY 4.0 · Review, September 26, 2023 Review of: "Modified Hawking radiation of Schwarzschild-like black hole in bumblebee gravity model" Nan Li1 1 Northeastern University Nan Li1 Potential competing interests: No potential competing interests to declare. This paper studies the modified Hawking radiation in the bumblebee gravity model, and also considers the generalized uncertainty principle and the quantum-corrected entropy of the Schwarzschild black hole at the same time. This paper is clearly written, and the structure is well organized. However, there are several problems, as listed below. The authors should first carefully deal with these issues before this paper can be considered for publication. 1. After providing some necessary background knowledge of the three special coordinate systems, the final results are always directly given, without any detailed derivation! The authors should present what they do in this paper and what is new compared to previous literature. For example, How did the authors obtain the results in Eqs. (9), (10), and (11)? I cannot find the derivation or any reference. How did the authors obtain the results in Eqs. (19) and (20)? Actually, these are the essential results to obtain the tunneling probability in Eq. (21), but I cannot find their calculations at all. In contrast, Eq. (18) is trivial, unnecessary to be shown. How did the authors obtain the results in Eqs. (29), (31), and (32)? Again, the derivation of these important results is absent. How did the authors obtain the results in Eqs. (29), (31), and (32)? Again, the derivation of these important results is absent. The detailed derivation of Eq. (52) must be provided, because it is the most important result in this subsection. The detailed derivation of Eq. (52) must be provided, because it is the most important result in this subsection. 2. What is $\tilde{r}_h$ in Eqs. (19), (20), and (21)? Is it the same as $r_h$? 3. In Eq. (25), should the coordinate $r$ be $r^\ast$? Also, should all $r$ in this subsection be $r^\ast$? Otherwise, why did the authors introduce this new coordinate $r^\ast$? 3. In Eq. (25), should the coordinate $r$ be $r^\ast$? Also, should all $r$ in this subsection be $r^\ast$? Otherwise, why did the authors introduce this new coordinate $r^\ast$? 4. In the line above Eq. (26), there are two partial derivative $\partial_v$. Should they be $\partial_\nu$? 5. What is ${\bf e}$ in Eq. (39), (42), (43), and (44)? Is it just ${\rm e}$? 6. In addition, there are several typos in this paper. In Eq. Qeios ID: 49HAI8 · https://doi.org/10.32388/49HAI8 Review of: "Modified Hawking radiation of Schwarzschild-like black hole in bumblebee gravity model" (1), there is Newton constant $G_N$, but $G_N$ is set to be 1 throughout this paper. Qeios ID: 49HAI8 · https://doi.org/10.32388/49HAI8 1/2 Qeios, CC-BY 4.0 · Review, September 26, 2023 ere should be "-2 Im" in both the exponential functions in the denominator and numerator in Eq. (32) In Eqs. (32) and (60), "exp" should be "\exp", and "Im" should be "{\rm Im}". In Eqs. (32) and (60), "exp" should be "\exp", and "Im" should be "{\rm Im}". In Eq. (37), $rh$ should be $r_h$. Throughout this paper, the derivative operator "d" should be expressed in one form, $d$ or ${\rm d}$ Qeios ID: 49HAI8 · https://doi.org/10.32388/49HAI8 2/2
https://openalex.org/W3195334570	https://www.aging-us.com/article/203426/pdf	English	null	Bu-Shen-Zhu-Yun decoction induces PRLR deubiquitination and JAK2/STAT5 activation via CSN5 in vitro	Aging	2,021	cc-by	8,751	ABSTRACT Purpose: To determine the effect of Bu-Shen-Zhu-Yun Decoction (BSZY-D) on the kisspeptin through JAK2/STAT5 signaling pathway in hyperprolactinemia (HPRL) infertility. Method: SD rats were treated with BSZY-D for cerebrospinal fluid (CSF) extraction. GT1-7 cells were subjected to different treatments. The phosphorylation levels of JAK2 and STAT5, and the expressions of PRLR and kisspeptin of GT1-7 cells in different groups were detected by western blot, RT-qPCR and immunofluorescence. The expressions of CSN5 and GATA1 and other molecular features were checked by western blot, RT-PCR, co- immunoprecipitation and renilla luciferase activity. Results: The phosphorylation levels of JAK2 and STAT5, and the expressions of PRLR and kisspeptin in the HPRL group were significantly decreased, and these changes could be reversed after BSZY-D treatment. In addition, the presence of PRLR deubiquitination was detected in the HPRL group, which could be reversed by shRNA- CSN5, suggesting that BSZY-D played a role through targeting CSN5. The binding level of GATA1 and CSN5 promoter in the HPRL group was significantly decreased, but elevated in the HPRL (BSZY-D/CSF) group (P < 0.05). Conclusion: BSZY-D improved the transcription activity of GATA1 and increased the binding of GATA1 and CSN5. BSZY-D was involved in the deubiquitination of PRLR, which contributes to alleviating the symptoms of HPRL infertility. Hua Feng1,2, Huifang Zhou3,&, Jianxia Lu1, Qing Zhang4, Xingran Tang5, Yujie Shang5 Hua Feng1,2, Huifang Zhou3,&, Jianxia Lu1, Qing Zhang4, Xingran Tang5, Yujie Shang5 1Institute of Rehabilitation, Jiangsu Vocational College of Medicine, Yancheng 224008, Jiangsu Province, China 2Department of Gynecology, Nanjing University of Chinese Medicine, Nanjing 224005, Jiangsu Province, China 3Department of Gynecology, Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing 210029, Jiangsu Province, China 4School of Clinical Medicine Jiangsu Vocational College of Medicine, Yancheng 224008, Jiangsu Province, China 5Nanjing University of Chinese Medicine, Nanjing 224005, Jiangsu Province, China Correspondence to: Huifang Zhou; email: zhouhuifang2011301@163.com, https://orcid.org/0000-0001-5208-8727 Keywords: Bu-Shen-Zhu-Yun decoction, hyperprolactinemia infertility Received: December 2, 2020 Accepted: May 11, 2021 Published: August 23, 2021 Copyright: © 2021 Feng et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. AGING 2021, Vol. 13, No. 16 AGING 2021, Vol. 13, No. 16 www.aging-us.com www.aging-us.com AGING 2021, Vol. 13, No. 16 Research Paper Bu-Shen-Zhu-Yun decoction induces PRLR deubiquitination and JAK2/STAT5 activation via CSN5 in vitro Hua Feng1,2, Huifang Zhou3,&, Jianxia Lu1, Qing Zhang4, Xingran Tang5, Yujie Shang5 1Institute of Rehabilitation, Jiangsu Vocational College of Medicine, Yancheng 224008, Jiangsu Province, China 2Department of Gynecology, Nanjing University of Chinese Medicine, Nanjing 224005, Jiangsu Province, China 3Department of Gynecology, Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing 210029, Jiangsu Province, China 4School of Clinical Medicine Jiangsu Vocational College of Medicine, Yancheng 224008, Jiangsu Province, China 5Nanjing University of Chinese Medicine, Nanjing 224005, Jiangsu Province, China Correspondence to: Huifang Zhou; email: zhouhuifang2011301@163.com, https://orcid.org/0000-0001-5208-8727 Keywords: Bu-Shen-Zhu-Yun decoction, hyperprolactinemia infertility Received: December 2, 2020 Accepted: May 11, 2021 Published: August 23, 2021 Copyright: © 2021 Feng et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. www.aging-us.com AGING 2021, Vol. 13, No. 16 Research Paper Bu-Shen-Zhu-Yun decoction induces PRLR deubiquitination and JAK2/STAT5 activation via CSN5 in vitro Hua Feng1,2, Huifang Zhou3,&, Jianxia Lu1, Qing Zhang4, Xingran Tang5, Yujie Shang5 1Institute of Rehabilitation, Jiangsu Vocational College of Medicine, Yancheng 224008, Jiangsu Province, China 2Department of Gynecology, Nanjing University of Chinese Medicine, Nanjing 224005, Jiangsu Province, China 3Department of Gynecology, Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing 210029, Jiangsu Province, China 4School of Clinical Medicine Jiangsu Vocational College of Medicine, Yancheng 224008, Jiangsu Province, China 5Nanjing University of Chinese Medicine, Nanjing 224005, Jiangsu Province, China Correspondence to: Huifang Zhou; email: zhouhuifang2011301@163.com, https://orcid.org/0000-0001-5208-8727 Keywords: Bu-Shen-Zhu-Yun decoction, hyperprolactinemia infertility Received: December 2, 2020 Accepted: May 11, 2021 Published: August 23, 2021 Copyright: © 2021 Feng et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. INTRODUCTION value more than 1.4 nmol/L should be diagnosed as HPRL [2]. The pathogenesis of HPRL includes the physiological, pathological, and pharmacological factors [3], among which the physiological factors include diet, sleep, gestation, nipple stimulation and breastfeeding; common pathological factors mainly include hypothalamic tumors, pituitary or ectopic prolactinoma, and acromegaly; as for pharmacological factors, some anesthetic (cocaine) and Chinese herbal medication (Angong Niuhuang Pill) can induce the Hyperprolactinemia (HPRL) is caused by a variety of factors and characterized by the significant elevation of serum prolactin (PRL). It is a syndrome of reproductive-endocrine disorder in the hypothalamic- pituitary axis, with relatively high morbidity [1]. At present, although there is no consistent detection method or reference range among different hospitals, it is widely accepted that the subject with serum PRL AGING 20418 www.aging-us.com JAK2/STAT5 pathway in a short time. On the other hand, it can lead to the ubiquitination, endocytosis, and degradation of PRLR, causing inhibition of PRL- mediated JAK2/STAT5 pathway in a long period. Excessive PRL cannot bind to its receptor, thus the expressions of JAK2/STAT5 pathway and its downstream kisspeptin are suppressed. CSN5, a type of deubiquitinating enzymes, can inhibit the degradation of PRLR through deubiquitination. We analyzed the transcription factors of CSN5 in the GENERADAR website (https://www.gcbi.com.cn/gcanalyze/html/gene radar/index), and found that GATA1 is one of the transcription factors of CSN5. Hence, the improvement of the deubiquitination level of PRLR in patients with HPRL is the key for the treatment of infertility, miscarriage, irregular menstruation and so on. Therefore, the illustration of BSZY-D underlying mechanism in HPRL is the crucial part. However, no research has reported the relationship between PRLR deubiquitination and BSZY-D. HPRL. The main clinical symptoms include galactorrhea, menstrual disorders, hirsutism, and even infertility [4]. Nowadays, the medication such as bromocriptine is mostly applied in clinical practice. Although the application of bromocriptine has a certain curative effect, the side effects such as dizziness, nausea, vomiting, and resistance to dopamine receptor agonists cannot be ignored. Therefore, it is necessary to explore more safe and effective therapeutic strategies for patients with HPRL infertility. The prescription of Bu-Shen-Zhu-Yun decoction (BSZY-D) created by Professor Guicheng Xia, is composed of yam, vinegar bupleurum, antlers tablets, red white peony root, cornus, dodder, salvia, angelica, amethyst, poria, fried cortex moutan, etc. This decoction has been proved to regulate menstruation and assist pregnancy [5]. INTRODUCTION BSZY-D has been widely applied in gynecological diseases, such as luteal phase defect, anovulatory infertility, and polycystic ovarian syndrome [5–7]. However, the efficacy of BSZY-D in HPRL infertility remains elusive. In the current study, we hypothesized that BSZY-D could improve the HPRL infertility through PRLR deubiquitination, targeting JAK2/STAT5 pathway and its downstream kisspeptin of hypothalamus. Our purpose is to explore the underlying molecular mechanism of HPRL infertility, thereby providing the new theoretical basis for the potential treatment application in clinic. Kisspeptin, a polypeptide hormone encoded by KISS-1 gene, can regulate the female reproductive endocrine functions. Kisspeptin modulates the secretion of gonadotropin-releasing hormone (GnRH) through the feedback effects of estradiol, and subsequently is involved in the regulation of reproductive and growth hormones in the reproductive system [8]. Hence, kisspeptin contributes to maintaining the stability of the hypothalamus-pituitary-ovarian (HPO) axis. A previous study has reported that BSZY-D can regulate the secretion of serum progesterone, follicle-stimulating hormone, luteinizing hormone, and estradiol through kisspeptin/GPR54 pathway, thereby improving luteal function and promoting embryo implantation [9]. The regulation of BSZY-D on PRLR/JAK2/STAT5 pathway via CSF in GT1-7 The level of PRLR in different GT1-7 cell groups were detected by western blot assay. As shown in Figure 3, compared with the Control group, the cytomembrane PRLR level in the HPRL group was significantly inhibited, but the total protein level of PRLR remained stable. Compared with the HPRL group, the protein level of PRLR in the HPRL (BSZY-D/CSF) group was significantly upregulated, which suggested that BSZY-D reversed the downregulation of PRLR protein level via HPRL (P < 0.05). Besides, we found that the PRLR ubiquitination level was increased in the HPRL group, however, there was deubiquitination of PRLR in the HPRL (BSZY-D/CSF) group (P < 0.05). It was indicated that HPRL caused the PRLR ubiquitination, but BSZY-D induced the deubiquitination of PRLR (Supplementary Figure 5). The phosphorylation levels of JAK, STAT5, and the levels of PRLR and kisspeptin in different groups were detected by western blot (Figure 2). Our data indicated that compared with the Control group, the phosphorylation levels of JAK2 and STAT5, and the levels of PRLR and kisspeptin in the HPRL group were decreased significantly (P < 0.05). Compared with the HPRL group, the phosphorylation levels of AK2 and STAT5, and the levels of PRLR and kisspeptin in the HPRL (BSZY-D/CSF) group were significantly elevated (P < 0.05), indicating that BSZY-D reversed the impact of HPRL on the downregulation of the PRLR/JAK/STAT5 pathway. Similarly, the kisspeptin mRNA expression in the HPRL group was significantly decreased, however, the PRLR mRNA expression was not downregulated significantly; compared with the HPRL group, the kisspeptin mRNA expression in the HPRL (BSZY- D/CSF) group was significantly increased, suggesting Compared with the Control group, the PRLR protein level was downregulated in the HPRL group, but there was no difference in total protein level. Moreover, Figure 1. Effect of PRL on PRLR-JAK-STAT5 pathway in GT1-7. The phosphorylation levels of JAK, STAT5 and the levels of PRLR and kisspeptin in GT1-7 from different groups were detected by western blot assay, and representative bands were shown in (A). The phosphorylation levels of JAK (B) and STAT5 (C), and the level of PRLR (D), and the level of kisspeptin (E) were normalized to control. The results were presented as mean ± SD (n = 3). p < 0.05, p < 0.01 vs. Control group. Figure 1. Effect of PRL on PRLR-JAK-STAT5 pathway in GT1-7. Effect of PRL on PRLR/JAK2/STAT5 pathway in GT1-7 The viability of GT1-7 cells under different concentrations of PRL treatment was detected, and the results showed that PRL (400 ng/ml) has a significant inhibitory effect on the viability of GT1-7 cells, compared with the control group (P < 0.05). No effects have been found in any concentration of CFS on the viability of GT1-7 cells (Supplementary Figure 2). Hence, we used PRL at the concentration of 0 ng/mL, 12.5 ng/mL, 25 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, and CFS at a concentration of 10%. Then the phosphorylation levels of JAK, STAT5, and the levels of PRLR and kisspeptin of GT1-7 cells in different groups were detected by western blot (Figure 1). Our data represented that compared with the Control group, the phosphorylation levels of JAK2 and STAT5, and the levels of PRLR and kisspeptin in the PRL (100 ng/ml and 200 ng/ml) group were significantly decreased (P < 0.05). Consistently, the mRNA expression of kisspeptin was significantly declined in the PRL (100 ng/ml and 200 ng/ml) group, but no apparent change was detected in PRLR mRNA expression (Supplementary Figure 3). PRL binds to its receptor PRLR and forms a complex that can further activate the Janus kinase 2 (JAK2). Subsequently, the activated JAK2 leads to its downstream signal transducer and activator of transcription 5 (STAT5) [10]. Phosphorylated STAT5 protein forms homodimers or heterodimerizes with other STAT family members, relating to the nucleus translocation and transactivation of the molecular target. JAK2/STAT5 signaling pathway is involved in the development of HPRL, and PRL/PRLR-JAK2/STAT5 signaling pathway is widely explored in cell pro- liferation, differentiation, and immune regulation. It can also initiate the transcription of certain target genes, and enable the expressions of ovarian and endometrial related receptors. High PRL level elevates the combination of PRL and PRLR. On the one hand, it can activate the downstream AGING 20419 www.aging-us.com Therefore, PRL was concentration-dependent. The higher PRL had a more significant effect, so we took 200 ng/mL PRL for the subsequent experiments. that BSZY-D reversed the downregulation trend of kisspeptin through HPRL (Supplementary Figure 4). Therefore, PRL was concentration-dependent. The higher PRL had a more significant effect, so we took 200 ng/mL PRL for the subsequent experiments. The regulation of BSZY-D on PRLR/JAK2/STAT5 pathway via CSF in GT1-7 AGING 20421 www.aging-us.com compared with the HPRL group, the PRLR protein level was augmented in the HPRL (BSZY-D/CSF) group, suggesting that CSF reversed the downregulation of PRLR protein level through HPRL (P < 0.05) (Figure 4). compared with the HPRL group, the PRLR protein level was augmented in the HPRL (BSZY-D/CSF) group, suggesting that CSF reversed the downregulation of PRLR protein level through HPRL (P < 0.05) (Figure 4). with the Control group, the binding of PRLR and β- Trcp was significantly increased in the HPRL group, and the binding of PRLR and CSN5 was decreased; compared with the HPRL group, the binding of PRLR and CSN5 in the HPRL (BSZY-D/CSF) group was increased (P < 0.05). It was suggested that BSZY-D played a role by promoting the binding of PRLR and CSN5. The PRLR protein level of GT1-7 cells at different time points were detected using western blot. Compared with the Control + CHX group, the PRLR protein level was decreased significantly in the HPRL group. Compared with the HPRL + CHX group, the PRLR level in the HPRL (BSZY-D/CSF) + CHX group was significantly upregulated (P < 0.05). The results demonstrated that the PRLR degradation would be accelerated under the high PRL condition, and this degradation was inhibited after the addition of BSZY-D (Figure 5). The levels of CSN5 and GATA1 in GT1-7 were detected by western blot (Figure 6). Our results represented that the CSN5 and GATA1 protein levels were significantly decreased in the HPRL group compared with the Control group. Compared with the HPRL group, the levels of CSN5 and GATA1 in the HPRL (BSZY-D/CSF) group were significantly elevated (P<0.05). In line with the results of western blot, the RT-PCR data showed that the CSN5 mRNA expression was significantly suppressed in the HPRL group compared with the Control group, and the GATA1 mRNA expression did not change significantly. Besides, compared with the HPRL group, the CSN5 The regulation of BSZY-D on PRLR/JAK2/STAT5 pathway via CSF in GT1-7 The phosphorylation levels of JAK, STAT5 and the levels of PRLR and kisspeptin in GT1-7 from different groups were detected by western blot assay, and representative bands were shown in (A). The phosphorylation levels of JAK (B) and STAT5 (C), and the level of PRLR (D), and the level of kisspeptin (E) were normalized to control. The results were presented as mean ± SD (n = 3). p < 0.05, p < 0.01 vs. Control group. AGING 20420 www.aging-us.com Figure 2. Effect of CSF on PRLR-JAK-STAT5 pathway in GT1-7. The phosphorylation levels of JAK, STAT5 and the levels of PRLR and kisspeptin in GT1-7 from different groups were detected by western blot assay, and representative bands were shown in (A). The phosphorylation levels of JAK (B) and STAT5 (C), and the level of PRLR (D), and the level of kisspeptin (E) were normalized to control. The results were presented as mean ± SD (n = 3).p < 0.01 vs. Control group, #p < 0.05, ##p < 0.01 vs. HPRL group. Figure 2. Effect of CSF on PRLR-JAK-STAT5 pathway in GT1-7. The phosphorylation levels of JAK, STAT5 and the levels of PRLR and kisspeptin in GT1-7 from different groups were detected by western blot assay, and representative bands were shown in (A). The phosphorylation levels of JAK (B) and STAT5 (C), and the level of PRLR (D), and the level of kisspeptin (E) were normalized to control. The results were presented as mean ± SD (n = 3).p < 0.01 vs. Control group, #p < 0.05, ##p < 0.01 vs. HPRL group. Figure 3. The level of PRLR in GT1-7 from different groups was detected by western blot assay, and representative bands were shown in (A). The levels of PRLR (B, C) were normalized to control. The results were presented as mean ± SD (n = 3). p < 0.01 vs. Control group, #p < 0.05, ##p < 0.01 vs. HPRL group. Figure 3. The level of PRLR in GT1-7 from different groups was detected by western blot assay, and representative bands were shown in (A). The levels of PRLR (B, C) were normalized to control. The results were presented as mean ± SD (n = 3). p < 0.01 vs. Control group, #p < 0.05, ##p < 0.01 vs. HPRL group. The changes of CSN5 expression in GT1-7 cells treated with different reagents It was indicated that BSZY-D reversed the downregulation effect of HPRL on CSN5. The changes of CSN5 expression in GT1-7 cells treated with different reagents Co-IP was applied to test the expressions of β-Trcp, PRLR and CSN5 (Supplementary Figure 6). Compared Figure 4. The immunofluorescence staining of PRLR in GT1-7. Figure 5. The level of PRLR in GT1-7 at different times was detected by western blot assay. The levels of PRLR were normalized to control. (A) Representative blot of PRLR level. (B) Quantitative relative gray values of PRLR protein level. The results were presented as mean ± SD (n = 3). p < 0.01 vs. Control group, ##p < 0.01 vs. HPRL group. Figure 4. The immunofluorescence staining of PRLR in GT1-7. Figure 4. The immunofluorescence staining of PRLR in GT1-7. Figure 5. The level of PRLR in GT1-7 at different times was detected by western blot assay. The levels of PRLR were normalized to control. (A) Representative blot of PRLR level. (B) Quantitative relative gray values of PRLR protein level. The results were presented as mean ± SD (n = 3). p < 0.01 vs. Control group, ##p < 0.01 vs. HPRL group. Figure 5. The level of PRLR in GT1-7 at different times was detected by western blot assay. The levels of PRLR were normalized to control (A) Representative blot of PRLR level (B) Quantitative relative gray values of PRLR protein level The results were presented as mean Figure 5. The level of PRLR in GT1-7 at different times was detected by western blot assay. The levels of PRLR were normalized to control. (A) Representative blot of PRLR level. (B) Quantitative relative gray values of PRLR protein level. The results were presented as mean ± SD (n = 3). p < 0.01 vs. Control group, ##p < 0.01 vs. HPRL group. AGING 20422 www.aging-us.com decreased; in turn, compared with the HPRL group, the CSN5 promoter activity in the HPRL (BSZY-D/CSF) group was significantly upregulated. Correspondingly, the binding of GATA1 and CSN5 promoter in the HPRL group was significantly decreased, but the binding of GATA1 and CSN5 promoter in the HPRL (BSZY-D/CSF) group was significantly elevated (P < 0.05) (Supplementary Figure 8). After the transfection of lentivirus plasmids, the CSN5 protein level was significantly inhibited in the shRNA-CSN5 group, suggesting that the shRNA-CSN5 plasmid reduced the expression of CSN5 in GT1-7 cells (P < 0.05) (Supplementary Figure 9). mRNA expression was significantly increased in the HPRL (BSZY-D/CSF) group (P<0.05) (Supplementary Figure 7). Effect of lentivirus plasmids shRNA-CSN5 on PRLR/JAK/STAT5 pathway in GT1-7 Luciferase activity was detected by measuring the renilla luciferase activity of GT1-7 cells transiently transfected with the CSN5 luciferase promoter (Figure 7). Compared with the Control group, the CSN5 promoter activity in the HPRL group was significantly Figure 6. The levels of CSN5 and GATA1 in GT1-7 were detected by western blot assay, and representative bands were shown in (A). The levels of CSN5 (B) and GATA1 (C) were normalized to control. The results were presented as mean ± SD (n = 3).p < 0.01 vs. Control group, #p < 0.05, ##p < 0.01 vs. HPRL group. Figure 6. The levels of CSN5 and GATA1 in GT1-7 were detected by western blot assay, and representative bands were shown in (A). The levels of CSN5 (B) and GATA1 (C) were normalized to control. The results were presented as mean ± SD (n = 3).p < 0.01 vs. Control group, #p < 0.05, ##p < 0.01 vs. HPRL group. Figure 7. Luciferase activity measured and normalized according to renilla luciferase activity in GT1-7 cells transiently transfected with the CSN5 luciferase promoter. The results were presented as mean ± SD (n = 3). p < 0.01 vs. Control group, ##p < 0.01 vs. HPRL group. Figure 7. Luciferase activity measured and normalized according to renilla luciferase activity in GT1-7 cells transiently transfected with the CSN5 luciferase promoter. The results were presented as mean ± SD (n = 3). p < 0.01 vs. Control group, ##p < 0.01 vs. HPRL group. AGING 20423 www.aging-us.com After the confirmation of transfection efficiency, the impact of shRNA-CSN5 on PRLR/JAK/STAT5 signaling pathway in GT1-7 cells of different treatment groups was detected by western blot. As shown in Figure 8, compared with the Control group, the phosphorylation levels of JAK2 and STAT5, and the protein levels PRLR and kisspeptin in the HPRL (BSZY-D/CSF) + shRNA-NC group were significantly decreased (P < 0.05). After knocking down CSN5 gene, the above-mentioned protein levels could be reversed. This result indicated that BSZY-D enhanced the PRLR/JAK/STAT5 signaling pathway through CSN5. Besides, there existed an increasing ubiquitination level of PRLR in the HPRL group, which could be reversed by knocking down shRNA-SCN5. It was suggested that BSZY-D played a role through targeting CSN5 (Supplementary Figure 10). DISCUSSION Previous researches of traditional Chinese medicine Bu- Shen-Zhu-Yun Decoction (BSZY-D) mainly focus on infertility caused by luteal phase defect. Recently, it is reported that BSZY-D can reduce the prolactin (PRL) Figure 8. Effect of shRNA-CSN5 on PRLR-JAK-STAT5 pathway in GT1-7. The phosphorylation levels of JAK, STAT5 and the levels of PRLR and kisspeptin in GT1-7 from different groups were detected by western blot assay, and representative bands were shown in (A). The phosphorylation levels of JAK (D) and STAT5 (E), and the level of PRLR (B, C), and the level of kisspeptin (F) were normalized to control. The results were presented as mean ± SD (n = 3). p < 0.05, p < 0.01 vs. Control group. Figure 8. Effect of shRNA-CSN5 on PRLR-JAK-STAT5 pathway in GT1-7. The phosphorylation levels of JAK, STAT5 and the levels of PRLR and kisspeptin in GT1-7 from different groups were detected by western blot assay, and representative bands were shown in (A). The phosphorylation levels of JAK (D) and STAT5 (E), and the level of PRLR (B, C), and the level of kisspeptin (F) were normalized to control. The results were presented as mean ± SD (n = 3). p < 0.05, p < 0.01 vs. Control group. AGING www.aging-us.com 20424 level in patients with luteal phase defect and hyperprolactinemia (HPRL) in clinic practice. Hence, our purpose in this study is to investigate the underlying mechanism of BSZY-D in decreasing PRL. In this study, we identified that HPRL could downregulate the levels of PRLR and kisspeptin, and phosphorylation levels of JAK2 and STAT5. BSZY-D reversed the effect of HPRL on the downregulation of the PRLR/ JAK2/STAT5 pathway. The potential mechanism was that excessive PRL cannot bind to its receptor under high PRL microenvironment, thus the expressions of JAK2/STAT5 pathway and kisspeptin were suppressed. Besides, CSN5 inhibited the degradation of PRLR through deubiquitination, and targeted its transcription factors GATA 1. These changes could be reversed after BSZY-D treatment. We proved that BSZY-D increased the binding level of CSN5 and GATA 1, and induced the deubiquitination of PRLR, thus increasing the expressions of JAK2/STAT5 pathway and its downstream signaling kisspeptin. Based on our study, BSZY-D can be regarded as an effective Chinese medicine to treat HPRL infertility in clinic (Figure 9). using vitro fertilization [11]. Several ingredients of BSZY-D including dioscoreae rhizome, cervi cornu, bupleuri radix and paeoniae radix alba, etc. www.aging-us.com DISCUSSION have been proved to improve the uterine receptivity and pregnancy rate in mice [12, 13]. Xiaofei Jiang et al. have demonstrated that BSZY-D can upregulate the expressions of integrin α5 and β3 by inhibiting the estrogen and progesterone receptor, meanwhile, improve the endometrial receptivity during embryo implantation and consequently enhance the pregnancy rates [14]. In the current study, we found that BSZY-D reserved the molecular and pathway changes caused by HPRL. The elevation of PRLR, kisspeptin, phosphorylated JAK2, and STAT5 levels were significantly reserved by BSZY-D treatment, as well as the decline of the PRLR ubiquitination. Prolactin (PRL) is a type of polypeptide hormone secreted by the anterior pituitary gland. PRL is balanced by PRL releasing factor (PRF) and PRL inhibiting hormone (PIH) [15, 16]. In the normal physio- logical condition, the increased transcription activity of GATA1 induces the transcription of its downstream CSN5 gene, leading to the upregulation of CSN5. CSN5 plays a role in mediating the deubiquitination of PRLR, blocking its endocytosis and degradation, and maintaining the normal function of PRLR. JAK2/STAT5 is the downstream signaling pathway of PRLR, and the activation of JAK2/STAT5 can elevate the expression of kisspeptin. In the presence of pathological HPRL condition, HPRL can reduce the The theory of Chinese medicine believes that the kidney affects reproduction, which is the innate foundation and the root of vitality. Ovulation relies on several factors, such as kidney, liver, spleen, qi, blood, yin and yang [7]. A previous study of meta-analysis has represented that BSZY-D can significantly elevate the pregnancy rate to 50%, comparing to the pregnancy rate of 30% Figure 9. The effects of BSZY-D on activating GATA 1 and CSN5, inducing PRLR deubiquitination and increasing the expressions of JAK2/STAT5 signaling pathway and kisspeptin. Hence, BSZY-D had an efficient role in HPRL infertility improvement. Figure 9. The effects of BSZY-D on activating GATA 1 and CSN5, inducing PRLR deubiquitination and increasing the expressions of JAK2/STAT5 signaling pathway and kisspeptin. Hence, BSZY-D had an efficient role in HPRL infertility improvement. AGING 20425 www.aging-us.com receptivity of the endometrium, and suppress the ovarian function, consequently causing ovulation disorders [17]. Recently, it is reported that the proper concentration of PRL can promote the growth of endometrial cells and blastocyst adhesion. However, the deregulated PRL concentration has an inhibitory effect, which causes follicle maturation disorder, ovulation disorder and decreased endometrial receptivity, thus eventually leading to infertility [18]. DISCUSSION The underlying mechanism can be explained by an abnormal level of PRLR ubiquitination. The overexpressing PRL augments the activity of CSN5, a type of deubiquitinating enzyme, which induces the deubiquitination of PRLR. Hence, the downstream JAK2/STAT5 signaling pathway is inhibited, as well as kisspeptin. In the current study, we identified that BSZY-D could reversely elevate the transcription activity of GATA1, increase the binding of GATA1 and CSN5, and then reserve the downstream expressions at a certain level. could increase the expressions of JAK/STAT5 and kisspeptin via inducing the deubiquitination of PRLR, thereby alleviating the clinic manifestations of HPRL infertility. We hope our novel work could establish a theoretical basis on the molecular mechanism of BSZY- D in HPRL infertility, meriting further investigation of potential treatment application. Cell culture and treatment GT1-7 cell line was purchased from Millipore (Schwalbach, Germany). GT1-7 cells were cultured and maintained in DMEM medium (GIBCO, Grand Island, USA) containing penicillin (final concentration 100U/mL) (Sigma-Aldrich, St. Louis, MO, USA), streptomycin (final concentration 100μg/mL) (Sigma- Aldrich, St. Louis, MO, USA), 2mM L-Glutamine (Millipore, Schwalbach, Germany) and 10% fetal bovine serum (FBS) (Hyclone, Logan, UT, USA). The cells were digested when reaching 90% confluence. Kisspeptin is a type of polypeptide encoded by the KISS-l gene. It is mainly distributed in the hypothalamus, pituitary, ovary, placenta, etc. Recent studies have represented that kisspeptin plays an important role in the initiation of puberty, menstrual cycle regulation, ovulation induction, and reproductive cycle regulation [19]. Some scholars have indicated that kisspeptin may have a certain correlation with pregnancy implantation, and further hypothesized it is also related to abortion [20]. In the current study, our results showed that HPRL could downregulate the expression of kisspeptin, which might impair the physiological role of kisspeptin in maintaining the normal productive function. Interestingly, kisspeptin expression could be reversed after BSZY-D treatment. Besides, in line with the changes of kisspeptin, the expression of PRLR/JAK/STAT5 signaling pathway showed the similar trend. Medicine and extraction preparation BSZY-D was concentrated to 2g/mL and stored at 4° C. In brief, the medicinal materials had been immersed in water for 30 min. Firstly, the deer horn slices were decocted and boiled for 20 min, and then added with the remaining medicinal materials. The decoction at a concentration of 2 g/mL was collected and stored at 4° C. A total of 75 female SD rats of specific pathogen-free (SPF) grade (aged 8 weeks and weighing 220 ± 20 g) were purchased from Animal Model Center of Nanjing University [license No: SCXK (Su) 2018- 0008], and raised in the SPF environment in the Experimental Animal Center of Nanjing Chinese Medicine University. The rats were reared at 40%- 70% humidity and 21-25° C, maintained in a 12 h light/dark cycle. The experimental operations were approved by the Animal Ethics Committee of Nanjing Chinese Medicine University [approval No: SYXK (Su) 2014-0001]. In this study, we proved that BSZY-D partly recovered the expressions of JAK/STAT5 and kisspeptin via inducing the deubiquitination of PRLR and targeting CSN5. The limitation of this study is that we did not clarify the possible molecular mechanism between CSN5 and PRLR deubiquitination, which will be investigated as a key point in our further study. Cerebrospinal fluid was prepared as follows: BSZY-D of 2 g/mL was given to normal female SD rats 1 mL/kg twice a day. After anesthesia, the neck skin of each rat was disinfected with 75% ethanol, and the foramen magnum was exposed. The micro-injector was pierced into the cistern magna and 100 uL cerebrospinal fluid (CSF) was slowly extracted. After centrifugation at 2000 r/min for 15 min, the supernatant was collected and stored at -20° C. The time point of CSF collection was after the estrus. CONCLUSIONS Altogether, we explored the underlying mechanism of BSZY-D in HPRL infertility. We demonstrated that BSZY-D could improve the transcription activity of GATA1 and increase the binding of GATA1 and CSN5. Also, the activated deubiquitinating enzyme CSN5 AGING www.aging-us.com 20426 8) Experiment 8: Normal group (Control group): 10% blank cerebrospinal fluid, cultured for 72 hours. 9) Experiment 9: Cerebrospinal fluid containing traditional Chinese medicine a concentration of 10% + MG132 [HPRL (BSZY-D/CSF) group]: 10% BSZY-D containing cerebrospinal fluid culture + 200ng/mL PRL + 10uM MG132 inhibitor, cultured for 72h. Cerebrospinal fluid containing traditional Chinese medicine at a concentration of 10% intervention [HPRL (BSZY-D/CSF) group]: 10% BSZY-D containing medicated cerebrospinal fluid culture + 200 ng/mL PRL, cultured for 72h. Cerebrospinal fluid containing traditional Chinese medicine at a concentration of 10% intervention [HPRL (BSZY-D/CSF) group]: 10% BSZY-D containing medicated cerebrospinal fluid culture + 200 ng/mL PRL, cultured for 72h. Cerebrospinal fluid containing traditional Chinese medicine at a concentration of 10% intervention [HPRL 5) Experiment 5: Prolactin intervention group (HPRL group): 10% blank cerebrospinal fluid + 200 ng/mL PRL, cultured for 72 h. Normal group + MG132 (Selleck Chemicals, Houston, Texas, USA) (Control + MG132 group): 10% blank cerebrospinal fluid + 10 uM MG132 inhibitor, cultured for 72h. Cerebrospinal fluid containing traditional Chinese medicine a concentration of 10% intervention [HPRL (BSZY-D/CSF) group]: 10% BSZY-D containing medicated cerebrospinal fluid culture + 200 ng/mL PRL, cultured for 72h. PRL intervention group + MG132 (HPRL + MG132 group): 10% blank cerebrospinal fluid + 200 ng/mL PRL + 10 uM MG132 inhibitor, cultured for 72h. PRL intervention group + MG132 (HPRL + MG132 group): 10% blank cerebrospinal fluid + 200 ng/mL PRL + 10 uM MG132 inhibitor, cultured for 72h. 9) Experiment 9: Experimental groups PRL intervention group + cycloheximide CHX (HPRL + CHX group): 10% blank cerebrospinal fluid + 200 ng/mL PRL + 10uM CHX inhibitor. Protocols in different experimental groups were shown as follows: Cerebrospinal fluid containing traditional Chinese medicine at a concentration of 10% + cycloheximide CHX [HPRL (BSZY-D/CSF) + CHX group]: 10% BSZY-D containing medicated cerebrospinal fluid culture + 200 ng/mL PRL + 10 uM CHX inhibitor. Cerebrospinal fluid containing traditional Chinese medicine at a concentration of 10% + cycloheximide CHX [HPRL (BSZY-D/CSF) + CHX group]: 10% BSZY-D containing medicated cerebrospinal fluid culture + 200 ng/mL PRL + 10 uM CHX inhibitor. 1) Experiment 1: 0% CSF (medicated cerebrospinal fluid), 5% CSF (medicated cerebrospinal fluid), 10% CSF (medicated cerebrospinal fluid) and 20% CSF (medicated cerebrospinal fluid). 2) Experiment 2: 0 ng/mL, 12.5 ng/mL, 25 ng/mL, 50 ng/mL, 100 ng /mL, 200 ng/mL and 400 ng/mL PRL. The time points for detection were 0h, 2h, 4h, 12h, 24h, respectively. 3) Experiment 3: 0 ng/mL, 12.5 ng/mL, 25 ng/mL, 50 ng/mL, 100 ng /mL and 200 ng/mL PRL. 7) Experiment 7: Normal group + MG132 (Control + MG132 group): 10% blank cerebrospinal fluid + 10 uM MG132 inhibitor, cultured for 72h. 4) Experiment 4: Normal group (Control group): 10% blank cerebrospinal fluid, cultured for 72 h. PRL intervention group + MG132 (HPRL + MG132 group): 10% blank cerebrospinal fluid + 200 ng/mL PRL + 10 uM MG132 inhibitor, cultured for 72h. PRL intervention group (HPRL group): 10% blank cerebrospinal fluid + 200 ng/mL PRL, cultured for 72 h. Cerebrospinal fluid containing traditional Chinese medicine at a concentration of 10% + MG132 [HPRL (BSZY-D/CSF) +MG132 group]: 10% BSZY-D containing medicated cerebrospinal fluid culture + 200 ng/mL PRL + 10 uM MG132 inhibitor, cultured for 72h. Cerebrospinal fluid containing BSZY-D intervention group (BSZY-D/CSF group): 10% BSZY-D containing medicated cerebrospinal fluid, cultured for 72 h. Cerebrospinal fluid containing BSZY-D intervention group (BSZY-D/CSF group): 10% BSZY-D containing medicated cerebrospinal fluid, cultured for 72 h. Cerebrospinal fluid containing traditional Chinese medicine at a concentration of 10% [HPRL (BSZY- D/CSF) group]: 10% BSZY-D containing cerebrospinal fluid culture + 10% blank cerebrospinal fluid + 200 ng/mL PRL, cultured for 72h. 8) Experiment 8: 6) Experiment 6: Cerebrospinal fluid containing traditional Chinese medicine a concentration of 10% + ShRNA-CSN5 [HPRL (BSZY-D/CSF) + ShRNA-CSN5 group]: after the cells were stably transfected with CSN5-knockdown Normal group + cycloheximide (CHX) (Seleck Chem, Shanghai, China) (Control + CHX group): 10% blank cerebrospinal fluid + 10uM CHX inhibitor. AGING www.aging-us.com 20427 lentivirus, added 10% BSZY-D containing medicated cerebrospinal fluid culture + 200 ng/mL PRL, cultured for 72h. lentivirus, added 10% BSZY-D containing medicated cerebrospinal fluid culture + 200 ng/mL PRL, cultured for 72h. Real-time PCR The total RNA was extracted from GT1-7 cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA then underwent reverse transcription using the iScript cDNA kit (Bio-Rad, Hercules, CA, USA), followed by analysis using real-time PCR (RT-PCR) with the SsoFast Eva Green Super Mix (Bio-Rad, Hercules, CA, USA) on Roche Light Cycler 480 system. Cerebrospinal fluid containing traditional Chinese medicine a concentration of 10% + ShRNA- NC [HPRL (BSZY-D/CSF) + ShRNA-NC group]: after the cells were stably transfected with NC lentivirus, added 10% BSZY-D containing medicated cerebrospinal fluid culture + 200 ng/mL PRL, cultured for 72h. Relative mRNA expressions of kisspeptin, PRLR, CSN5, GATA1, GAPDH were calculated by the comparative cycle threshold (CT) method, with expression of GAPDH as the internal reference. The 2−∆∆Ct method was used for analysis. The specific primers applied for RT-PCR reaction are shown in Table 1. Relative mRNA expressions of kisspeptin, PRLR, CSN5, GATA1, GAPDH were calculated by the comparative cycle threshold (CT) method, with expression of GAPDH as the internal reference. The 2−∆∆Ct method was used for analysis. The specific primers applied for RT-PCR reaction are shown in Table 1. Co-immunoprecipitation (Co-IP) The GT1-7 cells were treated as described in Experiment 5, 7 and 9. Then the cell precipitation was added with RIPA lysis buffer. Protein A/G-agarose microspheres were washed twice with PBS, and prepared into 50% protein A/G-agarose working solution with PBS. Next, 50% protein A/G agarose working solution was added into the samples at a ratio of 100 μL per 1 mL. The protein A/G-agarose microspheres were discarded by centrifugation. BCA assay was applied to test the protein concentration. Afterwards, 5 μL antibody (PRLR150) (IgG antibody as the control group) was added for incubation at 4° C overnight and the final volume was 500 μL. Next day, 100 μL protein A/G-agarose microspheres was added to capture the antigen-antibody complex at room temperature for 1h. The protein A/G-agarose microsphere antigen-antibody complex was collected after centrifugation and washed with PBS. The supernatant was collected, and the loaded samples were boiled for 5 min to precipitate. The obtained samples were detected by western blot using antibodies (β-Trcp, 4394, Abcam, Cambridge, UK), as described in 3.4. Western blot Total protein was extracted from GT1-7 cells using RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China). The tissue lysates were centrifuged at 12,000 rpm for 5 min at 4° C, and the supernatants were collected for further analysis. The bicinchoninic acid (BCA) assay was applied to detect the concentration of protein samples. Then, 60 μg protein samples per lane were separated using 8% SDS-PAGE and then transferred onto PVDF membranes (0.45 μm, Millipore, Schwalbach, Germany). The membranes were blocked in Tris-buffered saline Tween containing 5% skim milk for 1h at room temperature and then incubated overnight at 4° C with primary antibodies: Anti-phos- JAK2 (Tyr1007/1008) antibody (3771), Anti-JAK2 antibody (3230), Anti-phos-STAT5 (Tyr694) antibody (9314), Anti-STAT5 antibody (94205), Anti-NA/K ATP antibody (3010) (CST, Boston, USA); Anti-PRLR antibody (ab170935), Anti-kisspeptin antibody (ab19028) (Abcam, Cambridge, UK); Anti-PRLR antibody (ABIN152720) (IP), Anti-GATA1 antibody (ABIN2704828) (antibodies-online, Beijing, China). Next day, these membranes were washed with PBS and incubated with secondary antibodies for 1h at room temperature. After rinsing, the proteins were detected by enhanced chemiluminescence. The protein levels were quantified by densitometry using Image-Pro Plus 6.0 and normalized to the corresponding GAPDH level. CCK8 assay The GT1-7 cell density was adjusted to 5000 cells/well, seeded into a 96-well plate, and cultured in the incubator at 37° C with 5% CO2 for 24 h. Afterwards, the original culture solution was discarded and the cells were washed twice with phosphate-buffered saline (PBS). According to Experiment 1 or 2, GT1-7 cells were treated differently. Each experiment group had 3 parallel wells. After 72h of incubation, each well was added with 10 μL CCK-8 (Beyotime Biotechnology, Shanghai, China) solution and incubated for 2 h in an incubator. The optical density (OD) value of the liquid in each well was measured by a microreader (Berthold LB941) at the wavelength of 450 nm. Table 1. Primers used in quantitative real-time PCR and shRNA-CNS5 sequence Primers Sequences (5’→3’) kisspeptin F- CTCTGTGTCGCCACCTATGG R- AGGCTTGCTCTCTGCATACC PRLR F- GACTCAAGGGGGCAAAGTCA R- CACCTCCACAGAGAAGCGTT CSN5 F- GCCTTGAGAGTCTATCACCACT R- TGATGATCATGGTCTCGCCG GATA1 F- TTGGGATCACCCTGAACTCG R- GGTTGAACCTGGGCTTGTTG GAPDH F- CATCACTGCCACCCAGAAGACTG R- ATGCCAGTGAGCTTCCCGTTCAG ShRNA-CSN5 CCGGAGCGCAGAGTATCGATGAAATCTCG AGATTTCATCGATACTCTGCGCTTTTTTG Table 1. Primers used in quantitative real-time PCR and shRNA-CNS5 sequence. Chromatin immunoprecipitation assay (ChIP) The cells were treated with 1% paraformaldehyde and incubated at 37° C for 10min. The cells were resuspended in SDS lysis buffer and broken by ultrasonic, and the supernatant was collected. CHIP diluent was added with protease inhibitor, and this solution was used to dilute the cell supernatant. Then 200 μL cell supernatant was added into 1800 μL ChIP diluent, making the final volume as 2 mL. The 10 μL cell supernatant dilution was regard as input control. Protein A Agarose/salmon sperm DNA was added to the supernatant, followed by antibody (GATA1) incubation overnight. Then 200μL eluent was added to the protein A Agarose/antibody complex, and the supernatant was collected. Next, 20 μL 5MNaCl was added to each 500 μL eluent and subjected to water bath at 65° C for 4h, followed by 10 μL 0.5M EDTA, 20 μL 1M Tris-HC1 and 2 μL proteinase K at a 45° C-water Construction of luciferase reporter gene plasmid bath for 1h. After filtered by DNA clean-up Column, the fluid was collected, and the qPCR was performed as described as 3.6. The full length of CSN5 (NM001277101) promoter was detected, combining with the target gene prediction software (Jaspar), to find the interaction site of CSN5 promoter and acting site of GATA1. Primer 5 was applied to design primers, making PCR product containing CSN5 promoter and GATA1 predicted binding site 1 (GGATGT). Construction of lentivirus Based on the construction of vector and identification of Colony PCR (Supplementary Figure 1), the bacterial solution with correct sequencing was seeded into 10 mL LB liquid medium containing Amp antibiotic and incubated, and then the plasmid was extracted. The GT1-7 cells were seeded into a 24-well plate and prepared for transfection. Then 0.8 μg plasmid was added to 50 μL Opti-MEM medium (GIBCO, Grand Island, USA) or 2.0 μL lipofectamine 2000 (Thermo Fisher Scientific, NY, USA) at room temperature for 5min, respectively. The two diluents were mixed for 5 min, and then 100 μL mixture was added into each well of the cell plate for 12 h-incubation at 37° C. After 72 h of transfection, the infective efficiency could be determined. According to the protocol of Experiment 8, each well of 96-well plate was added with 100 μL reporter gene cell lysate, followed by centrifugation at 15000rpm for 5 min to collect the supernatant. The sample was detected by a microplate reader with an interval of 2 s and a measurement time of 10 s. Then 100 μL luciferase detection reagent was added into 100 μL sample for RLU measurement. Statistical analysis All data were expressed as means ± SEM. Assessment of statistical significance was done using statistical analysis software SPSS (IBM SPSS Statistics 21; SPSS Inc., Chicago, IL, USA). For comparisons between experimental groups, one-way ANOVA and Tukey’s test were applied. All the P values were two-sided and the differences were considered statistically significant at P < 0.05. Immunofluorescence The cells were fixed with 10% paraformaldehyde for 20 min at room temperature. The fixed cells were blocked with 1% bovine serum albumin (BSA) for 30 min and incubated with the corresponding primary antibody (PRLR 1:20) at 4° C overnight, followed by washing with PBST for 10 min. Then the secondary antibody was added for 2h-incubation at room temperature, followed by the 4’, 6-diamidino-2-phenylindole (DAPI) staining for 15 min, and observation under a laser confocal microscope (400 ×). AGING 20428 www.aging-us.com Table 1. Primers used in quantitative real-time PCR and shRNA-CNS5 sequence. Primers Sequences (5’→3’) kisspeptin F- CTCTGTGTCGCCACCTATGG R- AGGCTTGCTCTCTGCATACC PRLR F- GACTCAAGGGGGCAAAGTCA R- CACCTCCACAGAGAAGCGTT CSN5 F- GCCTTGAGAGTCTATCACCACT R- TGATGATCATGGTCTCGCCG GATA1 F- TTGGGATCACCCTGAACTCG R- GGTTGAACCTGGGCTTGTTG GAPDH F- CATCACTGCCACCCAGAAGACTG R- ATGCCAGTGAGCTTCCCGTTCAG ShRNA-CSN5 CCGGAGCGCAGAGTATCGATGAAATCTCG AGATTTCATCGATACTCTGCGCTTTTTTG Ethical statement The experimental operations were approved by the Animal Ethics Committee of Nanjing Chinese Medicine University (approval No: SYXK (Su) 2014-0001). AGING www.aging-us.com 20429 20429 https://doi.org/10.1159/000493406 &This corresponding author has a verified history of publications using a personal email address for correspondence. 10. Sinha S, Sharma S, Vora J, Shah H, Srivastava A, Shrivastava N. Mucuna pruriens (L.) DC chemo sensitize human breast cancer cells via downregulation of prolactin-mediated JAK2/STAT5A signaling. J Ethnopharmacol. 2018; 217:23–35. https://doi.org/10.1016/j.jep.2018.02.006 PMID:29427634 https://doi.org/10.1530/JOE-18-0108 9. Trevisan CM, Montagna E, de Oliveira R, Christofolini DM, Barbosa CP, Crandall KA, Bianco B. Kisspeptin/GPR54 System: What Do We Know About Its Role in Human Reproduction? Cell Physiol Biochem. 2018; 49:1259–76. AUTHOR CONTRIBUTIONS 5. Li L, Jiang H, Wei X, Geng D, He M, Du H. Bu Shen Zhu Yun Decoction Improves Endometrial Receptivity via VEGFR-2-Mediated Angiogenesis. Evid Based Complement Alternat Med. 2019; 2019:3949824. https://doi.org/10.1155/2019/3949824 PMID:32082394 Huifang Zhou finished study design, Hua Feng, Jianxia Lu, Qing Zhang finished experimental studies, Hua Feng, Xingran Tang Yujie Shang finished data analysis, Hua Feng finished manuscript editing. All authors read and approved the final manuscript. 6. Bei L, Hui-fang ZH, Bo-ru ZH, Xiao-fei JI, Jian-ya XU, Tong XI, Jin-jun SH. Regulation of BuShen ZhuYun Decotion on gonadotropin in rat pituitary cells. Journal of Medical Postgraduates. 2018; 703–08. ACKNOWLEDGMENT We would like to acknowledge the reviewers for their helpful comments on this paper. 7. Jiang X, Zhou H, Shi M, Zhou B, Liu B, Yuan Y, Shan J, Xu J, Xie T. Bu-shen-zhu-yun decoction promotes synthesis and secretion of FSHβ and LHβ in anterior pituitary cells in vitro. Biomed Pharmacother. 2018; 102:494–501. CONFLICTS OF INTEREST The authors declare that they have no conflicts of interest. REFERENCES 1. Bernard V, Young J, Binart N. Prolactin - a pleiotropic factor in health and disease. Nat Rev Endocrinol. 2019; 15:356–65. 1. Bernard V, Young J, Binart N. Prolactin - a pleiotropic factor in health and disease. Nat Rev Endocrinol. 2019; 15:356–65. 8. Harter CJ, Kavanagh GS, Smith JT. The role of kisspeptin neurons in reproduction and metabolism. J Endocrinol. 2018; 238:R173–83. https://doi.org/10.1530/JOE-18-0108 PMID:30042117 8. Harter CJ, Kavanagh GS, Smith JT. The role of kisspeptin neurons in reproduction and metabolism. J Endocrinol. 2018; 238:R173–83. 8. Harter CJ, Kavanagh GS, Smith JT. The role of kisspeptin neurons in reproduction and metabolism. J Endocrinol. 2018; 238:R173–83. https://doi.org/10.1530/JOE-18-0108 PMID:30042117 Sponsored by Project of National Natural Science Foundation of China (No. 81473713 and 81774354); Phase III Project of Advantageous Discipline of Traditional Chinese Medicine (No. ZYX03KF005); Leading Talents of Traditional Chinese Medicine in Jiangsu Province (No. SLJ0202). Sponsored by Project of National Natural Science Foundation of China (No. 81473713 and 81774354); Phase III Project of Advantageous Discipline of Traditional Chinese Medicine (No. ZYX03KF005); Leading Talents of Traditional Chinese Medicine in Jiangsu Province (No. SLJ0202). https://doi.org/10.1038/s41574-019-0194-6 11. Ried K, Stuart K. Efficacy of Traditional Chinese Herbal Medicine in the management of female infertility: a systematic review. Complement Ther Med. 2011; 19:319–31. https://doi.org/10.1016/j.ctim.2011.09.003 PMID:22036524 11. Ried K, Stuart K. Efficacy of Traditional Chinese Herbal Medicine in the management of female infertility: a systematic review. Complement Ther Med. 2011; 19:319–31. 2. Arefi S, Fallah N. Comparison of pct after vaginal and oral bromocriptine use in women with hyperprolactinemia and infertility. J Reprod Infertil. 2002; 3:36–41. 3. Capozzi A, Scambia G, Pontecorvi A, Lello S. Hyperprolactinemia: pathophysiology and therapeutic approach. Gynecol Endocrinol. 2015; 31:506–10. https://doi.org/10.3109/09513590.2015.1017810 PMID:26291795 3. Capozzi A, Scambia G, Pontecorvi A, Lello S. Hyperprolactinemia: pathophysiology and therapeutic approach. Gynecol Endocrinol. 2015; 31:506–10. https://doi.org/10.3109/09513590.2015.1017810 PMID:26291795 12. Wen-Jie Y, Jing Y, Tai-Lang Y. [Effects of zhuyun recipe on the endometrial receptivity in mice with blastocyst implantation dysfunction and ovulation stimulation]. Zhongguo Zhong Xi Yi Jie He Za Zhi. 2012; 32:1554–57. PMID:23359985 4. Gasco V, Grottoli S. Physiopathology, Diagnosis, and Treatment of Hyperprolactinemia. In: Casanueva F, Ghigo E, editors. Hypothalamic-Pituitary Diseases. Endocrinology. Cham: Springer. 2018. https://doi.org/10.1007/978-3-319-44444-4_15 13. Yu N, Yang J, Yin T. Extracts from a traditional Chinese herbal remedy (Zhuyun recipe) improve endometrial receptivity in mice with embryonic implantation dysfunction and ovulation stimulation. J Ethnopharmacol. 2011; 137:389–95. AGING www.aging-us.com 20430 20430 https://doi.org/10.1016/j.jep.2011.05.037 18. Goffin V, Touraine P. The prolactin receptor as a therapeutic target in human diseases: browsing new potential indications. Expert Opin Ther Targets. 2015; 19:1229–44. https://doi.org/10.1517/14728222.2015.1053209 PMID:26063597 14. Zhou HF, Li AP, Tan Y. [Effects of bushen zhuyun recipe on protein expressions of estrogen receptor, progesterone receptor and integrin alpha5 and beta3 in endometrium of rats at the implantation stage]. Zhongguo Zhong Xi Yi Jie He Za Zhi. 2009; 29:628–31. PMID:19852297 19. Clarke H, Dhillo WS, Jayasena CN. Comprehensive Review on Kisspeptin and Its Role in Reproductive Disorders. Endocrinol Metab (Seoul). 2015; 30:124–41. https://doi.org/10.3803/EnM.2015.30.2.124 PMID:26194072 15. Borba VV, Zandman-Goddard G, Shoenfeld Y. Prolactin and Autoimmunity. Front Immunol. 2018; 9:73. https://doi.org/10.3389/fimmu.2018.00073 PMID:29483903 20. Hu KL, Zhao H, Yu Y, Li R. Kisspeptin as a potential biomarker throughout pregnancy. Eur J Obstet Gynecol Reprod Biol. 2019; 240:261–66. https://doi.org/10.1016/j.ejogrb.2019.07.016 PMID:31344665 16. Kataria J, Gill GK, Kaur M. Interrelationship of thyroid hormones, obesity, and prolactin in infertile women. Asian J Pharm Clin Res. 2018; 11:136. https://doi.org/10.22159/ajpcr.2017.v11i1.20861 17. Salvatori R. Dopamine agonist withdrawal in hyperprolactinemia: when and how. Endocrine. 2018; 59:4–6. https://doi.org/10.1007/s12020-017-1469-0 PMID:29124662 AGING 20431 www.aging-us.com SUPPLEMENTARY MATERIALS Supplementary Figures Supplementary Figure 1. The schematic diagram of lentiviral vector. Supplementary Figure 1. The schematic diagram of lentiviral vector. Supplementary Figure 2. Inhibitory effect of PRL or CSF in GT1-7 cells. (A) The change of cell vitality effected by different PRL concentration levels. (B) The change of cell vitality effected by different CSF concentration levels. Results were expressed as mean ± SD (n=3). p<0.01 vs Control group. Supplementary Figure 2. Inhibitory effect of PRL or CSF in GT1-7 cells. (A) The change of cell vitality effected by different PRL concentration levels. (B) The change of cell vitality effected by different CSF concentration levels. Results were expressed as mean ± SD (n=3). p<0.01 vs Control group. AGING 20432 www.aging-us.com Supplementary Figure 3. Effect of the mRNA expressions of PRLR and kisspeptin in GT1-7. The mRNA expressions of PRLR and kisspeptin were detected by RT-PCR. The mRNA expressions of PRLR (A) and kisspeptin (B) were normalized to control. The results were presented as mean ± SD (n = 3). p < 0.01, vs. Control group. Supplementary Figure 3. Effect of the mRNA expressions of PRLR and kisspeptin in GT1-7. The mRNA expressions of PRLR and kisspeptin were detected by RT-PCR. The mRNA expressions of PRLR (A) and kisspeptin (B) were normalized to control. https://doi.org/10.1016/j.jep.2011.05.037 The results were presented as mean ± SD (n = 3). p < 0.01, vs. Control group. Supplementary Figure 4. Effect of the mRNA expressions of PRLR and kisspeptin in GT1-7. The mRNA expressions of PRLR and kisspeptin were detected by RT-PCR. The mRNA expressions of PRLR (A) and kisspeptin (B) were normalized to control. The results were presented as mean ± SD (n = 3). p < 0.01 vs. Control group, ##p < 0.01 vs. HPRL group. Supplementary Figure 4. Effect of the mRNA expressions of PRLR and kisspeptin in GT1-7. The mRNA expressions of PRLR and kisspeptin were detected by RT-PCR. The mRNA expressions of PRLR (A) and kisspeptin (B) were normalized to control. The results were presented as mean ± SD (n = 3). p < 0.01 vs. Control group, ##p < 0.01 vs. HPRL group. AGING 20433 www.aging-us.com www.aging-us.com Supplementary Figure 5. The ubiquitin level of PRLR was measured with Co-IP. (n = 3). Supplementary Figure 5. The ubiquitin level of PRLR was measured with Co-IP. (n = 3). AGING 20434 www.aging-us.com pplementary Figure 6. The level of β-Trcp, PRLR, and CSN5 were measured with Co-IP (n = 3). (A) Representative blots of Co-IP ween PRLR andβ-Trcp. (B) Representative blots of Co-IP between PRLR and CSN5. (C) Quantitative relative binding level of RLR andβ-Trcp. Quantitative relative binding level of RLR and CSN5. Supplementary Figure 6. The level of β-Trcp, PRLR, and CSN5 were measured with Co-IP (n = 3). (A) Representative blots of Co-IP between PRLR andβ-Trcp. (B) Representative blots of Co-IP between PRLR and CSN5. (C) Quantitative relative binding level of RLR andβ-Trcp. (D) Quantitative relative binding level of RLR and CSN5. Supplementary Figure 7. Effect of the mRNA expressions of CSN5and GATA1 in GT1-7. The mRNA expressions of CSN5 and GATA1 were detected by RT-PCR. The mRNA expressions of CSN5 (A) and GATA1 (B) were normalized to control. The results were presented as mean ± SD (n = 3). p < 0.01 vs. Control group, ##p < 0.01 vs. HPRL group. Supplementary Figure 7. Effect of the mRNA expressions of CSN5and GATA1 in GT1-7. The mRNA expressions of CSN5 and GATA1 were detected by RT-PCR. The mRNA expressions of CSN5 (A) and GATA1 (B) were normalized to control. The results were presented as mean ± SD (n = 3). p < 0.01 vs. Control group, ##p < 0.01 vs. HPRL group. https://doi.org/10.1016/j.jep.2011.05.037 AGING 20435 www.aging-us.com Supplementary Figure 8. Chromatin immunoprecipitation (ChIP) assay on the promoter of GATA1. (A) Representative blot of ChIP assay on GATA1. (B) Quantitative relative binding of GATA1 to CSN5 promoter. The results were presented as mean ± SD (n = 3). p < 0.01 vs. Control group, ##p < 0.01 vs. HPRL group. Supplementary Figure 8. Chromatin immunoprecipitation (ChIP) assay on the promoter of GATA1. (A) Representative blot of ChIP assay on GATA1. (B) Quantitative relative binding of GATA1 to CSN5 promoter. The results were presented as mean ± SD (n = 3). p < 0.01 vs. Control group, ##p < 0.01 vs. HPRL group. Supplementary Figure 9. The level of CSN5 in GT1-7 was detected by western blot assay and representative bands were shown in (A). The level of CSN5 (B) was normalized to control. The results were presented as mean ± SD (n = 3). p < 0.01 vs. shRNA-NC group. Supplementary Figure 9. The level of CSN5 in GT1-7 was detected by western blot assay and representative bands were shown in (A). The level of CSN5 (B) was normalized to control. The results were presented as mean ± SD (n = 3). **p < 0.01 vs. shRNA-NC group. AGING www.aging-us.com Supplementary Figure 10. The ubiquitin level of PRLR was measured with Co-IP (n = 3). Supplementary Figure 10. The ubiquitin level of PRLR was measured with Co-IP (n = 3). AGING www.aging-us.com
https://openalex.org/W4295925565	https://aladabj.uobaghdad.edu.iq/index.php/aladabjournal/article/download/2711/3327	Arabic	null	التعلیم الالكتروني وأثره على جودة التعليم العالي في العراق	Al-ādāb	2,022	cc-by	26,058	University of Religions and denominations Faculty of Media and communications DOI: https://doi.org/10.31973/aj.v1i142.2711 / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) 2022 / م1444 هـ Abstract: Keywords: e-learning, quality of higher education, Iraqi universities, faculty b Keywords: e-learning, quality of higher education, Iraqi universities, faculty members 543 / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) الباحث علي أحمد امين الطباطبائي ماجستير في ادارة االعالم والعالقات جامعة بغداد– كلية اللغات )(ملخَص البحث هدفت هذه الدراسة الةا الرفةعل وةا الةع الرفوةكت انل رع رة امة رال ّمة نسةك ّ فة ّالجّمفّت الفعاقك مدى تأثيعه وا ج دال الرفوةكت الفةّل اكةّل لة مةة جرة راةع ة هيئ الرةدرس و فةمرت الةدا ت اسسّسة لوف وكة الرفوك كة ال ةّفاية وةا رفةّرررّ لةذا تةت ّ إ ةةةةداد اسةةةةر ره خّفةةةة ورةةةةت ةةةةت الةةةة ا لةةةة اف لوفي ةةةة ال رةةةةّرال ف ةةةة ةةةةة ت ةةةةجيا . ال حاّت ح ل ل الرمةةةع ال ةةةل ال ةةةّل وةةةا ي ةةة م رةةة مةةةة313 ) تدرس ةةة مةةةة سةةةّتذال الجّمفةةةّت . Abstract: This study aimed to identify the reality of e-learning in general, and especially in Iraqi universities, and its impact on the quality of higher education. It concentrates on the point of view of the faculty members as the main supporter of the educational process and those who maintain its sobriety. A questionnaire is prepared for them and distributed randomly to the selected sample. In addition to recording notes about it, the current research is limited to a sample of (313) professors of Iraqi universities in the province of Baghdad (University of Baghdad, and Al-Mustansiriya University). The researcher adopted the survey method, one of the types of descriptive analysis method as a design for the research, and the research community consisted of university professors within the borders of the geographical area of the province of Baghdad, and the universities of Baghdad and Al-Mustansiriya were chosen to represent the sample of the current study. Teaching, and general evidence for teachers about e- learning, the pros and cons of e-learning, in addition to the obstacles of e-learning. The questionnaire’s paragraphs were statistically analyzed, and the questionnaire procedures took nearly two months, and the researcher analyzed the results he reached using the t-test, to verify the hypothesis Research and to calculate the significance of the differences between the averages to identify the effectiveness of e-learning and its impact on the quality of higher education in Iraqi universities, and found a statistically significant difference at the level of significance (0.05), it appeared that the role of e-learning is one of the reasons for the low quality of education in Iraqi universities, by comparing Between the arithmetic average and the hypothetical average, where the role of e-learning is negatively reflected on the reality of the quality of higher education because it does not require that attention And thinking may sometimes be a unilateral connection, in light of the results of the research, the researcher concluded that e-learning in Iraq is in dire need of elements and effort in order to contribute to improving the reality of higher education in Iraqi universities, due to the weak infrastructure that prevents leaving the old ways of education. Abstract: الريئ الردرس ك المقدمة نفةةد انتمةةّل هةةت سةة ن رةةّ ورةةّ ا ر ةةّل لةةدم جةةه ال لةةّ ا ر ةةّر ل تمةةةّل اسةةةر دامّت فةةة م جةةةّنت م رومةةة مثةةةا ت ةةةّدل اسف ةةةّر العسةةةّفا ال فو مةةةّت تفةةةد الف وكةةة الرفوك كة وكةة اتمةةّلك ت ةع ل مرةة م انتمةةّل ل ةّ ت رّجةةه مةةة تمّ ةا و اسةةل الف مةةّت العمةة الرةةة تلةةة ا حةةةد رةة اع انتمةةةّل مةةةة حيةةةل الوغةة اّ ر ةةةّر الةةةدر المةةةم ا ثّاةةة وكةةة اتمةةةّل لماةةة ن ةةةر دم ةةة مةةةة ا لردرس ةةة اللّلةةةب الومةةةل سةةةيو ل قةةةا العسةةةّل مةةةة ال مةةدر إلةةا ال روقةة اةةّخر ل اند ار هةة اتمةةّل خيةةع لماةة مةةة خةة ل لغةة ا شةةّرال ال ع ةة اسففةةّل سمةة انتمةةّل لوف وكةة الرفوك كةة اأرةةه اتمةةّل ج فةة الةةذل ن ةة ل وةةية مج ة مةة اسفةعاد ةد العجةة ع إلةا ال مةّدر ال رمة فةة وة م انتمةّل امةم ّمةة عربية ل وك انتمّل تر ل مة ّفع ه ال مدر العسّل ال سيو ال رقبا رجع المدى) د ملّاق تو الف ّفع مع الف وك الرفوك ك نقّوا ال مدر الردرس العسّل ال رج الرفوك ال سيو هة رعسقة الرةدرس ال ةرقبا هة اللّلةب ا خيةعا رجةع المةدى فةة قكةةّ مةةدى فرةةت اللّلةةب لو ةةّدال الفو كةة الرةة درسةةرّ مةةة خةة ل لةة عربةةية ل انتمةةّل عةةؤدل د را مر ةةّ فةة الف وكةة الرفوك كةة فةةم لةة ت ّدلكةة فّنتمةةّل نفربةةع ال سةةكط ال ةةا . Abstract: ) الفعاقك ف م ّفا اغداد جّمف اغداد) و فمرّ الجّمف اسم الجّمف ال ر معس ات ذ ال ّحل ال رج ال حد ر اع م رج الر ويةا ال فةم تمة ك ّ لو ةل تةأل مجر ةةع ال ةةل مةةة سةةّتذال الجّمفةةّت لةة ة حةةد د العلفةة الجغعادكةة ل ّفاةة اغةةداد جةةعى اخركّر جّمفر اغداد ال ر معس لر ثا ي الدراس ال ّلكة ةد ال ّحةل اسةر ّر م رة مة44) فقعال و ا م ّ ر ش وت البكّرّت اس لك ن ّ الريئة الردرس ةك اكّرةّت ّمة لوردرس يية ح ل الرفوكت انل رع ر م ر ال رّت الر تؤثع وا جة دال خةدمّت الرفوةكت الفةةّل م ةة ر سةةوبكّت الرفوةةكت انل رع رةة ف ةة ةةة م ةة ر مف لةةّت الرفوةةكت انل رع رةة حُووةةت فقةةةعات انسةةر ّر إحمةةةّفكّ اسةةةرغعلت اجةةعا ات انسةةةر ّر مّنقةةّر اللةةةرعسة حوةةةا ال ّحةةل ال رةةّفج الرةة ت فةةا إليرةةّ اّسةةرف ّل انخر ةةّر الرةةّفt-test ) لور قةةم مةةة فعلةةك ال ل ل ّ دنلة المةع ب وةية ال ر سةلّت لورفةعل وةا فّ وكة الرفوةكت انل رع رة ثةعه وا جة دال الرفوةكت الفةّل فة الجّم فةّت الفعاقكة ف جةد فةعب ل دنلة إحمةّفك ةد م ةر ى دنل0,05 ) ظرع إل د ر الرفوكت انل رع ر حد س ّ تدر ج دال الرفوكت ف الجّمفّت الفعاقك مة خ ل مقّرر وية ال ر سط ال ّو ال ر سةط المعلة إ عة ف د ر الرفوةكت انل رع ر سو ّ وا الع ج دال الرفوكت ا لفّل ل ره نعرلوب ل الرمّ ا الرم يع لد ن ة ل ف افض انحكّل اتمّل احّدل الجّرب ف ل ررّفج ال ةل سةر رج ال ّحةل إل الرفوةكت انلکرع ری فةی الفةعاب ا ّجة مّسة الةا مق مةّت جرةد حرةا ن ةرت فة ت ةية الةع الرفوةكت 544 / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) 2022 / م1444 هـ الفةةّلی فةةی الجّمفةةّت الفعاقكةة لةة ل ةةف الب كةة الر ركةة الرةة ت ةة ل د ل تةةع اللةةعب .القدن اّلرفوكت الکلمااات الماتاحيااة ّ: الرفوةةكت انل رع رةة جةة دال الرفوةةكت الفةةّل الجّمفةةّت الفعاقكةة ةة . Abstract: ف وك الرفوكت فةة ت ال ّجةة مو ةة الةةا م او ةة الرلةة ر ةةّل تةةأثيع الرلةة ر ال ر لةة ج وةةا الةةع الرفوةكت فةة رقةا هةةذا القلةةّع مةة اللعافةةم ال سةةك ك الةا وةةدافا رعافةةم حدعثة اةةّلرفوكت حرةةا خةةعل ل ةةّ ممرةة م الرفوةةكت ا ل رع رةة رخةةت ل هةةذا ال ةة ع اسةةر دم فةة م رمةة القةةعل الرّسةةع لةةع إن شةةّع اسةةر دامه فةة المرةةعال الرةة رافقةةت جّف ةة ر رةةّ حيةةل ا ربةةع البةةدعا لورفوةةكت .الرقويدل لةةذا تةةدفف ّ ال ّجةة د مةةّ نجةةّد وةةدافا حوةة ل فةة الق ةةّنّ ات انهر ةةّم ال ّشةةع ا كةةّال ا ر ّل رخت ا الرردعدات الر نفّر م رّ ال جر ع إن ل م أل الرفوكت تفد ا ثّا خةط اح ةةع ن ن ةةة الررةةّ ل ورةةّ حيةةل نفةةد الرقمةةيع فةة توقةة الفوةةت لةةه مةةعد د سةةوب وةةا حكةةّال المةةعد ال جر ةةع ل ةة ل الرفوةةةكت مةةة اسمةة ر ال ّلغةة انه كةةة فةة حكةةّال اللةةف سفربةةةع انداال ال ر ف م أل م اجر الرك مة لبا الد ل الر ت كلع وا انلرمّد الر ت رو لة ال ال ح ةا توة ال ف لةّت الرة ت ة ل د ل انسةر عار اةّلرفوكت افةد تفلةا سةبا ال كةّال جة عا 545 / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) ا جعا ات ال ر ف لو د مة اررلّر ال اّ الفةّل ةّل ال ةا فة الر جةه ر ة انسةرمّدال مةة اللة الفّل كةة تق كةةّت الر افةا فةة الةةدخ ل لوفةةّلت انفرعالة اسةةر عار الف وكةة الرفوك كةة . Abstract: د ل ارقلّع اسر ّل ال يعال الدراسك بع الرفوكت انل رع ر تک ة ه ك هذا ال ل ف ان ّح مّ ه البدعا الذل اتجرت إلكه ال ؤس ّت الرفوك ك ف ا اقّ وا الدراس د ل الرةأثع اةّ جعا ات ال لّفكة لةد فةّعع ر رةّ ةّل البةدعا ال ةةا هةة الرفوةةكت ا ل رع رةة اكةةّل مةةدى تةةأثيعه وةةا جةة دال الرفوةةكت الفةةّل اّ ر ةةّره اللةةغا اللةةّخا لو رمةةية فةةة ان رةة ان خيةةعال حيةةل شةةّع ممرةة م الرفوةةكت ا ل رع رةة اةةّلر امة مةةةع اررلةةّر فةةّعع ر رةةّ الةةذل ت ةةبب ورفليةةا ّفةة ممّفةةةا ال كةةّال حرةةا نن ةةكع مجرةة د اللو فة اسةر ّل دراسةررت ةّل نوةد مةة الر جةه ر ة ات ةّع اللةعب انل رع ركة فة إنمةّل ال ةةةّدال الدراسةةةك لولو ةةة ةةةا مةةةع مةةةّ عرفوةةةم اّلف وكةةة الرفوك كةةة مةةةة إجةةةعا انخر ةةةّرات لّمةةة ّال ةةةةؤت عات ال ةةةةد ات الفو كةةةة الةةةةد رات الردرسبكةةةة م ةةةةرغوية وةةةةذل مةةةةّ تقدمةةةةه لرةةةةت ت ل جكةةةة انتمّل ش انررعرةت مةة خةدمّت اةعامج مرلة رال ت ةّ د وةا الر افةا مةّ وةية اللّلةب الردرس لغعض ل ن ل الرفوكت انل رع ر البدعا ة الرفو.كت الرقويدل سفد للةّع الرفوةكت فة م لقة اللةعب اس سةط مةة الج ارةب الرة لةت نجةعل ويرةّ وكة الر سةةا الجةةذرل مةةة ال مةةّيكت القدن ةة إلةةا اللعافةةم ال دعثةة ال ر فةة فةة د ل الفةةّلت ال رلةة رال مة حيل اسر دام الرق كّت ال دعث ف ّف ممّفا ال كّال ففوا سةبيا ال ثةّل اةّت ا ة م الي م ن ا اّ م العل مفر دا وةا تقّرةّت انتمةّل ال دعثة الرة فةعت وةا ال روقة ال ؤس ا مك ال ثيع مةة الجرةد ال ةّل ةّ ّرةت وكةه ال سةّفا الرقويدنة ةذل هة الرفوكت الرقويدل د مقّررره ا ّ ع فعه الرفوكت انل رع ر رجد ال ع اس ل ن رةّل الةا الجرةد الف ةةّ الرقيةةد افّمةةا ال مةةّل ال ةةّل ف ةة ةةة ال مةةّرسا الرةة تر وةةا سةة ات الدراسةة وةةا توةة المع لةةّت رجةةدهّ فةة الرفوةةكت ا ل رع رةة وثةةع مع رةة لةةا تفقيةةدا لةةد ت ةة ل مفد مةة .ف افض ممّفا الف وك الرفوك ك ف وك الذهّ اننّ الا الجّمفّت مة جا مفعف ل الف وك الرفوك ك ت يع اّلل ا الذل ن ة لرّ ت قيم هدافرّ وا فم الرفوكت انل رع ر ّل لرذه العسّل جّرب مرت ف قكةّ تةأثيع الرفوةكت انل رع رة وةا جةة دال الرفوةةكت الفةةّل اخركةةّر مرغيةةع الجّمفةةّت الفعاقكةة في ةة لو ةةل مةةة خةة ل وكةةّل ر ل ّ الريئ الردرس ك ف الرفوكت انل رع ر م ةّ تلوةب ةا اسةربكّل مر ة ل مةة م ةّ ر ةةدال لغةةعض دراسةة ال رةةّت الرةة تةةؤثع وةةا جةة دال خةةدمّت الرفوةةكت الفةةّل ال ةةوبكّت اهةةت ال ف لةةّت الرةة ت اجةةه الرفوةةكت انل رع رةة حيةةل تلوةةب هةةذا اسمةةع جرةةد مةةة لبةةا ال ّحةةل فةة الذهّ الا الجّمفّت الفعاقك مة اجا ت. Abstract: سع ر خ انسر ّر وا الفي ال رردف الردرس لغعض ل ن ل الرفوكت انل رع ر البدعا ة الرفو.كت الرقويدل سفد للةّع الرفوةكت فة م لقة اللةعب اس سةط مةة الج ارةب الرة لةت نجةعل ويرةّ وكة الر سةةا الجةةذرل مةةة ال مةةّيكت القدن ةة إلةةا اللعافةةم ال دعثةة ال ر فةة فةة د ل الفةةّلت ال رلةة رال مة حيل اسر دام الرق كّت ال دعث ف ّف ممّفا ال كّال ففوا سةبيا ال ثةّل اةّت ا ة م الي م ن ا اّ م العل مفر دا وةا تقّرةّت انتمةّل ال دعثة الرة فةعت وةا ال روقة ال ؤس ا مك ال ثيع مةة الجرةد ال ةّل ةّ ّرةت وكةه ال سةّفا الرقويدنة ةذل هة الرفوكت الرقويدل د مقّررره ا ّ ع فعه الرفوكت انل رع ر رجد ال ع اس ل ن رةّل الةا الجرةد الف ةةّ الرقيةةد افّمةةا ال مةةّل ال ةةّل ف ةة ةةة ال مةةّرسا الرةة تر وةةا سةة ات الدراسةة وةةا توةة المع لةةّت رجةةدهّ فةة الرفوةةكت ا ل رع رةة وثةةع مع رةة لةةا تفقيةةدا لةةد ت ةة ل مفد مةة .ف افض ممّفا الف وك الرفوك ك ف وك الذهّ اننّ الا الجّمفّت مة جا مفعف ل الف وك الرفوك ك ت يع اّلل ا الذل ن ة لرّ ت قيم هدافرّ وا فم الرفوكت انل رع ر ّل لرذه العسّل جّرب مرت ف قكةّ تةأثيع الرفوةكت انل رع رة وةا جةة دال الرفوةةكت الفةةّل اخركةةّر مرغيةةع الجّمفةةّت الفعاقكةة في ةة لو ةةل مةةة خةة ل وكةةّل ر ل ّ الريئ الردرس ك ف الرفوكت انل رع ر م ةّ تلوةب ةا اسةربكّل مر ة ل مةة م ةّ ر ةةدال لغةةعض دراسةة ال رةةّت الرةة تةةؤثع وةةا جةة دال خةةدمّت الرفوةةكت الفةةّل ال ةةوبكّت اهةةت ال ف لةةّت الرةة ت اجةةه الرفوةةكت انل رع رةة حيةةل تلوةةب هةةذا اسمةةع جرةةد مةةة لبةةا ال ّحةةل فةة الذهّ الا الجّمفّت الفعاقك مة اجا ت. Abstract: سع ر خ انسر ّر وا الفي ال رردف 546 / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) سف د الر جه ر ال مةّيكت ال دعثة فة ل للةّع مةة للّ ةّت الف ةا لة ل ةّ نلةرده الفّلت ف الفمع ال ّل مة لم ال وةا ال ةر ى الر لة ج ثةعت الة ا الةج وةا ج كةع ّج ارةةةب ال كةةةّال م رةةةّ الفو كةةة انلرمةةةّدن وةةةا تفةةةدت لك ةةة ل تأثيعهةةة وةةةا الج ارةةةب الثقّدكةةة انجر ّعكةة هةة مةةّ ت ةة ا اّلف ل ةة لك رةةا مجةةّل الرعاكةة الرفوةةكت الجّرةةب اسهةةت فةة مةةدى .تأثيع الر ل جكّ وا هذا الجّرب ال رت ف ال كّال لةةةدمت تق كةةةّت ال ّسةةة سةةةّفا انتمةةةّل ال دعثةةة خدمةةة فةةة مجةةةّل تلةةة سع الرفوةةةكت ّانل رع ر ف ّل ان ر ّد سّاق وا سّفا ان م ال عفة ال ة ع فة الةرفوت مةة خة ل ول الدر البعامج الردرسبك الر ّرت مقرمعال ف حي رّ وا الردرس ّت الف ة عس افةد ل لقةةت ال داثةة اا لرةةّ وةةا للةةّع الرفوةةكت جفوةةت مةةة ال ةةرا ل نجوةة المةةعد فةة م لةةه سرفوت بع ش ا ررعرت اسلعاص الرفوك. ك لورفوكت ا ل رع ر ممّيكت دعدال ت ل ح ب ال ع اللعسق الر تقدم مة خ لرّ ال ةّدال الفو كةة فةةّلرفوكت ةةة افةةد حةةد توةة ال مةةّيكت الةةذل ن ةة ل و ةة ية مةةّ م ّشةةع ةةة رعسةةم اسرف ّل ال مّت الرفوك ك خيع م ّشع مة خ ل ملّهدال انلعاص الرفوك ك مقّرع الميةةةدع لو ّلةةةعات ال ةةةجو م ةةة قّ ل ةةةا رةةة ع م انةةةّ سةةةوبكّت تؤشةةةع وةةةا فةةةم ربكفةةة ال رميد سلوم وكه ان ةّ الرفوةكت ال ر قةا الةذل ن ة ل مةة خة ل اسةرف ّل انجرة ال الو حكة ال اسةةيب ال لةة الر اتةة الذ كةة ن ةةّل الةةا لةة الرفوةةكت ال ةةدمج الةةذل نج ةةع مةةّ وةةية اللعافم القدن اّلرفو كت المم اللعافم ال دعث بع ش انررعرت ه مّتم وه القكّدات الجّمعكةةة فةةة الفةةةعاب مةةةة اجةةةا ال مةةةّم وةةةا رفةةةّر الرفوةةةكت مةةةة خةةة ل خةةةذ افةةةض ال ةةة اد . Abstract: اّللعافم الرقويدن انخعى بع ال مّت انل رع رك ن ّ د الرفوكت انل رع رة فئةّت دعةدال مةة ال جر ةع فة ال مة ل وةا فعفة اسةر ّل دراسةةةةررت افةةةةد ل تةةةةع خوةةةةبرت مقّ ةةةةد الدراسةةةة سسةةةة ّ ترفوةةةةم اّل لةةةةع انلرمةةةةّدل ةةةةدم انسرقعار اسم حيل ت ة ال فض مة الذعة جبعترت الاع ل ال عكلةك وةا ةدم إو ةّل م ةةيعترت الفو كةة اّلدراسةة الف ةةا فةة لةةت احةةد ل ةةّ لةةه مةةة م يةة ات فةة تةة فيع ال عسةة فةة ال مّل ال.ّل دم الرقيد ت فيع الرفوت ال ا م ر ع مدى ال كّال ع قةةةةةةا ان ر ةةةةةةّد انررقةةةةةةّل الةةةةةةا الرفوةةةةةةكت انل رع رةةةةةة معهةةةةةة ل اةةةةةةّلاع ل انلرمةةةةةةّدن انجر ّعك لوبودال خّف تو الر لت تور م وع ب الرل ر الر ت رّل الا و كة ت ركة م ةةّهج مرلةة رال جرةة ال حدعثةة شةة اررعرةةت فّفقةة ال ةةع مةةة جةةا الر ةة ل مةةة الرفوةةكت . Abstract: الرقويدل الا الرفوكت انل رع ر اّلل ا ال لو راةةعا ل ةةّ ن ثوةةه الرفوةةكت الفةةّل مةةة اه كةة اّلغةة فةة تلةة سع الةةع ال جر ةةع ل ةة ل هةةذا ن ّ د الرفوكت انل رع رة فئةّت دعةدال مةة ال جر ةع فة ال مة ل وةا فعفة اسةر ّل دراسةةةةررت افةةةةد ل تةةةةع خوةةةةبرت مقّ ةةةةد الدراسةةةة سسةةةة ّ ترفوةةةةم اّل لةةةةع انلرمةةةةّدل ةةةةدم انسرقعار اسم حيل ت ة ال فض مة الذعة جبعترت الاع ل ال عكلةك وةا ةدم إو ةّل م ةةيعترت الفو كةة اّلدراسةة الف ةةا فةة لةةت احةةد ل ةةّ لةةه مةةة م يةة ات فةة تةة فيع ال عسةة فةة ال مّل ال.ّل دم الرقيد ت فيع الرفوت ال ا م ر ع مدى ال كّال 547 / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) 2022 / م1444 هـ اّل ةةةوب وةةةا حكةةةّال المةةة عد ف ةةةّل نوةةةد مةةةة انهر ةةةّم اةةةّلج دال الرةةة تفةةةد مةةةة هةةةت اسد ات فةةة ت ية ر عك الرفوكت حيل ت ثا الج دال الي م ف معرّ ال ّل ا ثّا ال قكةّ الةذل عبةية مدى الر ام ال ؤس ّت الرفوك ك ور قيم هدافرّ ال فّعيع ال اجب ت ميذهّ ل ة وعامج ّل ك مفةةدال لةةذل حيةةل ترةةرت الجةة دال ا ةةدخ ت ال ؤس ةة الرفوك كةة اّلم ةةّ ات ال ر ثوةة اّسو كةة القّ ةةةّت الدراسةةةك ال ربةةةعات ف ةةة ةةةة ال ةةةّهج الدراسةةةك الردرس ةةةيية اللو ةةة ر عكةةة ال ةة ا الفو كةة و ةةةّ اد انهر ةةّم وروةة الج ارةةب ثةةع الةة ا إنجةةةّو وةةا م عجةةّت توةة ال ؤس ّت الر ود رهّ تد ت للّ ّت ا. لد ل ال روم :التعليم االلكتروني ه ر ع مة الرفوكت ن وم فعص جدعةدال لورفوةكت مةدى ال كةّال فة ل لةت ف ل م ّل هذا ن ثا مع ر ف الف وك الرفوك ك ا ّ نجفورّ وثةع ففّلكة سةع لور كةا مع احركّجّت مرلو ّت الفمع. المعسجّت2014 ص167 ). Abstract: المر ل2019 ص148 .) مشكلة الدراسة عر ةةّ ل م لةة ع هةةذا ال ةةل انهر ةةّم اةةّلرفوكت انل رع رةة خّفةة افةةد اس مةة الفّل كةة ّالرةة اجرّحةةت الفةةّلت جّف ةة ر رةةّ) الةةذل جفةةا مةةة الرفوةةكت انل رع رةة شةة ه اّلاةةّهعال ل ةة حققةةه مةةة د ر انجةةّو مر يةة فةة ال ةةّ دال وةةا اسةةر ّل ال ةةيعال الرفوك كةة حيةةل وةةدت ال ّجةةة ال و ةةة نسةةةر ف ّل هةةةذه الرقّرةةة الرةةة هةةة لك ةةةت ليةةةدال اليةةة م ل ةةةة سةةةع ّل مةةةة ال ت ببت اس لّع ال عافق نررلّر فّعع ر رةّ فة ل عرمةم الج كةع وةا اسةر دام الرفوةكت انل رع رةة اّ ر ةةّره ال ةةا اسمثةةا فةة بةة ر هةةذه ال عحوةة حرةةا اةةّت اللةةغا اللةةّخا لج كةةع ال ّحثية ال رمية ف مجّل الرفوةك ت وةا حرةا تفةدى لة الةا اهر ةّم اللةع ّت الفّموة فة ال جّل العل ل ل تل سع اسرف ّل هذه الرقّرّت سفد اسرف ّل ال سّفا الر ل جك ه . ال ال ّر ال ف قكّ تل ر ال اّم الرفوك مة حيل الر ي اسر دام مفّعيع الج دال نفر ةد الرفوةكت ا ل رع رةة ن خيةعا وةا ا ل ةة رد ال لةعل الةذل وةةد ره ن رةّل الةا تلةة سع م ر ع حيل شرد الفعاب مؤخعا دخ ل الرقّرّت ال سّفا العل كة ا ة ل الةد ل ال جةّ رال لةه . اسمع الذل جفا مة المف ا لدى ال فض الر كا مع هذا اسمع إل الرفوكت ا ل رع ر له د ر ف ت ع ال سّفا اللعافم الرفوك ك فة لةت قكّسة الةا جرد فّفدال بيعال لألسرّ اللّلب لةذا مةة ال ةع رل جةدا ا ر ةّد هةذه الرقّرة لةفرّ وةا فةةم العةسةة ال ةةرقبوك لوقةةّف ية وةةا مةة ر الرفوةةكت س ةةة ال ت ةة ل الةة ط ال ةةّفد فةة الرفوةةكت م رميدعة وذل مةة ثةعال ت ة ع الرجةّر الرة اسةرف وت خة ل هةذه ال عحوة فة م روة د ل الفةةّلت قكةةّ الةةع الرفوةةكت ج دتةةه مةةة جرةة راةةع اسسةةرّ الةةذل ن ةة ل حةةعص فةة ممةةو . Abstract: التعليم االلكتروني: ه اسر دام الر ل جكّ ال دعث الر تفر د سّسٌّ وا ال رّرات ال م لورفّمةةةا مةةةع شةةة ال فو مةةةّت الد لكةةة افةةةض ال سةةةّفط الر ل جكةةة مثةةةا الميةةةدع ةةة رمعار ٌّ الروكم سةة ل الرفوك ةة اسل ةةّر المةة ّعك مةةة جةةا الرمّ ةةا وةةية اللةة اسسةةّتذال إل رع ركةة د ل الرقيد ا د د ال مّل ال ّل. ّمع2014 ص26 ). التعلااايم االلكتروناااي: هةةة رعسقةةة اور ّرسةةة نمةةةّل ويئةةةّت الةةةرفوت ال فّفةةةعال الرةةة ترمةةة اّلرمةة كت الجيةةد الرمّ وكةة الر ع ةة حةة ل ال ةةرفوت سل فةةعد فةة ل م ةةّل مةةّل ةةة رعسةةم انررمةةةّع مةةةة ال مةةةّفر ال مةةةّدر ال رةةة فعال فةةة الفد عةةةد مةةةة الرق كةةةّت العل كةةة ف ةةة ةةةة اسر ةةةةةةةةةّ اسخةةةةةةةةةعى مةةةةةةةةةة ال ةةةةةةةةة اد الرفوك كةةةةةةةةة ال ّسةةةةةةةةة لبيئةةةةةةةةةّت الةةةةةةةةةرفوت ال مرةةةةةةةةة ح ال ةةةةةةةةةعل ال ب. الف يعل2011 24 .) ا لجودة الشاملة كما عرفتها "منظمة الجودة البريطانية:" ه المو م ا دارس لو ؤس الر ّتةةةةدر مةةةةة خ لرةةةةّ ت قيةةةةم ةةةةا مةةةةة احرك جةةةةّت ال ةةةةررو ةةةةذل ت قيةةةةم هةةةةدال ال لةةةةع ع مفّ . رل ال2012 ص23 .) التعليم العالي: ه ال بدع لو فعف ال رج لوقّدال انل ل لرمّد الةد ر ال ّسةت فة رفةّه ال جر ع سفد ال يع الم عل لو جر ع. وي ار2010 ص41 .) الجامعاااة كماااا يقااادمها "ساااتانلي كاااا:" هةةة الرةةة تقةةة دم ال ةةةّفع الفّمةةة هةةة المّ ةةةا لو يةةةع انجر ةةةّ مر ررةةّ خوةةةم لةة ال ةةةا مةةةّهعال مثقمةة تلةةجكع ال لةةةّر ال دركةة ةةد اللو ةةة ت ةة ل ةةّ ال ع كةة انجر ّعكةة تؤسةة لةةع ااط مةةّ وةةية الرفوةةكت انورةةداف الثّر ل. فعارةة ري مّل2010 ص32 .) 548 / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) 2022 / م1444 هـ عضو هيئة التدريس: هة ةا مةة نقة م اّلرةدر س فة الجّمفة مةة ح وة شةرّدال الةد ر راه ال ّج ريع س ّهت ف ت قيم هدال الجّمف . Abstract: الف وك الرفوك ك ه مّ وي ّ مفعفره مة جر راع اسسرّ مة خ ل ت دعد ال ف لّت اننجّوكّت مة ّخ ل تأثيع ل وا ج دال الرفوكت مّ اسم ر الر رافقت هذه الرجعا الر مة خ لر ها ن ةةر ع تلبيةةم الرفوةةكت انل رع رةة فةة الجّمفةةّت الفعاقكةة كاكةة تلةة سع هةةذا ال ممةةا ال رةةت اةةّلعخت مةةة ةةدم جةة د الب كةة الر ركةة الرةة تفربةةع ال ةةّج الةةعفك فةة ال رةة ض و الةةع الرفوةةكت خّفةة فةة مجةةّل الرفوةةكت انل رع رةة الةةّف الةةا ردافةة شةة انررعرةةت امةة رال ّمةة الرةة ثعت سوب كّ وا ج دال انتمّل الر افا ف او ّل الغّنة الرة جةدت مةة جوةه نوةد مةة انسةر عار وةد ت ال ؤس ةّت الرفوك كة ال ة ارد ال لةعس الفّموة فة دن مة الرفوةكت انل رع رة ال ر ثوةة اّسسةةّتذال الم يةةية ف ةة ةةة جةة د م رةة ى و ةة خيةةع الةةذل ا رةةّد وكةةه اللّلةةب 549 / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) عر ّشةةةا مةةةع الثةةة ر ال الفو كةةة الرّفوةةة فةةة تلبكقةةةّت اسجرةةة ال ا ل رع ركةةة ّل اسةةةيب الر اتةةة . ال ل مع معا ّال تلبيم مفّعيع الج دال اسوّدن ك ال ؤال العفك اسسئو المععك ال ؤال العفك مةّ ثةع الرفوةكت انل رع رة وةا جة دال الرفوةكت الفةّل فة الجّمفةّت الفعاقكة مةة جرة راةةع ّ هيئ الردرس ؟ اسسئو المععك 1 - مّ خمّفر الرفوكت انلکرع ر ؟ 2 - مّ ال رّت الر تؤثع وا ج دال خدمّت الرفوكت الفّل ؟ 3 - مّ الف ل وية الرفوت انلکرع ر الرفوكت الفّل ؟ 4 - مّ ت دنّت الرفوكت انل رع ر ؟ س اسسئو المععك 1 - مّ خمّفر الرفوكت انلکرع ر ؟ 2 - مّ ال رّت الر تؤثع وا ج دال خدمّت الرفوكت الفّل ؟ 3 - مّ الف ل وية الرفوت انلکرع ر الرفوكت الفّل ؟ 4 - مّ ت دنّت الرفوكت انل رع ر ؟ اسسئو المععك 1 - مّ خمّفر الرفوكت انلکرع ر ؟ 2 - مّ ال رّت الر تؤثع وا ج دال خدمّت الرفوكت الفّل ؟ 3 - مّ الف ل وية الرفوت انلکرع ر الرفوكت الفّل ؟ 4 - مّ ت دنّت الرفوكت انل رع ر ؟ أهداف الدراسة 1 - ال ل ة ثع ارف ّسّت افّد اسر دام ال جّمفّت الفعاقك لورفوكت انل رع ر ثةع لة . Abstract: لد لمت ال رّفج ل ال ر سط ال وة لرقةدعع ّ هيئ الردرس وا مقكّ اسر ّر انتجّهّت ر اسةر دام د ات الةرفوت ا ل رع رة ةةةد فةةةعاد الفي ةةة لةةةد ووةةة لةةةدرال3.64 ) إ تبةةةية ل اتجّهةةةّت ةةةّ هيئةةة الرةةةدرس ر ةةة مرلو ةةّت اسةةر دام د ات الةةةرفوت ا ل رع رةةة إنجّوكةة ودرجةةة مر سةةل . ظرةةةعت ال رةةةّفج ةةة ةةةدم جةةة د فةةةع ب ات دنلةةة إحمةةةّفك وةةةية مر سةةةط تقةةةدععات ةةةّ هيئةةة الرةةةدرس ر ةةة مرلو ةةةّت اسةةةر دام د ات الةةةرفوت ا ل رع رةةة تفةةة ى ل رغيةةةعات: الجةةة الجّمفةةة سةةة ات ال بةةةعال وي ةةةّ ظرةةةعت فةةةع ب ات دنلةةة إحمةةةّفك نسةةةرجّاّت ةةةّ هيئةةة الرةةةدرس تفةةة ى ل رغيع الدرج الفو ك لمةّلج ةّ هيئة الرةدرس ودرجة سةرّ . لةدمت الدراسة ةدد مةة الر فةةةكّت ال قرعحةةةّت ه رةةةّ: الف ةةةا وةةةا رفةةةع مرةةةّرات ةةةّ هيئةةة الرةةةدرس فةةة مجةةةّل اسةةةةر دام د ات الةةةةرفوت ا ل رع رةةةة تلةةةةجكفرت وةةةةا اسةةةةر دامرّ تةةةة فيع ال رلو ةةةةّت ال ّدنةةةة . ال ّلك ال ّس نسر دام د ات الرفوت ا ل رع ر 2 - و ةةّ ترج ةةد ه كةة هةةذا ال ةةل فةة ت ةةوكط ال ةة ل لةة ع جةة دال الرفوةةكت و فةةمه الغّنةة اسسّسةةك مةةة الف وكةة الرعا سةة س ثةةا هةةذا اسمةةع قكةةّ ل ةةدى رفةةّر الرفوةةكت الفةةّل فةة الفةةةعاب لرةةةه افةةةد لةةة وةةةا جةةة دال ال ةةةعسج البيئةةة ال كلةةة اةةةه مةةةة خةةة ل م ّرسةةة .ت ممه اّلل ا الذل ن ن بب لعر وا ال جر ع 3 - إل ال ةةةل نفةةةةد مةةةة ال ةةةة ا ال يداركةةة سةةةةك ل مرةةةرت لورفةةةةعل وةةةا الف لةةةة الرةةة تةةةةعاط الجّمفّت الفعاقك اّلرفوكت ا ل رع ر اكّل ج كع ال رغيعات مةدى انسةر دام ال رةّت الرةةةة تةةةةؤثع وةةةةا جةةةة دال خةةةةدمّت الرفوةةةةكت ا لفةةةةّل ال ةةةةوبكّت مف لةةةةّت انسةةةةر دام) الرةةةة ن رقمة رةّ ال ةل سل الرفوةكت انل رع رة تةّثيعه وةا جة دال الرفوةكت الفةّل نقر ةة . Abstract: ال ل ه نجّد ان ّح ح ل ل 4 - مة خ ل الدراس الفو كة لرةذا ال ةل فةم مة رج و ة نفلة ل ةّ تمة ر الةج ن ةرت فةةة مةةةدى ت ظكةةةا الرفوةةةكت انل رع رةةة اّللةةة ا اسمثةةةا د ر لةةة وةةةا جةةة دال م عجةةةّت . الرفوكت الفّل مة خ ل دراس هذا اسمع فم قكّ الردرس يية لو وبكّت ال ج دال الدراسات السابقة هةدفت دراسة ر فةت الف لة2017 ) ال سةة م "اتجّهةّت ةّ هيئة الرةدرس فةة الجّمفّت المو لي ك ر مرلو ّت اسر دام " د ات الرفوكت ا ل رع ر ف الف وك الرفوك ك، إلةةا الرفةةعل وةةا اتجّهةةّت ةةّ هيئةة الرةةدرس فةة الجّمفةةّت المو ةةلي ك ر ةة مرلو ةةّت اسر دام د ات الرفوت ا ل رع ر ف الف وك الرفوك كة مةة جرة راةعهت اسةر دمت الدراسة ال ةةة رج ال فةةةم الر ويوةةة ت رةةةت ي ةةة الدراسةةة مةةة ة286 ) ةةة هيئةةة الرةةةدرس مةةةة جةةةّمفر اسلمةةةا اس هةةةع اغةةة ال ر قةةةت وةةةيرت اسةةةر ّر م رةةة مةةةة31 ) فقةةةعال فةةة الممةةةا الدراس الثّر لوفّم الجّمف2015 / 2016 م. لد لمت ال رّفج ل ال ر سط ال وة لرقةدعع ّ هيئ الردرس وا مقكّ اسر ّر انتجّهّت ر اسةر دام د ات الةرفوت ا ل رع رة ةةةد فةةةعاد الفي ةةة لةةةد ووةةة لةةةدرال3.64 ) إ تبةةةية ل اتجّهةةةّت ةةةّ هيئةةة الرةةةدرس ر ةةة مرلو ةةّت اسةةر دام د ات الةةةرفوت ا ل رع رةةة إنجّوكةة ودرجةةة مر سةةل . ظرةةةعت ال رةةةّفج ةةة ةةةدم جةةة د فةةةع ب ات دنلةةة إحمةةةّفك وةةةية مر سةةةط تقةةةدععات ةةةّ هيئةةة الرةةةدرس ر ةةة مرلو ةةةّت اسةةةر دام د ات الةةةرفوت ا ل رع رةةة تفةةة ى ل رغيةةةعات: الجةةة الجّمفةةة سةةة ات ال بةةةعال وي ةةةّ ظرةةةعت فةةةع ب ات دنلةةة إحمةةةّفك نسةةةرجّاّت ةةةّ هيئةةة الرةةةدرس تفةةة ى ل رغيع الدرج الفو ك لمةّلج ةّ هيئة الرةدرس ودرجة سةرّ . لةدمت الدراسة ةدد مةة الر فةةةكّت ال قرعحةةةّت ه رةةةّ: الف ةةةا وةةةا رفةةةع مرةةةّرات ةةةّ هيئةةة الرةةةدرس فةةة مجةةةّل اسةةةةر دام د ات الةةةةرفوت ا ل رع رةةةة تلةةةةجكفرت وةةةةا اسةةةةر دامرّ تةةةة فيع ال رلو ةةةةّت ال ّدنةةةة . Abstract: ال ّلك ال ّس نسر دام د ات الرفوت ا ل رع ر / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) 2022 / م1444 هـ 2022 / م1444 هـ / جمةل الداب( اجملدل1 ) العدد142 (أيلول) 2022 / م1444 هـ 2 - و ةةّ ترج ةةد ه كةة هةةذا ال ةةل فةة ت ةةوكط ال ةة ل لةة ع جةة دال الرفوةةكت و فةةمه الغّنةة اسسّسةةك مةةة الف وكةة الرعا سةة س ثةةا هةةذا اسمةةع قكةةّ ل ةةدى رفةةّر الرفوةةكت الفةةّل فةة الفةةةعاب لرةةةه افةةةد لةةة وةةةا جةةة دال ال ةةةعسج البيئةةة ال كلةةة اةةةه مةةةة خةةة ل م ّرسةةة .ت ممه اّلل ا الذل ن ن بب لعر وا ال جر ع 3 - إل ال ةةةل نفةةةةد مةةةة ال ةةةة ا ال يداركةةة سةةةةك ل مرةةةرت لورفةةةةعل وةةةا الف لةةةة الرةةة تةةةةعاط الجّمفّت الفعاقك اّلرفوكت ا ل رع ر اكّل ج كع ال رغيعات مةدى انسةر دام ال رةّت الرةةةة تةةةةؤثع وةةةةا جةةةة دال خةةةةدمّت الرفوةةةةكت ا لفةةةةّل ال ةةةةوبكّت مف لةةةةّت انسةةةةر دام) الرةةةة ن رقمة رةّ ال ةل سل الرفوةكت انل رع رة تةّثيعه وةا جة دال الرفوةكت الفةّل نقر ةة . ال ل ه نجّد ان ّح ح ل ل 4 - مة خ ل الدراس الفو كة لرةذا ال ةل فةم مة رج و ة نفلة ل ةّ تمة ر الةج ن ةرت فةةة مةةةدى ت ظكةةةا الرفوةةةكت انل رع رةةة اّللةةة ا اسمثةةةا د ر لةةة وةةةا جةةة دال م عجةةةّت . الرفوكت الفّل مة خ ل دراس هذا اسمع فم قكّ الردرس يية لو وبكّت ال ج دال الدراسات السابقة هةدفت دراسة ر فةت الف لة2017 ) ال سةة م "اتجّهةّت ةّ هيئة الرةدرس فةة الجّمفّت المو لي ك ر مرلو ّت اسر دام " د ات الرفوكت ا ل رع ر ف الف وك الرفوك ك، إلةةا الرفةةعل وةةا اتجّهةةّت ةةّ هيئةة الرةةدرس فةة الجّمفةةّت المو ةةلي ك ر ةة مرلو ةةّت اسر دام د ات الرفوت ا ل رع ر ف الف وك الرفوك كة مةة جرة راةعهت اسةر دمت الدراسة ال ةةة رج ال فةةةم الر ويوةةة ت رةةةت ي ةةة الدراسةةة مةةة ة286 ) ةةة هيئةةة الرةةةدرس مةةةة جةةةّمفر اسلمةةةا اس هةةةع اغةةة ال ر قةةةت وةةةيرت اسةةةر ّر م رةةة مةةةة31 ) فقةةةعال فةةة الممةةةا الدراس الثّر لوفّم الجّمف2015 / 2016 م. Abstract: وا ج دال الرفوكت الفّل قكّ افّده مة جر راع ّ هيئ الردرس 2 - الرفةعل وةةا تةةأثيعات اسةر دام الرفوةةكت انل رع رةة وةةا د ر الجّمفةّت الفعاقكةة فةة جةة دال .الرفوكت الفّل اافّد ل مة جر راع الردرس يية 3 - ال ل ل وا ال. د افع الفّم ال فعدك نسر دام ال ؤس ّت الرفوك ك لورفوكت انل رع ر 4 - . الرفعل وا تأثيع اسر دام الرفوكت انل رع ر وا ال الع الفو 5 - وكّل ارف ّسّت اسر دام الجّمفّت الفعاقك لورفوكت انل رع رة وةا ظكمة الر لةئ لولو ة 1 - ت ةة ه كة ال ةل فةة الةد ر الةذل ن ثوةةه الرفوةكت انل رع رة مةةة جرة الفي ة ال ةةرردف ه تدرس الجّمفّت الفعاقك اّلعخت مة جة د اا ةّا فة هةذا ال جةّل سفةد اللةغا اللةّخا لو ةّحثية فة اة رة اسخيةعال إن ل مج ةا اسا ةّا ال ةّاق لةت تةدر ارف ّسةّت اسةةةر دام هةةةذا ال ةةة ع مةةةة ال رفوةةةكت وةةةا جةةة دال الرفوةةةكت الفةةةّل قكةةةّ لةةة مةةةة جرةةة راةةةع الردرس يية مدى تأثيعه وا م عجّت الرفوكت الفّل ثعهّ وا ال جر ع لذا فإل ال رّفج الرة سةك عل ورةّ هةذا ال ةل سةر ل مر ة اّلةّف جّرةب رة فة دوكةّت هةذا ال جةةّل مةةة خةة ل دراسةة الرةةأثيعات لورفوةةكت انل رع رةة مةة ّدى فّفةةدال لةة مةةة جرةة راةةع ةة . الريئ الردرس ك 550 550 / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) 2022 / م1444 هـ 551 2 - و ةةّ ترج ةةد ه كةة هةةذا ال ةةل فةة ت ةةوكط ال ةة ل لةة ع جةة دال الرفوةةكت و فةةمه الغّنةة اسسّسةةك مةةة الف وكةة الرعا سةة س ثةةا هةةذا اسمةةع قكةةّ ل ةةدى رفةةّر الرفوةةكت الفةةّل فةة الفةةةعاب لرةةةه افةةةد لةةة وةةةا جةةة دال ال ةةةعسج البيئةةة ال كلةةة اةةةه مةةةة خةةة ل م ّرسةةة .ت ممه اّلل ا الذل ن ن بب لعر وا ال جر ع 3 - إل ال ةةةل نفةةةةد مةةةة ال ةةةة ا ال يداركةةة سةةةةك ل مرةةةرت لورفةةةةعل وةةةا الف لةةةة الرةةة تةةةةعاط الجّمفّت الفعاقك اّلرفوكت ا ل رع ر اكّل ج كع ال رغيعات مةدى انسةر دام ال رةّت الرةةةة تةةةةؤثع وةةةةا جةةةة دال خةةةةدمّت الرفوةةةةكت ا لفةةةةّل ال ةةةةوبكّت مف لةةةةّت انسةةةةر دام) الرةةةة ن رقمة رةّ ال ةل سل الرفوةكت انل رع رة تةّثيعه وةا جة دال الرفوةكت الفةّل نقر ةة . Abstract: ال ّلك ال ّس نسر دام د ات الرفوت ا ل رع ر 551 / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) 2022 / م1444 هـ ةة جة دال إدارال الرفوةكت هةةدفت دراسة رةدى ن يةا م ةد 2014 ) ال سة م "م ةةر ى جةة دال إدارال الرفوةةكت ا ل رع رةة فةة جّمفةة القةةد ال مر حةة مةةة جرةة راةةع ةةّ هيئةةةّت الردرس ف فع رّ ش ّل ال م الغعاك " إلةا مفعفة م ةر ى جة دال إدارال الرفوةكت ا ل رع رة ف جّمف القد ال مر ح مة جر راع ّ هيئّت الردرس ف فع رةّ شة ّل ال ةم الغعاك ف ر ثع ا مة مر غيعات الج ال ؤها الفو ال وك م ةّل ال ة ة) فة ّم ةةر ى جةة دال إدارال الرفوةةكت ا ل رع رةة فةة جّمفةة القةةد ال مر حةة مةةة جرةة راةةع ةة هيئّت الردرس فة فع رةّ شة ّل ال ةم الغعاكة ت ة ل مجر ةع الدراسة مةة ةّ هيئةّت الردرس ف فع ع جّمف القد ال مر ح شة ّل ال ة م الغعاكة ةددهت721 ) اخريةع مة رت ي ةة لةة افك تر ةة ل مةةةة105 ) ةة اسةةر دم فةةة هةةةذه الدراسةة ال ةة رج ال فةةم فةةةرت تم كت اسر ّر لج ةع البكّرةّت ت ة ت63 ) فقةعال ت ةت فة خ ة مجةّنت تةت الر قةم مةةةة فةةةدب اسداال ث ّترةةةّ ثةةةت ةةةت وةةةا فةةةعاد ي ةةة الدراسةةة مةةةة ثةةةت تةةةت ت ويةةةا البكّرةةةّت اّسةةر دام ال ر سةةلّت ال ةةّوك ال رةة انر عافةةّت ال عكّرسةة ت ويةةا الر ةةّعة اسحةةّدل .) اخر ّر ت ت ّ لةةةت دراسةةة امةةة ر فةةةا خويةةةا المةةةكم، 2015 ) ال سةةة م "اتجةةةّه ةةةّ هيئةةة الردرس اجّمف القد ال مر ح ر الرفوكت ا ل رع ر لره امّ وكة الةذات" إلةا مفعفة اتجّه ّ هيئ الردرس اجّمف القد ال مر ح ا ّفاّت خ ال ر الرفوكت انل رع رة و ّ هدفت إلا الرفعل إلا م ر ى فّ وك الذات لدعرت إلّف إلةا ال لة ةة المةع ب وةية مر سلّت انتجّه فّ وك الذات لدعرت فقّ ل رغيعات المعع ال ع انجر ّ الر مر ال ؤهةةةا الفو ةةة سةةة ات ال بةةةعال) ةةةّ هةةةدفت إلةةةا وكةةةّل الف لةةة وةةةية انتجةةةّه ر ةةة الرفوةةةكت انل رع رةة فّ وكةة الةةذات لةةدى ةةّ هيئةة الرةةدرس اجّمفةة القةةد ال مر حةة ا ّفاةةّت خ ال. Abstract: ال ل ه نجّد ان ّح ح ل ل هةدفت دراسة ر فةت الف لة2017 ) ال سةة م "اتجّهةّت ةّ هيئة الرةدرس فةة الجّمفّت المو لي ك ر مرلو ّت اسر دام " د ات الرفوكت ا ل رع ر ف الف وك الرفوك ك، إلةةا الرفةةعل وةةا اتجّهةةّت ةةّ هيئةة الرةةدرس فةة الجّمفةةّت المو ةةلي ك ر ةة مرلو ةةّت اسر دام د ات الرفوت ا ل رع ر ف الف وك الرفوك كة مةة جرة راةعهت اسةر دمت الدراسة ال ةةة رج ال فةةةم الر ويوةةة ت رةةةت ي ةةة الدراسةةة مةةة ة286 ) ةةة هيئةةة الرةةةدرس مةةةة جةةةّمفر اسلمةةةا اس هةةةع اغةةة ال ر قةةةت وةةةيرت اسةةةر ّر م رةةة مةةةة31 ) فقةةةعال فةةة الممةةةا الدراس الثّر لوفّم الجّمف2015 / 2016 م. لد لمت ال رّفج ل ال ر سط ال وة لرقةدعع ّ هيئ الردرس وا مقكّ اسر ّر انتجّهّت ر اسةر دام د ات الةرفوت ا ل رع رة ةةةد فةةةعاد الفي ةةة لةةةد ووةةة لةةةدرال3.64 ) إ تبةةةية ل اتجّهةةةّت ةةةّ هيئةةة الرةةةدرس ر ةةة مرلو ةةّت اسةةر دام د ات الةةةرفوت ا ل رع رةةة إنجّوكةة ودرجةةة مر سةةل . ظرةةةعت ال رةةةّفج ةةة ةةةدم جةةة د فةةةع ب ات دنلةةة إحمةةةّفك وةةةية مر سةةةط تقةةةدععات ةةةّ هيئةةة الرةةةدرس ر ةةة مرلو ةةةّت اسةةةر دام د ات الةةةرفوت ا ل رع رةةة تفةةة ى ل رغيةةةعات: الجةةة الجّمفةةة سةةة ات ال بةةةعال وي ةةةّ ظرةةةعت فةةةع ب ات دنلةةة إحمةةةّفك نسةةةرجّاّت ةةةّ هيئةةة الرةةةدرس تفةةة ى ل رغيع الدرج الفو ك لمةّلج ةّ هيئة الرةدرس ودرجة سةرّ . لةدمت الدراسة ةدد مةة الر فةةةكّت ال قرعحةةةّت ه رةةةّ: الف ةةةا وةةةا رفةةةع مرةةةّرات ةةةّ هيئةةة الرةةةدرس فةةة مجةةةّل اسةةةةر دام د ات الةةةةرفوت ا ل رع رةةةة تلةةةةجكفرت وةةةةا اسةةةةر دامرّ تةةةة فيع ال رلو ةةةةّت ال ّدنةةةة . Abstract: لر قيم هدال هذه الدراس لّم ال ّحل اإ داد اسر ّر ل تجّه فّ وك الذات ت رت ي الدرا س مة90 ) ا مة ّ هيئ الردرس اجّمف القد ال مر ح ا ّفاّت خةةة ال امع رةةةّ ال ةةة تةةةت ت ويةةةا البكّرةةةّت اّسةةةر دام البعرةةةّمج ا حمةةةّفSPSS ) لةةةد :ّت فةةةةةوت الدراسةةةةة إلةةةةةا مج ةةةةة مةةةةةة ال رةةةةةّفج ه رةةةةة1 - إل درجةةةةة انتجةةةةةّه ر ةةةةة الرفوةةةةةكت ا ل رع رةة لةةدى ةةّ هيئةة الرةةدرس اجّمفةة القةةد ال مر حةة لمةةع ع خةة ال ال ةة ّرةةت وةة ل ر ةةب مقةةداره81.1 )% 2 - الدرجةة ال وكةة لمّ وكةة الةةذات لةةدى ةةّ هيئةة الرةةدرس اجّمفةةة القةةةد ال مر حةةة لمةةةع ع خةةةة ال ال ةةة ّرةةةت وةةة ل ر ةةةب80.5 ) إ ت فةةةةوت% الدراس إلا ل فّ وكة ت اةكت الةذات جةّ ت فة ال عت ة اس لةا وة ل ر ةب83.8 ) توةا% لةة الثقةة اّلةةذات وةة ل ر ةةب77.5 ) ثةةت توةةا لةة تم ةةيا ال رةةّم المةةع وةة ل ر ةةب% 76.4 )% 3 - ت جد ل رعدن ات دنل إحمّفك د م ر ى دنلµ ³ 0.05 ) وية 552 / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) ّاتجّه ةّ هيئة الرةدرس اجّمفة القةد ال مر حة ر ة الرفوةكت انل رع رة ف وكة الةذات .لدعرت ّ مةةةّ ةةةة مفعفةةة تةةةأثيع ت ل جكةةةّ الرفوةةةكت ا ل رع رةةة وةةةا وكةةة الرمّ ةةةا وةةةية اسسةةةر اللو هدفت دراس ويو اوة م هّر 2015) ) ال س م " الةع اسةر دام ت ل جكةّ الرفوةكت ا ل رع ر اّلجّمف الج افعسة : دراسة اسر لةّدك لفي ة مةة اسسةّتذال اجّمفة لّفة دل معاةّح ّ رلو ر جّ" إلا ا جّا ة الر ّةل العفك ال ر ثا ف : كا عؤثع اسةر دام ت ل جكة الرفوكت ا ل رع ر وا وك الرمّ ا وية اسسرّ اللو ف جّمف لّفةدل معاةّح رلوة ؟ : لإلجّا وا هذا الر ّةل رعحت ال ّحث ث ا سئو فععك1 . مّ اسر دامّت ت ّل جكة الرفوكت اسوثع اهر ّمةّ مةة رةعل اسسةّتذال اجّمفة لّفةدل معاةّح رلوة الرةدرس ال ةل . ال لةةةةع انل رع رةةةة )؟2 . مةةةةّ ال سةةةةّفا الر ل جكةةةة اسوثةةةةع اسةةةةر دامّ مةةةةة رةةةةعل اسسةةةةّتذال اجّمفةةة لّفةةةدل معاةةةّح؟3 ّ. Abstract: كةةةا عةةةؤثع هةةةذا انسةةةر دام وةةةا وكةةة الرمّ ةةةا وةةةية اسسةةةر اللّلةةةةةب؟ لقةةةةةد ات فةةةةةت ال ّحثةةةةة مج ةةةةة مةةةةةة ا جةةةةةعا ات شةةةةة وت ال ةةةةةدخا ال سةةةةةي ل ج الرمّ وكةةةة العم سةةةة ) ال ةةةة رج ال فةةةةم اّن ر ةةةةّد وةةةةا افةةةةض اسد ات لج ةةةةع ال فو مةةةةّت ال حاةة اسةةر ّرال انسةةربكّل) فةة ن إلةةا ال رةةّفج ت ويةةا البكّرةةّت ال رفوقةة ا ةةا ال رةةّفج . تت تلبيم هذه اسداال وا ي مة اسسّتذال ف ا مة وك الفو م ا ر ةّرك انجر ّعكة وكةةة الفوةةة م انلرمةةةّدن وةةة م الرجةةةّرال الر ةةةييع وكةةة اةدا الوغةةةّت اسج بكةةة اجّمفةةة لّفةةدل معاةةةّح رلوةة ال ر ثوةةة فةةة394 سةةرّ تةةةت اخركةةةّر الفي ةة الل قكةةة انحر ّلكةةة حيةل تةت ت سةع79 اسةةر ّرال اسةربكّل اسة ّرعجع م رة56 اسةةر ّرال اسةربكّل. ت فةوت ررةةّفج :الدراس إلا ل اسسّتذال نف و ل وةا اسةر دام ت ل جكةّ الرفوةكت ا ل رع رة هةذا راجةع إلةا اّلعخت الجر د ال بذ ل مة رعل الجّمف ا م ص تل سع الرفوكت الجّمف ل او الرلة ر الفةةّل إ إن رةةه ن الةةت ف ةةعال تلبيةةم الرفوةةكت ا ّل رع رةة اجّمفةة لّفةةدل معاةةّح فةة وةةداعرر الجّمفةةة فةةة معحوةةة اررقّلكةةة لةةةذل فرةةة ت ةةةّ ل جّمفةةة لّفةةةدل معاةةةّح رلوةةة تةةة فيع ةةةا ال سةةةّفا ال دعثةةة القكةةةّم وةةةد رات ت س كةةة لألسةةةّتذال فةةة هةةةذا ال جةةةّل لةةة ل جةةة د افةةةض ال قةةّفر ال لةةّوا فةة افةةض وكةةّت الجّمفةة مةةة وي رةةّ ةةدم تةة فع اللةة الف ب تكةة الةة ا وّل ف الجّمف رقر اليد الفّمو الر ت رع وا ت ية ال دمّت م ةّ م الجرة د م ّ نف ل الجّمف مّ الت افيدال ر ّ مّ ة ت قيم الج دال ف الرفوكت الفّل مة الجّرةب الرق هذا مّ ن ل د ل تلبيم الرفوةكت انل رع رة ففوكةّ فة الجّمفة شةي ع الف و كة الة ا . Abstract: اسع مة خ ل انرمرّح وا الجّمفّت الد لك 553 / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) 2022 / م1444 هـ منهجية الدراسة ا ر دت الدراس ال رج ال فم الر ويوة هة حةد م ةّهج ال ةل الفو ة الةذل عةدر ّال الع الاّهعال م ل ع ال ل ّ ه ف الفرّ سررت ال ل فيرّ و فمرّ فمّ دقكقة ّمة جا ال ف ل الا اسر رّج ت ت رت ف الرل سع الرغييع هذه الا اهع ت ل ربكعك اجر ّعك سكّسةك الف عار2013 ص66 ) ت ثوةت ال ةد د ال مّركة لوفةّمية الدراسةية 2019 - 2020 / 2020 - 2021) فةةةةةةةة ال جّمفةةةةةةةةّت الفعاقكةةةةةةةةة ل ّفاةةةةةةةة اغةةةةةةةةداد اغةةةةةةةةداد ّال ر مةةةعس ) ت ثوةةت ال ةةد د ال لةةةعس ل روةة ح وةةة انلقةةة الفو كةةة مةةة الجّمفةةةّت ارمةةة الذ ع ّرت ال د د انجعافك ه الفي ةّت الفلة افك لومئة ال ةرردف ت ثوةت رعسقة ت ميةذ ال ةةل حيةةل نفربعرةة ع ال ةةل م ةةة سمةة مةةة ال ةة ا ال فةةاك مةةة خةةة ل اسةةرف ّل م رج ال ج هة دراسة انجةّد ال قةّفم مةة خة ل ج ةع البكّرةّت ال ّشةعال مةة مجر ة ع ي ة الدراس الذل عرلوب خبعال ف الر لكط الر ويا الرم يع لغعض ال ف ل الا ال رّفج تف د ال ا ال ك مة الدم اللعافم ال رف و ف ال ل الغّنة الرة جةدت مةة اجورةّ هة ال م ل وا ال فو مّت مةة مج ة مةة انفةعاد الة ا م ّشةع س ةر دم ال ة رج ال ة ّ وا رل ب اسع ف ال جّنت الرّلك وت انجر ّع دارال اس ّل الفو م ال كّسك وت انلرمةةّد الرعاكةة وةةت الةة م ان م) اوةة ةة م2013 ص297 ) ّرةةت رعسقةة تج كةةةةع ال فو مةةةةّت مةةةةة خةةةة ل اسةةةةر ّرال انسةةةةربكّل ت ةةةة ت م ةةةةّ ر م ةةةةددال ل ج ةةةة مةةةةة انسرم ّرات ال غوق الر تف اسئو هدال ال ل ّ مبي ة فة ال ةّ ر اسرافة هة وكّرّت ّم لوردرس يية ح ل الرفوكت انل رع رة ال رةّت الرة تةؤثع وةا جة دال خةدمّت الرفوةةكت الفةةّل ال ةةوبكّت ال ف لةةّت) ا ةةّ ل ال ّحةةل هةة مةةة لةة ة الفةةّموية فةة مجةةّل الفي ال رردف سر ل معالبر ه ل و الرفّما مع الرفوكت انل رع ر اكةّل مو ظّتةه حة ل . عرض نتائج االستبانة و حليلها و اسيرها عرض نتائج االستبانة و حليلها و اسيرها مة جا ت قيةم هةذا الرةدل جةعى تلبيةم انسةر ّر وةا فةعاد ي ة م رة مةة 313 ) تدرس ةةة مةةةة الجّمفةةةّت الفعاقكةةة حيةةةل ووةةة مجر ةةةع الدراسةةة1683 ) تدرس ةةة مةةةة م روةةة اسلقّ الفو ك خعجت ال لوفي فم لةّر ل "وة عاعة" هة مفّدلة مةة وثةع اللعافةم ال ةرف و ل ةّ حجةت الفي ةة انحمةّفك فةم ر ة الثقةة95 إ ظرةعت ررةّفج ال ةةل% إل د ر الرفوك ت ا ل رع ر حد سة ّ تةدر جة دال الرفوةكت فة الجّمفةّت الفعاقكة مةة خة ل ّ مقّررةة وةةةية مر سةةةط ال ةةةّو ال ر سةةةط المعلةة ال د ر الرفوةةةكت انل رع رةةة عةةة ف سةةةو وةةا الةةع جةة دال الرفوةةكت فةة الجّمفةةةّت الفعاقكةة قكةةّ لةة مةةة جرةة راةةع ةةّ هيئةة الرةةةدرس ةةةّ ظرةةةعت ررةةةّفج ال ةةة ل إل ه ةةةّ تةةةأثيعا الةةة ّ لورفوةةةكت انل رع رةةة ا ةةةةدى اسةةرف ّل خمةةّفر الرفوةةكت مةةة خةة ل ال قّررةة وةةية ال ر سةةط ال ةةّو ال ر سةةط المعلةة سفوةا ال ّحةل ال سةةبب لة فةة ارفةدام البةةدافا فة ظةا تملةة جّف ة ر رةةّ دفةع اّلقةةّف ية وةةا ال الةةع الرعاةة ل الةةا ات ةةّ الرفوةةكت انل رع رةة لغةةع ض ت ةةدل اس مةة ةةدم لةةكّع الفةةّم الدراس األا ال ّفع ةّ ظرةعت ال رةّفج ل ه ّلة تةأثيع الةج وةا الةع الرفوةكت الفةّل ا ؤشعات الج دال عرأثع الع الرفوكت الفّل ا ؤشعات الج دال سم ةع ال ّحةل لرةذا المةعض اةأل ال الةةةع الفو ةةة ال ةةةّتج ةةةة ال عجةةةّت الفو كةةة لو ؤس ةةةّت اسوّدن كةةة مةةةدى تةةةأثيعه اةةةّلرفوكت .انل رع ر كاك نف د اّل مع وا ال الع الفو اّل مو وا ال جر ع . حليل عالقة االر باط واالنحدار بين متغيرات البحث واختبار الارضية األصلية المعلةك اسفةوك: ت جةد لة ارت ةّ ات دنلة مف سة وةية الرفوةكت انل رع رة ثةعه . Abstract: ل منهجية الدراسة د ات تج كع ال فو مّت 1 - رعسق ال ر 2 - انررعرت 3 - ال رب ال لب صدق وثبات االستبانة ّوو مفّما المةّ رر ة-.033 0762 ) مةة الف ّفةع ال ّلغة44 ) سةؤال لة ة م ّ ر انسر ّر تت ةعض انسةر ّر وةا ةدد مةة ال ةية فة مجةّل ان ة م رعافةم .الردرس لبكّل فدب انسربكّل د ات تج كع ال فو مّت 1 - رعسق ال ر 2 - انررعرت 3 - ال رب ال لب صدق وثبات االستبانة ّوو مفّما المةّ رر ة-.033 0762 ) مةة الف ّفةع ال ّلغة44 ) سةؤال لة ة م ّ ر انسر ّر تت ةعض انسةر ّر وةا ةدد مةة ال ةية فة مجةّل ان ة م رعافةم .الردرس لبكّل فدب انسربكّل صدق وثبات االستبانة ّوو مفّما المةّ رر ة-.033 0762 ) مةة الف ّفةع ال ّلغة44 ) سةؤال لة ة م ّ ر انسر ّر تت ةعض انسةر ّر وةا ةدد مةة ال ةية فة مجةّل ان ة م رعافةم .الردرس لبكّل فدب انسربكّل 554 / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) / مةل الآداب( اجملدل1 ) العدد142 (أ يلول) 2022 / م1444 هـ عرض نتائج االستبانة و حليلها و اسيرها مة جا ت قيةم هةذا الرةدل جةعى تلبيةم انسةر ّر وةا فةعاد ي ة م رة مةة 313 ) تدرس ةةة مةةةة الجّمفةةةّت الفعاقكةةة حيةةةل ووةةة مجر ةةةع الدراسةةة1683 ) تدرس ةةة مةةةة م روةةة اسلقّ الفو ك خعجت ال لوفي فم لةّر ل "وة عاعة" هة مفّدلة مةة وثةع اللعافةم ال ةرف و ل ةّ حجةت الفي ةة انحمةّفك فةم ر ة الثقةة95 إ ظرةعت ررةّفج ال ةةل% إل د ر الرفوك ت ا ل رع ر حد سة ّ تةدر جة دال الرفوةكت فة الجّمفةّت الفعاقكة مةة خة ل ّ مقّررةة وةةةية مر سةةةط ال ةةةّو ال ر سةةةط المعلةة ال د ر الرفوةةةكت انل رع رةةة عةةة ف سةةةو وةةا الةةع جةة دال الرفوةةكت فةة الجّمفةةةّت الفعاقكةة قكةةّ لةة مةةة جرةة راةةع ةةّ هيئةة الرةةةدرس ةةةّ ظرةةةعت ررةةةّفج ال ةةة ل إل ه ةةةّ تةةةأثيعا الةةة ّ لورفوةةةكت انل رع رةةة ا ةةةةدى اسةةرف ّل خمةةّفر الرفوةةكت مةةة خةة ل ال قّررةة وةةية ال ر سةةط ال ةةّو ال ر سةةط المعلةة سفوةا ال ّحةل ال سةةبب لة فةة ارفةدام البةةدافا فة ظةا تملةة جّف ة ر رةةّ دفةع اّلقةةّف ية وةةا ال الةةع الرعاةة ل الةةا ات ةةّ الرفوةةكت انل رع رةة لغةةع ض ت ةةدل اس مةة ةةدم لةةكّع الفةةّم الدراس األا ال ّفع ةّ ظرةعت ال رةّفج ل ه ّلة تةأثيع الةج وةا الةع الرفوةكت الفةّل ا ؤشعات الج دال عرأثع الع الرفوكت الفّل ا ؤشعات الج دال سم ةع ال ّحةل لرةذا المةعض اةأل ال الةةةع الفو ةةة ال ةةةّتج ةةةة ال عجةةةّت الفو كةةة لو ؤس ةةةّت اسوّدن كةةة مةةةدى تةةةأثيعه اةةةّلرفوكت .انل رع ر كاك نف د اّل مع وا ال الع الفو اّل مو وا ال جر ع عرض نتائج االستبانة و حليلها و اسيرها خ ل قكّ ل مة جر راع ّ هيئ الردرس المةةةعض اس ل: ت جةةةد لةةة ارت ةةةّ ات دنلةةة مف سةةة وةةةية الرفوةةةكت انل رع رةةة د ره وةةةا . مرّرت اللو ج دال الرفوكت الفّل مة جا ت قيم هذه المعلك ا ر د وا م ر ال رّت الر تؤثع وا ج دال خدمّت الرفوكت الفّل سوبكّت الرفوكت ا ل رع ر تت تلبيةم وةا فةعاد ي ة ال ةل ال ّلغة313 ) تدرس ةة إ لةةّم ال ّحةةل اّسةةر عال ال ر سةةط ال ةةّو انر ةةعال ال عكةةّرل القك ةة الرّفكةة إ وو ال ر سط ال ّو15,38 ) اّر عال معكّرل لدره4,25 ) وي ّ وو ال ر سط المعل وةةةية وةةةا ر ةةة15,85 ) الةةةا ر ةةة14,91 ) لغةةةعض مفعفةةة دنلةةة المةةةعب وي ر ةةةّ تةةةت اسةةرف ّل انخر ةةّر الرةةّفt-test ) إ ووغةةت القك ةة الرّفكةة ال ةة ا64,01) م ةةر ى دنلةةة0,05 ) درجةةة حعسةةة312 ) ظرةةةع ل ه ّلةةة تةةةأثيع الةةةج لورفوةةةكت انل رع رةةة تأثيعه وا ج دال الرفوكت الفّل , ّ م لج ف الجد ل رلت2 ) ررّفج انخر ةّر الرةّفt- test ال رغيعات حجت الفي ال ر سط ال ّو انر عال ال عكّرل مفّما ّ انرت درج ال عس القك الرّفك م ر ى الدنل ال ر الثّر 313 15,71 3,79 136. 312 64,01 0,05 ال ر الثّلل 7,91 2,02 المةةةعض الثةةةّر : ت جةةد لةةة ارت ةةةّ ات دنلةةة مف سةةة وةةةية ال ةة ارد ال ّلكةةة جةة دال الرفوةةةكت . الفّل مة جا ت قيم هذا المعض ا ر ةد ال ّحةل وةا مرغيةع ال ر ة م ة ر مف لةّت الرفوةكت ا ل رع رةة ربةةم انسةةر ّر وةةا فةةعاد ي ةة ال ةةل ال ّلغةة313)تدرس ةة مةةة الجّمفةةّت الفعاقكةة إ لةةّم ال ّحةةل اّسةةر عال ال ر سةةط ال ةةّو انر ةةعال ال عكةةّرل القك ةة الرّفكةة إ تةةت اسةةر عال ال رةةّفج ال بي ةة فةة الجةةد ل1 ) اّسةةرف ّل ال سةةّفا ا حمةةّفك انسةةرفّر اّل قي ةة ا حمةةّفك لوفوةة م انجر ّعكةة ال م ةةكSPSS ) لر قيةةم هةةدال ال ةةل ال ةةّل حيل ع ف د ر الرفوكت انل رع ر سو ّ وا الع جة دال الرفوةكت فة الجّمفةّت الفعاقكة مةة . خ ل قكّ ل مة جر راع ّ هيئ الردرس المةةةعض اس ل: ت جةةةد لةةة ارت ةةةّ ات دنلةةة مف سةةة وةةةية الرفوةةةكت انل رع رةةة د ره وةةةا . عرض نتائج االستبانة و حليلها و اسيرها وا ج دال الرفوكت الفّل ف الجّمفّت الفعاقك مةةة جةةا ت قيةةم هةةذا الرةةدل تةةت تلبيةةم انسةةر ّر وةةا فةةعاد ي ةة م رةة مةةة313 ) تدرس مة الجّمفّت الفعاقك اخذ مرغيع ال ر ال ر الثّر ال رّت الر تؤثع وةا ج دال خدمّت الرفوكت الفّل إ لّم ال ّحل اّسر عال ال ر سط ال ّو انر عال ال عكّرل القك ةة الرّفكةة إ ووةة ال ر سةةط ال ةةّو13,17 ) اةةّر عال معكةةّرل لةةدره3,65 ّ) وي ةة وو ال ر سط المعل وية وا ر13,58 ) الا ر12,76 ) لغعض مفعفة دنلة المةةةةعب وي ر ةةةةّ تةةةةت اسةةةةرف ّل انخر ةةةةّر الرةةةةّفt-test ) إ ووغةةةةت القك ةةةة الرّفكةةةة ال ةةةة ا 63,70 ) م ر ى دنل0,05 ) درج حعس312 ) ظرع إل د ر الرفوكت ا ل رع ر حةةد سةة ّ تةةدر جةة دال الرفوةةكت الجّمفةةّت الفعاقكةة ةةّ م لةةج فةة الجةةد ل رلةةت1 ) ررةةّفج انخر ّر الرّفt-test . مةةة جةةا ت قيةةم هةةذا الرةةدل تةةت تلبيةةم انسةةر ّر وةةا فةةعاد ي ةة م رةة مةةة313 ) تدرس مة الجّمفّت الفعاقك اخذ مرغيع ال ر ال ر الثّر ال رّت الر تؤثع وةا ج دال خدمّت الرفوكت الفّل إ لّم ال ّحل اّسر عال ال ر سط ال ّو انر عال ال عكّرل القك ةة الرّفكةة إ ووةة ال ر سةةط ال ةةّو13,17 ) اةةّر عال معكةةّرل لةةدره3,65 ّ) وي ةة وو ال ر سط المعل وية وا ر13,58 ) الا ر12,76 ) لغعض مفعفة دنلة المةةةةعب وي ر ةةةةّ تةةةةت اسةةةةرف ّل انخر ةةةةّر الرةةةةّفt-test ) إ ووغةةةةت القك ةةةة الرّفكةةةة ال ةةةة ا 63,70 ) م ر ى دنل0,05 ) درج حعس312 ) ظرع إل د ر الرفوكت ا ل رع ر حةةد سةة ّ تةةدر جةة دال الرفوةةكت الجّمفةةّت الفعاقكةة ةةّ م لةةج فةة الجةةد ل رلةةت1 ) ررةةّفج انخر ّر الرّفt-test . 555 / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) 2022 / م1444 هـ ال رغيعات حجت الفي ال ر سط ال ّو انر عال ال عكّرل مفّما ّ انرت درج ال عس القك الرّفك م ر ى الدنل ال ر الثّر 313 15,71 3,79 299. 312 63,70 0,05 ال ر 2,54 615. عرض نتائج االستبانة و حليلها و اسيرها تةةت اسةةر عال ال رةةّفج ال بي ةة فةة الجةةد ل1 ) اّسةةرف ّل ال سةةّفا ا حمةةّفك انسةةرفّر اّل قي ةة ا حمةةّفك لوفوةة م انجر ّعكةة ال م ةةكSPSS ) لر قيةةم هةةدال ال ةةل ال ةةّل حيل ع ف د ر الرفوكت انل رع ر سو ّ وا الع جة دال الرفوةكت فة الجّمفةّت الفعاقكة مةة . خ ل قكّ ل مة جر راع ّ هيئ الردرس المةةةعض اس ل: ت جةةةد لةةة ارت ةةةّ ات دنلةةة مف سةةة وةةةية الرفوةةةكت انل رع رةةة د ره وةةةا . مرّرت اللو ج دال الرفوكت الفّل مة جا ت قيم هذه المعلك ا ر د وا م ر ال رّت الر تؤثع وا ج دال خدمّت الرفوكت الفّل سوبكّت الرفوكت ا ل رع ر تت تلبيةم وةا فةعاد ي ة ال ةل ال ّلغة313 ) تدرس ةة إ لةةّم ال ّحةةل اّسةةر عال ال ر سةةط ال ةةّو انر ةةعال ال عكةةّرل القك ةة الرّفكةة إ وو ال ر سط ال ّو15,38 ) اّر عال معكّرل لدره4,25 ) وي ّ وو ال ر سط المعل وةةةية وةةةا ر ةةة15,85 ) الةةةا ر ةةة14,91 ) لغةةةعض مفعفةةة دنلةةة المةةةعب وي ر ةةةّ تةةةت اسةةرف ّل انخر ةةّر الرةةّفt-test ) إ ووغةةت القك ةة الرّفكةة ال ةة ا64,01 ) م ةةر ى دنلةةة0,05 ) درجةةة حعسةةة312 ) ظرةةةع ل ه ّلةةة تةةةأثيع الةةةج لورفوةةةكت انل رع رةةة تأثيعه وا ج دال الرفوكت الفّل , ّ م لج ف الجد ل رلت2 ) ررّفج انخر ةّر الرةّفt- test ال رغيعات حجت الفي ال ر سط ال ّو انر عال ال عكّرل مفّما ّ انرت درج ال عس القك الرّفك م ر ى الدنل / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) 2022 / م1444 هـ ال رغيعات حجت الفي ال ر سط ال ّو انر عال ال عكّرل مفّما ّ انرت درج ال عس القك الرّفك م ر ى الدنل ال ر الثّر 313 15,71 3,79 299. 312 63,70 0,05 ال ر 2,54 615. ال ر الثّر 313 15,71 3,79 299. 312 63,70 0,05 ال ر 2,54 615. تةةت اسةةر عال ال رةةّفج ال بي ةة فةة الجةةد ل1 ) اّسةةرف ّل ال سةةّفا ا حمةةّفك انسةةرفّر اّل قي ةة ا حمةةّفك لوفوةة م انجر ّعكةة ال م ةةكSPSS ) لر قيةةم هةةدال ال ةةل ال ةةّل حيل ع ف د ر الرفوكت انل رع ر سو ّ وا الع جة دال الرفوةكت فة الجّمفةّت الفعاقكة مةة . عرض نتائج االستبانة و حليلها و اسيرها مرّرت اللو ج دال الرفوكت الفّل مة جا ت قيم هذه المعلك ا ر د وا م ر ال رّت الر تؤثع وا ج دال خدمّت الرفوكت الفّل سوبكّت الرفوكت ا ل رع ر تت تلبيةم وةا فةعاد ي ة ال ةل ال ّلغة313 ) تدرس ةة إ لةةّم ال ّحةةل اّسةةر عال ال ر سةةط ال ةةّو انر ةةعال ال عكةةّرل القك ةة الرّفكةة إ وو ال ر سط ال ّو15,38 ) اّر عال معكّرل لدره4,25 ) وي ّ وو ال ر سط المعل وةةةية وةةةا ر ةةة15,85 ) الةةةا ر ةةة14,91 ) لغةةةعض مفعفةةة دنلةةة المةةةعب وي ر ةةةّ تةةةت اسةةرف ّل انخر ةةّر الرةةّفt-test ) إ ووغةةت القك ةة الرّفكةة ال ةة ا64,01 ) م ةةر ى دنلةةة0,05 ) درجةةة حعسةةة312 ) ظرةةةع ل ه ّلةةة تةةةأثيع الةةةج لورفوةةةكت انل رع رةةة تأثيعه وا ج دال الرفوكت الفّل , ّ م لج ف الجد ل رلت2 ) ررّفج انخر ةّر الرةّفt- test مة جا ت قيم هذا المعض ا ر ةد ال ّحةل وةا مرغيةع ال ر ة م ة ر مف لةّت الرفوةكت ا ل رع رةة ربةةم انسةةر ّر وةةا فةةعاد ي ةة ال ةةل ال ّلغةة313 )تدرس ةة مةةة الجّمفةةّت الفعاقكةة إ لةةّم ال ّحةةل اّسةةر عال ال ر سةةط ال ةةّو انر ةةعال ال عكةةّرل القك ةة الرّفكةة إ 556 / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) 2022 / م1444 هـ 2022 / م1444 هـ ةجدمج م وو ال ر سط ال ّو15,36 ) اّر عال معكّرل لدره3,39 ) وي ّ وو ال ر سط المعل وةةةية وةةةا ر ةةة15,73 ) الةةةا ر ةةة14,98 ) لغةةةعض مفعفةةة دنلةةة المةةةعب وي ر ةةةّ تةةةت اسةةرف ّل انخر ةةّر الرةةّفt-test ) إ ووغةةت القك ةة الرّفكةة ال ةة ا79,95 ) م ةةر ى دنل0,05 ) درج حعس312 ) ظرع ل ه ّل تأثيع الج وا الةع الرفوةكت الفةّل ّا ّ ن ر ال ارد ال ّلك اّ ر ّرهّ احد ال ف لّت مّم تلبيم الج دال ف الرفوةكت الفةّل ة م لج ف الجد ل3 ) ررّفج انخر ّر الرّفt-test . ال رغيعات حجت الفي ال ر سط ال ّو انر عال ال عكّرل مفّما ّ انرت درج ال عس القك الرّفك م ر ى الدنل ال رالعااع 313 17,89 3,39 089. 312 79,95 0,05 ال ر 2,54 615. عرض نتائج االستبانة و حليلها و اسيرها عرةةةأثع الةةةع الرفوةةةكت الفةةةّل ا ؤشةةةعات الجةةة دال سم ةةةع ال ّحةةةل لرةةةذا المةةةعض اةةةأل ال الةةةع الفو ال ّتج ة ال عجةّت الفو كة لو ؤس ةّت اسوّدن كة مةدى تةأثيعه اةّلرفوكت انل رع رة كاك نف د اّل مع وا ال الع الفو اّل مو وا ال جر ع. المعض الثّلل : ت جد ل ارت ّ ات دنل مف س وية اسةر دام الرق كةّت انسةرعاتكجكّت . الرفوك ك ج دال الرفوكت الفّل مةة جةةا ت قيةةم هةذا المةةعض ا ر ةةد ال ّحةل وةةا مرغيةةع ال ر ة ال ةة ر ان ل وكّرةةّت ّمةة لوردرس ةةيية حةة ل الرفوةةكت انل رع رةة ربةةم انسةةر ّر وةةا فةةعاد ي ةة ال ةةل ال ّلغةة 313 ) تدرس ةةةةة مةةةةةة الجّمفةةةةةّت الفعاقكةةةةة إ لةةةةةّم ال ّحةةةةةل اّسةةةةةر عال ال ر سةةةةةط ال ةةةةةّو انر عال ال عكّرل الق ك الرّفك إ وو ال ر سط ال ّو28,56 ) اّر عال معكّرل لدره 3,98 ) وي ةةةّ ووةةة ال ر سةةةط المعلةةة وةةةية وةةةا ر ةةة29,00 ) الةةةا ر ةةةة28,11 ) لغةةعض مفعفةة دنلةة المةةعب وي ر ةةّ تةةت اسةةرف ّل انخر ةةّر الرةةّفt-test ) إ ووغةةت القك ةة الرّفكةةة ال ةةة ا126.85) م ةةةر ى دن لةةة0,05 ) درجةةة حعسةةة312 ) ظرةةةع ل ه ّلةة تةةأثيع الةةج وةةا الةةع الرفوةةكت الفةةّل ا ةةّ ن ةةر اسةةر دام الرق كةةّت انسةةرعاتكجكّت الرفوك ك اّ ر ّرهّ حد مق مّت الج دال فة الرفوةكت الفةّل ةّ م لةج فة الجةد ل رلةت4 ) ررّفج انخر ّر الرّفt-test . اسةةرف ّل الرفوةةكت انل رع رةة مةةة لبةةا ال ؤس ةةّت اسوّدن كةة مفعفةة مةةدى الف لةة الرةة . نجب لوردرس يية ل نق م ا ورّ خ ل الف وك الرفوك ك تأثيع ل ان ّ وا ال جر ع وو ال ر سط ال ّو15,36 ) اّر عال معكّرل لدره3,39 ) وي ّ وو ال ر سط المعل وةةةية وةةةا ر ةةة15,73 ) الةةةا ر ةةة14,98 ) لغةةةعض مفعفةةة دنلةةة المةةةعب وي ر ةةةّ تةةةت اسةةرف ّل انخر ةةّر الرةةّفt-test ) إ ووغةةت القك ةة الرّفكةة ال ةة ا79,95 ) م ةةر ى دنل0,05 ) درج حعس312 ) ظرع ل ه ّل تأثيع الج وا الةع الرفوةكت الفةّل ّا ّ ن ر ال ارد ال ّلك اّ ر ّرهّ احد ال ف لّت مّم تلبيم الج دال ف الرفوةكت الفةّل ة م لج ف الجد ل3 ) ررّفج انخر ّر الرّفt-test . عرض نتائج االستبانة و حليلها و اسيرها ج دال الرفوكت الفّل مة جا ت قيم هذا المعض ا ر د ال ّحل وا ال ر اس ل وكّرّت ّم لوردرس ةيية حةة ل الرفوةةكت انل رع رةة م ةة ر ال ف لةةّت ربةةم انسةةر ّر وةةا فةةعاد ي ةة ال ةةل ال ّلغةة 313 ) تدرس ةةةة مةةةةة الجّمفةةةةةّت الفعاقكةةةةة إ لةةةةةّم ال ّحةةةةل اّسةةةةةر عال ال ر سةةةةةط ال ةةةةةّو انر عال ال عكّرل ا لقك الرّفك إ وو ال ر سط ال ّو13,20 ) اّر عال معكّرل لدره 4 91 ) وي ةةةّ ووةةة ال ر سةةةط المعلةةة وةةةية وةةةا ر ةةة13 74 ) لةةةا ر ةةة12,65 ) لغةةعض مفعفةة دنلةة المةةعب وي ر ةةّ تةةت اسةةرف ّل انخر ةةّر الرةةّفt-test ) إ ووغةةت القك ةة الرّفك ال ا47,52 ) م ر ى د نل0,05 ) درج حعس312 ) ظرع ل ه ّل تأثيع الةج وةا الةع الرفوةكت الفةّل ا ةّ ن ةر اسةر دام الرق كةّت انسةرعاتكجكّت الرفوك كة و فةةةمرّ حةةد مق مةةةّت الجةة دال فةةة الرفوةةكت الفةةةّل ةةةّ م لةةج فةةة الجةةد ل رلةةت5 ) ررةةةّفج انخر ّر الرّفt-test . ال رغيعات حجت الفي ال ر سط ال ّو انر عال ال عكّرل مفّما ّ انرت درج ال عس القك الرّفك م ر ى الدنل ال ران ل 313 31,09 4,04 137. 312 47,52 0,05 ال ر العااع 17,89 3,39 البيانات األولية ألعضاء الهيئة التدريسية الج جد ل6 ) ع ال ال ئ س ارثا ال ال ئ س / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) 2022 / م1444 هـ ال رغيعات حجت الفي ال ر سط ال ّو انر عال ال عكّرل مفّما ّ انرت درج ال عس القك الرّفك م ر ى الدنل ال ران ل 313 31,09 4,04 175. 312 126.85 0,05 ال ر 2,54 615. المةةعض العااةةع: ت جةةد لةة ارت ةةّ ات دنلةة مف سةة وةةية القكةةّدات ا دارسةة فةة الجّمفةةّت . عرض نتائج االستبانة و حليلها و اسيرها ال رغيعات حجت الفي ال ر سط ال ّو انر عال ال عكّرل مفّما ّ انرت درج ال عس القك الرّفك م ر ى الدنل ال رالعااع 313 17,89 3,39 089. 312 79,95 0,05 ال ر 2,54 615. ا عرةةةأثع الةةةع الرفوةةةكت الفةةةّل ا ؤشةةةعات الجةةة دال سم ةةةع ال ّحةةةل لرةةةذا المةةةعض اةةةأل ال الةةةع الفو ال ّتج ة ال عجةّت الفو كة لو ؤس ةّت اسوّدن كة مةدى تةأثيعه اةّلرفوكت انل رع رة كاك نف د اّل مع وا ال الع الفو اّل مو وا ال جر ع. المعض الثّلل : ت جد ل ارت ّ ات دنل مف س وية اسةر دام الرق كةّت انسةرعاتكجكّت . الرفوك ك ج دال الرفوكت الفّل مةة جةةا ت قيةةم هةذا المةةعض ا ر ةةد ال ّحةل وةةا مرغيةةع ال ر ة ال ةة ر ان ل وكّرةةّت ّمةة لوردرس ةةيية حةة ل الرفوةةكت انل رع رةة ربةةم انسةةر ّر وةةا فةةعاد ي ةة ال ةةل ال ّلغةة 313 ) تدرس ةةةةة مةةةةةة الجّمفةةةةةّت الفعاقكةةةةة إ لةةةةةّم ال ّحةةةةةل اّسةةةةةر عال ال ر سةةةةةط ال ةةةةةّو انر عال ال عكّرل الق ك الرّفك إ وو ال ر سط ال ّو28,56 ) اّر عال معكّرل لدره 3,98 ) وي ةةةّ ووةةة ال ر سةةةط المعلةةة وةةةية وةةةا ر ةةة29,00 ) الةةةا ر ةةةة28,11 ) لغةةعض مفعفةة دنلةة المةةعب وي ر ةةّ تةةت اسةةرف ّل انخر ةةّر الرةةّفt-test ) إ ووغةةت القك ةة الرّفكةةة ال ةةة ا126.85) م ةةةر ى دن لةةة0,05 ) درجةةة حعسةةة312 ) ظرةةةع ل ه ّلةة تةةأثيع الةةج وةةا الةةع الرفوةةكت الفةةّل ا ةةّ ن ةةر اسةةر دام الرق كةةّت انسةةرعاتكجكّت الرفوك ك اّ ر ّرهّ حد مق مّت الج دال فة الرفوةكت الفةّل ةّ م لةج فة الجةد ل رلةت4 ) ررّفج انخر ّر الرّفt-test . اسةةرف ّل الرفوةةكت انل رع رةة مةةة لبةةا ال ؤس ةةّت اسوّدن كةة مفعفةة مةةدى الف لةة الرةة . نجب لوردرس يية ل نق م ا ورّ خ ل الف وك الرفوك ك تأثيع ل ان ّ وا ال جر ع 557 / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) 2022 / م1444 هـ ال رغيعات حجت الفي ال ر سط ال ّو انر عال ال عكّرل مفّما ّ انرت درج ال عس القك الرّفك م ر ى الدنل ال ران ل 313 31,09 4,04 175. 312 126.85 0,05 ال ر 2,54 615. المةةعض العااةةع: ت جةةد لةة ارت ةةّ ات دنلةة مف سةة وةةية القكةةّدات ا دارسةة فةة الجّمفةةّت . عرض نتائج االستبانة و حليلها و اسيرها ج دال الرفوكت الفّل مة جا ت قيم هذا المعض ا ر د ال ّحل وا ال ر اس ل وكّرّت ّم لوردرس ةيية حةة ل الرفوةةكت انل رع رةة م ةة ر ال ف لةةّت ربةةم انسةةر ّر وةةا فةةعاد ي ةة ال ةةل ال ّلغةة 313 ) تدرس ةةةة مةةةةة الجّمفةةةةةّت الفعاقكةةةةة إ لةةةةةّم ال ّحةةةةل اّسةةةةةر عال ال ر سةةةةةط ال ةةةةةّو انر عال ال عكّرل ا لقك الرّفك إ وو ال ر سط ال ّو13,20 ) اّر عال معكّرل لدره 4 91 ) وي ةةةّ ووةةة ال ر سةةةط المعلةةة وةةةية وةةةا ر ةةة13 74 ) لةةةا ر ةةة12,65 ) لغةةعض مفعفةة دنلةة المةةعب وي ر ةةّ تةةت اسةةرف ّل انخر ةةّر الرةةّفt-test ) إ ووغةةت القك ةة الرّفك ال ا47,52 ) م ر ى د نل0,05 ) درج حعس312 ) ظرع ل ه ّل تأثيع الةج وةا الةع الرفوةكت الفةّل ا ةّ ن ةر اسةر دام الرق كةّت انسةرعاتكجكّت الرفوك كة و فةةةمرّ حةةد مق مةةةّت الجةة دال فةةة الرفوةةكت الفةةةّل ةةةّ م لةةج فةةة الجةةد ل رلةةت5 ) ررةةةّفج انخر ّر الرّفt-test . البيانات األولية ألعضاء الهيئة التدريسية الج جد ل6 ) الج ع ال ال ئ س ارثا ال ال ئ س 169 54% 144 46% 558 / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) 2022 / م1444 هـ اللرّدال جد ل7 ) اللرّدال دوو م ّل مّج ريع د ر راه الفدد ال ال ئ س الفدد ال ال ئ س الفدد ال ال ئ س 20 6.40% 105 33.54% 188 60.06% البدائل والنسبة المئوية ال ر اس ل : وكّرّت ّم لوردرس يية ح ل الرفوكت انل رع ر جد ل رلت8 ) ت المقعات البدعا الر عار ال ال ئ س 1- هةةةةةةا ت جةةةةةةةد لّ ةةةةةةّت مر ممةةةةةةة فةةةةةةة الجّمف ت ّم الرفوكت انل رع ر. رفت 124 39.62% ن 189 60.38% 2- هةةا ت جةةد شةة إررعرةةت مرةة فعال امةة رال داف ةة س ّ الريئ. الردرس ك ف ال وك رفت 237 75.72% ن 76 24.28% 3- . عرض نتائج االستبانة و حليلها و اسيرها مّ مدى اجّدت نسر دام ال ّس ّم ر 147 46.96% جيد جدا 118 37.70% جيد 48 15.34% 4- مةةةّ مةةةدى اسةةةر دام ل ع ةةةّت ال ةةةل لو مةةة ل وةةةا مفو مةةةّت حةةة ل ال ةةةّدال الدراسةةةةك اإكةةةة إخ ةةةةّ ال ّلةةةةعال ورةةةةذه .ال فو مّت ام رال م ر عال 233 74.44% ّلويا الا حد م 70 22.36% ن اسر دم 10 3.20% 5- ا در اسرث ّفك لغةعض الر افةا ّمةةع اللو ةة لغةةعض ت ةةّدل ال بةةعات دك ةة .وي رت ام رال م ر عال 165 52.71% ّلويا الا حد م 113 36.11% ن اسر دم35 11.18% / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) 2022 / م1444 هـ اللرّدال جد ل7 ) اللرّدال دوو م ّل مّج ريع د ر راه الفدد ال ال ئ س الفدد ال ال ئ س الفدد ال ال ئ س 20 6.40% 105 33.54% 188 60.06% ة ال ئ ة ال ائل الن 559 20 6.40% 105 33.54% 188 60.06% البدائل والنسبة المئوية ال ر اس ل : وكّرّت ّم لوردرس يية ح ل الرفوكت انل رع ر جد ل رلت8 ) ت المقعات البدعا الر عار ال ال ئ س 1- هةةةةةةا ت جةةةةةةةد لّ ةةةةةةّت مر ممةةةةةةة فةةةةةةة الجّمف ت ّم الرفوكت انل رع ر. رفت 124 39.62% ن 189 60.38% 2- هةةا ت جةةد شةة إررعرةةت مرةة فعال امةة رال داف ةة س ّ الريئ. الردرس ك ف ال وك رفت 237 75.72% ن 76 24.28% 3- . مّ مدى اجّدت نسر دام ال ّس ّم ر 147 46.96% جيد جدا 118 37.70% جيد 48 15.34% 4- مةةةّ مةةةدى اسةةةر دام ل ع ةةةّت ال ةةةل لو مةةة ل وةةةا مفو مةةةّت حةةة ل ال ةةةّدال الدراسةةةةك اإكةةةة إخ ةةةةّ ال ّلةةةةعال ورةةةةذه .ال فو مّت ام رال م ر عال 233 74.44% ّلويا الا حد م 70 22.36% ن اسر دم 10 3.20% 5- ا در اسرث ّفك لغةعض الر افةا ّمةةع اللو ةة لغةةعض ت ةةّدل ال بةةعات دك ةة .وي رت ام رال م ر عال 165 52.71% ّلويا الا حد م 113 36.11% ن اسر دم 35 11.18% 6- م ّل . عرض نتائج االستبانة و حليلها و اسيرها ارسّل ال ّلعات لولو ّم 104 33.22% خّص 209 66.78% 7- اسر دام ال سّفا الر لك ك ف ام رال م ر عال 213 68.05% 559 / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) 2022 / م1444 هـ 560 . ال ّلعال انل رع رك ّلويا الا حد م 93 29.71% ن اسر دم 7 2.24% 8- :اللوب مة اللو ارسّل اج ّت وا البعسد انل رع ر 116 37.06% ال مّت الرفوك ك 195 62.31% م الع الر افا ّ انجر 2 0.63% 9- اخركّر م ر ى تفوك معت ط اّنهدال الرفوك ك الف ا وا .ّت قكقر ام رال م ر عال 230 73.49% ّلويا الا حد م 82 26.20% ن اسر دم 1 0.31% 10- اسر دام الوغ المم ا د شعح .ال ّدال ام رال م ر عال 169 53.99% ّلويا الا حد م 143 45.70% ن اسر دم 1 0.31% 11- ت فيع لّم ل ملو ّت ال قعر .ّ ال مّيكت ال عت ل ور ام رال م ر عال 211 67.42% ّلويا الا حد م 98 31.31% ن اسر دم 4 1.27% 12- اسر دام اسرعاتكجكّت تفوك ك حدعث . لرلجكع الل ام رال م ر عال 221 70.61% ّلويا الا حد م 89 28.44% ن اسر دم 3 0.95% 13- . البعرّمج ال ر ع ف الرفوكت انل رع ر ال مّت الرفوك ك 303 96.81% م الع الر افا ّ انجر 2 0.63% ّج كفر 8 2.56% 14- الرفّ ل مع مفو ية اخعسة , الر افا مع خبعا م ويية د ليية د ت ميذ ام رال م ر عال 146 46.64% ّلويا الا حد م 114 36.43% .ال ّدال 560 / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) / جمةل الداب( اجملدل1 ) العدد142 (أيلول) 2022 / م1444 هـ 56 البعرّمج الرفوك بع م مّت الرفوكت . انل رع ر ن اسر دم 53 16.93% 15- وا الردرس ال نغع ف رم ر ا انتجّهّت ا نجّوك ر الرفوكت . انل رع ر اتمم 105 33.54% م ّعد 131 41.86% ن اتمم 77 24.6% 16- نق م الرفوكت انل رع ر ا د رقر . عرض نتائج االستبانة و حليلها و اسيرها م الريئ الردرس ك اتمم 44 14.05% م ّعد 98 31.31% ن اتمم 171 54.64% 17- ع لد الرفوكت انل رع ر الر ل لدى ّ الريئ الردرس ك مة ارفدام .د رهت مقّرر اّلرفوكت الرقويدل اتمم 141 45.04% م ّعد 134 42.82% ن اتمم 38 12.14% 18- ّها ال ؤس الر تف ا ورّ و فمر ال ّل لّدرال وا انررقّل الا الرفوكت انل رع ر تع اللعب الرقويدن .اّلرفوكت رفت 58 18.54% ن 255 81.46% ال ر الثّر : ال رّت الر تؤثع وا ج دال خدمّت الرفوكت الفّل جد ل رلت9 ) ت المقعات البدعا الر عار ال ال ئ س ت ية م ر ى اللو ان ر ّد الةذات وا الرفوكت. اتمم 67 21.40% م ّعد 135 43.14% ن اتمم 111 35.46% 21- سةةةةةةّدال مرةةةةةةّرات اللو ةةةةةة فةةةةةة اسةةةةةةر دام ت و جكةةةةة ال ّسةةةةة تلةةةةة سع الرم يةةةةةع . ال قدل انودا اتمم 155 49.53% م ّعد 63 20.12% ن اتمم 95 30.35% 22- م ةةةةةةةةةةةّ دال اللو ةةةةةةةةةةة وةةةةةةةةةةةا انحرمةةةةةةةةةةةّم اّل فو مةةةةةةةّت انرةةةةةةةدفّع لوةةةةةةةرفوت رجةةةةةةةع اتمم 86 27.47% م ّعد 131 41.86% ال ر الثّر : ال رّت الر تؤثع وا ج دال خدمّت الرفوكت الفّل جد ل رلت9 ) ت المقعات البدعا الر عار ال ال ئ س ت ية م ر ى اللو ان ر ّد الةذات وا الرفوكت. اتمم 67 21.40% م ّعد 135 43.14% ن اتمم 111 35.46% 21- سةةةةةةّدال مرةةةةةةّرات اللو ةةةةةة فةةةةةة اسةةةةةةر دام ت و جكةةةةة ال ّسةةةةة تلةةةةة سع الرم يةةةةةع . ال قدل انودا اتمم 155 49.53% م ّعد 63 20.12% ن اتمم 95 30.35% 22- م ةةةةةةةةةةةّ دال اللو ةةةةةةةةةةة وةةةةةةةةةةةا انحرمةةةةةةةةةةةّم اّل فو مةةةةةةةّت انرةةةةةةةدفّع لوةةةةةةةرفوت رجةةةةةةةع اتمم 86 27.47% م ّعد 131 41.86% 561 / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) 2022 / م1444 هـ 562 .المدع ام رال م ّشعال ن اتمم 96 30.67% 23- لوةةةةة الر ومةةةةة ال ّلكةةةةة ل ةةةةةا مةةةةةة اللو ةةةةة الردرس ةةةةةةةة مةةةةةةةةة رّحكةةةةةةةة ال مةةةةةةةةّرسا اسسّسةةةةةةك الجّربكةةةةةة ال عافقةةةةةة لوف وكةةةةةة . الرفوك ك اتمم 141 45.47% م ّعد 98 31.40% ن اتمم 74 23.13% 24- مقّرا المج ال وية اللّلب الردرس اتمم 44 14.05% م ّعد 106 33.88% ن اتمم 163 52.07% 25- اسرث ّر ال لت لمف ا ال ف ل الا .الجّمفّت ا بب ا دحّم اللعسم اتمم 134 42.81% م ّعد 96 30.68% ن اتمم 83 26.51% 26- . عرض نتائج االستبانة و حليلها و اسيرها معا ّال المع ب المعدن وية اللو اتمم 39 12.46% م ّعد 90 28.75% ن اتمم 184 58.79% 27- ف ج الرفوكت انل رع ر ظّهعال لور ي . ل ر ل مفعف مفّعيع الج دال اسدا اتمم 42 13.42% م ّعد 118 37.70% ن اتمم 153 48.88% ال ر الثّلل: سوبكّت الرفوكت انل رع ر جد ل10 ) ت المقعات البدعا الر عار ال ال ئ س 28- الف لةةةةةةةة تةةةةةةةةؤثع وةةةةةةةةا ةةةةةةةةدم الر افةةةةةةةةا انجر ةةّ الجوةة لمرةةعال ر سوةة مةةّم جر ال ال ّس. اتمم 270 86.26% م ّعد 38 12.15% ن اتمم 5 1.59% 29- دم اسر دام ّف ال ا فة الرمّ ةا مع اللو. اتمم 226 72.21% م ّعد 45 14.38% ن اتمم 42 13.41% / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) 562 / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) 2022 / م1444 هـ 563 30- ن ةةةةةرغعب الرفوةةةةةكت انل رع رةةةةة ال لةةةةةت الجرةةةةةد لوردرس ةة لةةد نمةةا الةةا سةةّ ّت مرةةأخعال فةة .مرّاف ال اج ّت ال عسو انسرم ّرات اتمم 250 79.88% م ّعد 49 15.65% ن اتمم 14 4.47% 31- ن ت جةد خبةعال ّدكةة اّل ة لولو ة فةة اسر دام تق كّت الرفوكت انل رع ر. اتمم 218 69.65% م ّعد 46 14.69% ن اتمم 49 15.66% 32- ةةةةةةدم معا ةةةةةةّال راةةةةةةت القةةةةةةكت تعتيةةةةةةب ال مةةةةةةّيكت ل ةةةةةةةر ل توقةةةةةةة ال فو مةةةةةةةّت انشةةةةةةةرعا فةةةةةةة الةةةدر سفةةةد الرفوةةةكت انل رع رةةة خيةةةع ففةةةّل ف الرعاك الع حك اسخ قك. اتمم 267 85.31% م ّعد 42 13.42% ن اتمم 4 1.27% 33- الرةةةةةةةةةأثيع ال ةةةةةةةةةوب اللةةةةةةةةةف ر اّل ةةةةةةةةةيم الر تع ةد ارقلةّع انررعرةت ا الركةّر .ال رعاّف خ ل ول ال ّلعال اتمم 265 84.66% م ّعد 38 12.15% ن اتمم 10 3.19% ال ر العااع: مف لّت الرفوكت انل رع ر جد ل رلت11 ) ت المقعات البدعا الر عار ال ال ئ س 34- ةةدم جةة د معشةةد تفوك ةة ع جةةه كاكةة اسةةةةةةر دام الرق كةةةةةةّت ال ةةةةةةّ دال لورفوةةةةةةكت انل رع ر. اتمم 192 61.35% م ّعد 100 31.95% ن اتمم 21 6.70% 35- لو تلجكع اللو اجد ى الرفوكت انل رع ر. اتمم 158 50.48% م ّعد 110 35.15% ن اتمم 45 14.37% 36- ا م ّركّت ال ّدن ن ت ّسب ت فيا ّ اسرق ّل ال ّدال اّللعب ال ر ف ّل ك. عرض نتائج االستبانة و حليلها و اسيرها اتمم 175 55.92% م ّعد 97 30.99% ن اتمم 41 13.09% 37- ق ّت إدارس لقكّدات خيع اعك اتمم 63 20.12% / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) 563 / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) 2022 / م1444 هـ خيع مر لورل سع. م ّعد 156 49.85% ن اتمم 94 30.03% 38- ارقلةةةةةّع الركةةةةةّر ال رعاةةةةةةّف نفةةةةةد ّفقةةةةةّ مةةةةةةّم تلبيم الرفوكت انل رع ر ف ة انررعرت ردافرةةه ت ةة ل د ل الر افةةا ال ةةوكت نمةةّل . ال ّدال اّلم رال الم ك اتمم 254 81.16% م ّعد 51 16.29% ن اتمم 8 2.55% 39- ال م فةك ال ةعس م رةّ الرجة م القعفة وةا ال الةع العفك ة فة انررعرةت الرة تةةؤثع وا ال قعرات انل رع رك انمر ّرّت. اتمم 217 69.33% م ّعد 88 28.12% ن اتمم 8 2.55% 40- الر وم ّلك لرجري الب ك الر رك لرلبيم الرفوكت انل رع ر. اتمم 198 63.26% م ّعد 91 29.07% ن اتمم 24 7.67% 41- ّنفد الرفوكت انل رع ر بئّ الّدك. اتمم 129 41.21% م ّعد 103 32.91% ن اتمم 81 25.88% 42- ةةةةدم ا رةةةةةعال افةةةةةض الجرةةةةّت العسةةةة ك فةةةةة افض الةد ل اّللةرّدات ال حة ةة رعسةم الرفوةةكت انل رع رةة امةة رال ّمةة لبةةا جّف ةة ّو ر ر اتمم 213 68.05% م ّعد 89 28.44% ن اتمم 11 3.51% 43- ها ع جد اسركفّ م ّشع ف الرفوكت انل رع ر ؟ ع جد 70 22.36% نع جد 91 29.07% ّ ار 152 48.57% 44- مّ تقيكت ه ك الرفوكت ا ل رع ر لولّلب ؟ لعكا 130 41.53% مقب ل 153 48.89% جيد 30 9.58% / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) 564 / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) 2022 / م1444 هـ لمحة اريخية عن نشأة التعليم االلكتروني وماهومه نُفةةةد ممرةةة م الرفوةةةكت ا ل رع رةةة اأرةةةه اللعسقةةة لوةةةرفوت اّسةةةر دام ال سةةةّفط انل رع ركةةة فةةة وك رقا نمةّل ال فو مةّت سة الرفوةكت انل رع رة ور ة كّت خةعى ةّلرفوكت ةة افةد . عرض نتائج االستبانة و حليلها و اسيرها الرفوكت ال ر قا الرفوكت الذات الرفوكت انفرعال نفةة د تةةّر سخ الرفوةةكت انل رع رةة فةة وةةدانّت القةةعل الرّسةةع لةةع حةةية اسةةرف وت المةة ر ال ر ع ةة فةةة مجةةةّل الرفوةةكت لر ةةةا م ةةةا ال رةةب القةةةّف ية وةةةا الف وكةة الدراسةةك إ ّرةةت ال ةع هةة ال ةبب را تلةة ر مجةّنت الةةرفوت خّفة ااةةّل ال ةع الفّل كةة الثّركة ّرةةت ال ّجةة إلةةا ال فةة ل ل عةةية ال ج ةة د تةةدرسبرت ا ر ةةد فةة البدانةة وةةا تمةة سع الرةةدرس ّت الف عس اثرّ بع ال سّفا ال عفك ّلرومّ لك ل ل م ل ف مجّل الرفوكت انل رع ر ه مّ ّل مف ل اه لبا افد ال ع الفّل ك الثّرك لذا ن ّ الق ل إل ال ننّت ال ر دال اسمعس ك ّرت العافدال ف مجّل الرفوكت خيع الرقويدل إ ّرةت ه ةّ شةعاو مةّ وةية الجةك . الجّمفّت لغعض دمج ادخّل م ّدئ وت ال م ف الرفوت وةةد ت فةة سةةري كّت القةةعل ال ّلةة وةة ادر الرفوةةكت انل رع رةة مةةة خةة ل لةةع رمةة ص مبعمجةة وةةةا فةةم سلكةةةّت تؤسةة لورةةةدرسب الرةةةدرس و اسةةل ال ّسةةة حرةةةا ةةةّل لورفوةةةك ت . انل رع ر ف تو ال ق ه ك الرمّدن اّ ر ّره ج مة انلرمّد ال فعف د ر الرفوةةكت انل رع رةة الةة ا سةةوب وةةا ممرةة م الردرس ةةيية جفورةةت مةةّم ملةة و هةة ام ّركةة انسةةرغ ّ ةةة د رهةةت فةة ال جةةّل الرفوك ةة ل ةةة سةةع ّل مةةّ ثبةةت الف ةة مةةة توةة ال ةةّ ل هةة ارتقةةّع سةةفّر ال ر ل جكةةّ فةة توةة المرةةعال ف ةة ةةة ال ّجةة إلةةا الرمّ ةةا مةةع ال ةةرفوت فةة اسةةرعجّع ال فو مةةّت ةةّل للعافةةم اللةةعح مةةع م روةة اللةةعافج ال رفو ةة ال ةةبب .اةخع ف دم انسرغ ّ ة الردرس يية اد الف ا وا ال ن ل الرفوكت ا ل رع ر وثةع تمّ وكة فة ودانة ال مة الثةّر مةة القةةع ل الرّسةةع لةةع نسةةك ّ فةة ظرةة ر ال ّسةة الل مةة فةة ةةّم1977 الةةذل ةةّل الردل م ه ل ن ل تلغيوه م ّشع مة لبةا ال ةرميد م ةّ جفةا ال رر ةية فة مجةّل تلة سع وةةعامج الرفوةةكت انل رع رةة ل نق مةة ا ورأسةةك لّ ةةدال رفةةي لبةةعامجرت عةةرت تلةةغيورّ مةةة خةة ل . عرض نتائج االستبانة و حليلها و اسيرها ال ّس ظ ال و إل الذ الل ف و ل ل إن ال ّ لمحة اريخية عن نشأة التعليم االلكتروني وماهومه نُفةةةد ممرةةة م الرفوةةةكت ا ل رع رةةة اأرةةةه اللعسقةةة لوةةةرفوت اّسةةةر دام ال سةةةّفط انل رع ركةةة فةةة وك رقا نمةّل ال فو مةّت سة الرفوةكت انل رع رة ور ة كّت خةعى ةّلرفوكت ةة افةد . الرفوكت ال ر قا الرفوكت الذات الرفوكت انفرعال رس يية ة م ع اد الف ا وا ال ن ل الرفوكت ا ل رع ر وثةع تمّ وكة فة ودانة ال مة الثةّر مةة القةةع ل الرّسةةع لةةع نسةةك ّ فةة ظرةة ر ال ّسةة الل مةة فةة ةةّم1977 الةةذل ةةّل الردل م ه ل ن ل تلغيوه م ّشع مة لبةا ال ةرميد م ةّ جفةا ال رر ةية فة مجةّل تلة سع وةةعامج الرفوةةكت انل رع رةة ل نق مةة ا ورأسةةك لّ ةةدال رفةةي لبةةعامجرت عةةرت تلةةغيورّ مةةة خةة ل . ال ّس وا العخت مة ظر ر ال ّس إن ل ل لت ن ة وةا فةم الل ة ح الةذل ن ةفا إلكةه ال رمةةةية فةةة مجةةةّل الرفوةةةكت انل رع رةةة ا ةةةبب ال لةةة ت الم كةةة فةةة اخةةةر ل اللةةةع ّت ّال مة ف ل جرة ال انل رع ركة البةعامج هة مةّ جفةا انسةرمّدال مةة الرفوةكت ا ل رع رة حو ةة . سس ّ ارتمّع الر وم سرذا 565 / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) " ععفد تقعسع م ا اسمت ال ر دال لورعاك الفوت الثقّف الي رUNESCO , 2002 " الرل ر الرّرس لورفوكت انل رع ر ف ت و ا م ج ّةت: - . الجيا اس ل: را ال عاسو ّل نفر د وا ال ّدال ال لب بع ال عاس ت البعسدن ا ا - الجيا الثّر : را الروم س ل العادع سفر د وا ال ةّدال ال لب ة اسشةعر ال ة ال عفكةةةة الرفوةةةةكت ا ةةةةّ دال ال ّسةةةة اسلةةةةعاص ال دمجةةةة البةةةةل الروم سةةةة ر ا ا ةةةة . الرّت - الجيا الثّر : را الروم س ل العادع سفر د وا ال ةّدال ال لب ة اسشةعر ال ة ال عفكةةةة الرفوةةةةكت ا ةةةةّ دال ال ّسةةةة اسلةةةةعاص ال دمجةةةة البةةةةل الروم سةةةة ر ا ا ةةةة . الرّت - الجيةةةا الثّلةةةل: را ةةة ال سةةةّفط ال رفةةةددال سلةةة ا وةةةا انتمةةةّنت البكّركةةة ال ةةةؤت عات . عرض نتائج االستبانة و حليلها و اسيرها ال عفك اعامج اسل ّر الم ّعك - الج يةةا العااةةع: اسرا ةة ال عت ةة ال وةةا ا ررعرةةت سر ثةةا فةة الرفوةةكت ال ةةعل سفر ةةد وةةةا "ال سّفط ال رفددال الرمّ وك الر تق م ا ل العسّفا وا ش انتمّنتWWW " ه سةةةّفط إل رع ركةةة ن ت ام كةةة هةةةذا الجيةةةا عرةةةرت اةةةّسلعاص ال دمجةةة الرمّ وكةةة شةةة انتمّنت اّل بي تع ال ممةا الدراسة انفرعالة ال ر ةّت ال رةب انل رع ركة ل ا ةد البكّرّت د اللوب ال ّدث ال ّشعال. ال رد2018 ص296 .) ا فوائد التعليم االلكتروني 1 - .تقويا الر ّلكا 1 - .تقويا الر ّلكا 2 - .ال ف ل ف ال لت ال ّل ال ّسب 2 - .ال ف ل ف ال لت ال ّل ال ّسب 3 - . الفّل ك 4 - . و ّ مجر فّت داف ال ّرس 5 - ععفةةةةةةةع مةةةةةةةة اسةةةةةةةرث ّر اللةةةةةةةع ّت وةةةةةةة ا اللةةةةةةة الف ب تكةةةةةةة . مةةةةةةةّر ل ر ربيةةةةةةةعل 2019 28 ) 4 - . و ّ مجر فّت داف ال ّرس 5 - ععفةةةةةةةع مةةةةةةةة اسةةةةةةةرث ّر اللةةةةةةةع ّت وةةةةةةة ا اللةةةةةةة الف ب تكةةةةةةة . مةةةةةةةّر ل ر ربيةةةةةةةعل أنواع التعليم االلكتروني 1 - :الرفوةةكت ال رةة امة الةةذل نجفةةا ةة مةةة اللو ةة الردرس ةةيية مفةةّ فةة لةةت احةةد عر ةةّدل ل .ال فعف وا ال ط م ّ ن را مة وك الرفوت 2 - :الرفوكت خيع ال ر امة ّ ه ن را وا اللو إرج مرةّمرت الدراسةك الة ا اتة اّل لةت الةةةذل ع ف رةةةت مةةةة خةةة ل ان ر ةةةّد وةةةا البةةةعامج ال رةةة فعال وةةةا ا ررعرةةةت اسلةةةعاص ال دمج . و2021 ص86 .) ) 3 - :الرفوةةةكت ال ةةةدمج سلوةةةم وكةةةه الرفوةةةكت ال ةةةدمج سةةةرت اسةةةر دام الةةةرفوت الرقويةةةدل فةةة لّ ةةة المةة و ةةب م ةةددال اسةةر دام راةةت انتمةةّل انل رع رةة فةة الجةة اةخةةع مةةة رلةةل الرفوت. عرض نتائج االستبانة و حليلها و اسيرها الف يعل2011 ص25 .) 566 / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) 2022 / م1444 هـ أدوات التعليم االلكتروني د ات الرفوةةةكت انل رع رةةةة هةةة اسداال الرةةةة عةةةرت اسةةةةر دامرّ مةةةة لبةةةةا الردرس ةةة لغةةةةعض إنمةةةّل ال ةةةّدال الفو كةةة إلةةةا اللو ةةة بةةةع رةةة اع الرفوةةةكت انل رع رةةة ال رّحةةة تمةةة د ات الرفوكت انل رع ر ح ب ت ليت اسةر دام اسداال وةية رةعال وكة الرفوةكت الةرفوت ةّ مبةية :ف ادرّه 1 - د ات الرفوكت ال ر امة - ال ّدث ال ار الل مChatting .) - ال ةةؤت عات اأر ا رةةّ ال ةة عك ال ةة دال اّلمةة ر العسةة م) مج ةة ال قةةّ ) مؤت عات .) الميدع2 - د ات الرفوكت خي ع ال ر امة أدوات التعليم االلكتروني د ات الرفوةةةكت انل رع رةةةة هةةة اسداال الرةةةة عةةةرت اسةةةةر دامرّ مةةةة لبةةةةا الردرس ةةة لغةةةةعض إنمةةةّل ال ةةةّدال الفو كةةة إلةةةا اللو ةةة بةةةع رةةة اع الرفوةةةكت انل رع رةةة ال رّحةةة تمةةة د ات الرفوكت انل رع ر ح ب ت ليت اسةر دام اسداال وةية رةعال وكة الرفوةكت الةرفوت ةّ مبةية :ف ادرّه ة كت - ال ّدث ال ار الل مChatting .) - ال ةةؤت عات اأر ا رةةّ ال ةة عك ال ةة دال اّلمةة ر العسةة م) مج ةة ال قةةّ ) مؤت عات .) الميدع2 - د ات الرفوكت خي ع ال ر امة - ال ةةؤت عات اأر ا رةةّ ال ةة عك ال ةة دال اّلمةة ر العسةة م) مج ةة ال قةةّ ) مؤت عات .) الميدع2 - د ات الرفوكت خي ع ال ر امة - البعسد انل رع رE-mail . - البعسد انل رع رE-mail . - رقا ال ومّتF.T(File Transfer .) - ل ح ال لعاتBulletin Board).B.B - فم ّت ال سب ال ّوStatic Web Pages)S.W.P . س) - فم ّت الل الف ب تك / ال سب الرمّ وInteractive Web Pages)I.W.P . - ل ام ال دم / انفّدال ا ال ّ دالListserv . انتعا2019 ص97 .) :ّ ه ّ د ات ت ّهت ف و ّ ويئّت الرفوكت انل رع ر م ر - د ات ت ةةةّ د فةةة ت ةةة سة ال رةةة ى الرفوك ةةة : مثةةةا م الةةةع الةةةع ااط انجر ّعكةةة م الةةةع .ّالم ر الميدع ال د رّت ال س خيعه - فم ّت الل الف ب تك / ال سب الرمّ وInteractive Web Pages)I.W.P . عرض نتائج االستبانة و حليلها و اسيرها اا ) - فم ّت الل الف ب تك / ال سب الرمّ وtive Web Pages)I.W.Pاا - د ات ت ةةةّ د فةةة ت ةةة سة ال رةةة ى الرفوك ةةة : مثةةةا م الةةةع الةةةع ااط انجر ّعكةةة م الةةةع .ّالم ر الميدع ال د رّت ال س خيعه - اد ات ت ةةةةةةّ د فةةةةةة الر افةةةةةةا: تةةةةةةأت م وةةةةةة ل ظكمةةةةةة البعسةةةةةةد انل رع رةةةةةة مثةةةةةةا خدمةةةةةة Twitter .) - د ات ت ةةّ د فةة الرلةةبك : هةة خةةدمّت ت ةةّ د فةة راةةط ال رفو ةةية ال فو ةةية و ف ةةرت .ال فض لر ّدل ال بعات ال فو مّت .ال فض لر ّدل ال بعات ال فو مّت - د ات ت ةةةةّ د فةةةة فّ وكةةةة اسد ات ال ةةةةّاق : مثةةةةا اسةةةةر دام خ فةةةةّت ال الةةةةعRSS ) اسر دام العس مّت لر فكا ال مّدر ال روم . فّر2017 ص62 .) مة د ات الرفوكت انل رع ر وا فم را إدارال الرفوت - د ات ت ةةةةّ د فةةةة فّ وكةةةة اسد ات ال ةةةةّاق : مثةةةةا اسةةةةر دام خ فةةةةّت ال الةةةةعRSS ) اسر دام العس مّت لر فكا ال مّدر ال روم . فّر2017 ص62 .) مة د ات الرفوكت انل رع ر وا فم را إدارال الرفوت 1 - . المم ل الذ ك 2 - ال اج ةةّت انل رع ركةة : داال ت ةةة ال فوةةت مةةة قكةةّ م ةةر سّت الرم يةةع لةةدن ال عت لةة . اأهدال الرفوت المععك لودر 3 - ل ح ال قّ : م ّح لو ار وية ال فوت اللو اللو و ف رت.ال فض 4 - .انخر ّرات انل رع رك : داال لقكّ م عجّت الرفوكت مة خ ل انخر ّرات 567 / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) بة جم(جمل د )(ول)م 5 - ال ر ى الرمّ و : ه ال فو مّت ال فّرل الر عر رّ ال قعر الدراس الرة ترةدل الةةا ت قيةةم هةةدال تفوك كةة م لةة دال هةةذه ال فو مةةّت ال فةةّرل تقةةدم فةة شةة ّل مرفةةددال مّ ف لّلب امعل س ف س ف امعل. عرض نتائج االستبانة و حليلها و اسيرها انتعا2020 ص211 .) االختالف بين أنواع التعليم االلكتروني تارع ل ّ انخر فّت وية ر اع الرفوكت انل رع ر مةة خة ل ال مةّيكت الف ّفةع ال بي ة ف الجد ل درّه: و2021 ص88 ) الف مع الرفوكت انل رع ر الرفوكت الرقويدل ال ّدال الفو ك مة حيل ال ر ى الرم كت ) سو الفعض مرق مل ل تقويدن م د دال ر لك الج دال ثّور مرمّ ت قكّ ال رّفج توقّف ففب انحرمّم اّل فو مّت ّل مرمّ ت ال وم ال بك م م ّلك ال ف ّلك جدا مرمّ ت ال ع ر ّلك جدا مقيدال ان ر ّد وا ال م ّل جدا م د د رلّب ال ار و ر م و / الوك فعص ا وداع / انور ّر ّلك مرمّ ت ا أ يا 1 - .الرلجكع وا سّدال ال رفوت اقدراته ال ّف وا الرفوت ت ا م ؤ لك تفو ه 2 - عدفع ال رفوت وا ال ثّوعال. انسرق لك ل ط ال م 3 - .ن ج لو رفوت األ ن ل وثع فّ وك ثق و م ه ملّر ّ ف ف ع القعار 4 - ن ّ د ال رفوت وا تل سع ل ا د الرفوت ال ّفة اةه مةة لةدرال وةا الر بةؤ رةعح انسةئو ت لكج تو كر اسف ّر. 5 - ع لدى ال رفوت القدرال وا ال ل ال عاق الذاتكة مةة خة ل م رّافة معاجفة مةّ عةرت ارجّ ه مة هدال فّر2017 ص158 ) أهداف التعليم االلكتروني 1 - . ت ية م ر ى فّ وك الردرس يية سّدال ال بعال لدعرت ف إ داد ال اد الرفوك ك 2 - ال ف ل الا ممّدر ال فو مّت ت فيع ال ّدال الرفوك ك ام رترّ ا ل رع رك. 568 / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) 4 - .ال ّ دال ف فرت ال ّدال الرف م ورّ حيل ن رلكع اللّلب العج ع لودر ف ل لت 5 - سّدال ال اّسرغ ل ال لت ا ّ ع لدعرت القدرال وا ا وداع. عرض نتائج االستبانة و حليلها و اسيرها 6 - الر افةا مةع ال ؤس ةّت الفّل كة اإكة ال فة ل الةا ان ر ّدنة مةع توة ال ؤس ةّت لعفةع ال ر ى الفو ليع2017 ص80 .) معوقات التعليم اإللكتروني وسلبيا ه 1 - حةّج الوغة فةف اررّ: ل ة ل مفاةت ال فو مةةّت اسا ةّا ال رة افعال وةا شة انررعرةةت . ه اّلوغ انر وي س لذا فإل انسرمّدال ال ّمو سر ل مة رميب مة عرقة هذه الوغ 2 - خو. انررعرت مة العلّا 3 - . ال اج ال م ك مة جّرب اللو الردرس يية اتجّه الرفوكت انل رع ر 4 - . ّ اللف ر اّلف ل غكّ الرمّ ا انجر 5 - فف ا الرغييع ل ل انر ّل البكفره نن ب الرغييع وا مّ ا رّد وكةه نن ة ل لة اّت ّع سو م ّد وا نق سو ّ تجّه هذا الرغي.) يع مّ ت ا وة ال قّ م العاف 6 - مل ت مف لّت إدارس. 7 - مف لةةّت جةة د الميع سةةّت حيةةل تةةؤثع وةةا اسجرةة ال لةةّ ال ومّت سةةفّدال2015 ص241 - 243 .) 569 واقع التعليم االلكتروني في العراق شةةّع اررلةةّر ممرةة م الرفوةةكت انل رع رةة فةة الفةةعاب ةةد إ ةة ل حّلةة اللةة ارئ القمةة ى فعض ال اع اللّما افد سّدال ا فّاّت جعا تمل اةّ ر رةّ حيةل ا ة ت ال مة الفعاقكةة لةة ارال الرعاكةة الرفوةةكت الفةةّل ا ةةع رال إنجةةّد حوةة ل ل لةة و ةةدم د ام اللو ةة ا ةةبب إجةةةةعا ات ال جةةةةع المةةةة ةةةةدم لةةةةكّع ال ةةةة الدراسةةةةك وةةةةا اللو ةةةة ّرةةةةت ال قرعحةةةةّت ال و ل ف انررقّل . 2 أخذ من الموقع اإللكتروني لبرنامج االمم المتحدة االنمائي (- in - learning - stories/2020/09/e / https://www.iq.undp.org/content/iraq/ar/home iraq.html ) بتاريخ30 - 4 - 2021 . عرض نتائج االستبانة و حليلها و اسيرها اسر دام الرفوكت بع ال سّفط الرفوك ك مة خ ل ش انررعرت لةةت ن ةةة الرفوةةكت انل رع رةة ليةةد ال ةة ات انخيةةعال ل ةةة لوفةةعاب تجعاةة مةةع هةةذا ال مرةة م ل مة خ ل ملةع ع اوةة سةي ّ الةذل روقرةه م ا ة اسمةت ال ر ةدال لورعاكة الفوةت الثقّفة- م رةةب ع ر ةة الفةةعاب فةة ةةّم1 09 20 ةةّل هةةذا الرفةةّ ل مةةع ارال الرفوةةكت الفةةّل ال ةةل الفو مة جا تف س جة دال الرفوةكت وةا ج كةع ال ةر سّت د ةت اللةعاوّت وةية الجّمفةّت الفعاقك الجّمفّت خّرل الفعاب حيل ّرت ودان ال لع ع ف ثة ا جّمفةّت عاقكة هة 1 اخذ من الموقع االلكتروني لمكتب يونسكو العراق عبر الموقع (- office/single - this - office/about - http://www.unesco.org/new/ar/iraq view/news/launch_of_avicenna_virtual_campus_in_iraq/ ) بتاريخ30 - 4 - 2021 . 569 / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) 2022 / م1444 هـ / جمةل الداب( اجملدل1 ) العدد142 (أيلول) 2022 / م1444 هـ اغداد ال معال ف ح الدعة) رالرت مع ج ّمفة اوةة سةي ّ انفرعالةك فة م لقة اللةعب اس سط تت تدرسب مّ نقةّر18 تدرس ةكّ مةة الجّمفةّت ال رةّرال فة ةّل مةة لبةا ارافة خبةةعا مةةة مقةةع الي ر ةة الفةةّم م رةةب الي ر ةة فةة اغةةداد خ ةة مةةدراية مةةة مع ةة اوةةة سةةي ّ ال فعفةة فةة جّمفةة في دلاكةةّ رافةة مةةة جّمفةة الب لير كةة فةة فّل كّ اسةة ّركّ) ارال .الرفوكت الفّل ال ل الفو ف الج افع لدم وعرّمج اسمت ال ر دال ا ر ةّف حة ل الفةّلت فة ةّم2017 ملةع ع لرلبيةم راةّم الرفوةةكت انل رع رةة فةة جّمفةة ةةعا إ تةةت تةةدرسب الفةةّموية تلةة سع مرةةّراترت ف ةة ةةة ت سةةةةد سةةةةّتذال الجّمفةةةة اأحةةةةد ا اللعافةةةةم سةةةةّفا إدارال الرفوةةةةكت انل رع رةةةة تقّرةةةةّت إ ةةةةداد ال رةةة ى الرفوك ةةة الةةة ا ال رع رةةة وةةةا اثةةةع لةةة تةةةت تأسةةةك ل ل ةةةت م ةةةرر اةةةّلرفوكت انل رع ر ف الجّمف تل سع ل م م تف وذل ليرت انسرمّدال مةة هةذا ال لةع ع افةد ّتمل اّ ر رّ بع ال مّت ال ّف اّلج. عرض نتائج االستبانة و حليلها و اسيرها مف2 اا اغداد ال معال ف ح الدعة) رالرت مع ج ّمفة اوةة سةي ّ انفرعالةك فة م لقة اللةعب اس سط تت تدرسب مّ نقةّر18 تدرس ةكّ مةة الجّمفةّت ال رةّرال فة ةّل مةة لبةا ارافة خبةةعا مةةة مقةةع الي ر ةة الفةةّم م رةةب الي ر ةة فةة اغةةداد خ ةة مةةدراية مةةة مع ةة اوةةة سةةي ّ ال فعفةة فةة جّمفةة في دلاكةةّ رافةة مةةة جّمفةة الب لير كةة فةة فّل كّ اسةة ّركّ) ارال .الرفوكت الفّل ال ل الفو ف الج افع لدم وعرّمج اسمت ال ر دال ا ر ةّف حة ل الفةّلت فة ةّم2017 ملةع ع لرلبيةم راةّم الرفوةةكت انل رع رةة فةة جّمفةة ةةعا إ تةةت تةةدرسب الفةةّموية تلةة سع مرةةّراترت ف ةة ةةة ت سةةةةد سةةةةّتذال الجّمفةةةة اأحةةةةد ا اللعافةةةةم سةةةةّفا إدارال الرفوةةةةكت انل رع رةةةة تقّرةةةةّت إ ةةةةداد ال رةةة ى الرفوك ةةة الةةة ا ال رع رةةة وةةةا اثةةةع لةةة تةةةت تأسةةةك ل ل ةةةت م ةةةرر اةةةّلرفوكت انل رع ر ف الجّمف تل سع ل م م تف وذل ليرت انسرمّدال مةة هةذا ال لةع ع افةد ّتمل اّ ر رّ بع ال مّت ال ّف اّلج. مف2 الجودة الشاملة المبادئ واالعتماد وعالقتها بالتعليم العالي نفةةد إد ارد دن ةة ج رافةةد ف ةةعال الجةة دال اللةةّمو حيةةل رةة ر ارافةة لةةع رقلةة مةةة م ةةّدئ الج دال ت لج مّ عو م نجّد تلة سع ثقّفة الجة دال روةم وةا هةذه ال ةّدئ "جة هع الجة دال : ف الرفوكت" ترو ر دك ّ نأت 2 - . تب فو م الج دال اللّمو 4 - ّإرج .اس ّل ال اج ّت العب جدعدال ا 5 - .ت ية الج دال ا ررّجك خمض الر ّلكا ا 10 - .خوم ثقّف الج دال 11 - ت ية.الف وكّت 2 أخذ من الموقع اإللكتروني لبرنامج االمم المتحدة االنمائي (- in - learning - stories/2020/09/e / https://www.iq.undp.org/content/iraq/ar/home iraq.html ) بتاريخ30 - 4 - 2021 . 570 / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) 2022 / م1444 هـ عرمة الرفوةةكت انل رع رة ا رلو ةةّت شةع سّسةةك لرة فيع ال عكةة لة ّل الجةة دال : دكه معالبررّ مة هت هذه ال رلو ّت مّنأت 1 - ت فيع شع سّسك ف اللو ال ور قية ورذا ال ع مة الرفوكت ل ّل مدخ ت تفوك كة . م ّس 2 - ت لكط البعامج الرفوك ك وا ف ا . عرض نتائج االستبانة و حليلها و اسيرها ر اع ال فّرل ال فّفعال ال فو مّتك 3 - تةةةةة فيع شةةةةةع ر عكةةةةة فةةةةة الرفوةةةةةكت الةةةةةرفوت فةةةةة ال ةةةةةّدال الرفوك كةةةةة ال سةةةةةّفط الرفوك كةةةةة . الردرس ف ة البعمجكّت 4 - ت ميذ البعامج الر. فوك ك وا فم معاق دقكق 5 - تقيكت البعامج الرفوك ك ال ةرف و فةم سخةع ال ةرجدات الثقّدكة انجر ّعكة اسةر ص .الرغذن العاجف مة اجا ادخّل انف حّت الرل سع ا ن اأ ل ام رال م ر عال 6 - . تل سع دا ّ الريئ الردرس ك 7 - إ ةةّدال ال اةةع فةة ال اةةّم ا دارل الم ةة ت وةةكر الرفوةةكت انل رع رةة مةةة ةةا ا ّلةةّت الر تفعلا ت فيع ر عك الرفوكت لولو ال رد2018 ص298 .) : لغعض ت قيم ج دال الردرس يية نجب ل تر فع دد مة ال ّت لدعرت ه ّةت 1 - ال ّت الل مك : مةة خة ل امةر ال ع رة فة الرم يةع الرةأر فة إفةدار اسح ةّم ل ن رو الثق اّل م ل عرمرت اةخعسة سرقبورت ف ة ةة امةر مرةّرات انتمةّل .المفّل : لغعض ت قيم ج دال الردرس يية نجب ل تر فع دد مة ال ّت لدعرت ه ّةت 1 - ال ّت الل مك : مةة خة ل امةر ال ع رة فة الرم يةع الرةأر فة إفةدار اسح ةّم ل ن رو الثق اّل م ل عرمرت اةخعسة سرقبورت ف ة ةة امةر مرةّرات انتمةّل .المفّل 2 - ال مّنةةة الرعا سةةة الفو كةةة : هةةة ال فعفةةة اةةةّللعافم الرعا سةةة ال ّسةةة فةةة الرفّمةةةا مةةةع . اللو 2 - ال مّنةةة الرعا سةةة الفو كةةة : هةةة ال فعفةةة اةةةّللعافم الرعا سةةة ال ّسةةة فةةة الرفّمةةةا مةةةع . اللو 3 - ال مّنةّت ال ر كةة انتمةّلك : ل ن ةة ل الردرس ةة مةع اللو ةة ل عةدر ملةةّ ع اةخةةعسة . وكه ملّر اللو ا ال مثيعال لورم يع تقبا الرعاحّترت ال روم 4 - العغ ةة فةة الرفوةةكت ت ةةية م ةةر ى الرةةدرسب سلعارةةه م ةةة هةةت لةةا معت ةة و كةة تلةةجكع ال رّل الفو لرت ال م2017 ص169 .) 3 - ال مّنةّت ال ر كةة انتمةّلك : ل ن ةة ل الردرس ةة مةع اللو ةة ل عةدر ملةةّ ع اةخةةعسة . عرض نتائج االستبانة و حليلها و اسيرها الدافعك لورفوت مع عض ال ّدال العسق م م ال لولو 7 - ّ معا لا ف ّ الر ت ف لةع البعرةّمج الرفوك ة لغةعض ال فة ل إلكةه ا ةر ل مةة لبةا ل انحركّجّت ال ّف قا2014 ص393 - 395 .) طبيقات التعليم االلكتروني في مؤسسات التعليم العالي تقع تلبكقّت الرفوكت انل رع ر ل ة ويئّت الجيا الثّر سب2,0 ) سفد ال سب2,0 ) ع ّرال ة ت ق كّت ظرعت لجفا م الع انررعرت وثع دع ّمك كة تمّ وكة هة ال جة الثّركة مة ال سب حيل عر ة ال رميدعة مة هذه الرقّر ال دعث اّل لع ال لةّر ت ةّدل ال بةعات ال فو مّت مع ال ج ّت اسفعاد سر ي ال سب2,0 :ّ)ا مّفر دعدال م ر - خمّفر رمك : اخركّر لغ ال.رّا م ّرك الرفدعا ا لّف ال ذل - خمّفر شم س : ل مة خ ل د ات تلبكقّت ال ؤت عات ال عفكSkype . - خمّفر امعس : مثا مو مّت ال العRss . ) - خمّفر س عك : مة خ ل داال الرد سة الم تPodcast .) - رعافةةم جدعةةدال لو لةةّر لةةّ اس ل سةة لو لةةّر ية: إ إل ال ةةرميدعة هةةت مةةة عب ةة ل ال لع لك ال ّل مثا ال س ال عالWikipedia .) - تف س الرفوكت ا وداع: مثا م ع ّت ال ل وا سبيا ال ثّلGoogle .) - د ت انتمّل الرمّ وك : حيل تج ع اسفعاد ف ش ّت اجر ّعك مثاFacebook .) - تقويا الر ّلكا الف وك ال رفوك ك مقّرر اّل سب1,0 .) - ال ةر ل ال ةةع فة ال فةة ل إلةةا ال فو مةّت فةة ل لةةت ل م ةّل سر ةةت ا ةةر ى . ّ ّل مة اللّاع انجر - . اسر دام مج مر مة تلبكقّت الرفوكت انل رع ر - عض مقّرع الميدع بع تلبيمYoutube ) م2014 ص551 ) 5 - . مفّعيع الر ت ف البعمج مة لبا ال رفوت 6 - . عرض نتائج االستبانة و حليلها و اسيرها وكه ملّر اللو ا ال مثيعال لورم يع تقبا الرعاحّترت ال روم 3 - ال مّنةّت ال ر كةة انتمةّلك : ل ن ةة ل الردرس ةة مةع اللو ةة ل عةدر ملةةّ ع اةخةةعسة . وكه ملّر اللو ا ال مثيعال لورم يع تقبا الرعاحّترت ال روم 4 - العغ ةة فةة الرفوةةكت ت ةةية م ةةر ى الرةةدرسب سلعارةةه م ةةة هةةت لةةا معت ةة و كةة تلةةجكع ال رّل الفو لرت ال م2017 ص169 .) ا 4 - العغ ةة فةة الرفوةةكت ت ةةية م ةةر ى الرةةدرسب سلعارةةه م ةةة هةةت لةةا معت ةة و كةة تلةةجكع ال رّل الفو لرت ال م2017 ص169 .) 571 2022 / م1444 هـ / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) معايير الجودة في صميم المحتوى التعليمي الرق مي 1 - . الج دال الدل ف ال ر ى الفو 2 - .مرلو ّت العس ّلم يية ال رمية ف تم كت البعامج 3 - م ّدئ الرم كت مة خ ل ت ظكا اسل ال ةعض الة ر المة ر المة ت لقلةّت . الميدع ت ر ل وا سّليب جذ انرر ّه ل ت ل خواك اللّش معس لوفية 4 - ت دعةةةد اسهةةةدال ال لةةة ع اسرلةةةل ال رةةةّم الرفوك كةةة تفةةةد مةةةة مفةةةّعيع الرمةةة كت الرعاةة ل لو ةة رج ف ةة ةةة الرةةدرس ّت انهر ةةّم اّلرغذنةة العاجفةة مةةع لةةع رال اسةةرف ّل . سّليب لو ّ دال . سّليب لو ّ دال 5 - . مفّعيع الر ت ف البعمج مة لبا ال رفوت 6 - . عرض نتائج االستبانة و حليلها و اسيرها الدافعك لورفوت مع عض ال ّدال العسق م م ال لولو 7 - ّ معا لا ف ّ الر ت ف لةع البعرةّمج الرفوك ة لغةعض ال فة ل إلكةه ا ةر ل مةة لبةا ل انحركّجّت ال ّف قا2014 ص393 - 395 .) طبيقات التعليم االلكتروني في مؤسسات التعليم العالي تقع تلبكقّت الرفوكت انل رع ر ل ة ويئّت الجيا الثّر سب2,0 ) سفد ال سب2,0 ) ع ّرال ة ت ق كّت ظرعت لجفا م الع انررعرت وثع دع ّمك كة تمّ وكة هة ال جة الثّركة مة ال سب حيل عر ة ال رميدعة مة هذه الرقّر ال دعث اّل لع ال لةّر ت ةّدل ال بةعات ال فو مّت مع ال ج ّت اسفعاد سر ي ال سب2,0 :ّ)ا مّفر دعدال م ر - خمّفر س عك : مة خ ل داال الرد سة الم تPodcast .) - رعافةةم جدعةةدال لو لةةّر لةةّ اس ل سةة لو لةةّر ية: إ إل ال ةةرميدعة هةةت مةةة عب ةة ل ال لع لك ال ّل مثا ال س ال عالWikipedia .) - تف س الرفوكت ا وداع: مثا م ع ّت ال ل وا سبيا ال ثّلGoogle .) - د ت انتمّل الرمّ وك : حيل تج ع اسفعاد ف ش ّت اجر ّعك مثاFacebook .) - تقويا الر ّلكا الف وك ال رفوك ك مقّرر اّل سب1,0 .) - ال ةر ل ال ةةع فة ال فةة ل إلةةا ال فو مةّت فةة ل لةةت ل م ةّل سر ةةت ا ةةر ى . ّ ّل مة اللّاع انجر - . اسر دام مج مر مة تلبكقّت الرفوكت انل رع ر - عض مقّرع الميدع بع تلبيمYoutube ) م2014 ص551 .) 572 / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) 2022 / م1444 هـ عةةرت إرلةةّ تلبكقةةّت الرفوةةكت انل رع رةة اّسةةر دام لغةة تعميةة الةة ر ال رةةةعااطHTML ) هة اخرمةّر لةةHyper Text Markup Language ) ت ةر دم فة و ةّ ةا البةعامج : ال ّهج الرفوك ك تدار وك الرفوكت ا ل رع ر تلبكقّته فم راّمية - راةّم إدارال الةرفوتLMS ) هةة ع ةّرال ةة وعرةةّمجSoftware فة ت لو ةّ دال فةة إدارال مرّافة تقيةكت الرةدرسب الرفوةكت ال ةر ع ج كةع اسرلةل فة الجّمفةّت معاوة الرةةدرسب . عرض نتائج االستبانة و حليلها و اسيرها ه إحدى م ر دثّت الرفوكت انل رع ر - راّم إدارال ال ر ى الرفوكCMS ) ه مج مةة البعمجكةّت الرة ت ة ج ورمة كت ت دعل ل الع ال سب الرلبكقّت ال رفددال ا ل سّفط و2021 ص208 .) مة تلبكقّت الرفوكت انل رع ر ف مؤس ّت الرفوكت الفّل 1 - البعسةةةةةد انل رع رةةةةةe-mail ): هةةةةة ع ةةةةةّرال ةةةةةة ت ةةةةةّدل العسةةةةةّفا ال ثةةةةةّفم اّسةةةةةر دام ال ّس مةة خة ل شة ا ررعرةت سفةد البعسةد ا ل رع رة ال لة ال ان لةا فة الرفوةكت ل لت ع جد البعسةد ا ل رع رة ل ةّ جةدت شة انررعرةت مةة مبةعرات اسةر دام البعسةد :انل رع ر ف الرفوكت عةةرت إرلةةّ تلبكقةةّت الرفوةةكت انل رع رةة اّسةةر دام لغةة تعميةة الةة ر ال رةةةعااطHTML ) هة اخرمةّر لةةHyper Text Markup Language ) ت ةر دم فة و ةّ ةا البةعامج : ال ّهج الرفوك ك تدار وك الرفوكت ا ل رع ر تلبكقّته فم راّمية - راةّم إدارال الةرفوتLMS ) هةة ع ةّرال ةة وعرةةّمجSoftware فة ت لو ةّ دال فةة إدارال مرّافة تقيةكت الرةدرسب الرفوةكت ال ةر ع ج كةع اسرلةل فة الجّمفةّت معاوة الرةةدرسب . ه إحدى م ر دثّت الرفوكت انل رع ر ت إ - راّم إدارال ال ر ى الرفوكCMS) ه مج مةة البعمجكةّت الرة ت ة ج ورمة كت ت دعل ل الع ال سب الرلبكقّت ال رفددال ا ل سّفط و2021 ص208 .) ا مة تلبكقّت الرفوكت انل رع ر ف مؤس ّت الرفوكت الفّل 1 - البعسةةةةةد انل رع رةةةةةe-mail ): هةةةةة ع ةةةةةّرال ةةةةةة ت ةةةةةّدل العسةةةةةّفا ال ثةةةةةّفم اّسةةةةةر دام ال ّس مةة خة ل شة ا ررعرةت سفةد البعسةد ا ل رع رة ال لة ال ان لةا فة الرفوةكت ل لت ع جد البعسةد ا ل رع رة ل ةّ جةدت شة انررعرةت مةة مبةعرات اسةر دام البعسةد :انل رع ر ف الرفوكت - .سع ف ل العسّل مة ل م ّل ف الفّلت إ - .ووم م م لإلرسّل انسرق ّل - دم ج د سكط وية ال عسا ال رقبا م ّ عؤدل إلا . إلغّ ال اج ا دارس ال م ك - ّإم ّرك تف كت العس. ل ال احدال وا وثع مة جر - .ووم م م لإلرسّل انسرق ّل - دم ج د سكط وية ال عسا ال رقبا م ّ عؤدل إلا . عرض نتائج االستبانة و حليلها و اسيرها إلغّ ال اج ا دارس ال م ك - ّإم ّرك تف كت العس. ل ال احدال وا وثع مة جر - .إم ّرك حم ل ال رميد وا العسّل ف ال لت الذل ع ّس ه ا - .إم ّرك حم ل ال رميد وا العسّل ف ال لت الذل ع ّس ه ا - إم ّرك راط مومّت إلّدك اّلبعسد ا ل رع ر مثا ال م ص الم ر اسش ّل سةفّدال 2015 ص154 .) - إم ّرك راط مومّت إلّدك اّلبعسد ا ل رع ر مثا ال م ص الم ر اسش ّل سةفّدال 2015 ص154 .) 2 - :ّمج تلبكقّت شع ج جا لورفوكت م ر - م ع ال لGoogle . - م ع ال لGoogle . - م ع ال لGoogle . - خدم الرعجGoogle Translate . - ال ّا انل رع ركGoogle Drive . - خدم انتمّل الميدع سGoogle Meet . - المما الدراسGoogle Classroom . - ّال ّ ل ال ّف اف ا انسر ّر ت ويورGoogle Forms . - ّال ّ ل ال ّف اف ا انسر ّر ت ويورGoogle Forms . - ملغا الر جي ت ال عفك البل الYouTube . 3 - مج ةةة ال مةةة ال ربكةةة مةةةة شةةة ع مّن ع سةةة فت دةةةك الرةةة ت ةةةت مج ةةة مةةةة البعامج ال ربكWord, Excel, PowerPoint .) 573 2022 / م1444 هـ / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) 4 - . ّ سّفا الر افا انجر خطوات إقامة التعليم االلكتروني في المؤسسات التعليمية 1 - . ت فيع ال ّدر الذل نج ع وية ال بعتية الردرس الرق كّت ال دعث 2 - إدارال ح و ت عك شعح ال لع. عرض نتائج االستبانة و حليلها و اسيرها ع لدى ال رميدعة لبكّل اه يره ربكفره 3 - .) إ داد تجري الب ك الر رك لو لع ع ال رو مّت ال ّدن الرق ك 4 - تدرسب اللو الردرس يية وا اسر دام الرق كّت الرلبكقّت ال ا م ر ع ّمع2015 ص143 .) التقانات واألنظمة المستعملة في مجال التعليم االلكتروني 1 - وعرةةّمج سةةب سةة تةةWebCT :) ن ةةر دم مةةة لبةةا مّنقةةّر سنل ال ؤس ةةّت الرفوك كةة ّفةة وثةةع مةةة سةة فية د لةة عةةد ت رافةة لةةع لغةة م رةةّ ا رجوي سةة الفعاكةة ل اةةعا ل ةة نقدمةةه مةةة ويئةة خمةة اةةّسد ات مةةة ودانةة إ ةةداد ال قةةعر لرع ي ةةه وةةا ال اةةّم حرةةا فرةةعال .الرفوت 2 - وعرةةّمج وةة وةة ردBlackboard): نفةةد مةةة لةة ى اسرا ةة حيةةل نقةةدم فةةعص تفوك كةة ّمر مة خ ل ع ال اج الف افةم الرة ت اجةه ال ؤس ةّت الرفوك كة س رةّ ن ة اّل ع ر لّوويره لورل سع الر سع عد ت البعرّمج الوغّت انرجوي س الفعاك انس ّرك . عرض نتائج االستبانة و حليلها و اسيرها اننلّلك 3 - وعرةةّمج د يةةDokeos): ن ةةر دم مةةةة لبةةةا1200 مؤس ةةة تفوك كةة فةةة65 د لةةة س ةةةة لولّلةةةب ل ع لةةةع م رةةة ى تفوك ةةة ةةةّل الجةةة دال ت ةةةّرسة تمّ وكةةة مر افةةةم مةةةع SCORM عةةةةد ت34 لغةةةة مةةةةّ لوغةةةة الفعاكةةةة فقةةةةد تةةةةت تفعسةةةةب مّنقةةةةّر60.2 مةةةةة% ال رّ 3 - وعرةةّمج د يةةDokeos): ن ةةر دم مةةةة لبةةةا1200 مؤس ةةة تفوك كةة فةةة65 د لةةة س ةةةة لولّلةةةب ل ع لةةةع م رةةة ى تفوك ةةة ةةةّل الجةةة دال ت ةةةّرسة تمّ وكةةة مر افةةةم مةةةع SCORM عةةةةد ت34 لغةةةة مةةةةّ لوغةةةة الفعاكةةةة فقةةةةد تةةةةت تفعسةةةةب مّنقةةةةّر60.2 مةةةةة% .البعرّمج 4 - وعرّمج ت تعATutor :) ن رّ هذا البعرّمج اإم ّركة الر ةدعل الرغيةع ال ةعسع لو اجرةّت مة لبا اللو سد ت 30 .لغ م ر و الرعج الفعاك مفعا اّل ّما 5 - وعرةةةّمج مةةة دلMoodle): نُفةةةد مةةةة هةةةت ال مةةةّت ال ةةةرف و فةةة الفةةةّلت هةةة وعرةةةّمج حدعل ممر ح ال مدر ست ر اسرلل الرفوك ك ربت ووغPHP "" ه لغ وعمجك حةعال ممر ح ال مدر مجّرك انسرف ّل مم عال ت رف ا ف تلة سع تلبك قةّت ال سةب هة لغة خّفةةة اةةةّل الع الدع ّمك كةةة تةةةد ت مّنقةةةّر30 لغةةة رةةة رت هةةةذه ال مةةة مةةةة لبةةةا " انسرعالMartin Dougiamas " سقدم وعرّمج م دل خدمّت ل ّ نقّر40000 ال رّلةةب ل ةةا جّمفةة س ةةت رو ةة عر و ةة ل70 لغةة مةةة138 د لةة لةةذا نفربةةع البعرةةّمج ان ل فةة الرفوةةكت ان ل رع رةة حيةةل عةةدعع را ةة ال قةةعرات را ةة الرفوةةكت فةةّب اسةةر دام هذا البعرّمج لرما الا الرلبكقّت الر دسك الرق ك و2021 ص218 .) 574 / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) 6 - وعرةةةةةّمج ا دمةةةةة دEdmodo ): هةةةةة حةةةةةد تلبكقةةةةةّت انررعرةةةةةت مةةةةةة م مةةةةةّت الر افةةةةةا ّانجر ةةةةةّ ال ممةةةةة لورفوةةةةةكت ن ةةةةةّ د اللو ةةةةة فةةةةة انسةةةةةرمّدال مةةةةةة سةةةةة فا ا ةةةةة م انجر ّعك لرف س الرفوت ف إ داد المم ل الدراسك . عرض نتائج االستبانة و حليلها و اسيرها فّر2017 ص101 .) جارب وا جاهات الجامعات الدولية والعربية في مجال التعليم االلكتروني 1 - الرجعا اسمعس ك: تفد تق ك ال فو مّت لدى فةّرف القةعار فة ا دارال اسمعس كة مةة هةت ست ل ّنّ ف الرفوكت اسمعس ارلوم الرفوكت انل رع ر ف ّم1995 حيل او وةت امعس ّ ج كع خللرّ لرلبكقه ه ّ دال ملّرسع رافدال ف مجّل الرفوكت انل رع ر مثةا و ت مي سّتّ ردع تDyna Book LOBO ) تررت ج كفرّ ور فيع ش اسف . مة ال اسيب سرت تغذعررّ اّلبعمجكّت الرفوك ك 2 - الرجعاةة اسسةةرعالك: تجعاةة الرفوةةكت انل رع رةة مرمّ تةة مةةة ننةة إلةةا خةةعى ل جةة د ارال الرعاك الرفوكت ف ا نن فم نن ف ر رسّ دخوت هذه الرق كة فة ةّم1996 تفة د مةةة الرجةةّر المعسةةدال وةةا م ةةر ى الفةةّلت مةةة حيةةل ال ةةع اللةة لك حرةةا اشةةّد وةةذل .) رفك شع مّن ع س فت وا خير 3 - : الرجعاة ال دنة ّرخةت ال ةّح ال بيةعال الرة تر رةع ورةّ ةدا و فةمر وبةع ثةّر د لة فة الفّلت إن ل هذا اسمع لت نق حّف مّم الرفوت انل رع ر تفد مة هت ث ا د ل ف مجّل الرفوكت الجّمف ل ل الجّمفّت ال دن لرّ تجّر مر ي ال ف الرفوكت ا ل رع ر إ ّ سةر ت فة تمة كت وعمجكةّت مر ية ال ت ةة مفةد ممة ت ال ةّدال الرفوك كة مةة لةفر وةةا ا ررعرةةت الرفّمةةا مفرةةّ ا ةةر ل تفربةةع ةةدا رافةةد لورفوةةكت ةةة افةةد سةة ا داخةةا خّرل دا. ليع2017 ص169 - 173 .) 4 - : الرجعا البعسلّرك تفةد مةة الةد ل ال رقدمة فة اسةرف ّل را ة الةرفوت ا ل رع رة حيةل رلأت ال م البعسلّرك ّم2002 وعرّمج ملرع وية جّمف هيعتة ات هيئة تلة سع انلرمّد ال و ف اس رو دا لعاط الجّمفّت ال ر ي ال فيرّ اّلل ف ا ر ّ الفّلت ّإ نلرع فير60 ل رّلب مة الج ية مة20 .د ل ف الفّلت 5 - الرجعا اسل ّرك: ت رو وبع ملع ع مرل ر لوعاط ال سو وية ال حدات الرفوك كة تةت رالرةةّ اّللةة ّت الفّل كةة تريةةع ال جةةّل لر ةةّدل ال فو مةةّت دك ةةّ وي رةةّ انسةةرمّدال مةةة توةة الل ّت ف ت فيع ال فو مةّت ال ظكاكة تلة ر اسمةع لكلة ا الرفوةكت انل رع رة الرمةع . عرض نتائج االستبانة و حليلها و اسيرها وا تفوكت ّر ال ة رركج ال ثّف ال ّرك تقّر ال دل 6 - : الرجعاةةةة ال ةةةة سدن تفةةةةد ال ةةةة سد مةةةةة وثةةةةع الةةةةد ل تقةةةةدمّ فةةةة مجةةةةّل الرفوةةةةكت انل رع رةةةة نمر ورةةّ و كةة ت ركةة ل سةة ت ةةرف ا تقّرةةّت ّلكةة لةةد سةة قت ثيةةعا مةةة الةةد ل فةة هةةذا ال جةّل لرةذا تفربةع رافةةدال قكّدنة فة هةةذا ال ة ّر حيةل ترةةرت ال مة اهر ّمةّ اةةّلرفوكت 6 - وعرةةةةةّمج ا دمةةةةة دEdmodo ): هةةةةة حةةةةةد تلبكقةةةةةّت انررعرةةةةةت مةةةةةة م مةةةةةّت الر افةةةةةا ّانجر ةةةةةّ ال ممةةةةة لورفوةةةةةكت ن ةةةةةّ د اللو ةةةةة فةةةةة انسةةةةةرمّدال مةةةةةة سةةةةة فا ا ةةةةة م انجر ّعك لرف س الرفوت ف إ داد المم ل الدراسك . فّر2017 ص101 .) جارب وا جاهات الجامعات الدولية والعربية في مجال التعليم االلكتروني 1 - الرجعا اسمعس ك: تفد تق ك ال فو مّت لدى فةّرف القةعار فة ا دارال اسمعس كة مةة هةت ست ل ّنّ ف الرفوكت اسمعس ارلوم الرفوكت انل رع ر ف ّم1995 حيل او وةت امعس ّ ج كع خللرّ لرلبكقه ه ّ دال ملّرسع رافدال ف مجّل الرفوكت انل رع ر مثةا و ت مي سّتّ ردع تDyna Book LOBO ) تررت ج كفرّ ور فيع ش اسف . مة ال اسيب سرت تغذعررّ اّلبعمجكّت الرفوك ك 2 - الرجعاةة اسسةةرعالك: تجعاةة الرفوةةكت انل رع رةة مرمّ تةة مةةة ننةة إلةةا خةةعى ل جةة د ارال الرعاك الرفوكت ف ا نن فم نن ف ر رسّ دخوت هذه الرق كة فة ةّم1996 تفة د مةةة الرجةةّر المعسةةدال وةةا م ةةر ى الفةةّلت مةةة حيةةل ال ةةع اللةة لك حرةةا اشةةّد وةةذل .) رفك شع مّن ع س فت وا خير 3 - : الرجعاة ال دنة ّرخةت ال ةّح ال بيةعال الرة تر رةع ورةّ ةدا و فةمر وبةع ثةّر د لة فة الفّلت إن ل هذا اسمع لت نق حّف مّم الرفوت انل رع ر تفد مة هت ث ا د ل ف مجّل الرفوكت الجّمف ل ل الجّمفّت ال دن لرّ تجّر مر ي ال ف الرفوكت ا ل رع ر إ ّ سةر ت فة تمة كت وعمجكةّت مر ية ال ت ةة مفةد ممة ت ال ةّدال الرفوك كة مةة لةفر وةةا ا ررعرةةت الرفّمةةا مفرةةّ ا ةةر ل تفربةةع ةةدا رافةةد لورفوةةكت ةةة افةةد سةة ا داخةةا خّرل دا. عرض نتائج االستبانة و حليلها و اسيرها ليع2017 ص169 - 173 .) 4 - : الرجعا البعسلّرك تفةد مةة الةد ل ال رقدمة فة اسةرف ّل را ة الةرفوت ا ل رع رة حيةل جارب وا جاهات الجامعات الدولية والعربية في مجال التعليم االلكتروني 1 - الرجعا اسمعس ك: تفد تق ك ال فو مّت لدى فةّرف القةعار فة ا دارال اسمعس كة مةة هةت ست ل ّنّ ف الرفوكت اسمعس ارلوم الرفوكت انل رع ر ف ّم1995 حيل او وةت امعس ّ ج كع خللرّ لرلبكقه ه ّ دال ملّرسع رافدال ف مجّل الرفوكت انل رع ر مثةا و ت مي سّتّ ردع تDyna Book LOBO ) تررت ج كفرّ ور فيع ش اسف . مة ال اسيب سرت تغذعررّ اّلبعمجكّت الرفوك ك س ت 2 - الرجعاةة اسسةةرعالك: تجعاةة الرفوةةكت انل رع رةة مرمّ تةة مةةة ننةة إلةةا خةةعى ل جةة د ارال الرعاك الرفوكت ف ا نن فم نن ف ر رسّ دخوت هذه الرق كة فة ةّم1996 تفة د مةةة الرجةةّر المعسةةدال وةةا م ةةر ى الفةةّلت مةةة حيةةل ال ةةع اللةة لك حرةةا اشةةّد وةةذل .) رفك شع مّن ع س فت وا خير 3 - : الرجعاة ال دنة ّرخةت ال ةّح ال بيةعال الرة تر رةع ورةّ ةدا و فةمر وبةع ثةّر د لة فة الفّلت إن ل هذا اسمع لت نق حّف مّم الرفوت انل رع ر تفد مة هت ث ا د ل ف مجّل الرفوكت الجّمف ل ل الجّمفّت ال دن لرّ تجّر مر ي ال ف الرفوكت ا ل رع ر إ ّ سةر ت فة تمة كت وعمجكةّت مر ية ال ت ةة مفةد ممة ت ال ةّدال الرفوك كة مةة لةفر وةةا ا ررعرةةت الرفّمةةا مفرةةّ ا ةةر ل تفربةةع ةةدا رافةةد لورفوةةكت ةةة افةةد سةة ا داخةةا خّرل دا. عرض نتائج االستبانة و حليلها و اسيرها ) لغعض اسرفعاض م ّلعاترت وا اللو 10 - :تجعاةةةة د لةةةة للةةةةع لةةةةّم ال جوةةةة اس وةةةةا لورفوةةةةكت فةةةة د لةةةة للةةةةع ورةةةة فيع ال رةةةة ى ا ل رع ر مثا ال رب ا ل رع رك ال مّدر الرفوك ك الر ت ر ل وةا ر ااةط ل مة ّدر سّفط وا ش ا ررعرت ه مةّ ثةع وةا جة دال الرفوةكت الة ا انجةّو حيةل احروةت للع ال ع العااع وا م ر ى الفّلت ف ج دال الرفوكت لفّم2015 - 2016 . 11 - : الرجعاةة ال ةةف دن ن ثةةا الرفوةةكت انل رع رةة فةة مقدمةة اس لكةةّت لةةدعرّ ح ةةب تقعسةةع شع ويعس ل) ب ت فيع خدمّت الرفوكت ف الفّلت الذل عؤ د األ حجت ا رمّب وا ت ل جكّ الرفوكت نأت ل ة الرم كا اس وا ّل كّ لّمت ال ف دن افقد مذ عال تمّهت مةةع شةةع الرفوةةكت الرةةدرسب ا ل رع ر سةةك ّر ر) نقر ةة ور سةةا ّفةة ال ةةّهج الدراسةةك الا ر خ إل رع رك ة رعسم وعرّمج سك ّر ر الّذل عر ي افدال م انّ ه ر: - سةةر ل الرمةةةمج لج كةةع ال قةةةعرات مةةع إم ّركةةة ال ةةل الر ويةةا ثةةةعا ال ةةّدال الفو كةة مةةة خةةة ل م ع ةةةّت د ات ا ةةةل البعرةةةّمج تةةة فيع مج ةةة مةةةة ال ةةةّ دات مثةةةا ال ةةةعافط . الذه ك الميدع 9 - تجعاةة د لةة ا مةةّرات:الفعاكةة ال ر ةةدال ّرةةت وةةدانّت الرفوةةكت انل رع رةة مةةة خةة ل تب ةة ملةع ع تلةة سع م ةةّهج لرفوةةكت مةةّدال ال ّسة اةلةة لو عحوةة الثّر سةة ةةّم1990 الةةا ال ا ر ةةدت جّمفةة ا مةةّرات اّ ر ةةّد اةع ةةّد جرةةّ رسةة فةة الرفوةةكت الفةةّل وةةدع ةةة ال ّسب اةل ال ل اذل ف ج اللّلةب ا لجةّمف ن ةر دم وعرةّمج الةب وة رد ا ةا سر ل ذل اسر دام وعرّمجApple Tv . عرض نتائج االستبانة و حليلها و اسيرها وا تفوكت ّر ال ة رركج ال ثّف ال ّرك تقّر ال دل 575 / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) 2022 / م1444 هـ انل رع رةةة وةةةت ال ر ةةة لوريئةةة ال ةةة سدن لورفوةةةكت ةةةة افةةةد الرةةة تأس ةةةت ةةةّم1999 ّ مر ررةةةّ د ةةةت الرفوةةةكت ا ل رع رةةة فةةة الجّمفةةةّت سر ةةةع لرةةة31 ّ وكةةة ج مفةةة . وةةةّف 2009 ص105 - 106 .) انل رع رةةة وةةةت ال ر ةةة لوريئةةة ال ةةة سدن لورفوةةةكت ةةةة افةةةد الرةةة تأس ةةةت ةةةّم1999 ّ مر ررةةةّ د ةةةت الرفوةةةكت ا ل رع رةةة فةةة الجّمفةةةّت سر ةةةع لرةةة31 ّ وكةةة ج مفةةة . وةةةّف 2009 ص105 - 106 .) 7 - : الرجعا المي ك حققت المية لم ال ر عك ف مجّل الرفوكت انل رع ر ت روة ةة اقكة الد ل ل ررّ حققت تقدم ف مفدنت ال ف ت مةة الةد ل ال رقدمة تلة ر الرفوةكت انل رع رةة م ةةذ الفةةّم1999 الةة ا بيةةع ت جةةد شةةع ّت بيةةعال تف ةةا فةة مجةةّل الرفوةةكت انل رع رةة مةةة اللةةع ّت الف لةة الرةة تف ةةا فةة المةةية شةةع وةة وةة رد اسمعس كةة نما دد ال رميدعة مة الرفوكت ا ل رع ر إلا100 .موي ل 8 - : الرجعاةة الر دنةة تفةة د مةةة الةةد ل العافةةدال فةة فةة ّ البعمجكةةّت م ةةذ ودانةة الفقةةد اسخيةةع لورفةةةةّ ل ال ث ةةةةع ةةةةّم2003 وةةةةية شةةةةع مّن ع سةةةة فت الجّمفةةةةّت اللةةةةع ّت الر دنةةةة لفت مج مة ال لّرسع لد ت ت ل جكّ ال فو مّت انتمّنت فة الر ةد. الفّر 2015 ص42 - 43 .) 9 - تجعاةة د لةة ا مةةّرات:الفعاكةة ال ر ةةدال ّرةةت وةةدانّت الرفوةةكت انل رع رةة مةةة خةة ل تب ةة ملةع ع تلةة سع م ةةّهج لرفوةةكت مةةّدال ال ّسة اةلةة لو عحوةة الثّر سةة ةةّم1990 الةةا ال ا ر ةةدت جّمفةة ا مةةّرات اّ ر ةةّد اةع ةةّد جرةةّ رسةة فةة الرفوةةكت الفةةّل وةةدع ةةة ال ّسب اةل ال ل اذل ف ج اللّلةب ا لجةّمف ن ةر دم وعرةّمج الةب وة رد ا ةا سر ل ذل اسر دام وعرّمجApple Tv . عرض نتائج االستبانة و حليلها و اسيرها ليع2017 ص169 - 173 .) يع لر ص9 ) 4 - : الرجعا البعسلّرك تفةد مةة الةد ل ال رقدمة فة اسةرف ّل را ة الةرفوت ا ل رع رة حيةل رلأت ال م البعسلّرك ّم2002 وعرّمج ملرع وية جّمف هيعتة ات هيئة تلة سع انلرمّد ال و ف اس رو دا لعاط الجّمفّت ال ر ي ال فيرّ اّلل ف ا ر ّ الفّلت ّإ نلرع فير60 ل رّلب مة الج ية مة20 .د ل ف الفّلت 5 - الرجعا اسل ّرك: ت رو وبع ملع ع مرل ر لوعاط ال سو وية ال حدات الرفوك كة تةت رالرةةّ اّللةة ّت الفّل كةة تريةةع ال جةةّل لر ةةّدل ال فو مةةّت دك ةةّ وي رةةّ انسةةرمّدال مةةة توةة الل ّت ف ت فيع ال فو مةّت ال ظكاكة تلة ر اسمةع لكلة ا الرفوةكت انل رع رة الرمةع . وا تفوكت ّر ال ة رركج ال ثّف ال ّرك تقّر ال دل 6 - : الرجعاةةةة ال ةةةة سدن تفةةةةد ال ةةةة سد مةةةةة وثةةةةع الةةةةد ل تقةةةةدمّ فةةةة مجةةةةّل الرفوةةةةكت انل رع رةةةة نمر ورةةّ و كةة ت ركةة ل سةة ت ةةرف ا تقّرةةّت ّلكةة لةةد سةة قت ثيةةعا مةةة الةةد ل فةة هةةذا ال جةّل لرةذا تفربةع رافةةدال قكّدنة فة هةةذا ال ة ّر حيةل ترةةرت ال مة اهر ّمةّ اةةّلرفوكت 4 - : الرجعا البعسلّرك تفةد مةة الةد ل ال رقدمة فة اسةرف ّل را ة الةرفوت ا ل رع رة حيةل رلأت ال م البعسلّرك ّم2002 وعرّمج ملرع وية جّمف هيعتة ات هيئة تلة سع انلرمّد ال و ف اس رو دا لعاط الجّمفّت ال ر ي ال فيرّ اّلل ف ا ر ّ الفّلت ّإ نلرع فير60 ل رّلب مة الج ية مة20 .د ل ف الفّلت 5 - الرجعا اسل ّرك: ت رو وبع ملع ع مرل ر لوعاط ال سو وية ال حدات الرفوك كة تةت رالرةةّ اّللةة ّت الفّل كةة تريةةع ال جةةّل لر ةةّدل ال فو مةةّت دك ةةّ وي رةةّ انسةةرمّدال مةةة توةة الل ّت ف ت فيع ال فو مةّت ال ظكاكة تلة ر اسمةع لكلة ا الرفوةكت انل رع رة الرمةع . عرض نتائج االستبانة و حليلها و اسيرها وا ويئ الرفوكت ا ل رع ر اسر دام وعامجه 7 - ن رّل الرفوكت انل رع رة الةا ا ةداد م ةبم ادلة اوثةع مةة اجةا ت دعةد ّفةع الرمّ ةا . خ ل الدر 8 - لوةة جةة د م ربةةعات م رمةة نت ةةّم الدراسةة مةةة خةة ل الرفوةةكت انل ر ع رةة ال جةةدت فر ل م ر دم فة إ لةّ د رات ال ّسة اسةر دامرّ فة ت لةك اس ةّل ا دارسة فةة ال ؤس ةةةّت الرفوك كةة مثةةا اسةةر دام جرةة ال ال ّسةة مو قّترةةّ فةة ر ّ ةة ال رةةب . العس ك 9 - غكةةّ د ر الردرس ةة مقّررةة اةةّلرفوكت الرقويةةدل حيةةل ن ةةّ د ال رفو ةة ل اف ةةرت الةة فض فةة الر فةةا إلةةا ّإجّاةةّت م ّسةة ل ةةا ال لةة ت مةةة خةة ل ج ةةع البكّرةةّت ت ويورةة .ّ م ّللررّ تم يعه - إم ّرك إرلّ الفدعد مة انخر ةّرات تمة كج هةذه انخر ةّرات إل رع ركّ. عرض نتائج االستبانة و حليلها و اسيرها هّشةت2017 ص109 - 115 .) االستنتاجات 1 - دم ج د خواك مفعدك لورفوكت انل رع ر مة لبا انسّتذال اللو وا حرا ال ؤس ّت الرفوك كةة الرةة لةةت تم ةةع اةةأل ت رةةّر هةةذا ال ةة ع مةةة رةة اع الرفوةةكت لو اةةعال ا ةةر ى لةةا الةةا خعسج الرفوكت انل رع ر ل لفدم ان رعال ل ف.ض الد ل الرّدات الرفوكت ة افد 2 - الرفوةةكت انل رع رةة عةةؤثع وةةا جةة دال الرفوةةكت فةة الجّمفةةّت ح ةةب ر ل ةةّ الريئةة الردرس ةةك لةةة ل رةةه ن ن ةةةع ل ةةّ نقدمةةه الرفوةةكت الرقويةةدل مةةة الرمّ ةةةا الدلةة فةةة .إنمّل ال ّدال 1 - دم ج د خواك مفعدك لورفوكت انل رع ر مة لبا انسّتذال اللو وا حرا ال ؤس ّت الرفوك كةة الرةة لةةت تم ةةع اةةأل ت رةةّر هةةذا ال ةة ع مةةة رةة اع الرفوةةكت لو اةةعال ا ةةر ى لةةا الةةا خعسج الرفوكت انل رع ر ل لفدم ان رعال ل ف.ض الد ل الرّدات الرفوكت ة افد 2 - الرفوةةكت انل رع رةة عةةؤثع وةةا جةة دال الرفوةةكت فةة الجّمفةةّت ح ةةب ر ل ةةّ الريئةة الردرس ةةك لةةة ل رةةه ن ن ةةةع ل ةةّ نقدمةةه الرفوةةكت الرقويةةدل مةةة الرمّ ةةةا الدلةة فةةة .إنمّل ال ّدال 3 - ةةدم رغ ةة ال عاتةةب الفو كةة الفوكةةّ اةةّلرفوكت انل رع رةة خّفةة الةةذعة لةةت ت ةة ل وةةداعررت فةة سو الرفوكت مةع ودانة الثة رال الر ل جكة حيةل ع اجرة ل المةف ا فة م او ة الرلة رات ال ّفةةةو فةةة مجةةةّل تقّرةةةّت انتمةةةّل اسةةةرف ّل ال ّسةةة اّللةةة ا الةةةذل ن ةةة رت مةةةة الردرس مة خ ل ال مّت البعامج ال رّح ل ة رلّب الرفوكت ا ل رع ر. 4 - سر ل إجع ا انخر ّرات ل ة نقّووه دم ج د الثق وية الردرس اللّلب فة م لة ع إجةةةةعا انخر ةةةةّرات ا روةةةة شةةةة ّلرّ لةةةة لفةةةةدم ال ةةةةكلعال وةةةةا اللو ةةةة اث ةةةةّ اجةةةةعا ّ انمر ّرّت ل ر ل الغ ال ا م معد ج. 5 - . ارفدام ال رّرات ف اسرف ّل الرقّرّت ال دعث 6 - ارفدام الردرسب ال بم وا سخةع ال ةر دثّت فة ال جةّل الر لة ج مةة جةا الرفةعل . وا ويئ الرفوكت ا ل رع ر اسر دام وعامجه 6 - ارفدام الردرسب ال بم وا سخةع ال ةر دثّت فة ال جةّل الر لة ج مةة جةا الرفةعل . عرض نتائج االستبانة و حليلها و اسيرها ) لغعض اسرفعاض م ّلعاترت وا اللو 10 - :تجعاةةةة د لةةةة للةةةةع لةةةةّم ال جوةةةة اس وةةةةا لورفوةةةةكت فةةةة د لةةةة للةةةةع ورةةةة فيع ال رةةةة ى ا ل رع ر مثا ال رب ا ل رع رك ال مّدر الرفوك ك الر ت ر ل وةا ر ااةط ل مة ّدر سّفط وا ش ا ررعرت ه مةّ ثةع وةا جة دال الرفوةكت الة ا انجةّو حيةل احروةت للع ال ع العااع وا م ر ى الفّلت ف ج دال الرفوكت لفّم2015 - 2016 . 576 / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) / جمةل الداب( اجملدل1 ) العدد142 (أيلول) 2022 / م1444 هـ - إم ّرك إرلّ الفدعد مة انخر ةّرات تمة كج هةذه انخر ةّرات إل رع ركّ . هّشةت2017 ص109 - 115 .) االستنتاجات 1 - دم ج د خواك مفعدك لورفوكت انل رع ر مة لبا انسّتذال اللو وا حرا ال ؤس ّت الرفوك كةة الرةة لةةت تم ةةع اةةأل ت رةةّر هةةذا ال ةة ع مةةة رةة اع الرفوةةكت لو اةةعال ا ةةر ى لةةا الةةا خعسج الرفوكت انل رع ر ل لفدم ان رعال ل ف.ض الد ل الرّدات الرفوكت ة افد 2 - الرفوةةكت انل رع رةة عةةؤثع وةةا جةة دال الرفوةةكت فةة الجّمفةةّت ح ةةب ر ل ةةّ الريئةة الردرس ةةك لةةة ل رةةه ن ن ةةةع ل ةةّ نقدمةةه الرفوةةكت الرقويةةدل مةةة الرمّ ةةةا الدلةة فةةة .إنمّل ال ّدال 3 - ةةدم رغ ةة ال عاتةةب الفو كةة الفوكةةّ اةةّلرفوكت انل رع رةة خّفةة الةةذعة لةةت ت ةة ل وةةداعررت فةة سو الرفوكت مةع ودانة الثة رال الر ل جكة حيةل ع اجرة ل المةف ا فة م او ة الرلة رات ال ّفةةةو فةةة مجةةةّل تقّرةةةّت انتمةةةّل اسةةةرف ّل ال ّسةةة اّللةةة ا الةةةذل ن ةةة رت مةةةة الردرس مة خ ل ال مّت البعامج ال رّح ل ة رلّب الرفوكت ا ل رع ر. 4 - سر ل إجع ا انخر ّرات ل ة نقّووه دم ج د الثق وية الردرس اللّلب فة م لة ع إجةةةةعا انخر ةةةةّرات ا روةةةة شةةةة ّلرّ لةةةة لفةةةةدم ال ةةةةكلعال وةةةةا اللو ةةةة اث ةةةةّ اجةةةةعا ّ انمر ّرّت ل ر ل الغ ال ا م معد ج. 5 - . ارفدام ال رّرات ف اسرف ّل الرقّرّت ال دعث 6 - ارفدام الردرسب ال بم وا سخةع ال ةر دثّت فة ال جةّل الر لة ج مةة جةا الرفةعل . عرض نتائج االستبانة و حليلها و اسيرها العس ك 9 - غكةةّ د ر الردرس ةة مقّررةة اةةّلرفوكت الرقويةةدل حيةةل ن ةةّ د ال رفو ةة ل اف ةةرت الةة فض فةة الر فةةا إلةةا ّإجّاةةّت م ّسةة ل ةةا ال لةة ت مةةة خةة ل ج ةةع البكّرةةّت ت ويورةة .ّ م ّللررّ تم يعه 9 - غكةةّ د ر الردرس ةة مقّررةة اةةّلرفوكت الرقويةةدل حيةةل ن ةةّ د ال رفو ةة ل اف ةةرت الةة فض فةة الر فةةا إلةةا ّإجّاةةّت م ّسةة ل ةةا ال لةة ت مةةة خةة ل ج ةةع البكّرةةّت ت ويورةة .ّ م ّللررّ تم يعه 10 - ةدم جة د م ر ةةّت إل رع ركة فة الجّمفةةّت ت ةّ د اللو ة لو مةة ل وةا ال مةةّدر .مة اجا ارجّ ال ا .مة اجا ارجّ ال ا 577 / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) 2022 / م1444 هـ 11 - تةةةأثع ال ؤس ةةةّت الرفوك كةةة ا ؤشةةةعات الجةةة دال ا ةةةّ ن ةةةر م عجّترةةةّ جةةةعا اسةةةر دام الرفوكت ان ل رع ر م ّ ن بب تعاجع ا ر ى ال عسجية ل ل الرفوةكت انل رع رة لةت ن ة ل .مة ل ة رةس هدال تو ال ؤس ّت ا / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) 11 - تةةةأثع ال ؤس ةةةّت الرفوك كةةة ا ؤشةةةعات الجةةة دال ا ةةةّ ن ةةةر م عجّترةةةّ جةةةعا اسةةةر دام الرفوكت ان ل رع ر م ّ ن بب تعاجع ا ر ى ال عسجية ل ل الرفوةكت انل رع رة لةت ن ة ل .مة ل ة رةس هدال تو ال ؤس ّت 12 - عرةةةةدرا م ةةةةر ى الجةةةة دال لقوةةةة الرمرةةةةك ال عاق ةةةة مةةةةة الجرةةةةّت الفوكةةةةّ لةةةة ة الرفوةةةةكت . انل رع ر 12 - عرةةةةدرا م ةةةةر ى الجةةةة دال لقوةةةة الرمرةةةةك ال عاق ةةةة مةةةةة الجرةةةةّت الفوكةةةةّ لةةةة ة الرفوةةةةكت . عرض نتائج االستبانة و حليلها و اسيرها انل رع ر 13 - سةةّدال ال م ةةّنال لةةدى اللو ةة ارلةةغّلرت اةةأم ر جّربكةة ّلف ةةا سل الرفوةةكت انل رع رةة ن ن ثا ّ ّفق مّم الف ا خ ل لّت الدراس ا افدهّ ل ل افض ال ةّنت ش مةت امةةةرج اللّلةةةب لوبعرةةةّمج تةةةع انسةةةرّ نلةةةعح د ل ال ن ةةةر ع نلةةةّر اّلةةةدر سرفةةةذر اللّلةب افةدم ال لةّر ل ة خةط انررعرةت انرقلةّع ال ةر ع لولّلة ال رعاّفكة حيةةل تفربةةةةع ر دا ال م ا مةةةة انررعرةةةةت فةةةة الفةةةةعاب سةةةةّدال ارقلةةةةّع الركةةةةّر ال رعاةةةةّف مةةةةة هةةةةت ال ف لّت مّم رجّح الرفوكت ا ل رع ر. ا 14 - سّدال الفب وا الردرس ل ّ خوب لره ف الر افا مع روبره فة الةعد وةا .اسرم ّراترت افد ارررّ الدر مرّاف ا شعال وا ال ا 14 - سّدال الفب وا الردرس ل ّ خوب لره ف الر افا مع روبره فة الةعد وةا .اسرم ّراترت افد ارررّ الدر مرّاف ا شعال وا ال ا 15 - لوةة انرمةةّب ال ةة م ا ةةبب اس مةة ال ّلكةة ات ةةّع سكّسةة الرقلةة ثةةع وةةا ات ةةّع خلةة ات جةةّدال فةة رقةةا ال ؤس ةةّت الرفوك كةة مةةة الرفوةةكت ال ر ةةع لةةدعرّ وةةا فةةم اللعافةةم . الرقويدن الا الرفوكت انل رع ر ا 15 - لوةة انرمةةّب ال ةة م ا ةةبب اس مةة ال ّلكةة ات ةةّع سكّسةة الرقلةة ثةةع وةةا ات ةةّع خلةة ات جةةّدال فةة رقةةا ال ؤس ةةّت الرفوك كةة مةةة الرفوةةكت ال ر ةةع لةةدعرّ وةةا فةةم اللعافةةم . الرقويدن الا الرفوكت انل رع ر 16 - نقدم الرفوكت انل رع ر حا ل ل ت ال ة ال ة ّر ام ّركة اسةرقلّ ا ةداد ثيةعال . مة اللو 16 - نقدم الرفوكت انل رع ر حا ل ل ت ال ة ال ة ّر ام ّركة اسةرقلّ ا ةداد ثيةعال . عرض نتائج االستبانة و حليلها و اسيرها وا ويئ الرفوكت ا ل رع ر اسر دام وعامجه 7 - ن رّل الرفوكت انل رع رة الةا ا ةداد م ةبم ادلة اوثةع مةة اجةا ت دعةد ّفةع الرمّ ةا . خ ل الدر 7 - ن رّل الرفوكت انل رع رة الةا ا ةداد م ةبم ادلة اوثةع مةة اجةا ت دعةد ّفةع الرمّ ةا . خ ل الدر 8 - لوةة جةة د م ربةةعات م رمةة نت ةةّم الدراسةة مةةة خةة ل الرفوةةكت انل ر ع رةة ال جةةدت فر ل م ر دم فة إ لةّ د رات ال ّسة اسةر دامرّ فة ت لةك اس ةّل ا دارسة فةة ال ؤس ةةةّت الرفوك كةة مثةةا اسةةر دام جرةة ال ال ّسةة مو قّترةةّ فةة ر ّ ةة ال رةةب . عرض نتائج االستبانة و حليلها و اسيرها 578 / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) 24 - عرةةكج ال جةةّل انفرعالةة الةةذل ات ةةذه الرفوةةكت انل رع رةة مةةة إم ّركةة قةةد ال ةةؤت عات ال د ات الفو ك تقدنت ال مج ا رشّد تثق.كا ال جر فّت 25 - ام ّركةةة ل تقةةةدم ال ّتةةةب انسرلةةةّرس فةةة الجّمفةةةّت خةةةدمّترّ لو جر ةةةع بةةةع ات .البعامج ال ر ف ف الرفوكت انل رع ر انسرلّرات اللبك الر دسك الرعج ...الخ 26 - ت رّل الجّمفّت الفعاقك الا وذل ال سد مة الجرد ف تة فيع و كة ت ركة لةّدرال وةا تقدنت ال.رفوكت انل رع ر بدعا ة الرفوكت الرقويدل التوصيات 1 - الرثقكا ح ل الرفوكت انل رع ر اّلل ا الذل ن ج له ال ن ل ودعا مق ةع ةة الرفوةكت .الرقويدل 2 - لع مفّعيع لورفوكت ا ل رع ر الر مة شأررّ رفع م ةر ى جة دال الرفوةكت الفةّل الرة وةةةد رهّ تعفةةةع مةةةة اسةةةت ال ؤس ةةةّت الرف وك كةةة لغةةةعض دخ لرةةةّ لورمةةة كمّت ال ّف ةةة مةةةع . الجّمفّت الفّل ك 3 - د ت الردرس يية تذليا الفق ّت مّمرت مة جةا انلر ةّع اةّلرفوكت انل رع رة مةة خة ل تغييع ال مّيكت ح لةه نسةك ّ لةدى انسةرّ ال ال ةّر ل ة ررت سةّ ةا تغييةع و ة نلةع . ف مؤس ّت الرفوكت الفّل ت 4 - اللوب مةة اللةع ّت الرق كة ال ر ممة فة فة ّ البةعامج انل رع ركة مةة اسةر داا وعامج الغعض م رّ ال د مة ظّهعال الغ بع ال مّت ا ل رع رك. 5 - اّنلّف الا ج د رم ص لّر رك راد لوغ ل ةة مةة انف ةا لةع ميثةّب شةعل ع لع وكه اللّلب افدم الغ خ ل انمر ّرّت الغّنة م ةه سةّدال ّالثقة مةّ وةية انسةر . اللّلب هذا ود ره نل ا دافع مة جا ان ر ّد وا الرفوكت انل رع ر 6 - الف ةا وةةا إلّمةة الةةد رات الردرسبكةة لألسةةّتذال ال ة ظمية اللو ةة فةة الرفةةعل وةةا سخةةع الرقّرةةةةّت ال دعثةةةة فةةةة مجةةةةّل الرفوةةةةكت انل رع رةةةة مةةةةة اجةةةةا الرغوةةةةب وةةةةا ال لةةةة ت ال ف لّت الر . عرض نتائج االستبانة و حليلها و اسيرها مة اللو 17 - تبية ل لورفوكت انل رع رك ام ّرك ف ت فيع فعف الةرفوت لألفةعاد ل ةة فةّتررت فعفة .الدراس خ ل فرعال مفي مة حكّترت ه وذل ع فع الدراس مدى ال كّال ا 17 - تبية ل لورفوكت انل رع رك ام ّرك ف ت فيع فعف الةرفوت لألفةعاد ل ةة فةّتررت فعفة .الدراس خ ل فرعال مفي مة حكّترت ه وذل ع فع الدراس مدى ال كّال ا .الدراس خ ل فرعال مفي مة حكّترت ه وذل ع فع الدراس مدى ال كّال 18 - ن قةةم ت كةة الةة الثقةةّف الفو ةة لولو ةة س ةةّ دهت فةة تجةةّ ةةدم القةةدرال وةةا ال لّر الر نفّر م رّ اللو. ف ودان دراسررت الجّمعك 19 -الرفوكت الرقويدل ا بب ثعال عركج فعف ال لّر ال ا وبع ّ ّرت وكه ف 18 - ن قةةم ت كةة الةة الثقةةّف الفو ةة لولو ةة س ةةّ دهت فةة تجةةّ ةةدم القةةدرال وة ال لّر الر نفّر م رّ اللو. ف ودان دراسررت الجّمعك 18 - ن قةةم ت كةة الةة الثقةةّف الفو ةة لولو ةة س ةةّ دهت فةة تجةةّ ةةدم القةةدرال وةةا ال لّر الر نفّر م رّ اللو. ف ودان دراسررت الجّمعك 19 - عرةةكج فعفةة ال لةةّر الةة ا وبةةع ةةّ ّرةةت وكةةه فةة الرفوةةكت الرقويةةدل ا ةةبب ثةةعال . داد اللو الرقيد اّل لت ال دد لو ّلعال 20 - عر ةةبب الرفوةةكت انل رع رةة اّللةةف ر اّلف لةة لإكةةّ الرمّ ةةا انجر ةةّ وةةية اللو ةة . غكّ ا ح ّ اّل الع 20 - عر ةةبب الرفوةةكت انل رع رةة اّللةةف ر اّلف لةة لإكةةّ الرمّ ةةا انجر ةةّ وةةية اللو ةة . غكّ ا ح ّ اّل الع 22 - . الرأثيع ال وب وا و ّ المعد اّلم رال ال وك 23 - ام ّرك اسر دام ال مّت الرفوك ك ف إ لّ الد رات الردرسبك ف ّف ال جّنت. 23 - ام ّرك اسر دام ال مّت الرفوك ك ف إ لّ الد رات الردرسبك ف ّف ال جّنت. عرض نتائج االستبانة و حليلها و اسيرها ت اجه ال رميدعة مة الرفوكت انل رع ر 7 - الف ةا وةةا تغييةع ال ةةّهج الدراسةةك ا ةّ عر افةةم مةع اللعافةةم ال دعثةة ال ر فة فةة الرفوةةكت انل رع ر ل ل ال ّهج الرقويدن ت رّل الا ت سورّ الة ا مومةّت امةكغWord ) Power point ) PDF ) الةةا لةةت جرةةد هةةذا مةةّ نعر افةةم مةةع لةةت الردرس ةة ال دد اّلدر مرّاف اللو جعا ال ا الفو ك ف ة الر امّته ال كّتك ةا ّهذا نل ا ّفق مّم الرفوكت انل رع ر لذل مة انف ا الذهّ اّتجّه تغييةع ال ةّهج . م ّ ن را ا الردرس 4 - اللوب مةة اللةع ّت الرق كة ال ر ممة فة فة ّ البةعامج انل رع ركة مةة اسةر داا وعامج الغعض م رّ ال د مة ظّهعال الغ بع ال مّت ا ل رع رك. 579 / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) 2022 / م1444 هـ 8 - ت جكةةه ال ؤس ةةّت الرفو ك كةة ا ةةع رال ال ّفاةة وةةا ال ربةةعات الرةة ن ةةة انسةةرمّدال م رّ ف مجّل الرفوكت انل رع ر دم اسرف ّلرّ لألخعاض ا دارس. 9 - تف سةة د ر الردرس ةة مةةة خةة ل الر جكةةه اةةّلرفوكت ال ةةدمج الةةذل مةةة شةةأره رفةةع ال ةةر ى الفو ةة لولو ةة تفاةةكت د ر الردرس ةة افةةّد اللةةف ر ال ةةوب تجةة ّه الرفوةةكت انل رع رةة ارذه اللعسق ن ة حم اللّلب ف م أل ت دعد اللّلب ال ر ي مة خيعه مة اللو هذا الجّرةب تةأثع ا ةبب حةّنت الغة الرة عوجةأ إليرةّ اللو ة مةة جةا ال مة ل وةا .درجّت ت ّ سرت مع العاررت ال ر ي سة 10 - اسةةةر داا ال ر ةةةّت انل رع ركةةة تةةة فيع ال مةةةّدر ا لفو كةةة الةةة ا ال رع رةةة مقّررةةة اّل ر ّت ال ج دال ف الجّمفّت الر ت ّ د اللو ف إرجّ ا ثرت خ ل م يعترت . الدراسك 10 - اسةةةر داا ال ر ةةةّت انل رع ركةةة تةةة فيع ال مةةةّدر ا لفو كةةة الةةة ا ال رع رةةة مقّررةةة اّل ر ّت ال ج دال ف الجّمفّت الر ت ّ د اللو ف إرجّ ا ثرت خ ل م يعترت . عرض نتائج االستبانة و حليلها و اسيرها الدراسك 11 - لةةع رةسةة الةة لو ؤس ةةّت الرفوك كةة حةة ل الرفوةةكت ا ل رع رةة حرةةا ن ةة ل مةةة اسهدال الر ت فا الا ت قكقرّ مؤس ّت الرفوكت الفّل وا الا تقدعع ت دعد وكةّت ّمفي د ل خيعهّ ورلبيم الرفوكت ا ل رع ر ف حّل رجّح الرجعا سرف ت وا خيعه . مة الجّمفّت مة اجا ارجّح هذه الرجعا المعسدال وا م ر ى الجّمفّت الفعاقك 11 - لةةع رةسةة الةة لو ؤس ةةّت الرفوك كةة حةة ل الرفوةةكت ا ل رع رةة حرةةا ن ةة ل مةةة اسهدال الر ت فا الا ت قكقرّ مؤس ّت الرفوكت الفّل وا الا تقدعع ت دعد وكةّت ّمفي د ل خيعهّ ورلبيم الرفوكت ا ل رع ر ف حّل رجّح الرجعا سرف ت وا خيعه . مة الجّمفّت مة اجا ارجّح هذه الرجعا المعسدال وا م ر ى الجّمفّت الفعاقك 12 - سّدال انهر ّم اّلد ر العلّو مة لبا الجرّت الفوكّ ف ارال الرفوكت الفّل ال ةل الفو ةة حرةة ا ت ةة ل م عجةةّت الرفوةةكت الفةةّل اّللةة ا الةةذل نجفورةةّ مؤهوةة ل ةة ب الف ةةا هةةذا اسمةةع ح ةةب ر ل الردرس ةةيية تعاجةةع و ةةب خيةةع مقوقةة ل ةةة مةةؤثعال وةةا ر عكةة ال ةةةةعسجية خّفةةةة فةةةة انخرمّفةةةةّت الفو كةةةة الرةةةة ت رةةةةّل إلةةةةا در وكةةةة فةةةة ال ربةةعات الفو كةة هةةذا مةةّ ن ةة ل لةة ة الرفوةةكت ال ةةدمج الةةذل ت ةة ل دكةةه الةةدر ات اللّاع الفو الر ت رّل الا ج د اللّلب ف ال ربةع الة ا ح ة رل اقكة ال ة اد . خّف خيع انسّسك ت ل ال ا انل رع ر 13 - إل ام الردرس يية ا ع رال ت دعل رعافم الردرس اّسةر عار ةدم تقةدنت ال ةّدال العسقة احدال ت سد مة حّلة ال وةا ال مة ر لةد ى اللو ة ل ة ل الجوة مةّم جرةّ ال ّسة الرّت ال قّل لمرعات ر سو ع لد حّل سوبك تجّه الدراس خ ّع ال ّلةعات الة ا م ةةةةر ع لورمرةةةةك مةةةةة لبةةةةا لجةةةةّل م رمةةةة ترةةةةّاع م ةةةةأل الإكةةةةّ ةةةةدم ال ةةةة ر الةةةةا ّالمةةةم ل ا ل رع رةةة رفةةةع تقةةةّرسع الةةة ا د رل الةةةا ةةةّدال ال وكةةة اّ ر ّرهةةة ال ةةةؤ ل . عرض نتائج االستبانة و حليلها و اسيرها هذا اسمع ععا ظّهعال ال سّدال ال ّرك لو انو ك الجّمعك 17 - ت سةةكع رلةةّب الف ةةا فةة ل ةةّم الرفوةةكت ال ةةر ع فةة الجّمفةةّت الرةة تقةةدم خةةدمّت م رومةةة ةةة ح ةةةب انخرمةةةّص ففوةةةا سةةةبيا ال ثةةةّل نقةةة م الرفوةةةكت ال ةةةر ع فةةة وكةةة الوغّت جّمف اغداد اإلّم د رات تل سعس ف مجّل تفوكت الوغّت انج بك الا الجرّت ال مكةة حيةةل ن ةةة لرةةذا الق ةةت مةةة ت ةة سة فةةم ل لولو ةة الةةذعة لةةت ن وةة ا دراسةةررت انوّدن كةةة سسةةة ّ مرفةةةددال جفةةةا الدراسةةة ال رع ركةةة سةةةرت مةةة رت شةةةرّدال ت ةةةعل اسةةة ال اةةةإلعاررت ل ةةةةة بةةةةع وعرةةةةّمج و ةةةة م مةةةةر لرةةةذه المئةةةة م ةةةةرغوية مةةةةّ نقدمةةةةه الرفوةةةةكت .انل رع ر مة ام ّرك او ّل دراسررت ة افد 18 - الّمةةة المفّلكةةةّت ال ةةةؤت عات ال ةةةد ات الفو كةةة الةةة ا إل رع رةةة لولو ةةة حةةةثرت وةةةا ال لّر ورذه ال لّرّت الر ت ّ دهت وا ت لة معحوة الدراسة اّللة ا الةذل ع سةد مة ا.لرمّ ا انجر ّ دك ّ وي رت 18 - الّمةةة المفّلكةةةّت ال ةةةؤت عات ال ةةةد ات الفو كةةة الةةة ا إل رع رةةة لولو ةةة حةةةثرت وةةةا ال لّر ورذه ال لّرّت الر ت ّ دهت وا ت لة معحوة الدراسة اّللة ا الةذل ع سةد مة ا.لرمّ ا انجر ّ دك ّ وي رت 19 - د ةةةت العفةةةّر الفو كةةة ال ّفاةةة وةةةا سةةة ف الجّمفةةةّت الفعاقكةةة امةةةّم ال ا ةةةّت الفّل كةةة هةةةذا مةةةّنف د ر الفةةةعاب لةةة ة الرمةةة كمّت الد لكةةة فةةة مجةةةّل جةةة دال الرفوةةةكت الفةةّل مةةة خةة ل ال ةةل ال ةةر ع ةةة ال ةةوبكّت الرةة تعافةةم الف وكةة الرفوك كةة لةة ة الرفو. عرض نتائج االستبانة و حليلها و اسيرها اس ل وا العفّر الفو ك لو ؤس الرفوك ك 14 - ت جكةةه اللو ةة مةةة خةة ل رةسةةةّ اسل ةةةّم اةةّنلر ام اّلر ليرةةةّت ال ممةةة لوةةدر دم الرجّ وا ال لت ال ح وةذل اسمةع الةذل ن مة مةة الفةب وةا الردرس ة . ت اكت لره اّلل ا الذل ن دم ال مو الفّم 580 / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) 2022 / م1444 هـ 15 - ت مةةكر جةة مةةة ال ا رةة ال ّفةة اةةّلرفوكت الفةةّل وةةا د ةةت الرفوةةكت اسل رع رةة اّلل ا الذل نجفا الجّمفّت مؤهو لر سا راّم الرفوكت لدعرّ مةة الرفوةكت الرقويةدل الةا . الرفوكت انل رع ر 16 - اسرث ّر ال مّفر ال ية ات الرة ن رةّ ورةّ الرفوةكت انل رع رة خّفة انم ّركة فةة إلّمةة در ن ةةد اد مةةة اللو ةة تمةة ب اس ةةداد ال ةة ح ورةةّ فةة المةةم ل الرقويدنةة . عرض نتائج االستبانة و حليلها و اسيرها اااا نا 15 - االاربلغ ةریف لحميغ ال دا،ن اإللل رونل ولهاا، ا الهاار21 ن أ وا واساا راا،ج،، الاا دان الحيی االغ اله،هر،غ الدربل لات ر وال ع یعغ ط1 غ2020 . اااا 16 - لخ،رغ ب،ع ةف،الغ اال ا ، االلل رون،ل ميكن إ ا ي ل خعیر االنظماال ال دا،م،االغ لماا، غ ا ال ااروق لات ر وال ع یعغ ط1 غ2017 . ا 16 - لخ،رغ ب،ع ةف،الغ اال ا ، االلل رون،ل ميكن إ ا ي ل خعیر االنظماال ال دا،م،االغ لماا، غ ا ال ااروق لات ر وال ع یعغ ط1 غ2017 . ا ع 17 - ،سد، ،غ مع أحميغ ل، ل ف،یز السرط،ويغ اس خياو الح سعب واالن رنت فل ل،اا، ین ال رب،اال وال دااا،نغ لم، غ ا ال روق لات ر وال ع یعغ ط1 غ2015 . ع 17 - ،سد، ،غ مع أحميغ ل، ل ف،یز السرط،ويغ اس خياو الح سعب واالن رنت فل ل،اا، ین ال رب،اال وال دااا،نغ لم، غ ا ال روق لات ر وال ع یعغ ط1 غ2015 . ع 18 - لمسغ ل:خفا نمرغ إ ا ، الجع ، ال ،لال فل ال رب،ل وال دا،نغ لم، غ ا ل،ياء لات ر وال ع یعغط1 غ 2015 . 18 - لمسغ ل:خفا نمرغ إ ا ، الجع ، ال ،لال فل ال رب،ل وال دا،نغ لم، غ ا ل،ياء لات ر وال ع یعغط1 غ 2015 . 19 - ،ال للغ لس، م،ساان او غ هاايس م،ساان سااد،يغ إ ا ، الجااع ، ال اا،لال فاال الم سساا ال دا،م،االن لداا،ی،ر واخم،ه، ونم،مجغ لم، غ اليا المتهج،ل لات ر وال ع یعغ ط1 غ2017 . ا 19 - ،ال للغ لس، م،ساان او غ هاايس م،ساان سااد،يغ إ ا ، الجااع ، ال اا،لال فاال الم سساا ال دا،م،االن لداا،ی،ر واخم،ه، ونم،مجغ لم، غ اليا المتهج،ل لات ر وال ع یعغ ط1 غ2017 . ا ع ج 20 - لهنغ لج،ي سد،يغ لد،ی،ر ا:م،ن لت،صر ال دان بمس ع ل، ال دان االلل رونلغ بحااث لت ااع فاال لجااال م،لدل فاسخ،ن لذبح،ث والي اس، غ الدي الس، غ یت،ن،ر2014 . 20 - لهنغ لج،ي سد،يغ لد،ی،ر ا:م،ن لت،صر ال دان بمس ع ل، ال دان االلل رونلغ بحااث لت ااع فاال لجااال م،لدل فاسخ،ن لذبح،ث والي اس، غ الدي الس، غ یت،ن،ر2014 . 21 - لزللغ نم،ن م، غ ب،ئ، ال د ان ال ف،لا،لغ اله،هر،غ ا الفلر الدربلغ ط1 غ2014 . عرض نتائج االستبانة و حليلها و اسيرها ل 9 - أبع ل وغ م،ء لحمع غ لت،هج المحث اللمل والتعلل والمخ اطغ لم، غ ا المس،ر، لات اار وال ع یااعغ ط1 غ2013 . ا 9 - أبع ل وغ م،ء لحمع غ لت،هج المحث اللمل والتعلل والمخ اطغ لم، غ ا المس،ر، لات اار وال ع یااعغ ط1 غ2013 . ا 10 - الزنيغ ول،ي كضرغ ال :،ل،ن ال دا،م،ل والتعلعم،، ال دا،ن ب،ن التظریاال وال خم،ااالغ الریاا،ضغ ا الل اا،ب الج،لدل لات رغ ط1 غ2018 . اا 10 - الزنيغ ول،ي كضرغ ال :،ل،ن ال دا،م،ل والتعلعم،، ال دا،ن ب،ن التظریاال وال خم،ااالغ الریاا،ضغ ا الل اا،ب الج،لدل لات رغ ط1 غ2018 . اا 10 - الزنيغ ول،ي كضرغ ال :،ل،ن ال دا،م،ل والتعلعم،، ال دا،ن ب،ن التظریاال وال خم،ااالغ الریاا،ضغ ا الل اا،ب الج،لدل لات رغ ط1 غ2018 . اا 11 - ل، ك ج و نم،رجغ ال دان االلل رونل ن اساا راا،ج،، الی:اا،ل المد رفاال فاال الد:اار الرمماالغ ارمماال نع ، بتت سدع الهزانلغ الری،ضغ ا م،لدل الماك سدع لات رغ ط1 غ2019 . اإ 11 - ل، ك ج و نم،رجغ ال دان االلل رونل ن اساا راا،ج،، الی:اا،ل المد رفاال فاال الد:اار الرمماالغ ارمماال نع ، بتت سدع الهزانلغ الری،ضغ ا م،لدل الماك سدع لات رغ ط1 غ2019 . اإ ل 12 - لالغ لحمااي احماايغ التعلعم،اا، ال دااا،ن اإللل روناال ول خاماا، الجااع ، ال اا،لالغ االساالتي یلغ ا الفلاار الج،لدلغ ط1 غ2021 . اا ل 13 - االاربلغ ةریف لحمي ابراه،نغ ال دا،ن ب،ل خ،ن اساا را ا،ج،ل ال دااا،ن االلل روناال وأ وا الاا دانغ الهاا،هر،غ الدربل لات ر وال ع یعغ ط1 غ2019 . ا ل 13 - االاربلغ ةریف لحمي ابراه،نغ ال دا،ن ب،ل خ،ن اساا را ا،ج،ل ال دااا،ن االلل روناال وأ وا الاا دانغ الهاا،هر،غ الدربل لات ر وال ع یعغ ط1 غ2019 . ا ع ل 14 - فاا، غ نجاا ء لحماايغ لمااي الاارؤوف لحمااي اساام،ل،نغ ال دااا،ن االلل روناال لساا حي ، فاال التظریاال واالس راا،ج،لغ اله،هر،غ ل،لن الل دغ ط1 غ2017 . إا ع ل 14 - فاا، غ نجاا ء لحماايغ لمااي الاارؤوف لحمااي اساام،ل،نغ ال دااا،ن االلل روناال لساا حي ، فاال التظریاال واالس راا،ج،لغ اله،هر،غ ل،لن الل دغ ط1 غ2017 . إا نا 15 - االاربلغ ةریف لحميغ ال دا،ن اإللل رونل ولهاا، ا الهاار21 ن أ وا واساا راا،ج،، الاا دان الحيی االغ اله،هر،غ الدربل لات ر وال ع یعغ ط1 غ2020 . عرض نتائج االستبانة و حليلها و اسيرها كت انل رع ر م ّ ل ت ييم المج ات الر ت بب ف فلا هذه الرجعا 20 - ةةةدم اه ةةةّل الجّرةةةب الةةةذل مةةةة شةةةأره ال عةةةؤثع فةةة و ةةةّ ش مةةةك اللّلةةةب داخةةةا الجّمفةة مةةة خةة ل الر عكةة ال ةةر عال ا لةةّ ال بةةعال ال رعاو ةة لةةدى الردرس ةة اّ ر ةةّره م رلةةةةّر ل ةةةةّ ت ثوةةةةه الجّمفةةةةّت مةةةةة هيئةةةة اسرلةةةةّرس تقةةةة دم اسرلةةةةّرترّ لود لةةةة فةةةة ّفةةةة .ال جّنت حا لو ل ت الر ت اجه ال جر ع 21 - اسةةةةرغ ل ال ةةةةةّح الرةةةةة ت فعهةةةةّ ال مةةةةةّت الرةةةةة ت ةةةةر دم لةةةةة ة رلةةةةةّب الرفوةةةةةكت ا ل رع رةةة فةةةة الةةةةد رات الردرسبكةةةة لولو ةةةة ال ةةةعسجية مةةةةة جةةةةا م ةةةةّ دترت فةةةة ت كةةةة . مرّراترت ف لّت الفلا العاكعك المكاك 22 - تمف يةةا د ر ال ّتةةب انسرلةةّرس فةة الجّمفةةّت الفعاقكةة انسةةرمّدال مةةة وةةعامج الرفوةةكت انل رع ر ف تقدنت خدمّترّ لو رميدعة ا ّ ن ر الةد رات الرعج ة انسرلةّرات ة .ّح ب ت ممر 581 / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) / جمةل الآداب( اجملدل1 ) العدد142 (أ يلول) 2022 / م1444 هـ 4 - ضعا غ لحمع لمي الف ،حغ إ ا ، الجع ، ال ،لالغ اله،هر،غ المجمعلاال الدرب،اال لا ااي ید والت اارغ ط1غ 2012 . 5 - ،زا غ ا ی،ناا، وركاارو غ ال دااا،ن الداا،لل لخيلاال ال:اا،لا الداا،ون اصااعا صااي لاان حر اال معل،االغ الری،ضغ لل مل الدم،ل، لات ر وال ع یعغ ط1 غ2010 . ا ع ع 7 - ،،الف ويغ لحمي ،ظن حس،نغ أك م لهتل ال ي یسن اسل فل العظ،فاال الرساا،ل،ل لذساا ،م الجاا،لدلغ اإلل، ا الدرب،ل الم حي، غ ا افي لات رغ ط1 غ2019 . إ 8 - ،الدمرانیغ لمي الغتل لحمي اسم،ل،نغ لت،هج المحث الدملغ صتد،ءغ لر ااز م،لداال الداااعو وال لتعلعم،اا لال ،ب الج،لدلغ ط2 غ2013 . إ 8 - ،الدمرانیغ لمي الغتل لحمي اسم،ل،نغ لت،هج المحث الدملغ صتد،ءغ لر ااز م،لداال الداااعو وال لتعلعم،اا لال ،ب الج،لدلغ ط2 غ2013 . 8 - ،الدمرانیغ لمي الغتل لحمي اسم،ل،نغ لت،هج المحث الدملغ صتد،ءغ لر ااز م،لداال الداااعو وال لتعلعم،اا لال ،ب الج،لدلغ ط2 غ2013 . ل 9 - أبع ل وغ م،ء لحمع غ لت،هج المحث اللمل والتعلل والمخ اطغ لم، غ ا المس،ر، لات اار وال ع یااعغ ط1 غ2013 . عرض نتائج االستبانة و حليلها و اسيرها 21 - لزللغ نم،ن م، غ ب،ئ، ال د ان ال ف،لا،لغ اله،هر،غ ا الفلر الدربلغ ط1 غ2014 . ل ل ن 22 - ل،لرغ ط، ق لمي الرؤوفغ ال دا،ن لاان بدااي وال دااا،ن المف ااعح غ لماا، غ ا ال،اا، و ي الدام،اال لات اار وال ع یع غ ط1 غ2015 . اا 22 - ل،لرغ ط، ق لمي الرؤوفغ ال دا،ن لاان بدااي وال دااا،ن المف ااعح غ لماا، غ ا ال،اا، و ي الدام،اال لات اار وال ع یع غ ط1 غ2015 . اا ا 25 - ه،ةنغ لجيي یعنسغ ال دان االلل رونلغ اله،هر،غ ا هع المدرفل والمر لغ ط1 غ2017 . 582
https://openalex.org/W3101838266	https://www.confer.cz/metal/2020/download/3647-assessment-of-production-equipment-of-chosen-rolling-mill-in-poland.pdf	English	null	ASSESSMENT OF PRODUCTION EQUIPMENT OF chosen rolling mill IN POLAND	Metal ...	2,020	cc-by	4,052	ERROR: type should be string, got "https://doi.org/10.37904/metal.2020.3647 https://doi.org/10.37904/metal.2020.3647 Abstract Appropriate maintenance of machinery and production equipment allows for smooth production, eliminates \ndowntime related to failures, improves work safety and improves the productivity of an enterprise. Well-\nfunctioning machines allow for production of high-quality products. This is an important topic especially in \nenterprises of the metallurgical industry due to the specificity. They usually work in system 24/7, what is more, \nmachines and devices are used in the most effective way possible. In addition, an important factor is the ability \nto produce products with appropriate quality parameters, because many of these products are used, e.g. in \nvarious machines, vehicles, constructions or buildings. The purpose of the paper was to assess the production \ndevices of one of the rolling mills in Poland in terms of its condition and use. Chosen technical and \ntechnological indicators typical of metallurgical enterprises were used for this assessment. This research \ncovered 5 consecutive years. Slight fluctuations in the values of the research indicators were noted, which is \nwhy the machine breaks were additionally examined. Keywords: Rolling mill, production equipment, modernity, productivity ASSESSMENT OF PRODUCTION EQUIPMENT OF CHOSEN ROLLING MILL IN POLAND \n1Manuela INGALDI, 2Magdalena MAZUR \n1Czestochowa University of Technology, Faculty of Management, Czestochowa, Poland, EU, \nmanuela.ingaldi@wz.pcz.pl; manuela@gazeta.pl \n1Czestochowa University of Technology, Faculty of Management, Czestochowa, Poland, EU, \nmagdalena.mazur@wz.pcz.pl 1Czestochowa University of Technology, Faculty of Management, Czestochowa, Poland, EU, \nmanuela.ingaldi@wz.pcz.pl; manuela@gazeta.pl \n1Czestochowa University of Technology, Faculty of Management, Czestochowa, Poland, EU, \nmagdalena.mazur@wz.pcz.pl https://doi.org/10.37904/metal.2020.3647 ESSMENT OF PRODUCTION EQUIPMENT OF CHOSEN ROLLING MILL IN POLAN ASSESSMENT OF PRODUCTION EQUIPMENT OF CHOSEN ROLLING MILL IN POLAND\n1Manuela INGALDI, 2Magdalena MAZUR \n1Czestochowa University of Technology, Faculty of Management, Czestochowa, Poland, EU, \nmanuela.ingaldi@wz.pcz.pl; manuela@gazeta.pl \n1Czestochowa University of Technology, Faculty of Management, Czestochowa, Poland, EU, \nmagdalena.mazur@wz.pcz.pl May 20th - 22nd 2020, Brno, Czech Republic, EU 1. \nINTRODUCTION The maintenance department, which is responsible for maintaining the suitability of equipment for carrying out \nproduction processes, is part of every production system. Currently, this department is also part of the analysis \nof the effectiveness of processes that are part of the overall efficiency of implemented manufacturing \nprocesses [1]. The basic issue in this respect is the TPM concept, which includes the tasks of maintaining \nequipment for operation in processes, supervising the efficiency indicators of these machines, and the direct \nimpact on the production capacity and productivity of manufacturing processes [2,3]. The TPM system is one \nof the foundations of the Lean Management concept, which is based on the elimination of basic types of waste, \nalso those related to the operation of machinery and production equipment [4]. The technical condition and quality of used machines and devices directly affect the downtime in the \nimplementation of the production schedule. This element of waste in the production system has a significant \nimpact on the implementation of the assumed production plan and on the flexibility function of organized \nproduction process. Therefore, properly prepared strategies for the maintenance of machines is the basis for \nmaintaining the production capacity of the system for the production and realizations of orders in the planned \ntime periods [5]. The importance of machine maintenance strategies is enhanced by the high level of high-\nscale production automation and mechanization as well as the long service life of the used equipment [6,7]. Taking into account these elements and their importance, maintenance is the basis deciding about \nmanufacturing capabilities, but in accordance with the principles of Lean Management, they are also subject \nto the principles of optimization and adequate efficiency of operations that apply to all corporate resources, May 20th - 22nd 2020, Brno, Czech Republic, EU without exception. The main idea related to maintenance is to prepare parts and machines to do what is \nrequired in the allocated time and sizes, and to do it with less resources [8,9]. without exception. The main idea related to maintenance is to prepare parts and machines to do what is \nrequired in the allocated time and sizes, and to do it with less resources [8,9]. The TPM concept uses two basic indicators. Firstly, it is focused on minimizing waste in the production and \noperation of equipment from the point of view of the use of production capacities that have been planned. 1. \nINTRODUCTION Secondly, it focuses on the quality of created products, where the main goal is to achieve a zero level of \nproduct defects [10]. These two elements still allow reducing the amount of production scrap or reducing the \ndefect rate, no breakdown, no accidents, no waste during the process or replacement [11]. Numerous \npublications based on the implementation and use of the TPM concept show the benefits for the production \nsystem in terms of eliminating waste of time (according to the number of breakdowns as well as the number \nof production defects and the resulting corrective actions). Monitoring and calculating the main indicator of the \nefficiency and productivity of the use of the machine park also gives the company information at what stage of \nthe strategy implementation is, where weak points in the organization of production are, and as a consequence \nof further analysis also about way of possible improvement [12,13]. Metallurgical enterprises belong to those enterprises that work in a continuous system, which means that \nproduction devices are used 24/7. Maintenance and repairs of machines must be properly planned so as not \nto cause too long stoppages in production and reduction of the company's productivity and efficiency. The purpose of the paper was to assess the production devices of one of the rolling mills in Poland in terms of \nits condition and use. Chosen technical and technological indicators typical of metallurgical enterprises were \nused for this assessment. This research covered 5 consecutive years. 2. \nMETHODOLOGY An assessment of the rolling mill production equipment in one of the steelwork in Poland was conducted. The \nindicators for the research were chosen from the point of view of the specificity of the operation in the research \nenterprise and collected data. The estimation of the technical work equipment, which is used in the structures \nof the enterprise, illustrates the dependence of the replacement value of the machine park and labor inputs as \nwell as the work efficiency achieved with the use of these resources. This indicator is the result element of the \nimplemented TPM strategy and the principles of maintaining machinery and equipment in the technological \nline. The technical work equipment is the most general and most common indicator showing the technical level \nof an enterprise. This indicator is the ratio of value of fixed production assets to the number of employees [14]: 𝑇𝑒𝑐ℎ𝑛𝑖𝑐𝑎𝑙 𝑤𝑜𝑟𝑘 𝑒𝑞𝑢𝑖𝑝𝑚𝑒𝑛𝑡=\n𝑣𝑎𝑙𝑢𝑒 𝑜𝑓 𝑓𝑖𝑥𝑒𝑑 𝑝𝑟𝑜𝑑𝑢𝑐𝑡𝑖𝑜𝑛 𝑎𝑠𝑠𝑒𝑡𝑠\n𝑒𝑚𝑝𝑙𝑜𝑦𝑚𝑒𝑛𝑡 𝑇𝑒𝑐ℎ𝑛𝑖𝑐𝑎𝑙 𝑤𝑜𝑟𝑘 𝑒𝑞𝑢𝑖𝑝𝑚𝑒𝑛𝑡=\n𝑣𝑎𝑙𝑢𝑒 𝑜𝑓 𝑓𝑖𝑥𝑒𝑑 𝑝𝑟𝑜𝑑𝑢𝑐𝑡𝑖𝑜𝑛 𝑎𝑠𝑠𝑒𝑡𝑠\n𝑒𝑚𝑝𝑙𝑜𝑦𝑚𝑒𝑛𝑡 𝑇𝑒𝑐ℎ𝑛𝑖𝑐𝑎𝑙 𝑤𝑜𝑟𝑘 𝑒𝑞𝑢𝑖𝑝𝑚𝑒𝑛𝑡=\n𝑣𝑎𝑙𝑢𝑒 𝑜𝑓 𝑓𝑖𝑥𝑒𝑑 𝑝𝑟𝑜𝑑𝑢𝑐𝑡𝑖𝑜𝑛 𝑎𝑠𝑠𝑒𝑡𝑠\n𝑒𝑚𝑝𝑙𝑜𝑦𝑚𝑒𝑛𝑡 𝑚𝑒𝑛𝑡=\n𝑣𝑎𝑙𝑢𝑒 𝑜𝑓 𝑓𝑖𝑥𝑒𝑑 𝑝𝑟𝑜𝑑𝑢𝑐𝑡𝑖𝑜𝑛 𝑎𝑠𝑠𝑒𝑡𝑠\n𝑒𝑚𝑝𝑙𝑜𝑦𝑚𝑒𝑛𝑡\n \n \n \n \n \n \n (1) (1) The numerator \"may include all fixed assets or only the so-called active part of fixed assets (machinery and \ntechnical equipment without buildings)\" [15]. This part of the analysis of production resources is related to \ntechnical progress that may occur in the enterprise. The increase in value of equipment enhances the \nproduction force and in some way facilitates human work, which is expressed in higher productivity. When assessing the productivity of fixed assets or production assets, the research focuses primarily on \nassessing the relationship between fixed assets and the volume of production. Therefore, an important \nindicator that presents the efficiency relationship is the indicator showing the ratio of achieved production \neffects per 1 PLN of fixed assets involved in the production process [14]: 𝑃𝑟𝑜𝑑𝑢𝑐𝑡𝑖𝑣𝑖𝑡𝑦 𝑜𝑓 𝑓𝑖𝑥𝑒𝑑 𝑝𝑟𝑜𝑑𝑢𝑐𝑡𝑖𝑜𝑛 𝑎𝑠𝑠𝑒𝑡𝑠=\n𝑝𝑟𝑜𝑑𝑢𝑐𝑡𝑖𝑜𝑛\n𝑎𝑣𝑒𝑟𝑎𝑔𝑒 𝑣𝑎𝑙𝑢𝑒 𝑜𝑓 𝑓𝑖𝑥𝑒𝑑 𝑝𝑟𝑜𝑑𝑢𝑐𝑡𝑖𝑜𝑛 𝑎𝑠𝑠𝑒𝑡𝑠 \n \n \n \n (2) 3. \nRESULTS Table 1 presents summary of calculated indicators in the research period. To calculate these indicators, the \nvalue of production machinery and equipment of the chosen rolling mill was taken into account. Table 1 Summary of indicators in the research period [own study] Table 1 Summary of indicators in the research period [own study] \nIndicator \nYear 1 \nYear 2 \nYear 3 \nYear 4 \nYear 5 \nTechnical work equipment [PLN/person] \n509453.42 469646.47 398311.98 339327.97 299302.57 \nProductivity of fixed production assets [kg/PLN] \n2.49 \n2.62 \n3.66 \n4.82 \n4.25 \nFixed production assets involvement indicator [PLN/Mg] \n400.90 \n381.10 \n273.47 \n207.36 \n235.17 \nWorking time indicator for production equipment \n0.787 \n0.787 \n0.813 \n0.762 \n0.687 \nBreaks time indicator for production equipment \n0.213 \n0.213 \n0.187 \n0.238 \n0.313 In the case of the research rolling mill, the technical work equipment was systematically decreasing. This was \nrelated to changes in the means of production, as there were no major changes in the volume of employment. That is why this situation was related only to the general aging of the machine park and the lack of new \ninvestments in recent years. In the case of the research rolling mill, the technical work equipment was systematically decreasing. This was \nrelated to changes in the means of production, as there were no major changes in the volume of employment. That is why this situation was related only to the general aging of the machine park and the lack of new \ninvestments in recent years. In the case of the research rolling mill, the technical work equipment was systematically decreasing. This was \nrelated to changes in the means of production, as there were no major changes in the volume of employment. That is why this situation was related only to the general aging of the machine park and the lack of new \ninvestments in recent years. Productivity of fixed production assets and fixed production assets involvement indicator deserve a lot of \nattention. Despite a clear decrease in the value of production assets, these changes do not coincide with \nchanges in the production volume. What's more, it is possible to notice a continuous increase in productivity, \nexcept for last year of research period, when small decrease of the indicator was observed. This means that \ndespite the decrease in the value of production means and the lack of investment, the rolling mill is able to \nproduce the high amount of products. 𝑃𝑟𝑜𝑑𝑢𝑐𝑡𝑖𝑣𝑖𝑡𝑦 𝑜𝑓 𝑓𝑖𝑥𝑒𝑑 𝑝𝑟𝑜𝑑𝑢𝑐𝑡𝑖𝑜𝑛 𝑎𝑠𝑠𝑒𝑡𝑠=\n𝑝𝑟𝑜𝑑𝑢𝑐𝑡𝑖𝑜𝑛\n𝑎𝑣𝑒𝑟𝑎𝑔𝑒 𝑣𝑎𝑙𝑢𝑒 𝑜𝑓 𝑓𝑖𝑥𝑒𝑑 𝑝𝑟𝑜𝑑𝑢𝑐𝑡𝑖𝑜𝑛 𝑎𝑠𝑠𝑒𝑡𝑠 (2) Fixed production assets involvement indicator have an absorbent nature. It presents the opposite attitude to \nthe productivity of fixed production assets. It represents the amount of fixed assets involved per unit of \nproduction volume. In the literature it is often called the indicator of capital intensity or material intensity [16]. This indicator is calculated according to the following formula [17]: May 20th - 22nd 2020, Brno, Czech Republic, EU 𝐹𝑖𝑥𝑒𝑑 𝑝𝑟𝑜𝑑𝑢𝑐𝑡𝑖𝑜𝑛 𝑎𝑠𝑠𝑒𝑡𝑠 𝑖𝑛𝑣𝑜𝑙𝑣𝑒𝑚𝑒𝑛𝑡 𝑖𝑛𝑑𝑖𝑐𝑎𝑡𝑜𝑟=\n𝑎𝑣𝑒𝑟𝑎𝑔𝑒 𝑣𝑎𝑙𝑢𝑒 𝑜𝑓 𝑓𝑖𝑥𝑒𝑑 𝑝𝑟𝑜𝑑𝑢𝑐𝑡𝑖𝑜𝑛 𝑎𝑠𝑠𝑒𝑡𝑠\n𝑝𝑟𝑜𝑑𝑢𝑐𝑡𝑖𝑜𝑛 (3) Efficiency of use of production equipment is measured by the time of their work in the course of production. The most frequently calculated indicator is work time indicator for production equipment, presenting the ratio \nof the effective working time to the maximum possible to use working time [15]: 𝑊𝑜𝑟𝑘𝑖𝑛𝑔 𝑡𝑖𝑚𝑒 𝑖𝑛𝑑𝑖𝑐𝑎𝑡𝑜𝑟 𝑓𝑜𝑟 𝑝𝑟𝑜𝑑𝑢𝑐𝑡𝑖𝑜𝑛 𝑒𝑞𝑢𝑖𝑝𝑚𝑒𝑛𝑡=\n𝑠𝑢𝑚 𝑜𝑓 𝑡ℎ𝑒 𝑒𝑓𝑓𝑒𝑐𝑡𝑖𝑣𝑒 𝑤𝑜𝑟𝑘𝑖𝑛𝑔 𝑡𝑖𝑚𝑒\n𝑠𝑢𝑚 𝑜𝑓 𝑡ℎ𝑒 𝑚𝑎𝑥𝑖𝑚𝑢𝑚 𝑝𝑜𝑠𝑠𝑖𝑏𝑙𝑒 𝑡𝑜 𝑢𝑠𝑒 𝑤𝑜𝑟𝑘𝑖𝑛𝑔 𝑡𝑖𝑚𝑒 \n (4) (4) To illustrate the extent to which production machines and equipment were not used, the breaks time indicator \ncan be used. It can be calculated as follows [15]: 𝐵𝑟𝑒𝑎𝑘𝑠 𝑡𝑖𝑚𝑒 𝑖𝑛𝑑𝑖𝑐𝑎𝑡𝑜𝑟 𝑓𝑜𝑟 𝑝𝑟𝑜𝑑𝑢𝑐𝑡𝑖𝑜𝑛 𝑒𝑞𝑢𝑖𝑝𝑚𝑒𝑛𝑡=\n𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑏𝑟𝑒𝑎𝑘𝑠 ℎ𝑜𝑢𝑟𝑠\n𝑠𝑢𝑚 𝑜𝑓 𝑡ℎ𝑒 𝑚𝑎𝑥𝑖𝑚𝑢𝑚 𝑝𝑜𝑠𝑠𝑖𝑏𝑙𝑒 𝑡𝑜 𝑢𝑠𝑒 𝑤𝑜𝑟𝑘𝑖𝑛𝑔 𝑡𝑖𝑚𝑒 \n \n (5) (5) There are no specific values for individual indicators in the literature, because their level depends on many \nfactors. However, it is worth calculating them at regular intervals and comparing them to assess the overall \nsituation of the company. In addition, an analysis of the causes of breaks in the rolling mill was carried out. The percentage shares of \nindividual causes of breaks were calculated for each year in the analyzed period. 3. \nRESULTS The decrease of the indicator in year 5 was caused by a decrease in \norders for products, and thus a decrease in general production. The opposite situation was illustrated by fixed \nproduction assets involvement indicator. There was less and less fixed production assets for the production of \n1 Mg of products. With the exception of year 5, when production volume dropped dramatically, and thus a \nslight increase in the indicator was noted, which was caused by a decrease in demand. Working time indicator for production equipment informs about the rational use of production means. The \nhigher the value of the indicator is, the better the use of working time is. It can be seen that in year 1 and year May 20th - 22nd 2020, Brno, Czech Republic, EU 2 working time indicators were at the same level. In the following year, the rolling mill utilized the working time \nof the production means in over 0.813%, unfortunately in the last year only in almost 0.687%. Breaks time \nindicator for production equipment informs about level of breaks in working time. The high value of this indicator \nmeans low rationality of using the means of production. High value of this indicator was noted in year 5. This \nmeans that over 0.313 of the maximum possible to use time consists of different types of breaks in work of the \nrolling mean. However, one important thing should be emphasized that will be seen later in the further analysis. Research \nrolling mill produces different types of products, different dimensions of these products, which is associated \nwith the need for frequent changeover of the rolling mill. Additionally, the production plan includes periodic \ninspections and planned renovations of the rolling mill resulting from maintenance. Therefore, maximum \npossible to use time cannot be fully used. Fluctuations in the value of breaks time indicator for production equipment in individual years of the research \nperiod caused the need for additional research. The causes of breaks in work of the research rolling mill were \nanalyzed. Table 2 presents the causes and percentage shares of individual causes of breaks in the research \nperiod. 3. \nRESULTS Table 1 Summary of breaks in work of rolling mill in the research period, [%] [own study] \nCauses of breaks \nYear 1 \nYear 2 \nYear 3 \nYear 4 \nYear 5 \ntechnological \n73.08 \n84.21 \n87.71 \n76.46 \n75.05 \ndefects \n0.00 \n0.12 \n0.36 \n11.71 \n10.58 \nmechanical \n2.74 \n3.34 \n3.42 \n3.80 \n4.94 \nelectric \n4.67 \n3.64 \n3.20 \n3.26 \n4.09 \nenergetic \n1.83 \n1.15 \n0.95 \n1.51 \n1.40 \nexternal \n0.00 \n0.49 \n0.73 \n0.48 \n0.55 \norganizational, including: \n8.84 \n7.04 \n3.64 \n1.39 \n3.39 \n- lack of material \n0.00 \n3.28 \n2.40 \n0.00 \n0.00 \n- re-heating of material \n0.00 \n0.36 \n0.00 \n0.00 \n0.00 \n- other organizational reasons \n8.84 \n3.40 \n1.24 \n1.39 \n3.39 mmary of breaks in work of rolling mill in the research period, [%] [own study] Table 1 Summary of breaks in work of rolling mill in the research period, [%] [own study] Throughout the entire research period, technological breaks had the largest share in all breaks. As mentioned \nearlier, they are associated with all kinds of technological changes, primarily with machine as equipment \nchangeover but also inspections and repairs. However, some differences in the proportion of these breaks can \nbe seen. In year 2 and 3 the share of such breaks was around 85%, in the remaining years around 75%. This \nmeans that in the remaining years, the share of other, less desirable breaks increased. In year 4 and 5 a large share of breaks due to different production defects can be seen. This was related to \nthe increase in the number of defects during this period. They were caused by many factors: material defects \n(poor material composition) and technical factors resulting from the general condition of the equipment [13]. Mechanical breaks are associated with rolling mill breakdowns. However, there were no large differences in \nshare of this type of breaks. In the whole research period, this share did not exceed 5% of the total break time. This level of breaks was caused by the implementation of TPM principles and inspections and repairs at the \nrolling mill operation resulting from maintenance. A similar situation was noted in the case of electrical breaks, \ni.e. all the downtime associated with the lack of electricity or failures related to electricity. In the whole research \nperiod, this share was below 5%. Organizational breaks are noteworthy. Their large share was recorded in \nyear 1 and 2. 4. \nCONCLUSION The condition of production machines and devices influences not only the course of the production process, \nbut above all the quality of manufactured products. That is why proper management of machine exploitation is \nso important. Among the methods that are helpful in this case, it is necessary to highlight the TPM system \nfunctioning, among others, under the concept of Lean Management. TPM includes planned inspections and \nperiodic repairs to avoid waste related to e.g. breakdowns or defects of products [18,19]. The products from \nthe research rolling mill are used in many industries, e.g. in various types of constructions, machines, cars or \nbuildings. The proper quality of materials used in such constructions affects not only their quality but also the \nuser's safety. In the paper the use of rolling mill production machinery and equipment in one of Polish \nsteelworks was presented. Technical and technical indicators were used. The analysis showed that the \nproduction equipment was aging due to a lack of investment, but its overall condition should be considered \ngood. Despite the decrease in the value of fixed assets, it is possible to produce the right amount of products \nin accordance with orders. The working time of the machines was approx. 80%, except for the last research \nyear, when this level was below 70%. Nevertheless, it should be remembered that the breaks time should also \ninclude the time spent on the changeover of the rolling mill, so that a wide range of products can be produced. However, some investments in modernization of the machine park is needed because there has been an \nincrease in the number of defect. Such a careful approach to maintaining the machine park and thus ensuring \nthe safety of employees can also be useful in other industries, e.g. industrial soldering [20], biotechnology \n[21,22] or laser machining [23,24]. It may be also used in similar approaches to decision support [25] and \nevaluation methodology [26,27]. 3. \nRESULTS In the case of year 1, these were primarily other organizational breaks (breaks related to \nadditional days or free hours not previously planned). In case of year 2, apart from this reason, the lack of May 20th - 22nd 2020, Brno, Czech Republic, EU material also dominated, i.e. the lack of billets for production. In the same year there were breaks related to \nthe need to re-heat material before rolling. To avoid production defect and to modernize production, rolling mill \nmanagement should start investing more in production resources. Along with a decrease in the value of \nproduction means, their aging occurs, which results in greater system failure rate and more frequent defects. material also dominated, i.e. the lack of billets for production. In the same year there were breaks related to \nthe need to re-heat material before rolling. To avoid production defect and to modernize production, rolling mill \nmanagement should start investing more in production resources. Along with a decrease in the value of \nproduction means, their aging occurs, which results in greater system failure rate and more frequent defects. REFERENCES [1] \nNOWICKA-SKOWRON, M., ULEWICZ, R. Quality management in logistics processes in metal branch. In 24th \nInternational conference on metallurgy and materials, In METAL 2015: 24th Int. Conf. on Metallurgy and \nMaterials. Ostrava: TANGER, 2015, pp. 1707-1712. [1] \nNOWICKA-SKOWRON, M., ULEWICZ, R. Quality management in logistics processes in metal branch. In 24th \nInternational conference on metallurgy and materials, In METAL 2015: 24th Int. Conf. on Metallurgy and \nMaterials. Ostrava: TANGER, 2015, pp. 1707-1712. [2] \nMAZUR, M., MOMENI, H. Lean Production issues in the organization of the company - results. Production \nEngineering Archives. 2019, vol. 22, pp. 50-53, doi: 10.30657/pea.2019.22.10. [3] \nMASZKE, A., DWORNICKA, R., ULEWICZ, R. Problems in the Implementation of the Lean Concept at a Steel \nWorks - Case Study. MATEC Web of Conferences. 2018, vol. 183, doi: 10.1051/matecconf/201818301014. [4] \nMASZKE, A. The analysis of machine operation and equipment loss in ironworks and steelworks, Production \nEngineering Archives. 2017, vol. 17, pp. 45-48. doi: 10.30657/pea.2017.17.10. [5] \nULEWICZ, R., SELEJDAK, J., BORKOWSKI, S., JAGUSIAK-KOCIK, M., Process management in the cast iron \nfoundry. In METAL 2013: 22nd Int. Conf. on Metallurgy and Materials. Ostrava: TANGER, 2013, pp. 1926-1931. [6] \nSÖDERHOLM, P., NILSEN, T. Systematic risk-analysis to support a living maintenance programme for railway \ninfrastructure. Journal of Quality in Maintenance Engineering. 2017, vol. 23, no. 3, pp. 326-340, doi: \n10.1108/JQME-09-2016-0042. May 20th - 22nd 2020, Brno, Czech Republic, EU [7] \nGÁLOVÁ, K., RAJNOHA, R., ONDRA, P. The use of industrial lean management methods in the economics \npractice: An empirical study of the production companies in the Czech Republic. Polish Journal of Management \nStudies. 2018, vol. 17, iss. 1, pp. 93-104, DOI: 10.17512/pjms.2018.17.1.08. [8] \nALMEANAZEL, O.T.R. Total Productive Maintenance Review and Overall Equipment Effectiveness \nMeasurement. Jordan Journal of Mechanical and Industrial Engineering. 2010, vol. 4, no. 4, pp. 517-522. [9] \nWUDHIKARN, R. Implementation of overall equipment effectiveness in wire mesh manufacturing. In 2011 IEEE \nInternational Conference on Industrial Engineering and Engineering Management, IEEE, 2011, pp. 819-823. [10] \nLYSANOV, D.M., KARAMYSHEV, A.N., EREMINA, I.Il. Comparative Evaluation Of Quality Characteristics Of \nProcess Equipment. Astra Salvensis. 2017, year V, no. 10, pp. 217-223. [11] \nSHARMA, R.K., DINESH, K., PRADEEP, K. Manufacturing excellence through TPM implementation: a practical \nanalysis. Industrial Management & Data System. 2006, vol.106, pp. 256-280. [12] \nKNOP, K., MIKULOVÁ, P. REFERENCES Evaluation of the Level of Work Safety, Advancement and Efficiency of the Use of \nMachines and Devices on Labelling and Packaging Line and an Attempt to Compare the Results Using the \nModified McKinsey Matrix. System Safety: Human-Technical Facility-Environment. 2019, vol.1, iss. 1, pp. 189-\n196. [13] \nKLIMECKA-TATAR, D., DWORNICKA, R. The Assembly Process Stability Assessment Based on the Strength \nParameters Statistical Control of Complex Metal Products. In METAL 2019: 28th Int. Conf. on Metallurgy and \nMaterials. Ostrava: TANGER, 2019, pp. 709-714. [14] \nJANASZ, W., URBAŃCZYK, E., WAŚNIEWSKI, T. Gospodarka środkami trwałymi w przedsiębiorstwie. Warszawa: Państwowe Wydawnictwo Ekonomiczna, 1988. [15] \nSOBCZYK, G. (ed.) Ekonomika przedsiębiorstwa. Zbiór przykładów i zadań. Lublin: Wyd. UMCS, 1995. 6] \nGRABARA, A.S. Ekonomika i organizacja pracy w przedsiębiorstwie. Częstochowa: Skrypt uczelniany [16] \nGRABARA, A.S. Ekonomika i organizacja pracy w przedsiębiorstwie. Częstochowa: Skrypt uczelniany PCz, 1986\n[17]\nINGALDI M\nDZIUBA S T The Use of the FMEA Method for Qualitative Analysis of Steel Bars In METAL 2018: 7] \nINGALDI, M., DZIUBA, S.T. The Use of the FMEA Method for Qualitative Analysis of Steel Bars. In M\n27th Int. Conf. on Metallurgy and Materials. Ostrava: TANGER, 2018, pp. 1996-2001. [18] \nTABOR, J. Maintenance management and occupational safety in manufacturing organizations. Polish Journal of \nManagement Studies. 2014, vol. 10, iss. 2, pp. 225-235. [19] \nKRYNKE, M., MIELCZAREK, K., VAŠKO, A. Analysis of the Problem of Staff Allocation to Work Stations. Conf. Quality Production Improvement – CQPI. 2019, vol. 1, iss. 1, pp. 545-550, doi: 10.2478/cqpi-2019-0073. [20] \nPIETRASZEK, J., GADEK-MOSZCZAK, A., TORUNSKI, T. Modeling of Errors Counting System for PCB \nSoldered in the Wave Soldering Technology. Advanced Materials Research. 2014, vol.874, pp. 139-143. [21] \nSKRZYPCZAK-PIETRASZEK, E., PISKA, K., PIETRASZEK, J. Enhanced production of the pharmaceutically \nimportant polyphenolic compounds in Vitex agnus castus L. shoot cultures by precursor feeding strategy. Engineering in Life Sciences. 2018, vol. 18, pp. 287-297. [22] \nSKRZYPCZAK-PIETRASZEK, E., KWIECIEN, I., GOLDYN, A., PIETRASZEK, J. HPLC-DAD analysis of arbutin \nproduced from hydroquinone in a biotransformation process in Origanum majorana L. shoot culture. Phytochemistry Letters. 2017, vol. 20, pp. 443-448. [23] \nGADEK-MOSZCZAK, A., RADEK, N., WRONSKI, S., TARASIUK, J. Application the 3D Image Analysis \nTechniques for Assessment the Quality of Material Surface Layer Before and After Laser Treatment. Advanced \nMaterials Research-Switz. 2014, vol.874, pp.133-138. [24] \nRADEK, N., BARTKOWIAK, K. Laser treatment of electro-spark coatings deposited in the carbon steel substrate \nwith using nanostructured WC-Cu electrodes. Physics Procedia. 2012, vol. REFERENCES 39, pp. 295-301. [25] \nPACANA, A., PASTERNAK-MALICKA, M., ZAWADA. M., RADON-CHOLEWA, A. Decision support in the \nproduction of packaging films by cost-quality analysis. Przemysl Chemiczny. 2016, vol. 95, pp. 1042-1044. [26] \nBORKOWSKI, S., ULEWICZ, R., SELEJDAK, J., KONSTANCIAK, M., KLIMECKA-TATAR, D. The use of 3x3 \nmatrix to evaluation of ribbed wire manufacturing technology. In METAL 2012: 21st Int. Conf. on Metallurgy and \nMaterials. Ostrava: TANGER, 2012, pp. 1722-1728. [27] \nSYGUT, P., KLIMECKA-TATAR, D., BORKOWSKI, S. Theoretical Analysis of the Influence of Longitudinal Stress \nChanges on Band Dimensions During Continuous Rolling Process. Archives of Metallurgy and Materials. 2016, \nvol. 16, iss. 1, pp. 183-188, doi: 10.1515/amm-2016-0032."
https://openalex.org/W4377940325	https://www.scielo.br/j/rcbc/a/FW9HxzZkbDZZ7BWtRGxYTCz/?lang=en&format=pdf	English	null	Fecal microbiota transplantation and its repercussions in patients with melanoma refractory to anti-PD-1 therapy: scope review	Revista do Colégio Brasileiro de Cirurgiões	2,023	cc-by	8,996	Fecal microbiota transplantation and its repercussions in patients with melanoma refractory to anti-PD-1 therapy: scope review Transplante de microbiota fecal e suas repercussões em pacientes com melanoma refratário à terapia anti-PD-1: revisão de escopo Eduardo Cerchi Barbosa AcCBC-GO1 ; Eduarda Emília Cruz Bucar1 ; Gabriel Rodrigues Jubé1 ; Letícia Bonfim SilveiraNDbeSepF 1 ; Pedro Lucas Carneiro Ramos1 ; Vitor Ryuiti Yamamoto Moraes1 ; João Ormindo Beltrão Barros1,2 . 1 ; natália Cândido duailiBE silva1 ; PEdro Carvalho CamPos faria Eduardo Cerchi Barbosa AcCBC-GO1 ; Eduarda Emília Cruz Bucar1 ; Gabriel Rodrigues Jubé1 ; Letícia Bonfim SilveiraNDbeSepF 1 ; Pedro Lucas Carneiro Ramos1 ; Vitor Ryuiti Yamamoto Moraes1 ; João Ormindo Beltrão Barros1,2 . 1 ; natália Cândido duailiBE silva1 ; PEdro Carvalho CamPos faria A B S T R A C T A B S T R A C T Introduction: despite being extremely effective in some cases, up to 70% of patients with melanoma do not respond to anti-PD-1/ PD-L1 (primary resistance) and many of the responders eventually progress (secondary resistance). Extensive efforts are being made to overcome this resistance through new strategies, especially aimed at modulating the intestinal microbiota. Objective: to assess whether fecal microbiota transplantation (FMT), associated with immunotherapy, is beneficial in the clinical course of patients with refractory melanoma. Methods: this is a scope review, based on studies collected on the MEDLINE, ScienceDirect, The Cochrane Library, Embase and BMJ Journals; using the terms: “Antibodies, Monoclonal”; “Drug Resistance, Neoplasm”; “Fecal Microbiota Transplantation”; “Host Microbial Interactions”; “Immunotherapy”; “Melanoma”; and “Microbiota”. Clinical trials, in English, with relevant data on the subject and fully available were included. A cut-off period was not determined, due to the limited amount of evidence on the topic. Results: crossing the descriptors allowed the identification of 342 publications and, after applying the eligibility criteria, allowed the selection of 4 studies. From the analyses, it was observed that a considerable part of those studied overcame resistance to immune checkpoint inhibitors after FMT, with better response to treatment, less tumor growth and increased beneficial immune response. Conclusion: it is noted that FMT favors the response of melanoma to immunotherapy, translated into significant clinical benefit. However, further studies are necessary for the complete elucidation of the bacteria and the mechanisms involved, as well as for the translation of new evidence to oncological care practice. Keywords: Antibodies, Monoclonal. Immunotherapy. Melanoma. Microbiota. Fecal Microbiota Transplantation Melanoma is a neoplasm originating from the skin formed from the transformation of melanin- producing cells (melanocytes) and may appear in skin without previous lesions (60-80%) or with pigmented lesions (20-40%), such as melanocytic nevi3. Although it has a lower incidence when compared with non- melanomas, melanoma is the most important form of skin tumor, as it grows and spreads more quickly, has a higher risk of recurrence, and a worse prognosis, with a worldwide survival estimate in five years of 69%, 73% in developed countries and 56% in developing ones4,5. DOI: 10.1590/0100-6991e-20233490-en DOI: 10.1590/0100-6991e-20233490-en Review Article 1 - Universidade Evangélica de Goiás, Medicina - Anápolis - GO - Brasil 2 - Hospital Santa Casa de Anápolis, Cancerologia Cirúrgica - Anápolis - GO - Brasil 1 - Universidade Evangélica de Goiás, Medicina - Anápolis - GO - Brasil 2 - Hospital Santa Casa de Anápolis, Cancerologia Cirúrgica - Anápolis - GO - 1 - Universidade Evangélica de Goiás, Medicina - Anápolis - GO - Brasil INTRODUCTION C ancer is a state that involves both genetic and epigenetic interactions, culminating in a disorderly, autonomous, and uncontrollable proliferation of cells, which may eventually migrate to adjacent structures. This migratory growth characteristic belongs to invasive neoplasms, being called “malignant tumors”. There are more than 200 types of cancer, each with specific clinical and morphological characteristics, with melanoma being the third most common and the most lethal among skin cancers, with an estimated increase of 50% in new cases per year by 2040, according to data from the International Agency for Research on Cancer (IARC)1,2. C Among the risk factors for the development of this tumor, the inheritance of sun-sensitive skin stands out (Fitzpatrick skin types I and II – light skin, hair, and Rev Col Bras Cir 50:e20233490 Barbosa i Barbosa Fecal microbiota transplantation and its repercussions in patients with melanoma refractory to anti-PD-1 therapy: scope review eyes, and susceptibility to sunburn). Other factors include the presence of pigmented lesions, such as freckles, atypical nevi, or large numbers of common nevi (more than 50), intermittent sun exposure, sunburn (especially during childhood), artificial ultraviolet radiation (eg, use of tanning beds), proximity to the equator, and previous melanoma6,7. Positive family history for both melanoma and multiple atypical nevi is also considered a relevant factor. Mutations in the CDKN2A and CDK4 genes have been detected in some families with hereditary melanoma, conferring an approximately 60-90% increased risk for this neoplasm8,4. In addition, the immune status also seems to influence the development of melanoma, as this neoplasm is frequent in immunocompromised patients due to organ transplantation, human immunodeficiency virus (HIV) infection, hematologic malignancy, or use of immunosuppressants9. metastases), adjuvant treatment, systemic therapy, and/ or radiotherapy14. In cases of unresectable, metastatic cutaneous melanoma or with high rates of recurrence (stages III and IV), specific therapies should be considered, such as immunotherapy with immune checkpoint inhibitors (ICIs). Currently, the drugs most used in this treatment are the humanized monoclonal antibodies (mAbs) anti- PD-1 (pembrolizumab and nivolumab) and anti-PD-L1 (atezolizumab, avelumab, and durvalumab), which act by inhibiting the activity of the programmed cell death protein-1 (PD-1)15,16. These drugs bind to the PD-1 receptor, an immunological receptor present on the surface of tumor cells, preventing the receptor from interacting with specific ligands (PD-L1 and PD-L2). INTRODUCTION Blocking the PD-1 pathway signaling cascade inhibits the negative regulation of the immune system, reversing the suppression of T cells and culminating in the anti-tumor response17. The diagnostic suspicion, in general, is based on the identification of a new pigmented lesion (ab initio) or phenotypic alterations of a pre-existing melanocytic nevus. During the assessment, the mnemonic “ABCDE” has been shown to be useful: A – asymmetry; B – irregular edges; C – non-uniform coloring; D – diameter > 6mm; E – evolution, including rapid growth, pruritus, ulceration, and bleeding. The greater the positivity of these signs, the greater the probability of the lesion being a malignant melanoma. However, the definitive diagnosis is made through histopathological examination, which can identify the superficial disseminated, nodular, acral lentiginous, or lentigo maligna patterns10,11. Anti-PD-1/PD-L1 Anti-PD-1/PD-L1 demonstrated superior efficacy and a more favorable toxicity profile than other mAbs, such as ipilimumab, an inhibitor of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), also widely used in the therapeutic approach of melanoma18,19. However, although these blockers show extreme effectiveness in some cases, up to 70% of patients do not respond to PD-1 blockade (primary resistance) and many who do respond eventually progress (secondary resistance). Therefore, efforts are currently being made to understand in detail the mechanisms underlying resistance, aiming to elucidate biomarkers that can potentially help in identifying responders, as well as finding new strategies that can decrease anti-PD-1/PD- L1 resistance20-24. In terms of prognosis and treatment, staging the disease is essential, in accordance with the American Joint Commission on Cancer (AJCC) protocol, which divides patients into four distinct groups. The group is defined according to the phase the patient is in, in which stages I and II are considered clinically localized disease, stage III, locoregional disease, including individuals with metastases to regional or in-transit lymph nodes, and stage IV, comprising individuals with distant metastasis12. In search of advances in medicine, researchers returned to a Chinese practice dating back to the fourth century, during the Dong Jin dynasty, known as fecal microbiota transplantation (FMT). This practice consists of transferring fecal material from a healthy donor to a sick recipient, to repopulate the latter’s intestine. Despite being very old, FMT was scientifically reported for the first time in 1958, when it was successfully used in the treatment of four patients with pseudomembranous colitis25-27. Even in the face of apparent efficacy, only in the last ten years has FMT begun to be widely studied, After histopathological confirmation and staging, the treatment to be adopted is established13. The therapeutic management of melanoma includes different approaches, such as surgical intervention (excision with widened margins, sentinel lymph node investigation, lymphatic drainage, and resection of distant Rev Col Bras Cir 50:e20233490 2 Barbosa Fecal microbiota transplantation and its repercussions in patients with melanoma refractory to anti-PD-1 therapy: scope review Barbosa F l i this question, we carried out a broad search, aiming to minimize publication biases, in the following databases, in order of consultation: Medical Literature Analysis and Retrieval System Online (MEDLINE); ScienceDirect; The Cochrane Library; Excerpta Medica Database (EMBASE); and BMJ Journals. Anti-PD-1/PD-L1 During the search, we used the following Health Sciences Descriptors: “Antibodies, Monoclonal”; “Drug Resistance, Neoplasm”; “Fecal Microbiota Transplantation”; “Host Microbial Interactions”; “Immunotherapy”; “Melanoma”; and “Microbiota”. having proved to be beneficial, mainly when associated with other therapies, in various pathologies, including cancers such as gastric, colorectal, hepatocellular, pancreatic, breast, lung, and melanoma28-33. Therefore, due to the favorable pre-existing history, the present study aims to evaluate whether the ICI-FMT combined therapy positively impacts the clinical course of patients with melanoma refractory to isolated immunotherapy, representing a potential step during the therapeutic process of this neoplasm. METHODS The inclusion criteria of the studies were clinical trials with relevant data on the application of ICI-FMT therapy in patients with melanoma, research written only in English, and articles with the text available in full. We excluded articles outside thematic relevance and non-clinical studies, including research in animal and in vitro models, as well as those published in the form of letters to the editor, guidelines, books, literature reviews, dissertations, and case reports. A time cut-off period was not determined during the selection of studies, due to the limited amount of evidence on the subject. This is a descriptive study of exploratory nature in literature in the scope review modality, which is a research method that makes it possible to identify and analyze a set of scientific evidence to obtain a reliable understanding about a particular theme of study. This method makes it possible to summarize the state of knowledge in a given area, as well as pointing out gaps that need to be filled with new research. Therefore, it entails support for decision-making and improvement of clinical practice34. We used the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Extension for Scoping Reviews (PRISMA-ScR)34 strategy, through the following steps: (i) theme identification; (ii) formulation of the research question; (iii) collection of articles through searches on electronic platforms; (iv) eligibility of studies with application of inclusion and exclusion criteria; (v) assessment of the quality of evidence; (vi) elaboration of the collection instrument with the information to be extracted; (vii) data analysis, interpretation, synthesis, and discussion; and (viii) presentation of results. The final protocol was prospectively registered in the Open Science Framework platform. RESULTS The combination of search terms resulted in the identification of 342 publications. After removal of duplicates (n=27), 315 references were submitted to screening, with analysis of titles and abstracts, of which 302 were excluded by reviewers. The 13 remaining studies were read in full by two independent examiners for the application of the eligibility criteria, culminating in the final inclusion of four trials that met these criteria. The selected studies were published between 2019 and 2021. The research question was formulated based on the PICO strategy (Population, Intervention, Control, Outcome)35, where: Population – patients with melanoma refractory to immunotherapy with ICIs; Intervention – FMT + ICI; Control – ICIs monotherapy; Outcome – clinical benefit. Thus, we formulated the following guiding question: “Does the combined ICI- FMT therapy have a positive impact on the clinical course of patients with refractory melanoma?”. To answer After conducting the steps of identification, screening, and eligibility of the articles, it was possible to stratify and gather the main evidence related to the application of the ICI-FMT combined therapy in patients with melanoma. Studies extracted from each of the databases were recorded in a single Microsoft Excel 16.0 spreadsheet, with the aim of detecting repeated citations between platforms, forming the list of studies and assessing eligibility. Rev Col Bras Cir 50:e20233490 3 3 Barbosa i Barbosa Fecal microbiota transplantation and its repercussions in patients with melanoma refractory to anti-PD-1 therapy: scope review The results of clinical trials that investigated the impact of FMT on the effectiveness of the immune checkpoint inhibitor are summarized in Table 1. proliferation of T cells, as well as promoting immunological tolerance to self-antigens, preventing auto-inflammatory reactions and autoimmunity in the healthy host40,41. However, after continuous exposure and recognition of tumor antigens, tumor-specific effector T cells induce increased expression of PD-1 and secrete interferon-gamma (IFN-γ), a chemical signal that induces the expression of B7 H1 (PD-L1) in neoplastic cells. The PD-1/PD L1 interaction strongly suppresses T cell activation, disrupting its anti-tumor activity. This interruption of the T cell antineoplastic response, related to the anergy or “exhaustion” phenotype, represents a form of local immunocompromise that allows tumors to escape immunological surveillance42,43. PD-1/PD-L1 pathway as a therapeutic target It is estimated that its microbial population reaches 1011 to 1012 CFU/mL of intestinal contents, with about 700 different species of microorganisms, most of which are bacteria. However, although bacteria can be found throughout the GIT, the largest number resides in the colon56,57. Figure 1. PD-1/PD-L1 pathway blocking mechanism. The CD8+ T cell is activated after recognizing the tumor antigen presented by the MHC class I. After its activation, the CD8+ T cell releases IFN-γ, which binds to its receptor (IFN γR). From this connection, the tumor cell is induced to express PD-L1, which binds to the PD-1 protein, triggering an inhibitory effect against CD8+ T cells. Anti-PD 1 or anti PD L1 prevents the interac- tion between PD 1 and PD L1, suppressing the inhibitory action against CD8+ T cells and thus increasing the anti-tumor activity. PD-1, program- med cell death 1; anti-PD-1, PD-1 antibody; PDL-1, programmed-death ligand 1; anti-PDL-1, PDL-1 antibody; TCR, T cell receptor; MHC, major histocompatibility complex; IFN-γ; interferon-γ; IFN-γR, IFN- γ receptor; JAK1, janus kinase 1; JAK2, janus kinase 2; STAT-1, signal transducer and activator of transcription 1; IRF-1, interferon regulatory factor 1. Source: Adapted from Lei et al. (2020)24. In the ileum, the bacterial population is represented by 107 to 108 CFU/mL, consisting of facultative anaerobes, Enterobacteria, and obligate anaerobes, such as Veilonella, Enterococcus, Clostridium, Bacteroides, and Lactobacillus. On the other hand, in the large intestine, the population reaches the 1010 and 1011 CFU/mL and the most frequently found genera are Bacteroides, Bifidobacterium, Escherichia coli, Bacillus, Clostridium, Eubacterium, Fusobacterium, Peptostreptococcus, and Ruminococcus56-58. As a rule, facultative anaerobic bacteria such as E. coli, Enterococci faecalis, and E. faecium are the first to colonize the newborn’s GIT, due to the high level of oxygen that initially exists. As these microorganisms consume the gas, the medium becomes more suitable for restricted anaerobic bacteria such as Bacteriodes, Bifidobacterium, and Clostridium. The intestinal microbiota acquired in the postnatal period is composed of a vast diversity of bacteria and performs numerous functions in the human host56. Figure 1. PD-1/PD-L1 pathway blocking mechanism. The CD8+ T cell is activated after recognizing the tumor antigen presented by the MHC class I. After its activation, the CD8+ T cell releases IFN-γ, which binds to its receptor (IFN γR). From this connection, the tumor cell is induced to express PD-L1, which binds to the PD-1 protein, triggering an inhibitory effect against CD8+ T cells. PD-1/PD-L1 pathway as a therapeutic target PD-1 is a protein of the CD28 superfamily, characterized by generating negative signals when binding to PD-L1 and PD-L2 proteins. Both PD-1 and its ligands are widely expressed in different cell types (B cells, CD4+ and T CD8+ cells, natural killer cells, and dendritic cells) that physiologically act by limiting the activation and Table 1 - Selected clinical trials on the application of FMT associated with ICI in patients with refractory melanoma. Type of FMT- -associated immunotherapy Type of cancer Number of patients studied Number of patients with greater efficacy (%) Response to FMT in patients with greater efficacy Reference Anti-PD-1 Metastatic melanoma 5 3 (60%) Increased infiltration of functio- nal CD8+ T cells; increase in APCs (CD68+) in intestine and tumor; resis- tance to immunotherapy overcome Youngs- ter et al. (2019)36 Anti-PD-1 Metastatic melanoma 2 2 (100%) Increase in functional CD39+ and CD8+ T cells; decrease in dysfunctional PD1+, CD38+ and CD8+ T cells; resis- tance to immunotherapy overcome Maleki et al. (2020)37 Anti-PD-1 Metastatic melanoma 15 6 (40%) Higher number of functional CD8+ T cells; decrease in IL-8 producing mye- loid cells; resistance to immunotherapy overcome Davar et al. (2021)38 Anti-PD-1 Metastatic melanoma 10 3 (30%) Increased infiltration of functio- nal CD8+ T cells; increase in APCs (CD68+) in intestine and tumor; resis- tance to immunotherapy overcome Baruch et al. (2021)39 APCs: antigen-presenting cells. Source: Authors (2023). nical trials on the application of FMT associated with ICI in patients with refractory melanoma. Table 1 - Selected clinical trials on the application of FMT associated with ICI in patients with refractory melanoma. APCs: antigen-presenting cells. Source: Authors (2023). APCs: antigen-presenting cells. Source: Authors (2023). Blocking the PD-1/PD-L1 pathway (Figure 1) is considered one of the main targets of immunotherapy against cancer, with drugs already approved by the Food and Drug Administration (FDA), such as pembrolizumab, nivolumab, atezolizumab, and cemiplimab24,44-47. Table 2 highlights each drug, with its respective indications and main adverse effects. such as pembrolizumab, nivolumab, atezolizumab, and cemiplimab24,44-47. Table 2 highlights each drug, with its respective indications and main adverse effects. Rev Col Bras Cir 50:e20233490 4 Barbosa F l i Barbosa Fecal microbiota transplantation and its repercussions in patients with melanoma refractory to anti-PD-1 therapy: scope review (GIT). The GIT hosts the largest number and diversity of bacterial collections that colonize the human body. PD-1/PD-L1 pathway as a therapeutic target Anti-PD 1 or anti PD L1 prevents the interac- tion between PD 1 and PD L1, suppressing the inhibitory action against CD8+ T cells and thus increasing the anti-tumor activity. PD-1, program- med cell death 1; anti-PD-1, PD-1 antibody; PDL-1, programmed-death ligand 1; anti-PDL-1, PDL-1 antibody; TCR, T cell receptor; MHC, major histocompatibility complex; IFN-γ; interferon-γ; IFN-γR, IFN- γ receptor; JAK1, janus kinase 1; JAK2, janus kinase 2; STAT-1, signal transducer and activator of transcription 1; IRF-1, interferon regulatory factor 1. Source: Adapted from Lei et al. (2020)24. Among its various functions, the immunological stands out. The human intestinal mucosa is the main interface between the immune system and the external environment. The intestine is considered the largest immunological organ in the human body, housing about 80% of the cells of this system, being mainly responsible for the production of immunoglobulins essential for innate and adaptive immunity. In addition, it stimulates the immune system by recruiting immune cells and activating the function of epithelial cells, thus being able to modulate the organism’s multiple response pathways and tumor progression itself56,59-62. However, clinical data of anti-PD-1/PD L1 therapy show considerable rates of limited response, in which a large group of patients suffered primary resistance, not responding to therapy, and part of the responders developed acquired resistance over the course of treatment43. Extensive efforts are being made to overcome this resistance to therapy, especially in the approach to melanoma20-24. Intestinal Microbiota ADEs: adverse drug effects; NSCLC: non-small cell lung carcinoma; TCC: transitional cell carcinoma; cHL: classic Hodgkin’s lymphoma; RCC: renal cell carcinoma; HNSCC: head and neck squamous cell cancer; CRC: colorectal cancer; HCC: hepatocellular carcinoma; MPM: malignant pleural mesothe- lioma; BCC: basal cell carcinoma; SCC: squamous cell carcinoma. Source: Authors (2023). However, the microbiota is extremely dynamic and may be influenced by numerous factors. For example, most neoplasms reach extremes of age, when the structural ecology of the gut may be immature or disturbed by a lifetime’s exposure to environmental modifiers65. Furthermore, pathological states or specific therapies can also create an imbalance in the microbiota composition, increasing the influence of deleterious bacteria, as well as reducing the effectiveness of antineoplastic agents and exacerbating their toxicity. Therefore, the intestinal microbiota has been researched for the management of cancer, with the development of promising strategies, such as the modulation of the patients’ intestinal microorganisms’ content28-33. the use of feces as a therapy was described by Ge Hong, in China, in the fourth century, for the treatment of a variety of conditions, including diarrhea27,66,67. However, it was only in the 1950s that the practice was scientifically described by Eiseman et al. (1958)25, via fecal enema, for the treatment of pseudomembranous colitis, introducing FMT in conventional medicine. In general, the process initially involves selecting a donor with no personal or family history of metabolic, autoimmune, or malignant diseases, as well as screening for possible pathogens. The stool is then prepared by being mixed with water or normal saline, followed by a filtration step to remove any particulate matter. The mixture can be administered by nasogastric tube, nasojejunal tube, esophagogastroduodenoscopy, colonoscopy, retention enema, or lyophilized tablets. The choice of route depends on the institution facility, the physician’s experience, and the safety offered to the patient68. Intestinal Microbiota Moreover, the intestinal microbiota also influences the effectiveness of numerous drugs. With regard to antineoplastic agents, one of the first drugs to prove the influence of the intestinal microbiota on its response was cyclophosphamide. With the advancement The intestinal microbiota, previously referred to as flora, is a dynamic mixture of microorganisms, whose composition varies along the gastrointestinal tract Rev Col Bras Cir 50:e20233490 5 Barbosa i Barbosa Fecal microbiota transplantation and its repercussions in patients with melanoma refractory to anti-PD-1 therapy: scope review of high-throughput sequencing technologies, the importance of the intestinal microbiota in the modulation of chemotherapy drugs was listed and the search for new influenceable drugs is now increasingly recognized63,64. Table 2 - Applications and adverse effects of the main anti-PD-1/PD-L1 immunotherapies. References Antibody / drug Indications ADEs Garon et al. (2015)43 Martin-Liberal, et al. (2015)48 Ribas et al. (2016)49 Springman et al. (2020)50 Anti-PD-1 Pembrolizumab Melanoma, NSCLC, TCC, gastric cancer, cHL, RCC, HNSCC, esophageal cancer, CRC, endometrial cancer, HCC Fatigue, cough, nausea, pruritus, skin rash, decreased appetite, cold, arthralgia, diarrhea, infections Robert et al. (2015)45 Guo, Zhang, Chen (2017)51 Elias et al. (2017)52 Springman et al. (2020)50 Anti-PD-1 Nivolumab Melanoma, NSCLC, TCC, gastric cancer, cHL, RCC, HNSCC, esophageal cancer, HCC, MPM Skin rash, fatigue, dyspnoea, myalgia, decreased appetite, cough, nausea, cold Schmid et al. (2018)46 Elias et al. (2017)52 Tie et al. (2019)53 Anti-PD-L1 Atezolizumab Breast cancer, NSCLC, TCC Fatigue, decreased appetite, dysp- noea, cough, nausea, myalgia, cold, urinary infection, hypothyroidism, alopecia Migden et al. (2018)47 Goodman (2022)54 Sezer et al. (2021)55 Anti-PD-1 Cemiplimab BCC, SCC Diarrhea, fatigue, nausea, cold, chills, cough, diarrhea, pyrexia, hoarseness, pruritus, skin rash, backache ADEs: adverse drug effects; NSCLC: non-small cell lung carcinoma; TCC: transitional cell carcinoma; cHL: classic Hodgkin’s lymphoma; RCC: renal cell carcinoma; HNSCC: head and neck squamous cell cancer; CRC: colorectal cancer; HCC: hepatocellular carcinoma; MPM: malignant pleural mesothe- lioma; BCC: basal cell carcinoma; SCC: squamous cell carcinoma. Source: Authors (2023). Diarrhea, fatigue, nausea, cold, chills, cough, diarrhea, pyrexia, hoarseness, pruritus, skin rash, backache Anti-PD-1 Cemiplimab ADEs: adverse drug effects; NSCLC: non-small cell lung carcinoma; TCC: transitional cell carcinoma; cHL: classic Hodgkin’s lymphoma; RCC: renal cell carcinoma; HNSCC: head and neck squamous cell cancer; CRC: colorectal cancer; HCC: hepatocellular carcinoma; MPM: malignant pleural mesothe- lioma; BCC: basal cell carcinoma; SCC: squamous cell carcinoma. Source: Authors (2023). Influence of FMT on the response to anti-PD-1/PD- L1 in mice Given the evidence that the intestinal microbiota influenced the response to chemotherapy63,64, tests were carried out in mice with melanoma that would later be treated with FMT associated with immunotherapy. In the study by Sivan et al. (2015)69, two groups of genetically similar C57BL/6 mice from different locations were studied, the JAX group from the Jackson Laboratory and the TAC group from Taconic Farms, both of which presented differences in the intestinal microbial composition. Initially, melanoma cells were injected into both groups, and, before any other intervention, tumor growth was analyzed, which was more aggressive in TAC mice compared with JAX. Such responses were explained by divergences at the immunological level, the JAX group exhibiting a greater density of specific T cells against the tumor and a greater intratumoral accumulation of CD8+ T cells. After receiving treatment with PD-1 pathway inhibitors, significantly greater efficacy was observed in JAX mice. Aiming once again to determine the differences in intestinal microbial composition between immunotherapy responders and non-responders, Gopalakrishnan et al. (2018)72 showed that, in the R group (complete or partial response or stable disease for at least six months), there was a predominance of Faecalibacterium (belonging to the Ruminococcaceae family, order Clostridiales), while in the NR group (progressive or stable disease for less of six months), there was a predominance of Bacteroides thetaiotaomicron, Escherichia coli, and Anaerotruncus colihominis. In view of the results, the possible mechanisms by which the microbiota patterns could influence the response were described: in R patients, bacteria mainly exerted anabolic functions and biosynthesis of amino acids, in contrast to NR, in which predominantly catabolic functions were observed. Furthermore, in the R group, there was a Subsequently, to establish a cause-effect relationship, the fecal material of one of the JAX mice was transferred to TAC ones, generating a reduction in the tumor growth rate. In addition, some TAC mice were transplanted with JAX fecal microbiota associated with an anti-PD-L1, showing an even more effective response in tumor control. The analysis of the fecal material verified the predominance of bacteria of the genus Bifidobacterium (B. longum, B. breve, and B. adolescentis), which were found to be four hundred times more abundant in JAX mice. Fecal microbiota transplantation FMT consists of transferring a solution of fecal material from a donor to a recipient, aiming to directly change the microbial composition of the patient and confer benefits to his health. The first known report of Most of the clinical experience with FMT has been derived from the treatment of recurrent or refractory Rev Col Bras Cir 50:e20233490 6 Barbosa Fecal micr Barbosa Fecal microbiota transplantation and its repercussions in patients with melanoma refractory to anti-PD-1 therapy: scope review Based on the study by Sivan et al. (2015)69, new research was carried out aiming to list the differences in the constitution of the intestinal microbiota between individuals responsive (R) and non-responsive (NR) to immunotherapy, such as the studies by Frankel et al. (2017)70 and McCulloch et al. (2022)71. The former used a technique for sequencing the intestinal components of 39 individuals with metastatic melanoma before starting therapy with ICIs (ipilimumab, nivolumab, and pembrolizumab, either in monotherapy or combined). In general, all the R presented a microbiota enriched in Bacteroides caccae and Streptococcus parasanguinis, compared with the NR. In the second study, the analysis of the composition of the microbiota of 94 patients allowed the identification of the predominance of the following bacteria in group R: Ruminococcus (Mediterraneibacter) torques, Blautia (B. producta, B. wexlerae, and B. hansenii), Eubacterium rectale, Ruminococcus (Mediterraneibacter) gnavus, and Anaerostipes hadrus. On the other hand, in patients not responsive to anti-PD 1, a predominance of Prevotella spp., Oscillibacter spp., Alistipes spp., and Sutterellaceae spp. was observed. Subsequently, the fecal samples from these patients were taken for transcriptomic analysis, identifying a considerable increase of superoxide dismutase 2 (SOD2), pro-inflammatory cytokines (IL-1β and CXCL8), and transcription factors (NFKBIZ, NFKBIA, TNFAIP3, and LITAF) in NR. Clostridium difficile infection66,68. However, recent studies have listed its benefits, especially when associated with other therapies, in several pathologies, including cancers, such as gastric, colorectal, hepatocellular, pancreatic, breast, lung, and melanoma28-33. Influence of FMT on the response to anti-PD-1/PD- L1 in mice The presence of Bifidobacterium was strictly related to specific immune cytotoxicity against the tumor, translated by an exacerbated regulation of type-I IFN genes in antigen presenting cells (APCs) present in peripheral lymphoid organs, as well as a higher rate of maturation of dendritic cells and increased activity of effector CD8+ T cells69. Rev Col Bras Cir 50:e20233490 7 Barbosa i Barbosa i Barbosa Fecal microbiota transplantation and its repercussions in patients with melanoma refractory to anti-PD-1 therapy: scope review greater infiltration of CD8+ T cells in the tumor, as well as a greater diversity of immune cells. In order to confirm this relationship, fecal material from group R was transferred to germ-free mice, followed by inoculation of neoplastic melanoma cells and administration of PD 1 inhibitor. After two weeks, the transplanted mice evolved with a better response to therapy, lower tumor growth rate, and intestinal microbiota enriched with Faecalibacterium. In addition, they had a higher amount of CD8+ T cells, consistent with the results in human models. increase in the diversity of the intestinal microbiota (with a predominance of Ruminococcaceae, Bifidobacteriaceae, and Lachnospiraceae), a greater response to anti PD 1, an increase in CD8+ T cells activation, and a decrease in IL 8 producing myeloid cells. Therefore, it was shown that resistance to immunotherapy was overcome, as these patients, previously unresponsive to anti-PD-1 therapy, showed post-FMT clinical benefit, with tumor reduction and/or long-term disease stability. In addition, FMT has also been shown to modulate the levels of circulating chemical signals, since the R group showed a decrease in multiple cytokines associated with resistance to anti- PD-1 and an increase in biomarkers associated with a beneficial response to treatment. R patients were down regulated in circulating IL-8 as well as in tumor-producing IL-8 myeloid cells; IL-8 has been associated with low anti- PD-1 activity in several cancers, including melanoma. Matson et al. (2018)73 evaluated 42 patients with metastatic melanoma, of whom 26 responded to anti PD-1 treatment and 16 had disease progression. The most abundant bacteria among patients in group R were Bifidobacterium adolescentis, Bifidobacterium longum, Lactobacillus species, Klebsiella pneumoniae, Veillonella parvula, Parabacterioides shite, Collinsella aerofaciens, and Enterococcus faecium, while among NR patients they were Roseburia intestinalis and Ruminococcus obeum. Then human fecal material was transferred to germ-free mice, followed by inoculation of melanoma cells. Influence of FMT on the response to anti-PD-1/PD- L1 in mice They observed that the mice that received fecal material from the R group showed less tumor growth and greater infiltration of effector CD8+ T cells compared with those that received the microbiota from the NR group. The response to anti PD 1 therapy was also consistent with the response of human donors. However, there was a portion that did not have the same intestinal pattern as the donor, and there was no response to therapy. In view of this, they concluded that, although most mice mimic the donor’s response, certain bacteria may have different patterns of expansion and, therefore, generate changes in the recipient’s phenotype. Similar results were listed in the studies by Youngster et al. (2019)36 and Baruch et al. (2021)39, and in both there was a post-FMT increase in the infiltration of CD8+ T cells, as well as APCs (CD68+) in the intestine and in the tumor. Both examined therapy- resistant subjects with metastatic melanoma and defined resistance as the inability to achieve a lasting response to anti-PD-1 therapy. In the test performed by Youngster et al. (2019)36, three patients (60%) overcame resistance to immunotherapy. Furthermore, in this same study, there was a post-FMT increase in bacteria from the Paraprevotellaceae family, which have been associated with responsiveness to treatment, and a significant decrease in β-proteobacteria, which have been linked to resistance to treatment. In the phase 1 clinical trial carried out by Baruch et al. (2021)39, patients were treated with vancomycin and neomycin, aiming to eradicate their native microbiota, before receiving the lyophilized solution of fecal material associated with immunotherapy. They observed positive responses in three individuals, two partial and one complete. It is also worth noting that the patients with the highest response rate had an abundance of Ruminococcus (R. gnavus and R. callidus) and Bifidobacterium adolescentis, considered favorable to immunotherapy, while those with a lower response rate had a microbiota enriched in bacteria from the Clostridiaceae family. The researchers also showed an increase in the beneficial immune response. Influence of FMT on the response to anti-PD-1/PD-L1 in humans Thus, we haighlight t to conduct new randomized and controlled clini with a well-defined methodological design, pr follow-up time, representative sample, and low bias, to produce robust results and allow a more analysis of the benefits of this alternative therapy CONCLUSION This review presented consistent evidence regarding the use of ICI-FMT therapy in patients with refractory melanoma. However, it is worth mentioning its limitations. Among them, stands out the scarcity of studies that offer relevant data on the application of fecal microbiota transplantation associated with immunotherapy in individuals affected by melanoma. In addition, the few studies found also had limitations, mainly small sample size. Thus, we haighlight the need to conduct new randomized and controlled clinical trials, with a well-defined methodological design, prolonged follow-up time, representative sample, and low risk of bias, to produce robust results and allow a more in-depth analysis of the benefits of this alternative therapy. In the study carried out by Maleki et al. (2020)37, two individuals who had resistant melanoma underwent FMT from two different donors. Both patients showed an improvement in immune response, with an increase in the populations of CD39+ and CD8+ T cells, in addition to a decrease in the levels of dysfunctional PD1+, CD38+, and CD8+ T cells. Patient 1 also displayed stabilization of a large skin lesion. Some clinical trials aiming to evaluate the efficacy and safety of the ICI-FMT combination are in progress (Table 3). Despite the promising results of ICI-FMT therapy, there are still concerns about its long-term safety. In 2019, two independent clinical trials reported that two patients developed bacteremia by extended- spectrum β-lactamase-producing E. coli after receiving FMT from the same donor, leading to the death of one of them75. This study prompted the FDA to issue a safety bulletin warning of the risk of post-FMT infection. In addition, a recent retrospective cohort study analyzed donor stools and showed that six of the 66 individuals tested (9%) were positive for multidrug-resistant pathogens76. Influence of FMT on the response to anti-PD-1/PD-L1 in humans Based on the recognition of the influence of fecal transplantation associated with immunotherapy on the course of melanoma in mice, clinical trials were carried out with promising results. The trial conducted by Davar et al. (2021)38 analyzed the effect of FMT in 15 individuals with melanoma resistant to anti PD 1 therapy. After transplantation, six of the 15 patients (40%) showed an Rev Col Bras Cir 50:e20233490 8 Barbosa F l i Barbosa Fecal microbiota transplantation and its repercussions in patients with melanoma refractory to anti-PD-1 therapy: scope review Barbosa Fecal microbiota transplantation and its repercussions in patients with melanoma refractory to anti-PD-1 therapy: scope review Table 3 - Ongoing clinical trials with the application of combined ICI-FMT therapy in cancer patients (www.clinicaltrials.gov)74. NCT number Type of cancer Number of patients Intervention Stage NCT04521075 Melanoma; CPCNP 50 FMT + Nivolumab Phase 2 NCT04988841 Melanoma 60 MaaT013 + Ipilimumab + Nivolumab versus placebo + Ipilimumab + Nivolumab Phase 2 NCT03341143 Melanoma 20 FMT + Pembrolizumab Phase 2 NCT03772899 Melanoma 20 FMT + Pembrolizumab/Nivolumab Phase 1 NCT03353402 Melanoma 40 FMT + ICI Phase 1 NCT04577729 Melanoma 60 Allogeneic FMT + ICI versus autologous FMT + ICI Not applicable FMT: fecal microbiota transplantation; ICI: immune checkpoint inhibitor. Source: Authors (2023). In the study carried out by Maleki et al. 0)37, two individuals who had resistant melanoma rwent FMT from two different donors. Both patients wed an improvement in immune response, with an ase in the populations of CD39+ and CD8+ T cells, ddition to a decrease in the levels of dysfunctional +, CD38+, and CD8+ T cells. Patient 1 also displayed lization of a large skin lesion. Some clinical trials aiming to evaluate the acy and safety of the ICI-FMT combination are in ress (Table 3). Despite the promising results of ICI-FMT apy, there are still concerns about its long-term y. In 2019, two independent clinical trials reported two patients developed bacteremia by extended- β l d i E li f i i This review presented consistent regarding the use of ICI-FMT therapy in patie refractory melanoma. However, it is worth me its limitations. Among them, stands out the of studies that offer relevant data on the ap of fecal microbiota transplantation associat immunotherapy in individuals affected by me In addition, the few studies found also had lim mainly small sample size. CONCLUSION FMT has a positive effect on the response of melanoma to ICIs, translated into a significant clinical benefit. However, there is still no consensus regarding the specific bacteria that are associated with a superior response, although certain species belong to phylogenetically related groups. These disagreements can be explained by the use of different genomic sequencing techniques, as well as by geographic influences and dietary variations. Therefore, periodic screening of donor stools should be performed to strictly limit the spread of organisms that can lead to adverse events, which is especially relevant for immunocompromised patients. Additional clinical investigations, which allow a better understanding of the source, the procedure, and the phenotype of both the recipient and the donor are essential for the success of ICI-FMT combined therapy27. In addition, due to the limited amount of literature on the subject, it is not possible to determine the causal mechanism between the intestinal microbiota and the response to anti-PD-1/PD-L1 drugs. Nonetheless, we postulate that stimulation of responses by T cells Rev Col Bras Cir 50:e20233490 9 Barbosa i Barbosa i Barbosa Fecal microbiota transplantation and its repercussions in patients with melanoma refractory to anti-PD-1 therapy: scope review bacteria, the mechanisms involved, and the translation of new evidence to the care practice in oncology are indispensable. against microbial antigens is involved, which in turn aids the response against the tumor. In view of this, new studies aimed at the complete elucidation of the R E S U M O R E S U M O Introdução: apesar de extremamente eficaz em alguns casos, até 70% dos pacientes com melanoma não respondem aos anti-PD-1/PD- L1 (resistência primária) e muitos dos respondedores, eventualmente, acabam progredindo (resistência secundária). Extensos esforços estão sendo realizados para superar esta resistência através de novas estratégias, sobretudo, visando a modulação da microbiota intestinal. Objetivo: avaliar se o transplante de microbiota fecal (TMF), associado à imunoterapia, é benéfico no curso clínico do paciente com melanoma refratário. Métodos: trata-se de uma revisão de escopo, baseada em estudos coletados nas plataformas MEDLINE, ScienceDirect, The Cochrane Library, Embase e BMJ Journals; utilizando os descritores: “Antibodies, Monoclonal”; “Drug Resistance, Neoplasm”; “Fecal Microbiota Transplantation”; “Host Microbial Interactions”; “Immunotherapy”; “Melanoma”; e “Microbiota”. Foram incluídos ensaios clínicos, na língua inglesa, com dados relevantes sobre a temática e disponíveis integralmente. Não foi determinado um período de corte temporal, devido à quantidade limitada de evidências sobre o tema. Resultados: o cruzamento dos descritores permitiu a identificação de 342 publicações e, após a aplicação dos critérios de elegibilidade, permitiu a seleção de 4 estudos. A partir das análises, observou-se que grande parte dos estudados superaram a resistência aos inibidores do checkpoint imunológico pós-TMF, com melhor resposta ao tratamento, menor crescimento tumoral e aumento da resposta imunológica benéfica. Conclusão: nota-se que o TMF favorece a resposta do melanoma à imunoterapia, traduzido por benefício clínico significativo. Entretanto, novos estudos são necessários para a completa elucidação das bactérias e mecanismos envolvidos, bem como para que haja a translação das novas evidências para a prática assistencial oncológica. Palavras-chave: Anticorpos Monoclonais. Imunoterapia. Melanoma. Microbiota. Transplante de Microbiota Fecal 6. Colantonio S, Bracken MB, Beecker J. The association of indoor tanning melanoma in adults: systematic review and meta-analysis. J Am Acad Dermatol. 2014;70(5):847-57. doi: 10.1016/j. jaad.2013.11.050. alavras-chave: Anticorpos Monoclonais. Imunoterapia. Melanoma. Microbiota. Transplante de Microbiota Fecal. REFERENCES 1. Instituto Nacional de Câncer José Alencar Gomes da Silva. Estimativa 2020: incidência de câncer no Brasil. Rio de Janeiro: INCA. 2019. Disponível em: https://www.inca.gov.br/sites/ufu.sti.inca.local/files/ media/document/estimativa-2020-incidencia-de- cancer-no-brasil.pdf 7. Khan AQ, Travers JB, Kemp MG. Roles of UVA radiation and DNA damage responses in melanoma pathogenesis. Environ Mol Mutagen. 2018;59(5):438-60. doi: 10.1002/em.22176. 2. Arnold M, Singh D, Laversanne M, Vignat J, Vaccarella S, Meheus F, et al. Global Burden of Cutaneous Melanoma in 2020 and Projections to 2040. JAMA Dermatol. 2022;158(5):495-503. doi: 10.1001/jamadermatol.2022.0160. 8. Haenssle H, Mograby N, Ngassa A, Buhl T, Emmert S, Schön M, et al. Association of Patient Risk Factors and Frequency of Nevus-Associated Cutaneous Melanomas. JAMA Dermatol. 2016;152(3):291-8. doi: 10.1001/jamadermatol.2015.3775. 3. Eggermont AM, Spatz A, Robert C. Cutaneous melanoma. Lancet. 2014;383(9919):816-27. doi: 10.1016/S0140-6736(13)60802-8. 9. Kubica A, Brewer J. Melanoma in Immunosuppressed Patients. Mayo Clin Proc. 2012;87(10):991-1003. doi: 10.1016/j.mayocp.2012.04.018. 4. Dimitriou F, Krattinger R, Ramelyte E, Barysch M, Micaletto S, Dummer R, et al. The World of Melanoma: Epidemiologic, Genetic, and Anatomic Differences of Melanoma Across the Globe. Curr Oncol Rep. 2018;20(11):87. doi: 10.1007/s11912- 018-0732-8. 10. Antonio JR, Soubhia RMC, D’Avila SCGP, Caldas AC, Trídico LA, Alves FT. Correlation between dermoscopic and histopathological diagnoses of atypical nevi in a dermatology outpatient clinic of the Medical School of São José do Rio Preto, SP. An Bras Dermatol. 2013;88(2):199-203. doi: 10.1590/ S0365-05962013000200002. 5. Instituto Nacional de Câncer - INCA. Estatísticas de câncer. 2022. Disponível em: https://www.gov.br/ inca/pt-br/assuntos/cancer/numeros 11. Ahnlide I, Bjelerupp M, Nilsson F, Nielsen K. Validity 11. Ahnlide I, Bjelerupp M, Nilsson F, Nielsen K. Validity 10 Rev Col Bras Cir 50:e20233490 Barbosa Fecal micr Barbosa Fecal microbiota transplantation and its repercussions in patients with melanoma refractory to anti-PD-1 therapy: scope review of ABCD rule of dermoscopy in clinical practice. Acta Derm Venereol. 2016;96(3):367-72. doi: 10.2340/00015555-2239. images/Relatorios/2018/Relatorio_Ipilimumabe_ MelanomaMetastatico_FINAL.pdf 20. Spranger S, Bao R, Gajewski T. Melanoma-intrinsic β-catenin signalling prevents anti-tumour immunity. Nature. 2015;523(7559):231-5. doi: 10.1038/ nature14404. 12. Swetter S, Thompson J, Albertini M, Barker C, Baumgartner J, Boland G, et al. NCCN Guidelines® Insights: Melanoma: Cutaneous, Version 2.2021. J Natl Compr Canc Netw. 2021;19(4):364-76. doi: 10.6004/jnccn.2021.0018. 21. Koyama S, Akbay E, Li Y, Herter-Sprie G, Buczkowski K, Richards W, et al. Adaptive resistance to therapeutic PD-1 blockade is associated with upregulation of alternative immune checkpoints. Nat Commun. 2016;7(1):10501. doi: 10.1038/ ncomms10501. 13. Garbe C, Peris K, Hauschild A, Saiag P, Middleton M, Spatz A, et al. REFERENCES Diagnosis and treatment of melanoma. European consensus-based interdisciplinary guideline – Update 2012. Eur J Cancer. 2012;48(15):2375-90. doi: 10.1016/j.ejca.2012.06.013. 22. Zhang M, Yang J, Hua W, Li Z, Xu Z, Qian Q. Monitoring checkpoint inhibitors: predictive biomarkers in immunotherapy. Front Med. 2019;13(1):32-44. doi: 10.1007/s11684-018-0678- 0. 14. Burns D, George J, Aucoin D, Bower J, Burrell S, Gilbert R, et al. The Pathogenesis and Clinical Management of Cutaneous Melanoma: An Evidence-Based Review. J Med Imaging Radiat Sci. 2019;50(3):460-9. doi: 10.1016/j.jmir.2019.05.001. 23. Shergold A, Millar R, Nibbs R. Understanding and overcoming the resistance of cancer to PD-1/PD-L1 blockade. Pharmacol Res. 2019;145:104258. doi: 10.1016/j.phrs.2019.104258. 15. Schumacher T, Schreiber R. Neoantigens in cancer immunotherapy. Science. 2015;348(6230):69-74. doi: 10.1126/science.aaa4971. 16. Brasil. Ministério da Saúde. Secretaria de Ciência, Tecnologia, I. e I. E. Relatório de recomendação no 541 de 2020 - Terapia-alvo (vemurafenibe, dabrafenibe, cobimetinibe, trametinibe) e imunoterapia (ipilimumabe, nivolumabe, pembrolizumabe) para o tratamento de primeira linha do melanoma avançado não-cirúrgico e metastático. 2020. Disponível em: https:// docs.bvsalud.org/biblioref/2020/10/1123192/ relatorio_541_terapiaalvo_melanoma_final_2020. pdf 16. Brasil. Ministério da Saúde. Secretaria de Ciência, Tecnologia, I. e I. E. Relatório de recomendação no 541 de 2020 - Terapia-alvo (vemurafenibe, dabrafenibe, cobimetinibe, trametinibe) e imunoterapia (ipilimumabe, nivolumabe, pembrolizumabe) para o tratamento de primeira linha do melanoma avançado não-cirúrgico e metastático. 2020. Disponível em: https:// docs.bvsalud.org/biblioref/2020/10/1123192/ relatorio_541_terapiaalvo_melanoma_final_2020. pdf 24. Lei Q, Wang D, Sun K, Wang L, Zhang Y. Resistance Mechanisms of Anti-PD1/PDL1 Therapy in Solid Tumors. Front Cell Dev Biol. 2020;8. doi: 10.3389/ fcell.2020.00672. 25. Eiseman B, Silen W, Bascom G, Kauvar A. Fecal enema as an adjunct in the treatment of pseudomembranous enterocolitis. Surgery. 1958;44:854-9. 26. Hamilton MJ, Weingarden AR, Sadowsky MJ, Khoruts A. Standardized frozen preparation for transplantation of fecal microbiota for recurrent Clostridium difficile infection. Am J Gastroenterol. 2012;107(5):761-7. doi: 10.1038/ajg.2011.482. 17. Pardoll D. Cancer and the Immune System: Basic Concepts and Targets for Intervention. Semin Oncol. 2015;42(4):523-38. doi: 10.1053/j. seminoncol.2015.05.003. 17. Pardoll D. Cancer and the Immune System: Basic Concepts and Targets for Intervention. Semin Oncol. 2015;42(4):523-38. doi: 10.1053/j. seminoncol.2015.05.003. 27. Wang J, Kuo C, Kuo F, Wang Y, Hsu W, Yu F et al. Fecal microbiota transplantation: Review and update. J Formos Med Assoc. 2019;118 Suppl 1:S23-S31. 10.1016/j.jfma.2018.08.011. 18. Weber J, Mandala M, Del Vecchio M, et al. Adjuvant nivolumab versus ipilimumab in resected stage III or IV melanoma. N Engl Med. 2017;377(19):1824-35. doi: 10.1056/NEJMoa1709030. 28. Rossen N. Fecal microbiota transplantation as novel therapy in gastroenterology: A systematic review. REFERENCES World J Gastroenterol. 2015;21(17):5359-71. doi: 10.3748/wjg.v21.i17.5359. 19. Brasil. Ministério da Saúde. Secretaria de Ciência, T. e I. E. Relatório de recomendação no 391 de 2018 - Ipilimumabe para tratamento de pacientes com melanoma metastático. 2018. Disponível em: http://antigo-conitec.saude.gov.br/ 29. Wong S, Zhao L, Zhang X, Nakatsu G, Han J, Xu W, et al. Gavage of Fecal Samples From Patients With Colorectal Cancer Promotes Intestinal Carcinogenesis in Germ-Free and Conventional Rev Col Bras Cir 50:e20233490 11 Barbosa pe review Barbosa ope review Barbosa Fecal microbiota transplantation and its repercussions in patients with melanoma refractory to anti-PD-1 therapy: scope review Mice. Gastroenterology. 2017;153(6):1621-33. doi: 10.1053/j.gastro.2017.08.022. PD-1 therapy in melanoma patients. Science. 2021;371(6529):595-602. doi: 10.1126/science. abf3363. 30. Ma C, Han M, Heinrich B, Fu Q, Zhang Q, Sandhu M, et al. Gut microbiome–mediated bile acid metabolism regulates liver cancer via NKT cells. Science. 2018;360(6391):eaan5931. doi: 10.1126/ science.aan5931. 39. Baruch E, Youngster I, Ben-Betzalel G, Ortenberg R, Lahat A, Katz L, et al. Fecal microbiota transplant promotes response in immunotherapy-refractory melanoma patients. Science. 2021;371(6529):602- 9. doi: 10.1126/science.abb5920. 31. Pushalkar S, Hundeyin M, Daley D, Zambirinis C, Kurz E, Mishra A, et al. The Pancreatic Cancer Microbiome Promotes Oncogenesis by Induction of Innate and Adaptive Immune Suppression. Cancer Discov. 2018;8(4):403-16. doi: 10.1158/2159-8290. CD-17-1134. 40. Balar A, Weber J. PD-1 and PD-L1 antibodies in cancer: current status and future directions. Cancer Immunol Immunother. 2017;66(5):551-64. doi: 10.1007/s00262-017-1954-6. 41. Constantinidou A, Alifieris C, Trafalis DT. Targeting Programmed Cell Death -1 (PD-1) and Ligand (PD- L1): A new era in cancer active immunotherapy. Pharmacol Ther. 2019;194:84-106. doi: 10.1016/j. pharmthera.2018.09.008. 32. Chen D, Wu J, Jin D, Wang B, Cao H. Fecal microbiota transplantation in cancer management: Current status and perspectives. Int J Cancer. 2019;145(8):2021–31. doi: 10.1002/ijc.32003. 33. Di Modica M, Gargari G, Regondi V, Bonizzi A, Arioli S, Belmonte B, et al. Gut Microbiota Condition the Therapeutic Efficacy of Trastuzumab in HER2-Positive Breast Cancer. Cancer Res. 2021;81(8):2195-206. doi: 10.1158/0008-5472.CAN-20-1659. 42. Wang Y, Ma R, Liu F, Lee S, Zhang L. Modulation of Gut Microbiota: A Novel Paradigm of Enhancing the Efficacy of Programmed Death-1 and Programmed Death Ligand-1 Blockade Therapy. Front Immunol. 2018;9:374. doi: 10.3389/fimmu.2018.00374. 43. Sharma P, Hu-Lieskovan S, Wargo J, Ribas A. Primary, Adaptive, and Acquired Resistance to Cancer Immunotherapy. Cell. 2017;168(4):707-23. doi: 10.1016/j.cell.2017.01.017. 34. Tricco AC, Lillie E, Zarin W, O’Brien KK, Colquhoun H, Levac D, et al. 10.1056/NEJMoa1805131. 57. Tortora GJ, Funke BR, Case CL, Weber D, Bair WB. Microbiology: An Introduction, Global Edition. 13 ed. Pearson Education Limited; 2020. 48. Martin-Liberal J, Kordbacheh T, Larkin J. Safety of pembrolizumab for the treatment of melanoma. Expert Opin Drug Saf. 2015;14(6):957-64. doi: 10.1517/14740338.2015.1021774. 58. Sommer F, Bäckhed F. The gut microbiota – masters of host development and physiology. Nat Rev Microbiol. 2013;11(4):227-38. doi: 10.1038/ nrmicro2974. 49. Ribas A, Hamid O, Daud A, Hodi F, Wolchok J, Kefford R, et al. Association of Pembrolizumab With Tumor Response and Survival Among Patients With Advanced Melanoma. JAMA. 2016;315(15):1600. doi: 10.1001/jama.2016.4059. 59. Lopez A, Hansmannel F, Kokten T, Bronowicki J, Melhem H, Sokol H, et al. Microbiota in digestive cancers: our new partner?. Carcinogenesis. 2017;38(12):1157-66. doi: 10.1093/carcin/bgx087. 50. Springman S, Trapskin PJ, Hennes A, Vanderloo J. Perioperative Medication Management - Adult/ Pediatric - Inpatient/Ambulatory - Clinical Practice Guideline. UW Health, 2022. Disponível em: https://www.uwhealth.org/cckm/cpg/medications/ Perioperative-Medication-Management-Clinical- Pratice-Guideline---August-2022.pdf 60. Zitvogel L, Ma Y, Raoult D, Kroemer G, Gajewski T. The microbiome in cancer immunotherapy: Diagnostic tools and therapeutic strategies. Science. 2018;359(6382):1366-70. doi: 10.1126/science. aar6918. 61. Tilg H, Schmiderer A, Djanani A. Gut microbiome- immune crosstalk affects progression of cancer. Transl Gastroenterol Hepatol. 2018;3:34. doi: 10.21037/tgh.2018.06.02. 51. Guo L, Zhang H, Chen B. Nivolumab as Programmed Death-1 (PD-1) Inhibitor for Targeted Immunotherapy in Tumor. J Cancer. 2017;8(3):410-16. doi: 10.7150/ jca.17144. 62. Asmar N, Ibrahim T, Rey J. Checkpoint Inhibitors: Conquering Cancer with a Little (T)-Help from Our Microbial Friends. Dig Dis Sci. 2018;63(9):2177-9. doi: 10.1007/s10620-018-5188. 52. Elias A, Kasi P, Stauffer J, Thiel D, Colibaseanu D, Mody K, et al. The Feasibility and Safety of Surgery in Patients Receiving Immune Checkpoint Inhibitors: A Retrospective Study. Front Oncol. 2017;7:121. doi: 10.3389/fonc.2017.00121. 63. Iida N, Dzutsev A, Stewart C, Smith L, Bouladoux N, Weingarten R, et al. Commensal Bacteria Control Cancer Response to Therapy by Modulating the Tumor Microenvironment. Science. 2013;342(6161):967- 70. doi: 10.1126/science.1240527. 53. Tie Y, Yang H, Zhao R, Zheng H, Yang D, Zhao J et al. Safety and efficacy of atezolizumab in the treatment of cancers: a systematic review and pooled-analysis. Drug Des Devel Ther. 2019;13:523- 38. doi: 10.2147/DDDT.S188893. 64. Viaud S, Saccheri F, Mignot G, Yamazaki T, Daillère R, Hannani D, et al. The Intestinal Microbiota Modulates the Anticancer Immune Effects of Cyclophosphamide. Science. 2013;342(6161):971- 6. doi: 10.1126/science.1240537. 54. Goodman D. Cemiplimab and Cutaneous Squamous Cell Carcinoma: From Bench to Bedside. JPRAS Open. 2022;33:155-160. REFERENCES PRISMA Extension for Scoping Reviews (PRISMA-ScR): Checklist and Explanation. Ann Intern Med. 2018 Oct 2;169(7):467-73. doi: 10.7326/M18-0850. 44. Garon E, Rizvi N, Hui R, Leighl N, Balmanoukian A, Eder J, et al. Pembrolizumab for the Treatment of Non–Small-Cell Lung Cancer. N Engl J Med. 2015;372(21):2018-28. doi: 10.1056/ NEJMoa1501824. 35. Akobeng AK. Principles of evidence based medicine. Arch Dis Child. 2005;90(8):837-40. doi: 10.1136/ adc.2005.071761. 36. Youngster I, Baruch E, Katz L, Lahat A, Brosh- Nissimov T, Schachter J, et al. 90. Fecal Microbiota Transplantation in Metastatic Melanoma Patients Resistant to Anti-PD-1 Treatment. Open Forum Infect Dis. 2019;6(2):S7. doi: 10.1093/ofid/ofz359.014. 45. Robert C, Schachter J, Long G, Arance A, Grob J, Mortier L, et al. Pembrolizumab versus Ipilimumab in Advanced Melanoma. N Engl J Med. 2015;372(26):2521-32. doi: 10.1056/ NEJMoa1503093. 37. Maleki S, Lenehan J, Burton J, Silverman M, Parvathy S, El-Hajjar M, et al. P864 Combination of fecal microbiota transplantation from healthy donors with anti-PD1 immunotherapy in treatment- naïve advanced or metastatic melanoma patients. J Immunother Cancer. 2020;8(1):A11-A12. doi: 10.1136/LBA2019.17. 46. Schmid P, Adams S, Rugo H, Schneeweiss A, Barrios C, Iwata H, et al. Atezolizumab and Nab-Paclitaxel in Advanced Triple-Negative Breast Cancer. N Engl J Med. 2018;379(22):2108-21. doi: 10.1056/ NEJMoa1809615. 47. Migden M, Rischin D, Schmults C, Guminski A, Hauschild A, Lewis K, et al. PD-1 Blockade with Cemiplimab in Advanced Cutaneous Squamous-Cell Carcinoma. N Engl J Med. 2018;379(4):341-51. doi: 38. Davar D, Dzutsev A, McCulloch J, Rodrigues R, Chauvin J, Morrison R et al. Fecal microbiota transplant overcomes resistance to anti– Rev Col Bras Cir 50:e20233490 12 Barbosa Fecal microbiota transplantation and its repercussions in patients with melanoma refractory to anti-PD-1 therapy: scope review Barbosa F l i Barbosa Fecal microbiota transplantation and its repercussions in patients with melanoma refractory to anti-PD-1 therapy: scope review 10.1056/NEJMoa1805131. doi: 10.1016/j.jpra.2022.06.003. 65. Claesson M, Cusack S, O’Sullivan O, Greene-Diniz R, de Weerd H, Flannery E, et al. Composition, variability, and temporal stability of the intestinal microbiota of the elderly. Proc Natl Acad Sci U S A. 2010;108(1):4586-91. doi: 10.1073/ pnas.1000097107. 55. Sezer A, Kilickap S, Gümüş M, Bondarenko I, Özgüroğlu M, Gogishvili M, et al. Cemiplimab monotherapy for first-line treatment of advanced non-small-cell lung cancer with PD-L1 of at least 50%: a multicentre, open-label, global, phase 3, randomised, controlled trial. Lancet. 2021;397(10274):592-604. doi: 10.1016/S0140- 6736(21)00228-2. 66. Smits L, Bouter K, de Vos W, Borody T, Nieuwdorp M. Therapeutic Potential of Fecal Microbiota Transplantation. Gastroenterology. 2013;145(5):946-53. doi: 10.1053/j. gastro.2013.08.058. 56. Trabulsi L, Alterthum F, Rácz M, Martinez M, Campos L, Gompertz O et al. Microbiologia. 6 ed. São Paulo: Atheneu; 2015. 13 Rev Col Bras Cir 50:e20233490 13 Barbosa ope re ie Barbos Fecal microbiota transplantation and its repercussions in patients with melanoma refractory to anti-PD-1 therapy: scope review 10.1038/s41591-022-01698-2. 67. Zhang F, Luo W, Shi Y, Fan Z, Ji G. Should We Standardize the 1,700-Year-Old Fecal Microbiota Transplantation? Am J Gastroenterol. 2012;107(11):1755. doi: 10.1038/ajg.2012.251. 10.1038/s41591-022-01698-2. 72. Gopalakrishnan V, Spencer C, Nezi L, Reuben A, Andrews M, Karpinets T, et al. Gut microbiome modulates response to anti–PD-1 immunotherapy in melanoma patients. Science. 2018;359(6371):97- 103. doi: 10.1126/science.aan4236. 68. Dupont HL. Diagnosis and management of Clostridium difficile infection. Clin Gastroenterol Hepatol. 2013;11(10):1216-23. doi: 10.1016/j. cgh.2013.03.016. 73. Matson V, Fessler J, Bao R, Chongsuwat T, Zha Y, Alegre M, et al. The commensal microbiome is associated with anti–PD-1 efficacy in metastatic melanoma patients. Science. 2018;359(6371):104- 8. doi: 10.1126/science.aao3290. 69. Sivan A, Corrales L, Hubert N, Williams J, Aquino- Michaels K, Earley Z, et al. Commensal Bifidobacterium promotes antitumor immunity and facilitates anti– PD-L1 efficacy. Science. 2015;350(6264):1084-9. doi: 10.1126/science.aac4255. 74. ClinicalTrials. Clinicaltrials.gov. 2022. Disponível em: https://clinicaltrials.gov. 70. Frankel A, Coughlin L, Kim J, Froehlich T, Xie Y, Frenkel E, et al. Metagenomic Shotgun Sequencing and Unbiased Metabolomic Profiling Identify Specific Human Gut Microbiota and Metabolites Associated with Immune Checkpoint Therapy Efficacy in Melanoma Patients. Neoplasia. 2017;19(10):848- 55. doi: 10.1016/j.neo.2017.08.004. 75. DeFilipp Z, Bloom P, Torres Soto M, Mansour M, Sater M, Huntley M, et al. Drug-Resistant E. coli Bacteremia Transmitted by Fecal Microbiota Transplant. N Engl J Med. 2019;381(21):2043-50. doi: 10.1056/NEJMoa1910437. 76. Vendrik K, Terveer E, Kuijper E, Nooij S, Boeije- Koppenol E, Sanders I, et al. Periodic screening of donor faeces with a quarantine period to prevent transmission of multidrug-resistant organisms during faecal microbiota transplantation: a retrospective cohort study. Lancet Infect Dis. 2021;21(5):711-21. doi: 10.1016/S1473-3099(20)30473-4. 71. McCulloch JA, Davar D, Rodrigues RR, Badger JH, Fang JR, Cole AM et al. Intestinal microbiota signatures of clinical response and immune-related adverse events in melanoma patients treated with anti-PD-1. Nat Med. 2022;28(3):545-56. doi: Received in: 24/10/2022 Accepted for publication: 24/02/2023 Conflict of interest: no. Funding source: none. Mailing address: Eduardo Cerchi Barbosa E-mail: eduardo.cerchi27@gmail.com 14 14 Rev Col Bras Cir 50:e20233490
https://openalex.org/W4377293081	https://periodicos.uffs.edu.br/index.php/RIS/article/download/13597/8862	Portuguese	null	Estágio supervisionado em ensino de Ciências e Biologia: ações integradoras e interdisciplinares	Revista Insignare Scientia	2,023	cc-by	6,822	Resumo: Resumo: Os estágios supervisionados representam uma oportunidade de discussão, aproximação e compreensão da carreira docente e do campo de atuação dos professores, reconhecendo a realidade sociocultural dos espaços educativos, seus constituintes e nuances. Diante desse cenário, este artigo trata-se de relato de experiência descritivo, de abordagem qualitativa, cujo objetivo é explanar sobre o desenvolvimento do Projeto Integrador dos Estágios supervisionados (PIE) de um curso de licenciatura em Ciências Biológicas, ofertado por uma universidade estadual, situada na região dos Sertões de Crateús. O Projeto Integrador dos Estágios (PIE), foi desenvolvido ao longo do semestre letivo 2021.1 (setembro/2021 a janeiro/2022). Ele foi dividido em seis encontros ocorridos via aplicativo de web conferência Google Meet® ou pelo canal oficial do Programa Residência Pedagógica da instituição no Youtube®. Foram abordadas temáticas como: atuais documentos legais da educação brasileira (Base Nacional Comum Curricular – BNCC – e as Diretrizes Curriculares Referencial do Ceará – DCRC); utilização das tecnologias educativas no ensino de Ciências e Biologia; a educação contextualizada; os saberes e os fazeres docente; além da relação entre saúde física e mental em tempos de pandemia. Esse projeto visou, portanto, impactar em uma formação holística, integral e humana deste professor- aprendiz. Palavras-chave: Estágio; Formação de professores; Licenciatura. Palavras-chave: Estágio; Formação de professores; Licenciatura. Recebido em: 30/11/2022 Aceito em: 30/04/2023 Abstract: Vol. 6, n. 2. 2023 Edição Especial: Estágio Supervisionado e Prática de Ensino em Ciências aculdade de Educação de Crateús/Universidade Estadual do Ceará, Brasil Resumen: Las prácticas supervisadas son una oportunidad para discutir, abordar y comprender la carrera docente y el campo de trabajo de los docentes, reconociendo la realidad sociocultural de los espacios educativos, sus constituyentes y matices. Ante este escenario, este artículo es un relato de experiencia descriptivo, con enfoque cualitativo, cuyo objetivo es explicar el desarrollo del Proyecto Integrado de Pasantías Supervisadas (PIE) de una carrera de Licenciatura en Ciencias Biológicas ofrecida por una universidad estatal ubicada en la región. de los Sertões de Crateús. El Proyecto de Integración de Pasantías (PIE) se desarrolló durante el semestre académico 2021.1 (septiembre/2021 a enero/2022). Se dividió en seis encuentros que se realizaron a través de la aplicación de conferencias web Google Meet® oa través del canal oficial de la institución del Programa de Residencia Pedagógica en Youtube®. Se abordaron temas como: documentos legales actuales de la educación brasileña (Base Nacional Comum Curricular – BNCC – y Directrices Curriculares de Referencia de Ceará – DCRC); uso de tecnologías educativas en la enseñanza de Ciencias y Biología; educación contextualizada; los saberes y prácticas docentes; además de la relación entre la salud física y mental en tiempos de pandemia. Este proyecto pretendía, por tanto, incidir en una formación holística, integral y humana de este docente-aprendiz. Palabras-clave: Prácticas; Formación de profesores; Graduación. Palabras-clave: Prácticas; Formación de profesores; Graduación. Abstract: Abstract: Recebido em: 30/11/2022 Aceito em: 30/04/2023 492 Vol. 6, n. 2. 2023 Edição Especial: Estágio Supervisionado e Prática de Ensino em Ciências Supervised internships are an opportunity to discuss, approach and understand the teaching career and the field of work of teachers, recognizing the sociocultural reality of educational spaces, their constituents and nuances. Given this scenario, this article is a descriptive experience report, with a qualitative approach, whose objective is to explain the development of the Integrated Project of Supervised Internships (PIE) of a degree course in Biological Sciences offered by a state university located in the region. from the Sertões of Crateús. The Internship Integrating Project (PIE) was developed during the 2021.1 academic semester (September/2021 to January/2022). It was divided into six meetings that took place via the Google Meet® web conference application or through the institution's official channel of the Pedagogical Residency Program on Youtube®. Themes were addressed such as: current legal documents of Brazilian education (Base Nacional Comum Curricular – BNCC – and the Referential Curriculum Guidelines of Ceará – DCRC); use of educational technologies in Science and Biology teaching; contextualized education; the knowledge and teaching practices; in addition to the relationship between physical and mental health in times of a pandemic. This project aimed, therefore, to impact on a holistic, integral and human formation of this teacher- learner. Keywords: Internship; Teacher training; Graduation. alabras-clave: Prácticas; Formación de profesores; Graduación. ESTÁGIO SUPERVISIONADO: APROXIMAÇÕES INICIAIS Atividades práticas, que permitam o exercício da docência, sob supervisão, ainda durante a formação inicial dos professores (as), além de requisito legal (BRASIL, 2019), configura-se como etapa indispensável para a formação docente. Nesse contexto, o Recebido em: 30/11/2022 Aceito em: 30/04/2023 Recebido em: 30/11/2022 Aceito em: 30/04/2023 493 493 Vol. 6, n. 2. 2023 Edição Especial: Estágio Supervisionado e Prática de Ensino em Ciências estágio supervisionado nos cursos de licenciatura, apresenta-se como fundamental para uma formação docente ampla, articulada com a realidade e voltada para o desenvolvimento do trabalho docente (LÜDKE, 2013; GATTI, 2013). Esse momento formativo é necessário para a aprendizagem de um profissional que deseja realmente seguir e estar preparado para enfrentar os desafios de uma carreira docente. É durante o desenvolvimento dos estágios que os licenciandos se aproximam do futuro campo de atuação profissional, entrando em contato com a realidade sociocultural dos espaços educativos formais e não-formais, reconhecendo seus constituintes e nuances (FRACARO; HAEFLIGER, 2022; PINHEIRO JÚNIOR et al., 2022). Similarmente, para Lima (2009, p. 47), o desenvolvimento dos estágios em sua concepção mais ampla propõe-se a “instrumentalizar o estagiário para a reflexão sobre o seu fazer pedagógico mais abrangente e a sua identidade profissional”. Lima (2009) sugere que esse período de (form)ação para e na docência seja desenvolvido em três etapas: Lima (2009) sugere que esse período de (form)ação para e na docência seja desenvolvido em três etapas: i) Professor, Identidade e Memória Docente – acontecimento para apropriação da fundamentação teórico-epistemológicos, filosóficos e históricos da prática de ensino; ii) O professor e a escola em movimento – imergência reflexiva a partir do movimento real de aproximação com espaço escolar por meio das aprendizagens do contexto; iii) O Livro Didático e os Parâmetros Curriculares – fase de compreensão do material didático e das especificidades curriculares da área de conhecimento que o estudante esteja inserido (LIMA, 2009, p. 46). i) Professor, Identidade e Memória Docente – acontecimento para apropriação da fundamentação teórico-epistemológicos, filosóficos e históricos da prática de ensino; ii) O professor e a escola em movimento – imergência reflexiva a partir do movimento real de aproximação com espaço escolar por meio das aprendizagens do contexto; iii) O Livro Didático e os Parâmetros Curriculares – fase de compreensão do material didático e das especificidades curriculares da área de conhecimento que o estudante esteja inserido (LIMA, 2009, p. 46). ESTÁGIO SUPERVISIONADO: APROXIMAÇÕES INICIAIS Essas etapas possibilitam a construção da identidade docente assim como a compreensão do papel do professor na escola e no processo de ensino e aprendizagem, garantindo uma interação mútua entre licenciandos-professores supervisores-escola- universidade sedimentada na reflexão-ação. A Resolução CNE/CP Nº 2, de 20 de Dezembro de 2019 define as Diretrizes Curriculares Nacionais para a Formação Inicial de Professores para a Educação Básica e institui a Base Nacional Comum para a Formação Inicial de Professores da Educação Básica (BNC-Formação) (BRASIL, 2019), nela, os estágios supervisionados devem compor o Projeto Pedagógico do Curso (PPC) dos cursos de licenciatura, destinando-se, no mínimo, 400 (quatrocentas) horas para o estágio supervisionado, em situação de imersão de trabalho nas escolas. m: 30/11/2022 No Curso de Licenciatura em Ciências Biológicas representado neste manuscrito, são destinadas 408 horas de estágios, divididas em quatro disciplinas: Estágio No Curso de Licenciatura em Ciências Biológicas representado neste manuscrito, são destinadas 408 horas de estágios, divididas em quatro disciplinas: Estágio Recebido em: 30/11/2022 Aceito em: 30/04/2023 Recebido em: 30/11/2022 Aceito em: 30/04/2023 494 Recebido em: 30/11/2022 Aceito em: 30/04/2023 494 494 Vol. 6, n. 2. 2023 Edição Especial: Estágio Supervisionado e Prática de Ensino em Ciências Supervisionado no Ensino Fundamental, Estágio Supervisionado I, II e III no Ensino Médio, com carga horária de 102 horas cada, onde os alunos estagiários estarão em contato com o Ensino de Ciências e Biologia. Supervisionado no Ensino Fundamental, Estágio Supervisionado I, II e III no Ensino Médio, com carga horária de 102 horas cada, onde os alunos estagiários estarão em contato com o Ensino de Ciências e Biologia. Durante os estágios, os estudantes analisam a estrutura político-pedagógico- administrativa e o Projeto Político Pedagógico da escola, além de desenvolverem um projeto de ensino que será realizado ao longo do semestre, englobando o planejamento, as estratégias de integração da comunidade escolar, a observação e a regência das aulas (CEARÁ, 2012). Além disso, há também momentos de encontros presenciais com o professor orientador (responsável pelas disciplinas de estágios na Universidade), onde há aprofundamento teórico, discussões de temáticas inerentes à prática docente no ensino de Ciências e Biologia e socializações reflexivas das atividades desenvolvidas nas escolas. Todavia, devido à necessidade do ensino remoto emergencial em decorrência da pandemia da Covid-19, os encontros foram virtuais por meio de atividades síncronas. ESTÁGIO SUPERVISIONADO: APROXIMAÇÕES INICIAIS Nesse contexto, desenvolveu-se o Projeto Integrador dos Estágios (PIE), ao longo do semestre letivo 2021.1, o qual será descrito mais amplamente no tópico a seguir. A respeito dos estágios supervisionados em cursos de licenciatura durante a pandemia e a consequente adoção do ensino remoto emergencial, Souza e Ferreira (2020, p. 3) destacaram que: Além disso, há também momentos de encontros presenciais com o professor orientador (responsável pelas disciplinas de estágios na Universidade), onde há aprofundamento teórico, discussões de temáticas inerentes à prática docente no ensino de Ciências e Biologia e socializações reflexivas das atividades desenvolvidas nas escolas. Todavia, devido à necessidade do ensino remoto emergencial em decorrência da pandemia da Covid-19, os encontros foram virtuais por meio de atividades síncronas. Nesse contexto, desenvolveu-se o Projeto Integrador dos Estágios (PIE), ao longo do semestre letivo 2021.1, o qual será descrito mais amplamente no tópico a seguir. A respeito dos estágios supervisionados em cursos de licenciatura durante a pandemia e a consequente adoção do ensino remoto emergencial, Souza e Ferreira (2020, p. 3) destacaram que: [...] dentre os inúmeros impactos decorrentes da suspensão de aulas e de atividades presenciais, nos convoca a observar e pensar alternativas para a validação do componente estágio supervisionado nos cursos de licenciatura, à luz de estratégias que considerem as atividades formativas do ensino remoto, tanto já integrantes ao currículo do curso, ou propostas a partir da necessidade de replanejamento advinda da pandemia. Devido a isso, houve a necessidade de (re)planejar as atividades dos estágios em cursos de licenciatura para adaptação às condições remotas, Mendes, Maia e Biacon (2021, p. 88) destacam que a “adoção de modificações na condução e na realização dos Estágios em regime remoto já nos indica implicações na compreensão das/os estagiárias/os sobre a realidade escolar e o ato pedagógico de Ciências ou Biologia”. Para esses autores, o contexto de realização remota dos estágios “permite reflexões posteriores mais aprofundadas sobre os possíveis impactos na futura práxis docente de licenciandas/os em formação” (MENDES; MAIA; BIACON, 2021, p. 97). Recebido em: 30/11/2022 Aceito em: 30/04/2023 Recebido em: 30/11/2022 Aceito em: 30/04/2023 495 495 Vol. 6, n. 2. 2023 Edição Especial: Estágio Supervisionado e Prática de Ensino em Ciências Vol. 6, n. 2. ESTÁGIO SUPERVISIONADO: APROXIMAÇÕES INICIAIS 2023 Edição Especial: Estágio Supervisionado e Prática de Ensino em Ciências Dito isto, tratamos aqui de um relato de experiência descritivo, de abordagem qualitativa, cujo intuito é explanar sobre o desenvolvimento do projeto integrador dos estágios supervisionados de um curso de licenciatura em Ciências Biológicas, ofertado por uma universidade estadual, situada na região dos Sertões de Crateús. PROJETO INTEGRADOR DOS ESTÁGIOS (PIE) O Projeto Integrador dos Estágios (PIE), foi desenvolvido ao longo do semestre letivo 2021.1 (setembro/2021 a janeiro/2022). Sua idealização ocorreu no início do semestre, durante a semana pedagógica do curso, quando os professores orientadores dos estágios reuniram-se e propuseram realizar uma ação integradora e interdisciplinar, fazendo surgir o PIE. O PIE foi então definido como um momento de discussão de temáticas relacionadas diretamente ou transversalmente ao processo de ensino e aprendizagem, docência e/ou estrutura e funcionamento da educação básica. Ele foi dividido em seis encontros durante o decorrer do semestre, ocorridos via aplicativo de web conferência Google Meet® ou pelo canal oficial do PIRP/UECE (Programa Institucional de Residência Pedagógica da Universidade Estadual do Ceará) no Youtube®. Foram abordadas temáticas como: atuais documentos legais da educação brasileira (Base Nacional Comum Curricular – BNCC – e as Diretrizes Curriculares Referencial do Ceará – DCRC); utilização das tecnologias educativas no ensino de Ciências e Biologia; a educação contextualizada; os saberes e os fazeres docente; além da relação entre saúde física e mental em tempos de pandemia. O detalhamento do exposto em cada temática encontra-se no Quadro 1 a seguir. o em: 30/11/2022 Quadro 1 – Temáticas abordadas durante o desenvolvimento do Projeto Integrador dos Estágios supervisionados em ensino de Ciências e Biologia. Temática abordada Descrição da temática Base Nacional Comum Curricular (BNCC) Neste tópico foram apresentadas as concepções e conceitos que fundamentam a BNCC-EB. Para isto, realizou-se uma historização da proposta de implementação de uma Base Comum Nacional, em todo o território brasileiro e os marcos legais que regulamentam essa implementação, com destaque para o Art. 26 da Lei de Diretrizes e Bases da Educação-LDB, o Art. 14 das Diretrizes Curriculares nacionais-DCB e as metas 2, 3 e 8 do Plano Nacional de Educação-PNE. Discutiu- se ainda o contexto político e social no qual se deu a aprovação da BNCC, ocorrida, em 02 etapas distintas, sendo a primeira em dezembro de 2017, na parte referente a Educação infantil 496 Recebido em: 30/11/2022 Aceito em: 30/04/2023 Quadro 1 – Temáticas abordadas durante o desenvolvimento do Projeto Integrador dos Estágios supervisionados em ensino de Ciências e Biologia. Temática abordada Descrição da temática Base Nacional Comum Curricular (BNCC) Neste tópico foram apresentadas as concepções e conceitos que fundamentam a BNCC-EB. PROJETO INTEGRADOR DOS ESTÁGIOS (PIE) Nesse segundo encontro foram apresentados os desdobramentos que se seguiram a aprovação da BNCC, em especial o lançamento do Programa de Apoio à Implementação da Base Nacional Comum Curricular - ProBNCC, em abril de 2018, pelo Ministério da Educação, e instituído por meio da portaria Nº 331, que estabeleceu o regime de colaboração que seria instalado a partir daquele momento para que todos os Estados e o Distrito federal revisassem ou elaborassem seus currículos, alinhados a BNCC. Foram apresentados todos os regulamentos dos programas, a exemplo das Portaria MEC nº 756, de 3 de abril de 2019, para inserir aspectos específicos da implementação da BNCC para o Ensino Médio. No encontro, foram discutidos como a partir da implementação do programa, todos os Estados e o Distrito Federal iniciaram o processo de revisão e ou escrita de seus documentos curriculares e como este foi responsável pelo aporte financeiro do proBNCC, subsidiaram a formação das equipes de redatores e, cada Unidade da Federação. A segunda metade do encontro foi dedicada a apresentação do Documento Curricular do Estado do Ceará- DCRC, referente ao Ensino Fundamental discutindo-se, como documentos elaborados a luz da BNCC, podem configurar-se como espaços/oportunidades de resistências para que Estados e o DF pudessem imprimir suas marcas. Foram ainda apresentadas as concepções assumidas, tais como contextualização, interdisciplinaridade e como essas concepções ancoram a Autonomia docente e Gestão democrática das Escolas. A palestrante convidada iniciou sua explanação ressaltando fatores positivos relacionados às atividades físicas e estabelecendo relações entre saúde e o bem-estar físico, e o Ensino Fundamental e a segunda em dezembro de 2018 com aprovação do texto referente ao Ensino Médio. Foram apresentadas, comparadas e discutidas as diferenças entre as chamadas primeira e segundas versões, submetidas a consultas públicas e a terceira versão, que embora também tenha passado por consulta pública, apresentava escopo totalmente diferente dos documentos anteriores, com uma proposta formativa baseada em competências e habilidades. A partir dessas discussões foram apresentadas as estruturas presentes no documento final da BNCC, a organização das áreas e componentes curriculares, seguindo-se ao aprofundamento da área/componente Ciências da Natureza. A partir desse momento seguiu-se um estudo a miúde, com apresentação das competências específicas, Unidades temáticas, com detalhamento dos objetos de conhecimento e habilidades a estas associadas. PROJETO INTEGRADOR DOS ESTÁGIOS (PIE) Para isto, realizou-se uma historização da proposta de implementação de uma Base Comum Nacional, em todo o território brasileiro e os marcos legais que regulamentam essa implementação, com destaque para o Art. 26 da Lei de Diretrizes e Bases da Educação-LDB, o Art. 14 das Diretrizes Curriculares nacionais-DCB e as metas 2, 3 e 8 do Plano Nacional de Educação-PNE. Discutiu- se ainda o contexto político e social no qual se deu a aprovação da BNCC, ocorrida, em 02 etapas distintas, sendo a primeira em dezembro de 2017, na parte referente a Educação infantil Recebido em: 30/11/2022 Aceito em: 30/04/2023 496 Vol. 6, n. 2. 2023 Edição Especial: Estágio Supervisionado e Prática de Ensino em Ciências Vol. 6, n. 2. 2023 Edição Especial: Estágio Supervisionado e Prática de Ensino em Ciências e o Ensino Fundamental e a segunda em dezembro de 2018 com aprovação do texto referente ao Ensino Médio. Foram apresentadas, comparadas e discutidas as diferenças entre as chamadas primeira e segundas versões, submetidas a consultas públicas e a terceira versão, que embora também tenha passado por consulta pública, apresentava escopo totalmente diferente dos documentos anteriores, com uma proposta formativa baseada em competências e habilidades. A partir dessas discussões foram apresentadas as estruturas presentes no documento final da BNCC, a organização das áreas e componentes curriculares, seguindo-se ao aprofundamento da área/componente Ciências da Natureza. A partir desse momento seguiu-se um estudo a miúde, com apresentação das competências específicas, Unidades temáticas, com detalhamento dos objetos de conhecimento e habilidades a estas associadas. O encontro foi finalizado com uma análise crítica geral do documento, dos processos cognitivos presentes nas habilidades associadas as competências específicas do componente, assim como discussões de assuntos/conteúdos e conceitos historicamente tratados em ciências e biologia que foram deixados de fora da proposta aprovada pelo Conselho Nacional de Educação em dezembro de 2018. Nesse segundo encontro foram apresentados os desdobramentos que se seguiram a aprovação da BNCC, em especial o lançamento do Programa de Apoio à Implementação da Base Nacional Comum Curricular - ProBNCC, em abril de 2018, pelo Ministério da Educação, e instituído por meio da portaria Nº 331, que estabeleceu o regime de colaboração que seria instalado a partir daquele momento para que todos os Estados e o Distrito federal revisassem ou elaborassem seus currículos, alinhados a BNCC. PROJETO INTEGRADOR DOS ESTÁGIOS (PIE) Foram apresentados todos os regulamentos dos programas, a exemplo das Portaria MEC nº 756, de 3 de abril de 2019, para inserir aspectos específicos da implementação da BNCC para o Ensino Médio. No encontro, foram discutidos como a partir da implementação do programa, todos os Estados e o Distrito Federal iniciaram o processo de revisão e ou escrita de seus documentos curriculares e como este foi responsável pelo aporte financeiro do proBNCC, subsidiaram a formação das equipes de redatores e, cada Unidade da Federação. A segunda metade do encontro foi dedicada a apresentação do Documento Curricular do Estado do Ceará- DCRC, referente ao Ensino Fundamental discutindo-se, como documentos elaborados a luz da BNCC, podem configurar-se como espaços/oportunidades de resistências para que Estados e o DF pudessem imprimir suas marcas. Foram ainda apresentadas as concepções assumidas, tais como contextualização, interdisciplinaridade e como essas concepções ancoram a Autonomia docente e Gestão democrática das Escolas. A palestrante convidada iniciou sua explanação ressaltando fatores positivos relacionados às atividades físicas e estabelecendo relações entre saúde e o bem-estar físico, mental e social. Destacou a importância de se levar em Diretrizes Curriculares Referencial do Ceará (DCRC) Live PIRP/UECE - Impacto da atividade física na saúde mental em tempos de e o Ensino Fundamental e a segunda em dezembro de 2018 com aprovação do texto referente ao Ensino Médio. Foram apresentadas, comparadas e discutidas as diferenças entre as chamadas primeira e segundas versões, submetidas a consultas públicas e a terceira versão, que embora também tenha passado por consulta pública, apresentava escopo totalmente diferente dos documentos anteriores, com uma proposta formativa baseada em competências e habilidades. A partir dessas discussões foram apresentadas as estruturas presentes no documento final da BNCC, a organização das áreas e componentes curriculares, seguindo-se ao aprofundamento da área/componente Ciências da Natureza. A partir desse momento seguiu-se um estudo a miúde, com apresentação das competências específicas, Unidades temáticas, com detalhamento dos objetos de conhecimento e habilidades a estas associadas. O encontro foi finalizado com uma análise crítica geral do documento, dos processos cognitivos presentes nas habilidades associadas as competências específicas do componente, assim como discussões de assuntos/conteúdos e conceitos historicamente tratados em ciências e biologia que foram deixados de fora da proposta aprovada pelo Conselho Nacional de Educação em dezembro de 2018. PROJETO INTEGRADOR DOS ESTÁGIOS (PIE) O encontro foi finalizado com uma análise crítica geral do documento, dos processos cognitivos presentes nas habilidades associadas as competências específicas do componente, assim como discussões de assuntos/conteúdos e conceitos historicamente tratados em ciências e biologia que foram deixados de fora da proposta aprovada pelo Conselho Nacional de Educação em dezembro de 2018. Nesse segundo encontro foram apresentados os desdobramentos que se seguiram a aprovação da BNCC, em especial o lançamento do Programa de Apoio à Implementação da Base Nacional Comum Curricular - ProBNCC, em abril de 2018, pelo Ministério da Educação, e instituído por meio da portaria Nº 331, que estabeleceu o regime de colaboração que seria instalado a partir daquele momento para que todos os Estados e o Distrito federal revisassem ou elaborassem seus currículos, alinhados a BNCC. Foram apresentados todos os regulamentos dos programas, a exemplo das Portaria MEC nº 756, de 3 de abril de 2019, para inserir aspectos específicos da implementação da BNCC para o Ensino Médio. No encontro, foram discutidos como a partir da implementação do programa, todos os Estados e o Distrito Federal iniciaram o processo de revisão e ou escrita de seus documentos curriculares e como este foi responsável pelo aporte financeiro do proBNCC, subsidiaram a formação das equipes de redatores e, cada Unidade da Federação. A segunda metade do encontro foi dedicada a apresentação do Documento Curricular do Estado do Ceará- DCRC, referente ao Ensino Fundamental discutindo-se, como documentos elaborados a luz da BNCC, podem configurar-se como espaços/oportunidades de resistências para que Estados e o DF pudessem imprimir suas marcas. Foram ainda apresentadas as concepções assumidas, tais como contextualização, interdisciplinaridade e como essas concepções ancoram a Autonomia docente e Gestão democrática das Escolas. A palestrante convidada iniciou sua explanação ressaltando Diretrizes Curriculares Referencial do Ceará (DCRC) Diretrizes Curriculares Referencial do Ceará (DCRC) Diretrizes Curriculares Referencial do Ceará (DCRC) Live PIRP/UECE - Impacto da atividade física na saúde mental em tempos de Recebido em: 30/11/2022 Aceito em: 30/04/2023 Recebido em: 30/11/2022 Aceito em: 30/04/2023 497 Vol. 6, n. 2. 2023 Edição Especial: Estágio Supervisionado e Prática de Ensino em Ciências pandemia: um olhar sobre a saúde integral do indivíduo consideração não somente a ausência de doença, mas também o fato de que a qualquer momento, por mais que se esteja bem, eventuais lesões de natureza mental ou física podem agredir a saúde mental. PROJETO INTEGRADOR DOS ESTÁGIOS (PIE) Falou também sobre a combinação de pensamentos, percepções, emoções e comportamentos “anormais” que podem afetar as relações interpessoais, sociais, laborais, culturais, econômicas e ambientais. Destacou, ainda, alguns tipos de transtornos comuns na sociedade, tais como: depressão, transtorno afetivo bipolar; esquizofrenia; demência, dentre outros transtornos decorrentes de problemas psicológicos. Ao final, compreendeu-se que todos esses aspectos podem afetar fortemente as atividades acadêmicas e profissionais de estudantes e professores. Live PIRP/UECE - Diálogos sobre a docência: quais saberes? Quais fazeres? A palestrante iniciou a sua exposição fazendo questionamentos sobre o que identifica a profissão de professor. Destacou que é fundamental estabelecer um diálogo sobre o “fazer” e “ser” professor, refletindo e questionando sobre saberes e fazeres na profissão cotidiana como professor. Ressaltou a importância do envolvimento ativo do professor, buscando agir de forma inovadora, reflexiva, experimental e interativa, sempre com o objetivo de promover uma mudança em prol da promoção de conhecimento. Deixou claro que o processo formativo é dinâmico, não linear e depende da atividade profissional do professor. Deu destaque a três tipos de racionalidades: a técnica, a prática e a reflexiva. Quanto à racionalidade técnica, explicou que a mesma não é suficiente para a promoção de uma mudança de postura docente, já que: técnicas e métodos únicos não atendem qualquer realidade; não deve haver supremacia de conhecimentos teóricos sobre os saberes práticos; não deve haver uma predominância do trabalho individual; e não é adequado se basear em metas e objetivos externos como cernes do trabalho docente. Destacou que a racionalidade prática permite uma prática reflexiva pelo professor. Contudo, enfatizou que não basta refletir sobre a prática, mas é preciso colocar essa reflexão em um contexto crítico-reflexivo que representa a práxis docente, considerando as condições sociais, políticas e econômicas refletidas na prática do professor. Salientou a importância de reconhecer a epistemologia da prática, sendo necessário incluir a teoria a este pensamento, priorizando um acervo de experiências teórico-práticas. Forneceu também detalhes e referências educacionais no campo dos saberes docentes e das práticas pedagógicas e finalizou ressaltando que o ato de ensinar exige comprometimento, já que a atividade docente não é neutra (e sim intencional), devendo-se sempre buscar a transformação dos estudantes por meio do conhecimento. PROJETO INTEGRADOR DOS ESTÁGIOS (PIE) Tecnologias educacionais no ensino de Ciências e Biologia Foi abordado inicialmente o conceito das tecnologias digitais da informação e comunicação e contexto da sua utilização visto a nossa inserção em uma Sociedade do Conhecimento e de uma cultura digital, o que aproxima essas ferramentas tecnológicas do processo educacional. Assim, foram apresentadas algumas possibilidades de estratégias e recursos didáticos que podem ser utilizadas no processo de ensino e pandemia: um olhar sobre a saúde integral do indivíduo pandemia: um olhar sobre a saúde integral do indivíduo consideração não somente a ausência de doença, mas também o fato de que a qualquer momento, por mais que se esteja bem, eventuais lesões de natureza mental ou física podem agredir a saúde mental. Falou também sobre a combinação de pensamentos, percepções, emoções e comportamentos “anormais” que podem afetar as relações interpessoais, sociais, laborais, culturais, econômicas e ambientais. Destacou, ainda, alguns tipos de transtornos comuns na sociedade, tais como: depressão, transtorno afetivo bipolar; esquizofrenia; demência, dentre outros transtornos decorrentes de problemas psicológicos. Ao final, compreendeu-se que todos esses aspectos podem afetar fortemente as atividades acadêmicas e profissionais de estudantes e professores. A p Foi abordado inicialmente o conceito das tecnologias digitais da informação e comunicação e contexto da sua utilização visto a nossa inserção em uma Sociedade do Conhecimento e de uma cultura digital, o que aproxima essas ferramentas tecnológicas do processo educacional. Assim, foram apresentadas algumas possibilidades de estratégias e recursos didáticos que podem ser utilizadas no processo de ensino e aprendizagem mediados pela tecnologia. Por fim, convidamos Tecnologias educacionais no ensino de Ciências e Biologia Recebido em: 30/11/2022 Aceito em: 30/04/2023 498 Vol. 6, n. 2. 20 Edição Especial: Estágio Supervisionado e Prática de Ensino e à reflexão sobre as características do professor do futuro (ou seria presente?) e quem são os atores para que haja uma verdadeira (r)evolução educacional. Educação contextualizada Neste encontro, inicialmente foi apresentado a necessidade de pensarmos a Educação Contextualizada para a Convivência com o Semiárido Brasileiro, expondo sobre os estereótipos e preconceitos sobre as condições geoambientais e da população da região. Depois destacou-se as potencialidades e sobretudo a estratégias construídas ao longo da história, tanto pela população local, quanto pelos integrantes dos movimentos sociais, organização da sociedade civil, universidades, igrejas, etc. No momento seguinte discutiu-se sobre o histórico da Educação Contextualizada, bem como o seu conceito e elementos básicos dessa proposta. PROJETO INTEGRADOR DOS ESTÁGIOS (PIE) Após a realização das ações do projeto, os 19 estudantes regularmente matriculados nos quatro estágios (Estágio Supervisionado no Ensino Fundamental, Estágio à reflexão sobre as características do professor do futuro (ou seria presente?) e quem são os atores para que haja uma verdadeira (r)evolução educacional. Neste encontro, inicialmente foi apresentado a necessidade de pensarmos a Educação Contextualizada para a Convivência com o Semiárido Brasileiro, expondo sobre os estereótipos e preconceitos sobre as condições geoambientais e da população da região. Depois destacou-se as potencialidades e sobretudo a estratégias construídas ao longo da história, tanto pela população local, quanto pelos integrantes dos movimentos sociais, organização da sociedade civil, universidades, igrejas, etc. No momento seguinte discutiu-se sobre o histórico da Educação Contextualizada, bem como o seu conceito e elementos básicos dessa proposta. Por fim, foi apresentado experiências exitosas, utilizando uma que ocorreu no ensino básico e outra no curso de formação de professores. A ideia foi entender as aplicações básicas da Educação Contextualizada e instigar a comunidade acadêmica a colocá-la como prioridade nas pautas de propostas curriculares e na prática docente. Após a realização das ações do projeto, os 19 estudantes regularmente matriculados nos quatro estágios (Estágio Supervisionado no Ensino Fundamental, Estágio Supervisionado I, II e III no Ensino Médio, no qual os estagiários estarão desenvolvendo as atividades do 6º ao 9º ano; 1º ano, 2º ano e 3º ano, respectivamente) foram convidados a participar da avaliação do PIE. Para isso, foi enviado nos grupos das disciplinas, em um aplicativo de envio de mensagens, um formulário eletrônico no qual eles indicaram o grau de satisfação (em uma escala de Likert de 0 a 5, sendo 0 totalmente insatisfeito e o 5 totalmente satisfeito) para cada momento. Participaram voluntariamente deste passo 9 (nove) estudantes, sendo 5 do sexo masculino e 4 do sexo feminino, com idade variando de 20 a 27 anos. Para a maior parte dos professores em formação (88,9%) a participação e desenvolvimentos das ações obtiveram alto grau de satisfação (nota 5), sendo justificada na fala de alguns participantes, como posto a seguir. 499 Recebido em: 30/11/2022 Aceito em: 30/04/2023 Foi muito bem direcionada, contextualizada e os palestrantes mostraram domínio do conteúdo. Foram temas instigantes, importantes e provocativas (Estagiário 2). PROJETO INTEGRADOR DOS ESTÁGIOS (PIE) Por fim, foi apresentado experiências exitosas, utilizando uma que ocorreu no ensino básico e outra no curso de formação de professores. A ideia foi entender as aplicações básicas da Educação Contextualizada e instigar a comunidade acadêmica a colocá-la como prioridade nas pautas de propostas curriculares e na prática docente. Fonte: Elaborado pelos autores. Após a realização das ações do projeto, os 19 estudantes regularmente matriculados nos quatro estágios (Estágio Supervisionado no Ensino Fundamental, Estágio Supervisionado I, II e III no Ensino Médio, no qual os estagiários estarão desenvolvendo as atividades do 6º ao 9º ano; 1º ano, 2º ano e 3º ano, respectivamente) foram convidados a participar da avaliação do PIE. Para isso, foi enviado nos grupos das disciplinas, em um aplicativo de envio de mensagens, um formulário eletrônico no qual eles indicaram o grau de satisfação (em uma escala de Likert de 0 a 5, sendo 0 totalmente insatisfeito e o 5 totalmente satisfeito) para cada momento. Participaram voluntariamente deste passo 9 (nove) estudantes, sendo 5 do sexo masculino e 4 do sexo feminino, com idade variando de 20 a 27 anos. i d f f (88 9%) i i Vol. 6, n. 2. 2023 Edição Especial: Estágio Supervisionado e Prática de Ensino em Ciências à reflexão sobre as características do professor do futuro (ou seria presente?) e quem são os atores para que haja uma verdadeira (r)evolução educacional. Educação contextualizada Neste encontro, inicialmente foi apresentado a necessidade de pensarmos a Educação Contextualizada para a Convivência com o Semiárido Brasileiro, expondo sobre os estereótipos e preconceitos sobre as condições geoambientais e da população da região. Depois destacou-se as potencialidades e sobretudo a estratégias construídas ao longo da história, tanto pela população local, quanto pelos integrantes dos movimentos sociais, organização da sociedade civil, universidades, igrejas, etc. No momento seguinte discutiu-se sobre o histórico da Educação Contextualizada, bem como o seu conceito e elementos básicos dessa proposta. Por fim, foi apresentado experiências exitosas, utilizando uma que ocorreu no ensino básico e outra no curso de formação de professores. A ideia foi entender as aplicações básicas da Educação Contextualizada e instigar a comunidade acadêmica a colocá-la como prioridade nas pautas de propostas curriculares e na prática docente. Fonte: Elaborado pelos autores. PROJETO INTEGRADOR DOS ESTÁGIOS (PIE) Além de contribuir para a compreensão de fenômenos e conhecimentos científicos, estabelecer relações dos aspectos com o contexto em que vive Foi muito bem direcionada, contextualizada e os palestrantes mostraram domínio do conteúdo. Foram temas instigantes, importantes e provocativas (Estagiário 2). Além de contribuir para a compreensão de fenômenos e conhecimentos científicos, estabelecer relações dos aspectos com o contexto em que vive Recebido em: 30/11/2022 Aceito em: 30/04/2023 Recebido em: 30/11/2022 Aceito em: 30/04/2023 499 499 Vol. 6, n. 2. 2023 Edição Especial: Estágio Supervisionado e Prática de Ensino em Ciências Vol. 6, n. 2. 2023 Edição Especial: Estágio Supervisionado e Prática de Ensino em Ciências auxilia na compreensão e interação do aluno com as informações que estão sendo repassadas (Estagiário 3). Momento muito enriquecedor onde aprendemos várias formas de transmitir conhecimento de modo a integrar os alunos na sala, proporcionando um melhor ensino-aprendizagem (Estagiário 6). As proposições do PIE relacionam-se como formação humanística do licenciando, cuja centralidade é um processo de interação e integração, tangenciada pela colaboração e conhecimento. Esse tipo de aprendizagem formativa na prática favorece o desenvolvimento de múltiplas habilidades (procedimentais, conceituais e atitudinais) (LOPES et al., 2022), centrada numa alfabetização científica colaborativa que contribui no desenvolvimento profissional docente (FONSECA; GONÇALVES; SANTOS JÚNIOR, 2022). O desenvolvimento do projeto torna-se ainda mais relevante no momento histórico em que foi realizado: o ensino remoto emergencial. Nesse contexto, a ressignificação das práticas pedagógicas foi necessária, o que impactou diretamente na formação (inicial e continuada) dos professores. Neste percurso diversos desafios e barreiras precisaram ser transponíveis, como a falta de equipamentos e infraestrutura, apoio institucional e de formação (CLESAR; GIRAFFA, 2022; MENEZES; MOURA, 2021). O desenvolvimento do projeto torna-se ainda mais relevante no momento histórico em que foi realizado: o ensino remoto emergencial. Nesse contexto, a ressignificação das práticas pedagógicas foi necessária, o que impactou diretamente na formação (inicial e continuada) dos professores. Neste percurso diversos desafios e barreiras precisaram ser transponíveis, como a falta de equipamentos e infraestrutura, apoio institucional e de formação (CLESAR; GIRAFFA, 2022; MENEZES; MOURA, 2021). O PIE também se torna um espelho para a aplicação de uma prática docente interacionista, integrativa, colaborativa e interdisciplinar, como apontou o Estagiário 6, anteriormente. PROJETO INTEGRADOR DOS ESTÁGIOS (PIE) Neste ponto, Tonet (2006) reforça a necessidade de uma atividade educativa que contribua na formação de indivíduos autônomos, críticos, reflexivos e comprometidos para a construção de uma sociedade que por meio da formação integral possa transformar a realidade. O PIE também se torna um espelho para a aplicação de uma prática docente interacionista, integrativa, colaborativa e interdisciplinar, como apontou o Estagiário 6, anteriormente. Neste ponto, Tonet (2006) reforça a necessidade de uma atividade educativa que contribua na formação de indivíduos autônomos, críticos, reflexivos e comprometidos para a construção de uma sociedade que por meio da formação integral possa transformar a realidade. Essa realidade está em alinhamento à proposta educativa de uma educação global, integrando as diversas áreas do conhecimento, as dimensões intelectuais, sociais e cognitivas. Ações com essa característica acabam por promover uma forma autônoma, reflexiva, interdisciplinar e integradora, além de possibilitar livrar-se das amarras do ensino fragmento e descontextualizado com a realidade (BEANE, 2003; FAZENDA, 2014). Fato este corroborado por Nóvoa (2011), o qual destaca que a docência deve exercer a partilha de saberes e das práticas de modo colaborativo. Recebido em: 30/11/2022 Aceito em: 30/04/2023 Recebido em: 30/11/2022 Aceito em: 30/04/2023 500 500 Vol. 6, n. 2. 2023 Edição Especial: Estágio Supervisionado e Prática de Ensino em Ciências Vol. 6, n. 2. 2023 Edição Especial: Estágio Supervisionado e Prática de Ensino em Ciências REFERÊNCIAS BEANE, J. A. Integração Curricular: a essência de uma escola democrática. Currículo Sem Fronteiras, v.3, n.2, p. 91-110, 2003. BEANE, J. A. Integração Curricular: a essência de uma escola democrática. Currículo Sem Fronteiras, v.3, n.2, p. 91-110, 2003. BRASIL. Resolução CNE/CP 2/2019. Diário Oficial da União, Brasília, 15 de abril de 2020, Seção 1, p. 46-49, 2020. CEARÁ. Projeto pedagógico curso de Ciências Biológicas - modalidade licenciatura – FAEC/UECE. 2012. CONSIDERAÇÕES FINAIS O Projeto Integrado dos Estágios (PIE) trouxe aos estudantes licenciandos estagiários a oportunidade de um caro momento de discussão acerca de temáticas curriculares, metodológicas e integradoras do contexto educacional, ampliando o escopo dialógico da formação docente. As temáticas sobre os documentos curriculares (BNCC e DCRC), as tecnologias digitais no processo educacional, saberes e fazeres docente, educação contextualizada e saúde mental na pandemia são temas que permeiam a (form)ação docente, sendo fundamentalmente importantes sua abordagem e discussão nos cursos de licenciatura, em especial se forem realizadas de forma inte(g)rativa, contextualizada e colaborativa. No desabrochar do Estágio, esses tipos de ações são ainda mais profícuas, visto que este é um espaço de alinhamento da teoria e prática. Para mais, é uma possibilidade de promoção de atividades que proporcionem a reflexão da profissão docente e de temáticas que a rodeiam, possibilitando uma formação plural, integradora, humanística e holística do futuro professor, tornando-o capacitado para transformar uma sala de aula por vez, um aluno por vez, e contribuindo para reconstruir a realidade e alavancar a qualidade da educação brasileira. REFERÊNCIAS BEANE, J. A. Integração Curricular: a essência de uma escola democrática. Currículo Sem Fronteiras, v.3, n.2, p. 91-110, 2003. No desabrochar do Estágio, esses tipos de ações são ainda mais profícuas, visto que este é um espaço de alinhamento da teoria e prática. Para mais, é uma possibilidade de promoção de atividades que proporcionem a reflexão da profissão docente e de temáticas que a rodeiam, possibilitando uma formação plural, integradora, humanística e holística do futuro professor, tornando-o capacitado para transformar uma sala de aula por vez, um aluno por vez, e contribuindo para reconstruir a realidade e alavancar a qualidade da educação brasileira. REFERÊNCIAS BEANE, J. A. Integração Curricular: a essência de uma escola democrática. Currículo Sem Fronteiras, v.3, n.2, p. 91-110, 2003. No desabrochar do Estágio, esses tipos de ações são ainda mais profícuas, visto que este é um espaço de alinhamento da teoria e prática. Para mais, é uma possibilidade de promoção de atividades que proporcionem a reflexão da profissão docente e de temáticas que a rodeiam, possibilitando uma formação plural, integradora, humanística e holística do futuro professor, tornando-o capacitado para transformar uma sala de aula por vez, um aluno por vez, e contribuindo para reconstruir a realidade e alavancar a qualidade da educação brasileira. CEARÁ. Projeto pedagógico curso de Ciências Biológicas - modalidade licenciatura – FAEC/UECE. 2012. CLESAR, C.; GIRAFFA, L. A formação de professores a partir das vivências do ensino remoto: desafios, expectativas e possibilidades. Revista Insignare Scientia - RIS, v. 5, n. 5, p. 143-163, 2022. FAZENDA, I. A. C. (Org.). Interdisciplinaridade: pensar, pesquisar e intervir. São Paulo: Cortez, 2014. 501 Vol. 6, n. 2. 2023 Edição Especial: Estágio Supervisionado e Prática de Ensino em Ciências FONSECA, C.; GONÇALVES, S.; DOS SANTOS JR, J. Alfabetização científica, colaboração e desenvolvimento profissional: Investigando conexões em um grupo colaborativo de professoras dos anos iniciais. Revista Insignare Scientia - RIS, v. 5, n. 3, p. 226-242, 2022. FRACARO, A. R.; HAEFLIGER, C. K. M. Tornando-se professor: Um relato de experiência e vivência durante o Estágio Supervisionado II. Contraponto: Discussões científicas e pedagógicas em Ciências, Matemática e Educação, v. 3, n. 3, p. 92-106, 2022. GATTI, B. A. A formação inicial de professores para a educação básica: as licenciaturas. Revista USP, v. 100, p. 33-46, 2013. LIMA, M. S. L. O estágio nos cursos de licenciatura e a metáfora da árvore. Revista eletrônica pesquiseduca, v. 1, n. 01, p. 45-48, 2009. LOPES, A. et al. Percepções dos professores sobre interdisciplinaridade em um curso de formação continuada online. Revista Insignare Scientia - RIS, v. 5, n. 4, p. 1-17, 2022. LÜDKE, H. A. M. O lugar do estágio na formação de professores. Educação em Perspectiva, v.4, n.1, p. 111-133, 2013. MENDES, C. B.; MAIA, J. S. S.; BIANCON, M. L. Impactos do ensino remoto: a questão do Estágio Supervisionado Obrigatório em Ensino de Ciências e Biologia numa Universidade Estadual paranaense em tempos de pandemia. Momento: diálogos em educação, v. 30, n. 01, p. 78-98, jan./abr., 2021. MENEZES, J. B. F. de M.; MOURA, F. N. de S. O ensino em tempos de isolamento social: percepção de docentes de um curso de formação de professores. Olhar de Professor, [S. l.], v. 24, p. 1-10, 2021. DOI: 10.5212/OlharProfr.v.24.15836.046. SOUZA, E. M. F.; FERREIRA, L. G. Ensino remoto emergencial e o estágio supervisionado nos cursos de licenciatura no cenário da pandemia Covid 19. Rev. Tempos Espaços Educ., v.13, n. 32, p. 1-20. e-14290, jan./dez., 2020. PINHEIRO JÚNIOR, E. C. et al. O estágio supervisionado no curso de graduação em Ciências Biológicas: um relato de experiência. Educamazônia-Educação, Sociedade e Meio Ambiente, v. 15, n. 2, jul-dez, p. 188-209, 2022. TONET, I. Educação e formação humana. Ideação, v. 8, n. 9, p. 09-21, 2006. Recebido em: 30/11/2022 Aceito em: 30/04/2023 Recebido em: 30/11/2022 Aceito em: 30/04/2023 Recebido em: 30/11/2022 Aceito em: 30/04/2023 502
https://openalex.org/W4321004345	https://www.nature.com/articles/s41598-023-29439-6.pdf	English	null	Classification of electrically-evoked potentials in the parkinsonian subthalamic nucleus region	Scientific reports	2,023	cc-by	8,308	Classification of electrically‑evoked potentials in the parkinsonian subthalamic nucleus region OPEN Joshua Rosing 1, Alex Doyle 2, AnneMarie Brinda 1, Madeline Blumenfeld 1, Emily Lecy 2, Chelsea Spencer 1, Joan Dao 1, Jordan Krieg 1, Kelton Wilmerding 1, Disa Sullivan 1, Sendréa Best 1, Biswaranjan Mohanty 3, Jing Wang 3, Luke A. Johnson 3, Jerrold L. Vitek 3 & Matthew D. Johnson 1,4* Electrically evoked compound action potentials (ECAPs) generated in the subthalamic nucleus (STN) contain features that may be useful for titrating deep brain stimulation (DBS) therapy for Parkinson’s disease. Delivering a strong therapeutic effect with DBS therapies, however, relies on selectively targeting neural pathways to avoid inducing side effects. In this study, we investigated the spatiotemporal features of ECAPs in and around the STN across parameter sweeps of stimulation current amplitude, pulse width, and electrode configuration, and used a linear classifier of ECAP responses to predict electrode location. Four non-human primates were implanted unilaterally with either a directional (n = 3) or non-directional (n = 1) DBS lead targeting the sensorimotor STN. ECAP responses were characterized by primary features (within 1.6 ms after a stimulus pulse) and secondary features (between 1.6 and 7.4 ms after a stimulus pulse). Using these features, a linear classifier was able to accurately differentiate electrodes within the STN versus dorsal to the STN in all four subjects. ECAP responses varied systematically with recording and stimulating electrode locations, which provides a subject-specific neuroanatomical basis for selecting electrode configurations in the treatment of Parkinson’s disease with DBS therapy. Electrical stimulation within the nervous system is well known to generate evoked compound action potentials (ECAPs) whose features occur within milliseconds of stimulus onset and attenuate in amplitude over time1,2. This physiological activity, which is often detected from one or more recording electrodes positioned near the stimulating electrode, reflects the spatial summation of induced membrane polarization adjacent to the recording electrode(s)3–6. ECAP features are thought to be indicative of both direct activation of axons (immediate primary features) as well as synaptic and network-level modulation patterns (delayed secondary features)1,2. Such ECAP features have shown utility for assessing the degree of membrane polarization and thus target engagement with stimulation of the peripheral nerves7, spinal cord8–10, cochlea11–14, retina15,16, and deep brain regions2,17. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Methods li i l Preclinical subjects. This study investigated ECAPs from STN-DBS leads in four aged female rhesus macaque monkeys (macaca mulatta; Subjects Ne, Az, So, and Bl; 14.75, 18.5, 19.5, and 26 years old, respectively, at the time of the recordings). Procedures used in the study were approved by the University of Minnesota’s Institutional Animal Care and Use Committee, were performed following the United States Public Health Ser- vice policy for humane care and use of laboratory animals, and follow the recommendations in the ARRIVE guidelines. All animals received environmental enrichment, free access to water, and a wide variety of foods, including fresh fruits and vegetables. All effort was made to provide animals with adequate care and prevent discomfort during the study. Surgical procedures. Animals were imaged pre-operatively using a 7 T or 10.5 T human bore magnet with custom-designed head coils for non-human primates at the Center for Magnetic Resonance Research at the Uni- versity of Minnesota. Similar to previous studies21,22, DBS leads were implanted along an oblique mapping track that had the largest span of sensorimotor-responsive STN cell activity. The depth of the implant was designed to have electrodes within the sensorimotor STN and the region dorsal to the STN, containing the lenticular fasciculus23 (Fig. 1). DBS implant procedures. Subjects Ne and Az were implanted with a 6-channel directional DBS lead (Abbott Neuromodulation, 0.6 mm diameter), consisting of 2 rows and 3 columns of electrodes (0.75 mm height, 0.5 mm spacing between rows) with the top row clocked 60 degrees from the bottom row. Subject So was implanted with a 12-channel directional DBS lead (Heraeus Medical Components, 0.8 mm diameter), consist- ing of 4 rows and 3 columns of electrodes (0.5 mm height, 0.5 mm spacing) with no rotational offset. Subject Bl was implanted with a non-directional DBS lead (NuMed, 0.625 mm diameter) with 8 electrodes (one band electrode per row, 0.5 mm height, and 0.5 mm spacing). DBS lead wires were routed to another chamber to interface with an external neurostimulator (IZ2MH) and recording (PZ5) system (Tucker-Davis Technologies). A CT scan was taken post-implant and co-registered to the pre-operative MRI to estimate placement of the DBS lead implant with respect to the STN (Fig. 1), and this was confirmed with post-mortem block-face histological imaging. Methods li i l Split-band electrode orientation for Subjects Ne, Az, and So was determined using a post-mortem bub- ble test whereby the explanted lead, still integrated with the chamber and cap, was submerged in 0.9% saline and stimulated through with 1–5 mA DC current over 1–5 s to generate an electrolysis reaction and small bubble at the stimulated electrode. Range in current and time needed for electrolysis reaction depended on the lead. This was used to verify electrode connector maps, check for electrical shorts, and identify each electrode’s orientation relative to the the STN. ECAP stimulation protocol. In each subject, monopolar stimulus pulse trains (125 Hz) were delivered through a single electrode contact using biphasic pulse waveforms that alternated between cathodic or anodic first phase polarities. The first phase had a duration of 100 µs, and the second active recharge phase had a duration of 1 ms with a stimulus amplitude at 10% of the first phase’s amplitude. Using these waveforms and pulse trains, the overall stimulus amplitude was increased until sustained side effects were observed on the contralateral side (e.g., muscle contractions, dyskinesias, etc.). Subsequent stimulation trials were then capped at 25 µA below the side effect threshold as determined for each electrode on the DBS lead. During all stimulation trials, the subject was seated and alert while wide-band, monopolar ECAP recordings were collected from the other electrodes at a sampling rate of 48.8 kHz and in reference to cranial bone screws distributed over the parietal lobe. 0.6 mm a 0.75 mm 2 3 1 1 2 A B C A B C A C B b Az Bl Ne So c 10 mm 2 mm STN D L 2 3 A B C A C B 2 A B C 0.8 mm Figure 1. DBS lead localization within the STN. (a) Directional and non-directional DBS leads used in this study. (b, c) High-field 7 T or 10.5 T MRI and CT co-registration were used to approximate the position of each lead within the subthalamic nucleus. The exact electrode position (all leads) and orientation (for the directional leads) was identified through post-mortem block-face histology and a post-mortem bubble test. These positions are shown in the figure. So a Bl Figure 1. DBS lead localization within the STN. (a) Directional and non-directional DBS leads used in this study. Classification of electrically‑evoked potentials in the parkinsonian subthalamic nucleus region OPEN ECAP features have also been integrated as feedback signals to adjust therapies dynamically, includ- ing cochlear implants to streamline behavioral fitting procedures18 and spinal cord stimulation to account for changes in the distance between the stimulating electrode(s) and the spinal cord during activities of daily living9. Similarly, for DBS applications, knowing the spatial position and orientation of each electrode in the context of the targeted nucleus or fiber pathways can be helpful for fine-tuning stimulation settings19. Previous studies have shown that ECAP feature presence and prominence in the subthalamic nucleus (STN) is associated with Such ECAP features have shown utility for assessing the degree of membrane polarization and thus target engagement with stimulation of the peripheral nerves7, spinal cord8–10, cochlea11–14, retina15,16, and deep brain regions2,17. ECAP features have also been integrated as feedback signals to adjust therapies dynamically, includ- ing cochlear implants to streamline behavioral fitting procedures18 and spinal cord stimulation to account for changes in the distance between the stimulating electrode(s) and the spinal cord during activities of daily living9. Si il l f DBS li i k i h i l i i d i i f h l d i h g g p g y g Similarly, for DBS applications, knowing the spatial position and orientation of each electrode in the context of the targeted nucleus or fiber pathways can be helpful for fine-tuning stimulation settings19. Previous studies have shown that ECAP feature presence and prominence in the subthalamic nucleus (STN) is associated with therapeutic effectiveness in Parkinson’s disease17,20. However, the degree of spatial heterogeneity of ECAPs within and adjacent to the STN remains unclear. In this study, we investigated the spatiotemporal features of ECAPs in and around the STN in four non-human primates rendered parkinsonian with MPTP treatments and aligned those features to neuroanatomical borders around each DBS lead implant. 1Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN, USA. 2Department of Neuroscience, University of Minnesota, Minneapolis, MN, USA. 3Department of Neurology, University of Minnesota, Minneapolis, MN, USA. 4Institute for Translational Neuroscience, Department of Biomedical Engineering, University of Minnesota, 6‑134 Nils Hasselmo Hall, 312 Church Street SE, Minneapolis, MN 55455, USA. *email: john5101@umn.edu \| https://doi.org/10.1038/s41598-023-29439-6 \| https://doi.org/10.1038/s41598-023-29439-6 Scientific Reports \| (2023) 13:2685 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Current sweep experiments. To confirm the existence of ECAPs as neural responses as opposed to electrical artifacts, the current amplitude of the stimulus pulse train was varied randomly between no stimulation and 25 µA below the side effect threshold for each electrode. Current sweep data collection trials consisted of 5 s of stimulation followed by 5 s of no stimulation at each amplitude, with repeated measures of 5 trials per electrode. All current sweep data were collected in a parkinsonian condition. Strength‑duration sweep experiments. To further confirm that the signals were of neural origin, strength-duration relationships were assessed through ECAP recordings by systematically varying the current amplitude and pulse width of the charge-balanced, biphasic waveform. The protocol consisted of stimulating at (1) seven current ampli- tudes (12.5%, 25%, 37.5%, 50%, 62.5%, 75% and 87.5% of side-effect threshold) in the parkinsonian condition, and (2) five pulse widths per stimulus amplitude ranging from 40 to 160 µs for the first phase. Each trial consisted of 30 s of stimulation followed by 30 s without stimulation for each parameter combination. The presentation order of the parameters was randomized but kept consistent across electrodes and subjects (Az and So). Classification experiments. Stimulation settings for classifying electrode location were based on experiments running biphasic pulse waveforms at 125 Hz consisting of alternating presentations of a short phase (100 µs) of one polarity followed by a long recharge phase (1 ms) of the opposite polarity. In each subject, the electrode with the lowest side-effect threshold determined the peak stimulation amplitude (87.5% of that electrode’s side effect threshold) applied across all electrodes. This ensured that the within-subject classification would not be biased by varying stimulation amplitudes across electrodes. MPTP treatment and evaluation. Each primate was given a series of systemic injections of the neu- rotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). Following MPTP treatment, the parkinsonian motor sign severity was rated for each subject using a modified version of the Unified Parkinson’s Disease Rating Scale (mUPDRS), which consisted of 14 motor scores, quantified from 0 (no effect) to 3 (severe)24. The total motor score for each subject was used to determine the overall severity of parkinsonian motor signs, and these scores were measured at least five times. Averaged scores were 18.25/42 for Subject Ne (moderate), 8.47/42 for Subject Az (mild), 0/42 for Subject So (asymptomatic), and 10.4/27 for Subject Bl (moderate). ECAP processing. www.nature.com/scientificreports/ Data collected during current sweep and strength-duration sweep experiments were processed to remove the stimulation artifacts and residual noise (Fig. 2). First, baseline subtraction was applied to each interstimulus ECAP segment (8 ms long) by subtracting the amplitude of the first data point (0.4 ms before the stimulus pulse) from all subsequent data points in the ECAP segment. Next, segments were sorted based on cathodic-anodic or anodic–cathodic stimulus waveforms to ensure that the sample sizes for both stim- ulus waveforms were identical. For the current sweep experiments, all 5 s of ECAP recordings were averaged together, and for the strength-duration sweep experiments, the last 20 s of the 30 s long ECAP recordings were averaged together2. Averaging significantly reduced the electrical artifact as shown in Fig. 2. The resulting data were then smoothed (4-sample moving average over the first 1.17 ms and 16 sample moving average over the remaining portion of the segment). This two-part filtering approach avoided over-smoothing the primary fea- tures while still removing high frequency noise from the secondary features of the ECAP. g g q y y Some ECAP recordings, most notably in Subjects Az and So, contained a 0.8–1.0 kHz noise source that was consistent in amplitude over the entire peri-stimulus time window. The peri-stimulus data (from 1.25 to 7.4 ms) was passed through an IIR filter with 0.99 steepness, with the delayed filter onset designed to avoid generating artifacts in the primary features (Fig. S1). The processed data before and after 1.25 ms were then stitched together with a weighted averaging of 9 samples about the stitch to avoid discontinuities in the data (sample 1 was 90% unfiltered sample value plus 10% filtered sample value, and sample 9 was 10% unfiltered sample value plus 90% filtered sample value).ih i Data with this 0.8–1.0 kHz noise source were marked for filtering objectively as follows. The maximum spec- tral power in two bands (< 500 Hz and 700–1500 Hz) in each recording were calculated and used to define two conditions: (A) the maximum spectral power of the unfiltered < 500 Hz band was sufficiently greater (determined by a manually set threshold for each subject) than the maximum spectral power for the unfiltered 700–1500 Hz band, and (B) the maximum spectral power of the unfiltered < 500 Hz band was less than the maximum spectral power of the filtered < 500 Hz band. Methods li i l (b, c) High-field 7 T or 10.5 T MRI and CT co-registration were used to approximate the position of each lead within the subthalamic nucleus. The exact electrode position (all leads) and orientation (for the directional leads) was identified through post-mortem block-face histology and a post-mortem bubble test. These positions are shown in the figure. https://doi.org/10.1038/s41598-023-29439-6 Scientific Reports \| (2023) 13:2685 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ (a) An alternating sequence of cathodic leading and anodic-leading waveforms was applied through one electrode while ECAP recordings were collected through all adjacent electrodes. (b) Raw ECAP data were grouped, averaged, filtered, and separated into primary, early secondary, and late secondary features. 10 ms 1. Raw 2. Grouped ECAP Signal Processing 2 ms 3. Avg/Filter 2 ms 20 µV 70 mV b b ECAP Signal Processing 4. Feature Visualization Secondary Features ry Figure 2. Signal processing of ECAPs within and adjacent to the STN. (a) An alternating sequence of cathodic- leading and anodic-leading waveforms was applied through one electrode while ECAP recordings were collected through all adjacent electrodes. (b) Raw ECAP data were grouped, averaged, filtered, and separated into primary, early secondary, and late secondary features. from those two groups as samples and training in a leave-one-out approach. Accuracy was calculated from the classification error, was averaged across trials, and compared against chance (50%) to determine effectiveness of the classifier. The classifier was also trained on all data from three subjects and tested using all data from a fourth subject. from those two groups as samples and training in a leave-one-out approach. Accuracy was calculated from the classification error, was averaged across trials, and compared against chance (50%) to determine effectiveness of the classifier. The classifier was also trained on all data from three subjects and tested using all data from a fourth subject. www.nature.com/scientificreports/ If condition A was false, or if both conditions were true, the recording was marked for filtering. Data analysis and classification. ECAP feature windows were determined based on time segments con- taining similar ECAP responses within and across subjects. Primary features were the first negative and positive peaks to occur, typically within 0.6–1.6 ms of stimulus pulse onset. Secondary features were divided into early (1.6–3.8 ms) and late (3.8–7.4 ms) windows after a stimulus pulse. The separation between early and late win- dows was based on previous data showing a clear transition (at approximately 3.8 ms) between stimulus evoked neuronal spike inhibition and a return to a baseline spiking probability for peri-stimulus time histograms of STN neurons during STN-DBS (see25). For each ECAP window, the root mean square (RMS) of the data was calculated and then used as feature amplitudes for graphical comparisons, and as features in a linear discrimi- nant analysis classifier. The rationale for feature windows as opposed to defining specific peaks and troughs was based on observations that ECAP features differed in manifestation and in their exact timing across recording configurations for each subject.i i g j A linear classifier (MATLAB) was used to predict stimulation and recording electrode site locations for two ossible groups—STN/STN and LF/LF (lenticular fasciculus)—in all subjects. The classifier used all data points https://doi.org/10.1038/s41598-023-29439-6 Scientific Reports \| (2023) 13:2685 \| www.nature.com/scientificreports/ DBS cathodic- anodic anodic- cathodic a ECAP Response a 0 1 2 3 4 5 6 7 -50 0 50 100 Response (µV) Peristimulus Time (ms) 10 ms cathodic- anodic 1. Raw 4. Feature Visualization 2. Grouped ECAP Signal Processing 2 ms Late Early Secondary Features Primary Artifact 3. Avg/Filter 2 ms 20 µV 70 mV b Figure 2. Signal processing of ECAPs within and adjacent to the STN. (a) An alternating sequence of cathodic- leading and anodic-leading waveforms was applied through one electrode while ECAP recordings were collected through all adjacent electrodes. (b) Raw ECAP data were grouped, averaged, filtered, and separated into primary, early secondary, and late secondary features. 0 1 2 3 4 5 6 7 -50 0 50 100 Response (µV) Peristimulus Time (ms) 10 ms 1. Raw 4. Feature Visualization 2. Grouped ECAP Signal Processing 2 ms Late Early Secondary Features Primary Artifact 3. Avg/Filter 2 ms 20 µV 70 mV b Figure 2. Signal processing of ECAPs within and adjacent to the STN. Results Results Distributions and variability of ECAP features. Within-subject comparison of ECAPs showed high variability of responses across all stimulation parameters (Fig. 3). Variance was highest during the period of known artifact, and second highest during the earlier portion of the primary feature window. The primary fea- ture window’s variance formed a second prominence, suggesting a different source than that of the artifact’s vari- ance. Variance declined rapidly during the primary feature window for most subjects, then steadily declined over https://doi.org/10.1038/s41598-023-29439-6 Scientific Reports \| (2023) 13:2685 \| www.nature.com/scientificreports/ ECAP Response (µV) Variance in ECAP (µV) -50 0 50 100 Subject So Time (ms) Subject Bl Time (ms) Subject Az Time (ms) 10 -3 10 -2 10 -1 10 0 10 1 10 2 10 3 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 Subject Ne Time (ms) A B 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 Figure 3. Distributions of all ECAP stimulation/recording experiments across subjects. (a) Pooled recordings across tested stimulation pulse widths (subjects Az and So only), amplitudes (all subjects), and electrode configurations (all subjects) for a single recording day. (b) Variance across pooled recordings across the ECAP window. Black lines indicate edges of selected feature windows (primary: 0.6–1.6 ms, early secondary 1.6– 3.8 ms, late secondary 3.8–7.4 ms). ECAP Response (µV) -50 0 50 100 Subject Az A Subject So Subject Ne A B Variance in ECAP (µV) Time (ms) 10 -3 10 -2 10 -1 10 0 10 1 10 2 10 3 0 1 2 3 4 5 6 7 B Time (ms) Time (ms) 6 7 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 Figure 3. Distributions of all ECAP stimulation/recording experiments across subjects. (a) Pooled recordings across tested stimulation pulse widths (subjects Az and So only), amplitudes (all subjects), and electrode configurations (all subjects) for a single recording day. (b) Variance across pooled recordings across the ECAP window. Black lines indicate edges of selected feature windows (primary: 0.6–1.6 ms, early secondary 1.6– 3.8 ms, late secondary 3.8–7.4 ms). the early and late secondary feature windows. Most subjects showed much lower variance during the secondary feature windows than during the primary feature window. ECAP responses to varying stimulation amplitudes and pulse widths. Results Current sweep and pulse width sweep experiments in each of the four subjects showed that features of the ECAP response changed non- linearly to adjustments in stimulation parameters while the stimulation artifact increased linearly with increas- ing stimulation amplitude (Fig. 4). Features emerged gradually as stimulation amplitude was increased, with secondary features appearing only at stimulation amplitudes that were higher than those sufficient to evoke pri- mary features. Primary and secondary features also generally increased in amplitude with increasing stimulation amplitude (Fig. 4). ECAP response features also followed classical strength duration curves across stimulation amplitudes and pulse widths (Fig. S2). Spatial heterogeneity of ECAP features. The spatial ECAP response differences were readily visible and distinguished by a simple linear classifier (Fig. 5). This classifier, trained on all subjects, was capable of cor- rectly determining the stimulation and recording sites as being either both in the STN or both in LF with 95.2% accuracy (measured using the leave-one-out method). Furthermore, when trained on all data from three sub- jects and tested using data solely from a fourth, the classifier was able to accurately determine the locations of the fourth subject’s electrodes with 75% (subject Az), 81.25% (subject So), 100% (subject Bl), and 93.3% (subject Ne) accuracy. This suggests that the DBS ECAP response in both directional and ring electrode configurations can be used to gain an understanding of implant location and is generalizable across subjects. The main determining feature across all subjects was the primary feature amplitude (Fig. 5), though there were also visible differences in secondary features in subjects Az and So. Discussionh This study investigated the spatiotemporal features of ECAPs collected within and around the STN region in four MPTP-treated non-human primates who were chronically implanted with STN-DBS leads. The use of directional DBS leads in this study enabled multi-channel recordings within and dorsal to the STN in the same subject with electrode contact position and orientation verified with post-mortem histology. ECAP features in the STN region were observed to (a) follow a sigmoidal intensity curve with respect to stimulation amplitude; (b) exhibit strength-duration curve relationships; and (c) vary systematically by stimulation/recording electrode configurations. Findings (a) and (b) provide continued support for the neurobiological basis of ECAPs in the STN region, while finding (c) indicates an opportunity to use subject-agnostic analysis methods of ECAP responses to determine electrode positions using pooled multi-subject classifiers.il p g p ji Traditionally, ECAP features have been viewed as having specific peak and trough features1,2,4,17,26 that reflect detection of neural sources of convergent activity. Discussionh ECAP responses to increasing stimulation amplitudes across all subject (a–d). (subpanel i) Lead diagrams for each subject with stimulation electrode shown in red and recording site shown in blue. Example ECAP response to (subpanel ii) the highest stimulation amplitude tested in the MPTP-treated state and (subpanel iii) varying stimulation current amplitudes. RMS values across stimulation amplitudes from epochs containing the (subpanel iv) stimulation artifact and primary features and (subpanel part v) secondary features. varied depending on stimulation amplitude and electrode configuration, lead geometry, and implant location. Using time ranges rather than specific points when calculating ECAP features allowed for more broadly applica- ble data analyses such as the use of summary statistics (RMS values), rather than peak-to-trough amplitudes or prominences. Additionally, previous ECAP studies have relied on macroscale electrodes and bipolar recording configurations with two electrodes guarding a center stimulating electrode2,17,26. In this study, single-electrode ECAP responses were used to show visually separable features between the STN and regions dorsal to the STN, most prominently observed in the primary features. These features enabled classifying electrode location (within the subthalamic nucleus or within the lenticular fasciculus) with high accuracy across subjects. g y j Previous research has suggested that the likely source of the primary features are direct axonal activation of passing white matter fibers1, which would be in much greater number and more aligned in the LF than in the STN. This may provide one possible explanation for why the primary feature amplitude was larger in the LF/ LF than the STN/STN configurations. Though the ECAP response data did not support a method to determine DBS lead orientation, additional subjects and recordings spanning a broader set of implant locations would be helpful to tease apart the neuroanatomical origins of differences in directional ECAP responses (Figs. 5, S3). Additionally, the classifier was not used to predict whether a stimulating electrode was in the STN while the recording electrode was in the LF or vice versa given that stimulating and recording electrode pairs in the same region proved to have a much higher accuracy level.hi The ability of a linear classifier to accurately predict where a stimulation and recording site pair are located could be useful for programming DBS systems, especially as DBS lead designs become more complex and clini- cal monopolar reviews take longer to complete. Discussionh -50 0 50 100 ECAP (µV) a 0 1 2 3 4 5 6 7 Time (ms) 200 150 100 50 0 DBS Amplitude (µA) -50 0 50 100 ECAP (µV) c Subject Ne ii iii i -50 0 50 100 ECAP (µV) 0 50 100 150 200 DBS Amplitude (µA) 0 10 20 30 RMS (µV) a 0 1 2 3 4 5 6 7 Time (ms) 200 150 100 50 0 DBS Amplitude (µA) -50 0 50 100 ECAP (µV) 0 40 80 120 RMS (µV) 0 0.1 0.2 0.3 0.4 Artifact Magnitude (V) c artifact primary early secondary late secondary Subject Ne ii iii iv v i 0 50 100 150 200 DBS Amplitude (µA) 0 10 20 30 RMS (µV) 0 40 80 120 RMS (µV) 0 0.1 0.2 0.3 0.4 Artifact Magnitude (V) artifact primary early secondary late secondary iv v b -50 0 50 100 ECAP (µV) 50 100 150 200 DBS Amplitude (µA) 0 20 40 60 RMS (µV) b 0 1 2 3 4 5 6 7 Time (ms) 240 200 170 100 35 DBS Amplitude (µA) -50 0 50 100 ECAP (µV) 0 100 200 300 RMS (µV) 0 0.1 0.2 0.3 Artifact Magnitude (V) Subject Az 140 70 -50 0 50 100 ECAP (µV) b 0 1 2 3 4 5 6 7 Time (ms) 240 200 170 100 35 DBS Amplitude (µA) -50 0 50 100 ECAP (µV) d Subject Az 140 70 b a 50 100 150 200 DBS Amplitude (µA) 0 20 40 60 RMS (µV) ECAP (µV) 0 100 200 300 RMS (µV) 0 0.1 0.2 0.3 Artifact Magnitude (V) 2 RMS (µV) d c Time (ms) -50 0 50 100 ECAP (µV) d 0 1 2 3 4 5 6 7 Time (ms) 200 150 100 50 0 DBS Amplitude (µA) -50 0 50 100 Subject Bl Time (ms) -50 0 50 100 ECAP (µV) c 0 1 2 3 4 5 6 7 Time (ms) 488 406 244 163 81 DBS Amplitude (µA) -50 0 50 100 Subject So 325 DBS Amplitude (µA) 100 200 300 400 DBS Amplitude (µA) 0 10 20 RMS (µV) 10 30 70 90 RMS (µV) 0 0.2 0.4 Artifact Magnitude (V) 50 500 p (µ ) ( ) -50 0 50 100 ECAP (µV) 100 200 300 400 DBS Amplitude (µA) 0 10 20 RMS (µV) c 0 1 2 3 4 5 6 7 Time (ms) 488 406 244 163 81 DBS Amplitude (µA) -50 0 50 100 ECAP (µV) 10 30 70 90 RMS (µV) 0 0.2 0.4 Artifact Magnitude (V) Subject So 50 500 325 -50 0 50 100 ECAP (µV) 0 50 100 150 200 DBS Amplitude (µA) 0 20 30 40 RMS (µV) d 0 1 2 3 4 5 6 7 Time (ms) 200 150 100 50 0 DBS Amplitude (µA) -50 0 50 100 ECAP (µV) 0 50 150 200 RMS (µV) 0 0.1 0.2 Artifact Magnitude (V) Subject Bl 100 10 0 50 100 150 200 DBS Amplitude (µA) 0 20 30 40 RMS (µV) ECAP (µV) 0 50 150 200 RMS (µV) 0 0.1 0.2 Artifact Magnitude (V) 100 10 Figure 4. Discussionh In this study, the timing, location, and number of these features Scientific Reports \| (2023) 13:2685 \| https://doi.org/10.1038/s41598-023-29439-6 www.nature.com/scientificreports/ -50 0 50 100 ECAP (µV) 0 50 100 150 200 DBS Amplitude (µA) 0 10 20 30 RMS (µV) a 0 1 2 3 4 5 6 7 Time (ms) 200 150 100 50 0 DBS Amplitude (µA) -50 0 50 100 ECAP (µV) 0 40 80 120 RMS (µV) 0 0.1 0.2 0.3 0.4 Artifact Magnitude (V) -50 0 50 100 ECAP (µV) 50 100 150 200 DBS Amplitude (µA) 0 20 40 60 RMS (µV) b 0 1 2 3 4 5 6 7 Time (ms) 240 200 170 100 35 DBS Amplitude (µA) -50 0 50 100 ECAP (µV) 0 100 200 300 RMS (µV) 0 0.1 0.2 0.3 Artifact Magnitude (V) -50 0 50 100 ECAP (µV) 100 200 300 400 DBS Amplitude (µA) 0 10 20 RMS (µV) c 0 1 2 3 4 5 6 7 Time (ms) 488 406 244 163 81 DBS Amplitude (µA) -50 0 50 100 ECAP (µV) 10 30 70 90 RMS (µV) 0 0.2 0.4 Artifact Magnitude (V) -50 0 50 100 ECAP (µV) 0 50 100 150 200 DBS Amplitude (µA) 0 20 30 40 RMS (µV) d 0 1 2 3 4 5 6 7 Time (ms) 200 150 100 50 0 DBS Amplitude (µA) -50 0 50 100 ECAP (µV) 0 50 150 200 RMS (µV) 0 0.1 0.2 Artifact Magnitude (V) artifact primary early secondary late secondary Subject Az Subject Ne Subject Bl Subject So 100 10 50 500 325 140 70 ii iii iv v i Figure 4. ECAP responses to increasing stimulation amplitudes across all subject (a–d). (subpanel i) Lead diagrams for each subject with stimulation electrode shown in red and recording site shown in blue. Example ECAP response to (subpanel ii) the highest stimulation amplitude tested in the MPTP-treated state and (subpanel iii) varying stimulation current amplitudes. RMS values across stimulation amplitudes from epochs containing the (subpanel iv) stimulation artifact and primary features and (subpanel part v) secondary features. Discussionh 0 1 2 3 4 5 6 7 Time (ms) -0.5 0 0.5 1 Subject Az a STN/STN LF/LF ECAP (mV) b Early Feature (µV) Late Feature (µV) Primary Feature (µV) Late Early Primary 0 10 20 30 40 50 60 200 100 600 500 400 0 300 200 100 10 20 30 40 50 60 100 50 1000 600 0200 b Early Feature (µV Late Feature (µV) Primary Feature (µV) 10 20 30 40 50 60 100 50 1000 600 0200 c Early Feature (norm.) Late Feature (norm.) Primary Feature (norm.) 0 0.2 1 0.4 0.6 0.8 1 0.5 1 0.8 0.6 0.4 0 0.2 0 All Subjects b a c 0 1 2 3 4 5 6 7 Time (ms) -0.5 0 0.5 1 ECAP (mV) Subject So Primary Feature (norm.) 0.8 0.6 0.4 0.2 0 0 0 0.1 0.1 0.1 0.2 0.2 0.2 0.3 0.3 0.3 0.4 0.4 0.4 0.5 0.5 0.5 0.6 0.6 0.6 0.7 0.7 0.7 0.8 0.8 0.8 0.9 0.9 0.9 1 1 1 Early Primary Late d Time (ms) Primary 300 Late Early Primary 5 10 15 20 100 700 500 0 300 100 Subject Bl 0 1 2 3 4 5 6 7 Time (ms) -0.5 0 0.5 1 ECAP (mV) Time (ms) Early Figure 5. Classification of ECAP responses within and dorsal to the STN. (a) Example ECAP responses for stimulation/recording configurations with STN/STN or LF/LF from subjects Az, So, and Bl. Subject Ne was not included in this figure as all directional DBS electrodes were within the STN. Stimulation amplitudes in each case are similar but not identical within each subject. ECAP responses are shown at a scale for visualizing primary features (dark grey). Early (middle gray) and late (light gray) secondary features were considerably smaller and played a smaller role in differentiating brain regions. (b) Plots of the three RMS features for each subject used by the classifier showing separability of spatial categories (STN/STN, LF/LF) in the feature space. (c, d) RMS values (normalized within subjects) that are plotted together to show separability of spatial categories in feature space. for thalamic ECAP recordings suggested that the anodic-leading and cathodic-leading ECAP responses and therapeutic effect on tremor were largely similar26. Other studies using cochlear stimulation32 have indicated differences in ECAP responses between anodic-leading and cathodic-leading pulses. Discussionh Studies have shown that stimulation targeting specific regions about the STN can be helpful in the programming process. For example, the dorsal region above STN can be beneficial for the cardinal motor signs of PD including rigidity and bradykinesia27, but also helpful for sup- pressing stimulation-induced dyskinesias19. At the same time, stimulation of the lenticular fasciculus has been associated with worsening of gait parameters28 and worsening of freezing of gait29.hf g g p g g g This approach complements other techniques for identifying DBS lead position and offers some strong advan- tages. One common approach is to merge high-field pre-operative MR imaging, that can visualize boundaries of the STN30, with post-operative CT imaging showing the position of the lead within the context of the cranium and ventricles. Most DBS centers, however, do not have access to high-field MRI, and even with such imaging facilities the definitive location and orientation of the DBS lead implant can only be confirmed, as shown in this study, with post-mortem histology. Previous studies have also shown that resting-state local field potentials filtered in the beta-band (12–30 Hz) can be useful for intra-operative targeting of DBS lead implants in the dorsolateral ‘motor’ STN, but again the exact location of the spectral feature can vary by several millimeters, especially considering the necessity to use bipolar electrode montages31. In this way, the ECAP approach may be a valuable complement for clinicians to use with current methods.h p The use of alternating polarity stimulation as a means of canceling stimulation artifact meant the stimula- tion parameters differed slightly from what is used clinically. Discussionh As such, the ECAP signals recorded may reflect an amalgam of different sources evoked between waveform polarities. The study also used monopolar electrode configurations for stimulation and recording to maintain a higher spatial resolution for the ECAP responses. Bipolar stimulation montages are also known to create orientation-selective activation patterns33 on pathways in and around the STN, and bipolar recording montages can reject local signals that are common to both electrodes34. Some, but not all, recordings also contained high-frequency noise in the 0.7–1.5 kHz range, which necessitated offline filtering. While the approach removed most of the high-frequency noise, some recordings still had a small residual noise level remaining. By employing analysis techniques such as calculation of RMS values as our feature amplitude metric, we reduced the effect that any residual noise had on the analysis, since the noise was of a high enough frequency to have multiple periods within a given feature window and features were either positive enough or negative enough that the noise did not cause the signal to cross 0 (thus the RMS of the noise approached a net 0 change on the signal). Discussionh In some studies, using artifact removal hardware Scientific Reports \| (2023) 13:2685 \| https://doi.org/10.1038/s41598-023-29439-6 www.nature.com/scientificreports/ 0 1 2 3 4 5 6 7 Time (ms) -0.5 0 0.5 1 0 1 2 3 4 5 6 7 Time (ms) -0.5 0 0.5 1 ECAP (mV) Subject Bl Subject Az Subject So a b STN/STN LF/LF Early Feature (µV) Late Feature (µV) Primary Feature (µV) Late Early Primary 0 1 2 3 4 5 6 7 Time (ms) -0.5 0 0.5 1 Late Early Primary 0 0 0 0.1 0.1 0.1 0.2 0.2 0.2 0.3 0.3 0.3 0.4 0.4 0.4 0.5 0.5 0.5 0.6 0.6 0.6 0.7 0.7 0.7 0.8 0.8 0.8 0.9 0.9 0.9 1 1 1 Early Primary Late c d Early Feature (norm.) Late Feature (norm.) Primary Feature (norm.) 0 0.2 1 0.4 0.6 0.8 1 0.5 1 0.8 0.6 0.4 0 0.2 0 5 10 15 20 100 700 500 0 300 100 0 10 20 30 40 50 60 200 100 600 500 400 0 300 200 100 10 20 30 40 50 60 100 50 1000 600 0200 All Subjects ECAP (mV) ECAP (mV) Figure 5. Classification of ECAP responses within and dorsal to the STN. (a) Example ECAP responses for stimulation/recording configurations with STN/STN or LF/LF from subjects Az, So, and Bl. Subject Ne was not included in this figure as all directional DBS electrodes were within the STN. Stimulation amplitudes in each case are similar but not identical within each subject. ECAP responses are shown at a scale for visualizing primary features (dark grey). Early (middle gray) and late (light gray) secondary features were considerably smaller and played a smaller role in differentiating brain regions. (b) Plots of the three RMS features for each subject used by the classifier showing separability of spatial categories (STN/STN, LF/LF) in the feature space. (c, d) RMS values (normalized within subjects) that are plotted together to show separability of spatial categories in feature space. Conclusionh This study found several principles that govern ECAP responses to DBS targeted within and around the STN. Increased stimulation amplitude or pulse width primarily affected the amplitude of ECAP features so long as the stimulation parameter exceeded a threshold to produce a detectable response feature. In contrast, variation in stimulation and recording site configurations had a large effect on when and what features were present in the ECAP response. Importantly, primary feature amplitude provided a means to accurately distinguish electrode contacts within or dorsal to the STN, which will be useful for guiding the programming of STN-DBS systems in the future. https://doi.org/10.1038/s41598-023-29439-6 Scientific Reports \| (2023) 13:2685 \| www.nature.com/scientificreports/ References References 1. Kent, A. R. & Grill, W. M. Neural origin of evoked potentials during thalamic deep brain stimulation. J. Neurophysiol. 110, 826–843. https://doi.org/10.1152/jn.00074.2013 (2013). p g j ( ) 2. Schmidt, S. L., Brocker, D. T., Swan, B. D., Turner, D. A. & Grill, W. M. Evoked potentials reveal neural circuits engaged by human deep brain stimulation. Brain Stimul. 13, 1706–1718. https://doi.org/10.1016/j.brs.2020.09.028 (2020).hi 3. Buzsáki, G., Anastassiou, C. A. & Koch, C. The origin of extracellular fields and currents: EEG, ECoG, LFP and spikes. Nat. Rev. Neurosci. 13, 407–420. https://doi.org/10.1038/nrn3241 (2012). , p g ( ) 4. Kent, A. R. & Grill, W. M. Recording evoked potentials during deep brain stimulation: Development and validation of instrumenta- tion to suppress the stimulus artefact. J. Neural Eng. 9, 036004. https://doi.org/10.1088/1741-2560/9/3/036004 (2012). 4. Kent, A. R. & Grill, W. M. Recording evoked potentials during deep brain stimulation: Development tion to suppress the stimulus artefact. J. Neural Eng. 9, 036004. https://doi.org/10.1088/1741-2560 tion to suppress the stimulus artefact. J. Neural Eng. 9, 036004. htt 5. Cohen, L. T., Richardson, L. M., Saunders, E. & Cowan, R. S. C. Spatial spread of neural excitation in cochlear implant recipients: Comparison of improved ECAP method and psychophysical forward masking. Hear. Res. 179, 72–87. https://doi.org/10.1016/ S0378-5955(03)00096-0 (2003). ( ) ( ) 6. Tang, Q., Benítez, R. & Zeng, F.-G. Spatial channel interactions in cochlear implants. J. Neural Eng. 8, 046029. https://doi.org/10 1088/1741-2560/8/4/046029 (2011). 7. Parker, J. L., Shariati, N. H. & Karantonis, D. M. Electrically evoked compound action potential recording in peripheral nerves Bioelectron. Med. 1, 71–83. https://doi.org/10.2217/bem-2017-0005 (2018). p g 8. Parker, J. L. et al. Evoked compound action potentials reveal spinal cord dorsal column neuroanatomy. Neuromodul. Technol. Neural Interface 23, 82–95. https://doi.org/10.1111/ner.12968 (2020). eural Interface 23, 82–95. https://doi.org/10.1111/ner.12968 (2020) f p g 9. Russo, M. et al. Sustained long-term outcomes with closed-loop spinal cord stimulation: 12-month results of the prosp multicenter open-label avalon study. Neurosurgery 87, E485–E495. https://doi.org/10.1093/neuros/nyaa003 (2020). 10. Parker, J. L. et al. Electrically evoked compound action potentials recorded from Neural Interface 16, 295–303. https://doi.org/10.1111/ner.12053 (2013). 10. Parker, J. L. et al. Electrically evoked compound action potentials recorded from the sheep spinal cord. Neuromodul. Technol. Neural Interface 16, 295–303. https://doi.org/10.1111/ner.12053 (2013). 11. Jeon, E. K. et al. Comparison of electrically evoked compound action potential thresholds and loudness estimates for the stimuli used to program the advanced bionics cochlear implant. J. Am. Acad. Audiol. Received: 15 August 2022; Accepted: 6 February 2023 Received: 15 August 2022; Accepted: 6 February 2023 References Real-time suppression and amplification of frequency-specific neural activity using stimulation evoked oscil lations. Brain Stimul. 13, 1732–1742. https://doi.org/10.1016/j.brs.2020.09.017 (2020).i 20. Sanabria, D. E. et al. Real-time suppression and amplification of frequency-specific neural activity u lations. Brain Stimul. 13, 1732–1742. https://doi.org/10.1016/j.brs.2020.09.017 (2020).i 20. Sanabria, D. E. et al. Real time suppression and amplification of frequency specific neural activity using stimulation evoked lations. Brain Stimul. 13, 1732–1742. https://doi.org/10.1016/j.brs.2020.09.017 (2020).i p g j 1. Brinda, A. K. et al. Longitudinal analysis of local field potentials recorded from directional deep brain stimulation lead implants in the subthalamic nucleus. J. Neural Eng. 18, 046050. https://doi.org/10.1088/1741-2552/abfc1c (2021). p g j 21. Brinda, A. K. et al. Longitudinal analysis of local field potentials recorded from directio in the subthalamic nucleus. J. Neural Eng. 18, 046050. https://doi.org/10.1088/1741-255 g p g 22. Vitek, J. L. & Johnson, L. A. Understanding Parkinson’s disease and deep brain stimulation: Role of monkey models. Proc. Natl. Acad. Sci. 116, 26259–26265. https://doi.org/10.1073/pnas.1902300116 (2019). p g p 3. Miocinovic, S. et al. Computational analysis of subthalamic nucleus and lenticular fasciculus activation during therapeutic deep brain stimulation. J. Neurophysiol. 96, 1569–1580. https://doi.org/10.1152/jn.00305.2006 (2006). J p y , p g j ( ) 4. Imbert, C., Bezard, E., Guitraud, S., Boraud, T. & Gross, C. E. Comparison of eight clinical rating scales used for the assessment of MPTP-induced parkinsonism in the Macaque monkey. J. Neurosci. Methods 96, 71–76 (2000).i 24. Imbert, C., Bezard, E., Guitraud, S., Boraud, T. & Gross, C. E. Comparison of eight clinical rating of MPTP-induced parkinsonism in the Macaque monkey. J. Neurosci. Methods 96, 71–76 (2000).i 5. Meissner, W. et al. Subthalamic high frequency stimulation resets subthalamic firing and reduces abnormal oscillations. Brain 128 2372–2382. https://doi.org/10.1093/brain/awh616 (2005). g 6. Kent, A. R. et al. Measurement of evoked potentials during thalamic deep brain stimulation. Brain Stimul. 8, 42–56. https://doi org/10.1016/j.brs.2014.09.017 (2015).i g j ( ) 7. Butson, C. R., Cooper, S. E., Henderson, J. M. & McIntyre, C. C. Patient-specific analysis of the volume of tissue activated during deep brain stimulation. Neuroimage 34, 661–670. https://doi.org/10.1016/j.neuroimage.2006.09.034 (2007).tf 28. Goftari, M. et al. Parkinsonian gait effects with DBS are associated with pallido-peduncular axis activation. BioRxiv https://doi. org/10.1101/2021.10.13.464270 (2021). 29. Strelow, J. N. et al. Structural connectivity of subthalamic nucleus stimulation for improving freezing of gait. MedRxiv https://doi. org/10.1101/2021.07.01.21259612 (2021). g 0. He, C., Zhang, F., Li, L., Jiang, C. & Li, L. References 21, 016–027. https://doi.org/10.3766/jaaa.21.1.3 (2010). ( ) 12. Miller, C. A., Brown, C. J., Abbas, P. J. & Chi, S.-L. The clinical application of potentials evoked from the peripheral auditory system. Hear. Res. 242, 184–197. https://doi.org/10.1016/j.heares.2008.04.005 (2008). 3. Adenis, V. et al. ECAP growth function to increasing pulse amplitude or pulse duration demonstrates large inter-animal variability that is reflected in auditory cortex of the guinea pig. PLoS ONE 13, e0201771. https://doi.org/10.1371/journal.pone.0201771 (2018) that is reflected in auditory cortex of the guinea pig. PLoS ONE 13, e0201771. https://doi.org/10.1371/journal.pone.0201771 (2 14. Gärtner, L., Lenarz, T. & Büchner, A. Fine-grain recordings of the electrically evoked compound action potential amplitude gr l y g p g p g j p 14. Gärtner, L., Lenarz, T. & Büchner, A. Fine-grain recordings of the electrically evoked compound action potential amplitude growth function in cochlear implant recipients Biomed Eng Online 17 140 https://doi org/10 1186/s12938 018 0588 z (2018) l 4. Gärtner, L., Lenarz, T. & Büchner, A. Fine-grain recordings of the electrically evoked compound action potential amplitude growth function in cochlear implant recipients. Biomed. Eng. Online 17, 140. https://doi.org/10.1186/s12938-018-0588-z (2018). , , , , g g y p p p g function in cochlear implant recipients. Biomed. Eng. Online 17, 140. https://doi.org/10.1186/s12938-018-0588-z (2018). g g 5. Finn, W. E. & LoPresti, P. G. Wavelength and intensity dependence of retinal evoked responses using in vivo optic nerve recording IEEE Trans. Neural Syst. Rehabil. Eng. 11, 372–376. https://doi.org/10.1109/TNSRE.2003.819928 (2003). 6. Maynard, K. I., Yanez, P. & Ogilvy, C. S. Nitric oxide modulates light-evoked compound action potentials in the intact rabbit retina NeuroReport 6(6), 850–852 (1995). p 7. Gmel, G. E. et al. A new biomarker for subthalamic deep brain stimulation for patients with advanced Parkinson’s disease: A pilot study. J. Neural Eng. 12, 066013. https://doi.org/10.1088/1741-2560/12/6/066013 (2015). y g g 8. Somers, B., Long, C. J. & Francart, T. EEG-based diagnostics of the auditory system using cochlear implant electrodes as sensors Sci. Rep. 11, 5383. https://doi.org/10.1038/s41598-021-84829-y (2021).t 18. Somers, B., Long, C. J. & Francart, T. EEG-based diagnostics of the auditory Sci. Rep. 11, 5383. https://doi.org/10.1038/s41598-021-84829-y (2021).t p p g y ( ) 9. Goftari, M. et al. Pallidothalamic tract activation predicts suppression of stimulation-induced dyskinesias in a case study of Par kinson’s disease. Brain Stimul. 13, 1821–1823. https://doi.org/10.1016/j.brs.2020.09.022 (2020). p g j 0. Sanabria, D. E. et al. Data availability Th d d d y The datasets used and analyzed during the current study are available from the corresponding author on reason- able request. Acknowledgements ld l k h k h c o edge e ts We would like to thank the Neuromodulation Research and Technology Lab for helpful comments in preparation of this manuscript, and the RAR and veterinary staff at the University of Minnesota. Author contributions J.R.: Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Data Curation, Writ- ing—original draft, Writing—review & editing, Visualization. A.D.: Investigation, Writing—review & editing, Visualization. A.B.: Investigation, Writing—review & editing, Visualization. M.B.: Investigation, Writing—review & editing. E.L.: Investigation, Writing—review & editing. C.S.: Investigation, Writing—review & editing. J.D.: Investigation, Writing—review & editing. J.K.: Investigation, Writing—review & editing. K.W.: Investigation, Writing—review & editing. D.S.: Investigation, Writing—review & editing. S.B.: Investigation, Writing—review & editing. B.M.: Investigation, Writing—review & editing. J.W.: Investigation, Writing—review & editing. L.A.J.: Conceptualization, Investigation, Resources, Writing—review & editing, Supervision, Project administration. J.L.V.: Conceptualization, Resources, Writing—review & editing, Supervision, Project administration, Funding acquisition. M.D.J.: Conceptualization, Methodology, Validation, Formal analysis, Writing—original draft, Writ- ing—review & editing, Visualization, Supervision, Project administration, Funding acquisition. Funding Thi t d Funding This study was supported by the NIH-NINDS under Grants R01-NS094206, P50-NS098573, P50-NS123109, R01-NS037019, R37-NS077657, R01-NS058945, F31-NS108625 (to AD) and GRFP award (to AB). References Measurement of lead localization accuracy based on magnetic resonance imaging. Front Neurosci. 15, 63288 (2021). 1. Darcy, N. et al. Spectral and spatial distribution of subthalamic beta peak activity in Parkinson’s disease patients. Exp. Neurol. 2022 114150. https://doi.org/10.1016/j.expneurol.2022.114150 (2022).h p g j p 32. He, S., Teagle, H. F. B. & Buchman, C. A. The electrically evoked compound action potential: From laboratory to clinic. Front. Neurosci. 11, 1–10 (2017). 3. Slopsema, J. P. et al. Clinical deep brain stimulation strategies for orientation-selective pathway activation. J. Neural Eng. 15, 056029 https://doi.org/10.1088/1741-2552/aad978 (2018).i p g 34. Zhang, S., Connolly, A. T., Madden, L. R., Vitek, J. L. & Johnson, M. D. High-resolution local field potentials measured with deep brain stimulation arrays. J. Neural Eng. 15, 046019. https://doi.org/10.1088/1741-2552/aabdf5 (2018). https://doi.org/10.1038/s41598-023-29439-6 Scientific Reports \| (2023) 13:2685 \| www.nature.com/scientificreports/ © The Author(s) 2023 Competing interests h g The authors declare no competing interests. Additional informationh Additional information Supplementary Information The online version contains supplementary material available at https://doi.org/ 10.1038/s41598-023-29439-6. Additional information Supplementary Information The online version contains supplementary material available at https://doi.org/ 10.1038/s41598-023-29439-6. Correspondence and requests for materials should be addressed to M.D.J. Correspondence and requests for materials should be addressed to M.D.J. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. 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https://openalex.org/W1486260669	https://revistas.pucp.edu.pe/index.php/agendainternacional/article/download/3667/3646, https://revistas.pucp.edu.pe/index.php/agendainternacional/article/download/3667/3646/	es		Activismo judicial en la evolución del Derecho Internacional: hacia la configuración de un incipiente orden público internacional	Agenda internacional	2,010	cc-by	13,672	Agenda Internacional Año XVII, N° 28, 2010, pp. 75-102 ISSN 1027-6750 Activismo judicial en la evolución del Derecho Internacional: hacia la configuración de un incipiente orden público internacional Francisco Jiménez García 1. La dimensión jurídica de la actual comunidad internacional y el activismo judicial como test de su efectividad y coherencia La expresión «activismo judicial» en Derecho internacional no presenta necesariamente las connotaciones ni las dimensiones suscitadas en el ámbito nacional en cuanto poder de los jueces frente al poder legislativo,1 sino que con ella se pretende hacer referencia al papel que desempeña los jueces y tribunales en la aplicación efectiva de los principios estructurales o constitucionales consagrados en el ordenamiento normativo internacional, aun cuando tal aplicación no esté exenta de controversia y tensión. No se trata tanto de una dialéctica entre poderes, pues como es sabido en Derecho internacional no se puede hablar estrictamente de un poder legislativo y de un poder judicial en cuanto órganos superiores a los destinatarios de este ordenamiento. Es preferible hablar de un proceso de creación normativa que no concluye con la adopción o cristalización de la norma en cuestión, sino con el test del análisis de sus consecuencias. En este sentido, con la expresión activismo judicial se pretende analizar hasta qué punto el juez internacional deduce consecuencias jurídicas de los principios y normas declaradas en el ordenamiento como estructurales y de ahí inferir su coherencia.2 Se trata de la complementariedad necesaria o la interrelación 1 Sobre esta cuestión véase ZAPATERO, V. El arte de legislar. Cizur Menor: Thomson/Aranzadi, 2009, pp. 349-372. 2 Como se destaca en el Curso de Derecho Internacional Público de los profesores Julio D. GONZÁLEZ CAMPOS, Luis L. SÁNCHEZ RODRÍGUEZ y Andrés SÁENZ DE SANTA MARÍA (Madrid: Thomson/ Civitas, 2008, pp. 105-107), «en cuanto ordenamiento jurídico, el derecho internacional público constituye un conjunto organizado de principios y normas. Aquellos expresan ciertos valores jurídicos fundamentales 76 Francisco Jiménez García dialéctica entre la norma y el juez en la resolución del dilema de la coherencia del ordenamiento internacional como sistema jurídico. Como ha afirmado el profesor Sánchez Rodríguez, «el examen analítico de la jurisprudencia nos enfrenta al reto formidable e irrenunciable de explicar los mecanismos y metodologías a través de los cuales los enunciados necesariamente abstractos de las fórmulas normativas, así como la consideración teórica de los diversos intereses que se hallan en conflicto en el momento de su formulación, se concretan y solucionan controversialmente en la vida práctica, en la puesta en movimiento, en la dinámica de tales principios y normas. La interrelación dialéctica existente entre la formación y la aplicación normativas».3 También es verdad que el activismo judicial ha sido asociado a la fragmentación del Derecho internacional como consecuencia de la proliferación de tribunales internacionales de carácter sectorial y la inexistencia de un poder judicial supremo o constitucional que pudiera actuar como órgano de casación de las decisiones judiciales adoptadas por los tribunales sectoriales.4 Pero en este caso el factor determinante no es el pretendido activismo judicial, sino las características del nuevo orden internacional que se caracteriza, paradójicamente, por un reglamentarismo que hace que la norma internacional se proyecte no solo respecto de los Estados, sino directamente sobre la esfera de los derechos y obligaciones de los individuos sin que a la vez se articulen mecanismos generales de control judicial de tal actividad normativa, correspondiendo tal función a los tribunales internacionales sectoriales que controlan la legalidad del régimen jurídico singular afectado. Desde la perspectiva nacional se ha destacado que el resultado de la creciente internacionalización sin control de la que informan el sistema jurídico en su totalidad o en un determinado sector del mismo. Las normas, en cambio, contienen la prescripción de un comportamiento, una conducta que se exige, prohíbe o se permite a sus destinatarios. Ahora bien, la coherencia interna del ordenamiento determina que entre principios y normas exista una relación sustantiva». Los principios estructurales serían aquellos que expresan los valores jurídicos básicos que inspiran el ordenamiento internacional en un determinado momento de su evolución histórica, entre los que cabría citar los previstos en la Resolución 2625 (XXV) de la Asamblea General de las Naciones Unidas así como los relativos a la promoción y respeto de los derechos humanos, al derecho al desarrollo y a la protección del medio ambiente. «Estos principios poseen una doble función en el actual ordenamiento internacional. De un lado, por expresar valores generalmente aceptados por la comunidad internacional en su conjunto, informan las distintas estructuras jurídicas que hoy integran el derecho internacional […] .Y en ellos se encuentra, por tanto, el núcleo de las normas de ius cogens del ordenamiento internacional […]. De otro lado, constituyen verdaderos principios de organización de la sociedad de Estados, por ser indispensables para la existencia de unas relaciones pacíficas y de cooperación en dicho grupo social. Y por tanto, constituyen un límite importante al relativismo del derecho internacional». 3 Véase su «Prólogo» a la obra colectiva El poder de los jueces y el estado actual del Derecho Internacional. Análisis crítico a la jurisprudencia internacional (2000-2007), SÁNCHEZ RODRÍGUEZ, L. I., F.J. QUEL LÓPEZ, A. G. MARTÍN LÓPEZ (eds.). Bilbao: Universidad del País Vasco, 2010, p. 18. 4 Sobre esta cuestión véase SIMMA, B. «Universality of international law from perspective of a practitioner». EJIL 2009-2, pp. 265-297; HIGGINS, R. «A Babel of judicial voices? Ruminations from the Bench». ICLQ, 2006-4, pp. 791-804. Activismo judicial en la evolución del Derecho Internacional 77 creación de normas no puede ser otro que el debilitamiento del hilo capital que une al ciudadano con las normas y que es la fuente de la que nace su legitimidad.5 Desde el Derecho internacional se ha planteado el debate acerca de la aplicación de los principios del estado de derecho al propio sistema internacional, en particular con motivo de la potestad sancionadora antiterrorista del Consejo de Seguridad.6 Si bien es verdad que tal situación puede plantear problemas al universalismo y la unidad del Derecho internacional, sin embargo tal activismo judicial —incluido el nacional—7 coadyuvan a evitar anquilosamientos jurídicos que impiden comprender la nueva realidad existente y deslegitiman un sistema jurídico incapaz de articular los avances que declara normativamente con las decisiones prácticas que adopta. En realidad se trata del clásico debate en torno al fundamento voluntarista u objetivista del Derecho internacional. Para algunos autores, como P. Weil,8 el concepto de orden público internacional es en sí mismo incompatible con la estructura de las sociedad internacional en la que no existe una entidad superior a los Estados. El Derecho internacional presenta, según este autor, una doble crisis de normatividad derivada de elementos no voluntaristas. Por un lado cuantitativa, por la inflación de la juridicidad internacional debida a la acumulación del no-derecho o pre-derecho (soft-law y sus variantes), por otro lado, de carácter cualitativo fruto de la aparición de una «normatividad reforzada» (ius cogens y obligaciones erga omnes) que suponen un alto riesgo por sus dificultades de identificación y por el efecto desestabilizador que provoca en las estructuras de coexistencia y cooperación del actual sistema jurídico internacional. Para el profesor francés, esta práctica cercana al derecho («near-law») es reflejo del desarrollo de la sociedad internacional pero no constituye derecho,9 5 Cfr. Zapatero, ob. cit., p. 30. Informe del Secretario General, «Fortalecimiento y coordinación de las actividades de las Naciones Unidas orientadas a la promoción del estado de derecho», UN. Doc. A/63/226 de 6 de agosto de 2008. Sobre el estado de derecho en Derecho internacional véase L’Etat de Droit en Droit International, Colloque de Bruxelles, Pedone, Paris, 2009; CHESTERMAN, S. «An International Rule of Law?». New York University School of Law, April 11, 2008, Papers 70 <http://lsr.nellco.org/nyu/plltwp/papers/70>. Respecto a la aplicación de este principio a los respectivos Comités de sanciones del Consejo de Seguridad, véase JIMÉNEZ GARCÍA, F. «Tutela judicial efectiva, Pilares intergubernamentales de la Unión Europea y Naciones Unidas o viceversa». En A. Cuerda Riezu y F. Jiménez García (Dirs.). Nuevos desafíos del Derecho Penal Internacional. Madrid: Tecnos, 2009, pp. 411-463; HINOJOSA MARTÍNEZ, L. M. La financiación del terrorismo y las Naciones Unidas. Madrid: Tecnos, 2008. 7 Cabría destacar en este sentido, la jurisprudencia española generada en torno al asunto Scilingo (véase JIMÉNEZ GARCÍA, F. «El fin de la impunidad ante los crímenes internacionales». Tiempo de Paz, N° 93 2009, pp. 70-78). Sobre esta cuestión véase Benvenisti, E. «Reclaiming Democracy: the estrategic uses of foreign and international law by national Courts». AJIL 2008-2, pp. 241-274. 8 Cfr. su Curso General de La Haya, «Le Droit International en quête de son identité», RCADI 1992-VI, t. 237. 9 «Le near law peut ressembler au droit, il peut préceder le droit ou préfigurer le droit; il n’est pas du droit...l’acumulation de non-droit ou de pré-droit ne suffit à elle seule à créer du droit», véase ibíd. p. 243. 6 78 Francisco Jiménez García al igual que el ius cogens aparece como una doctrina necesaria pero imposible por su falta de identificación y de adaptación a la realidad jurídica internacional, sería simplemente un arma de disuasión, el arma nuclear del Derecho internacional, la caja vacía indispensable que nunca hay que llenar.10 Por el contrario, para otros autores, la concepción objetivista no niega el papel fundamental de la voluntad de los Estados en la positivación de las normas jurídicas internacionales, lo que sucede es que la razón de ser del Derecho transciende la voluntad unilateral del Estado. En este sentido, el profesor Carrillo Salcedo hace converger el relativismo del Derecho internacional, pues, los Estados existen per se y de facto sin que pueda estimarse que la soberanía es una delegación del Derecho Internacional, con las limitaciones que le impone el Derecho internacional contemporáneo, en particular, como consecuencia de la influencia de las organizaciones internacionales en la estructura del sistema jurídico internacional y la afirmación progresiva de nociones jurídicas como comunidad internacional, ius cogens y obligaciones erga omnes.11 En definitiva, se trata de observar cómo se resuelve la tensión entre la protección de los intereses colectivos reconocidos por el Derecho internacional a la comunidad internacional en su conjunto y los intereses individuales de los sujetos primigenios de este ordenamiento.12 2. El reconocimiento del ius cogens internacional por la Corte Internacional de Justicia: ¿el inicio de una nueva tendencia o inmovilismo con aportaciones simbólicas? A pesar de que el término de ius cogens (normas imperativas de Derecho internacional general) fue asumido por los artículos 53 y 64 de la Convención de Viena de 1969 sobre el derecho de los tratados y de que en la Sentencia de 1970 sobre el asunto de la Barcelona Traction Light & Power se declarara por parte de la Corte Internacional de Justicia (CIJ) la existencia de obligaciones erga omnes, respecto de 10 Cfr. ibíd. pp. 227-312. El símil de la caja vacía hace referencia a la reflexión de G. Abi-Saab acerca de que «even as an empty box, jus cogens is necessary, because if you do not have the box, you cannot put anything in it», Change and Stability in International Law Making. CASSESE, A. y J. WEILER (Dirs.), Berlin, 1988, p. 96. Respecto a las obligaciones erga omnes llega a la misma conclusión, constituye una teoría imprescindible e irresistible para la promoción de valores morales y solidarios que responde a una necesidad del mundo internacional de hoy; pero de otro lado, su defensa resulta imposible desde el punto de vista de las actuales estructuras del Derecho internacional, pues «quels que soient ses attraits, la théorie de l’obligation erga omnes exige une maîtrise et une élaboration qui jusqu’à présent font défaut». Ibíd., pp. 290-291. 11 Cfr. CARRILLO SALCEDO, J. A. «Droit International et Souveraineté des Etats». RCADI, 1996, t. 257. Del mismo autor, «El fundamento del Derecho internacional: algunas reflexiones sobre un problema clásico». REDI 1998-1, pp. 30-31. 12 Sobre esta cuestión véase VILLALPANDO, S. «The legal dimension of international community: how community interests are protected in international law». EJIL 2010-2, pp. 387-419. Activismo judicial en la evolución del Derecho Internacional 79 las cuales todos los Estados tienen un interés jurídico en su protección,13 este mismo Tribunal obstinadamente ha evitado este término hasta la Sentencia de 3 de febrero de 2006 dictada en el asunto de las Actividades armadas en el territorio del Congo (nueva demanda:2002) (República Democrática del Congo c. Ruanda).14 Hasta entonces había preferido las perífrasis relativas a los «principios intransgredibles de Derecho internacional consuetudinario» y a las «obligaciones erga ommes» declaradas en las opiniones consultivas sobre la Legalidad del uso o la amenaza de armas nucleares15 y las Consecuencias jurídicas de la construcción del Muro de Israel en territorio palestino ocupado,16 así como en los contenciosos relativos a Timor Oriental17 y la Aplicación de la Convención para la prevención y sanción del crimen de genocidio.18 Es más, en el asunto consultivo del Muro en territorio palestino ocupado, la CIJ asumió las consecuencias propias de la violación de este tipo de normas –positivadas en el artículo 41 del Proyecto de la Comisión de Derecho Internacional (CDI) sobre responsabilidad del Estado por hechos internacionalmente ilícitos–19, aun cuando en la práctica sus conclusiones hayan quedado en nada.20 En concreto la Corte declaró que Todos los Estados tienen la obligación de no reconocer la situación ilegal resultante de la construcción del muro en el territorio palestino ocupado, incluida Jerusalén oriental y sus alrededores. Asimismo tienen la obligación de no prestar ayuda ni asistencia para el mantenimiento de la situación creada por tal construcción. Incumbe también a todos los Estados, dentro del respeto por la Carta de las Naciones Unidas y el derecho internacional, velar por que se ponga fin a cualquier impedimento, resultante de la construcción del muro, para el ejercicio por el pueblo palestino de su derecho a la libre determinación. Además, todos los Estados partes en el Convenio de Ginebra relativo a la protección debida a las personas civiles en tiempo de guerra de 12 de agosto de 1949, tienen la obligación, dentro del respeto por la Carta de las Naciones Unidas y el derecho internacional, de hacer que Israel respete el derecho internacional humanitario incorporado en dicho Convenio. Por último, la Corte opina que las Naciones Unidas, y en especial la Asamblea General y el Consejo de Seguridad, deberían considerar qué medidas adicionales son necesarias 13 Cfr. ICJ Reports 1970, p. 32, párr. 33. Cfr. ICJ Reports 2006, pp. 32 y 53, párrs. 64 y 123. 15 Cfr. ICJ Reports 1996 (I), p. 257, párr. 79. 16 Cfr. ICJ Reports 2004, p. 199, párrs. 155 y 157. 17 Cfr. ICJ Reports 1995, p. 102, párr. 29. 18 Cfr. Aplicación de la Convención para la prevención y sanción del crimen de genocidio (Bosnia- Herzegovina c. Yugoslavia), Excepciones preliminares, C.I.J. Recueil 1996 (II), p. 616, párr. 31. 19 Texto aprobado por la CDI en su 53° período de sesiones, Documentos oficiales de la Asamblea General, 56 período de sesiones, Suplemento nº 10 (A/56/10), pp. 10-405. En su Resolución 56/83, de 12 de diciembre de 2001, la Asamblea General de las Naciones Unidas tomó nota de este texto. 20 Como reacción más destacada véase la Resolución ES-10/17 de la Asamblea General de las Naciones Unidas de 15 de diciembre de 2006. 14 80 Francisco Jiménez García para poner fin a la situación ilegal resultante de la construcción del muro y el régimen conexo, teniendo debidamente en cuenta la presente Opinión Consultiva.21 El principal órgano judicial de las Naciones Unidas ha asumido, pues, el término ius cogens tiempo después de que ya fuera reconocido por otros tribunales internacionales. El Tribunal Penal Internacional para la antigua Yugoslavia había invocado en 1998, en el asunto Furundzija, el carácter de ius cogens de la norma que prohíbe la tortura para fundamentar asimismo la aplicación del principio de justicia universal en la persecución y sanción de tal crimen.22 Esta decisión tuvo especial incidencia en el asunto Al-Adsani c. Reino Unido, resuelto el 21 de noviembre de 2001 por el Tribunal Europeo de Derechos Humanos (TEDH), en el que de forma incongruente se concluyó, por un lado, que si bien la prohibición de la tortura es una norma de ius cogens que ostenta dentro de la jerarquía normativa internacional, convencional y consuetudinaria, el rango más elevado; sin embargo, se afirmaba por otro lado, que esta posición solo sirve para los procesos penales y no para los procesos civiles por daños y perjuicios ocasionados en el extranjero.23 Por su parte, la Corte Interamericana de Derechos Humanos (CorteIDH) declaraba en su opinión consultiva de 17 de septiembre de 2003 sobre la Condición jurídica y derechos de los inmigrantes indocumentados, que el principio de igualdad y no discriminación había ingresado en el dominio del ius cogens,24 conclusión que era reiterada dos años después en el asunto de la Masacre de Mapiripán vs. Colombia precisando que […] en cumplimiento de dichas obligaciones, los Estados deben abstenerse de realizar acciones que de cualquier manera vayan dirigidas, directa o indirectamente, a crear situaciones de discriminación de jure o de facto, así como a adoptar medidas positivas para revertir o cambiar situaciones discriminatorias existentes en sus sociedades, en perjuicio de determinado grupo de personas. Esto implica el deber especial de protección que el Estado debe ejercer con respecto a actuaciones y prácticas de terceros que, bajo su tolerancia o aquiescencia, creen, mantengan o favorezcan las situaciones discriminatorias.25 21 Párrs. 159 y 160 de la Opinión Consultiva. Cfr. TPY Fiscal c. Furundzija, Sentencia de 10 de diciembre de 1998m IT-95-17/1 párrs. 153-156. 23 Cfr. TEDH asunto Al-Adsani c. Reino Unido, Sentencia de 21 de noviembre de 2001, párr. 61. En materia de inmunidades destaca también la Sentencia de 14 de febrero de 2002 de la CIJ en el asunto de la Orden de detención de 11 de abril de 2000 (República Democrática del Congo c. Bélgica) que mantiene también una interpretación restrictiva en cuanto a los límites de las inmunidades soberanas respecto a los crímenes internacionales. Sobre esta cuestión véase ESPÓSITO MASSICCI, C. Inmunidad del Estado y Derechos Humanos. Madrid: Civitas, 2007; JIMÉNEZ GARCÍA, F. «Justicia universal e inmunidades estatales: justicia o impunidad ¿Una encrucijada dualista para el Derecho Internacional?». ADI 2002, pp. 63-124. 24 CorteIDH, Opinión Consultiva OC-18/03 de 17 de septiembre de 2003, párr. 101. 25 CorteIDH, Sentencia de 15 de septiembre de 2005, Serie C Nº 134, párr. 178. 22 Activismo judicial en la evolución del Derecho Internacional 81 No obstante, el avance aludido por parte de la Corte Internacional de La Haya en el asunto de las Actividades armadas en el territorio del Congo (2006), se limita al hecho histórico de la invocación expresa del término ius cogens, pues el Tribunal se enroca en su clásica proclama relativa a la distinción entre la oponibilidad erga omnes de una norma y el carácter facultativo de la jurisdicción internacional, declarada en el asunto Timor Oriental, para no deducir consecuencias jurídicas de la existencia de una norma imperativa que en teoría conlleva la nulidad de pleno derecho de los actos que la contravenga. En este caso, el Estado demandante, la República del Congo, había argumentado que la reserva ruandesa al artículo IX de la Convención contra el genocidio, que reconocía la jurisdicción de la CIJ, resultaba nula, pues tal exclusión suponía la consagración de la impunidad de los Estados autores de tal crimen, cuando precisamente el objeto y fin de tal Convención es su erradicación, propósito al que se han comprometido todos los Estados Parte en virtud del artículo I. Se invoca además que la Convención contra el crimen del genocidio, en virtud de la propia jurisprudencia de este Tribunal, constituye una ley general de obligado cumplimiento para todos los Estados de la Comunidad internacional, incluida Ruanda, en la medida en que contiene normas de ius cogens y que de acuerdo con lo dispuesto en los artículos 53 y 64 del Convenio de Viena sobre el derecho de los tratados, declarativos de dicha noción, ha de entenderse como nulo de pleno derecho todo acto convencional contrario a esta categoría de normas. Por tal razón, la reserva ruandesa a dicha cláusula de arreglo judicial sería nula y carecería de todo efecto jurídico. Sin embargo la Corte de La Haya, volviendo la vista atrás a su jurisprudencia sobre la materia (Opinión consultiva sobre las Reservas a la Convención contra el crimen del genocidio de 195126 y su Sentencia sobre las excepciones preliminares dictada en el asunto de la Aplicación de la convención contra el crimen del genocidio, Bosnia-Herzegovina c. Yugoslavia27), afirma que no cabe duda de que los principios sustantivos de la Convención contra el genocidio constituyen principios que obligan a todos los Estados al margen de todo vínculo convencional. Los derechos y obligaciones consagrados por la Convención son derechos y obligaciones erga omnes integrantes de normas imperativas de ius cogens.28 No obstante y sin desviar su mirada del pasado, insiste en que tales reconocimientos no alteran su jurisprudencia sentada en 1995 en el asunto Timor Oriental29 en virtud de la cual la oponibilidad erga omnes de una norma y la regla del consentimiento a la jurisdicción del Tribunal son dos cuestiones totalmente diferentes, sin 26 CIJ Recueil 1951, p. 23. CIJ Recueil 1996, p. 616, párr. 31. 28 Cfr. CIJ Recueil 2006, p. 32, párr. 64. 29 Timor Oriental (Portugal c. Australia), CIJ Recueil 1995, p. 102, párr. 29, decisión recordada en su Providencia de 10 de julio de 2002 sobre medidas provisionales en el propio asunto de las Actividades armadas en territorio del Congo (RDC c. Ruanda), CIJ Recueil 2002, p. 241, párr. 57. 27 82 Francisco Jiménez García que por lo tanto la libertad reservista de los Estados se haya visto modificada ni un ápice por la calificación de ius cogens de la norma convencional reservada. Para el Tribunal, la evolución del Derecho internacional acerca del genocidio, en particular el artículo 120 del Estatuto de la Corte Penal Internacional que prohíbe las reservas al mismo, no ha modificado su doctrina general declarada en la Opinión consultiva de 1951 acerca de que las reservas a la competencia de la Corte Internacional de Justicia, prevista en el artículo IX, no resultan contrarias o incompatibles con el objeto y fin del tratado en la medida en que no afectan a las obligaciones sustantivas que derivan de esta Convención en relación con la prevención y sanción del crimen de genocidio.30 En el mismo sentido se pronuncia el Tribunal respecto a la reserva a la cláusula compromisoria de la Convención contra la discriminación racial al afirmar que el hecho de que una controversia se relacione con la inobservancia de una norma imperativa del Derecho internacional general no es fundamento suficiente para demostrar la competencia de la Corte al respecto, y que no existe ninguna norma imperativa que obligue a los Estados a aceptar dicha competencia para resolver controversias relativas a la Convención internacional sobre la eliminación de todas las formas de discriminación racial.31 El carácter self-restraint mostrado por esta decisión contó con la crítica de la Opinión particular conjunta de los jueces Higgins, Kooijmans, Elabary, Owada y Simma que estimaron que el principio consensualista aplicado por la Corte en su fallo no podía tener un alcance absoluto, subrayando que podría haber situaciones en que una reserva a una cláusula relativa al arreglo de controversias pudiera ser contraria al objeto y fin de un tratado, todo dependía de las circunstancias del caso. Requieren a tal efecto del Tribunal una interpretación más sutil de la Opinión consultiva de 1951 en la que se introdujera la evolución acaecida desde entonces en el Derecho internacional como consecuencia de la multiplicación de instrumentos internacionales sobre derechos humanos así como de la existencia de ciertas instancias judiciales y determinados órganos de control de estos tratados. De forma acertada recuerdan que en 1951 no existía como tal un corpus jurídico sobre derechos humanos hasta tal punto que la Convención contra el genocidio constituía prácticamente el único tratado internacional sobre la materia. Por ello resulta difícilmente concebible que en 2006 se pueda mantener los mismos criterios interpretativos que en 1951. La opinión conjunta de estos jueces se aparta expresamente del régimen interestatal de laissez-faire en materia de reservas proclamado en la Opinión consultiva de 1951, rechazando por irrealizable que, ante 30 31 Cfr. CIJ Recueil 2006, p. 32, párrs. 66-67. Cfr. ibíd., p. 35, párr. 78. Activismo judicial en la evolución del Derecho Internacional 83 la plétora de reservas formuladas a los tratados de derechos humanos, se pueda seguir manteniendo el esquema bilateral de aceptación/objeción, que en la mayoría de los casos obedece a consideraciones políticas, para apreciar la validez de las reservas formalizadas.32 Además, destacan el papel de los órganos judiciales y de los mecanismos convencionales de control de los tratados sobre derechos humanos que han limitado sustancialmente la libertad de los Estados en cuanto a la formulación de reservas a la competencia de los mismos. A tal efecto se hace referencia a la jurisprudencia del Tribunal Europeo de Derechos Humanos (sentencias Belilos de 1988; Loizidou de 1995), de la Corte Interamericana de Derechos Humanos (Opiniones consultivas sobre el Efecto de las reservas a la entrada en vigor de la Convención americana de 1982 y sobre las Restricciones a la pena de muerte 1983); así como la doctrina sentada por el Comité de Derechos Humanos de Naciones Unidas en el asunto Rawle Kennedy c. Trinidad y Tobago de 1999 y, principalmente, en su Observación General N° 24 de 2 de noviembre de 1994 en la que convergen las anteriores decisiones. Como es sabido, en tal Comentario General del Comité de Derechos Humanos de Naciones Unidas se estimó que las reservas destinadas a excluir los mecanismos de garantía y control establecidos en el Pacto de Derechos Civiles y Políticos, elementos básicos de la concepción del Pacto que están destinados a garantizar el disfrute de los derechos, resultaban incompatibles con el objeto y fin del tratado.33 Por estas razones y en virtud de la especial responsabilidad de los Estados en la ejecución de la Convención contra el genocidio, uno de los más graves crímenes conocidos, los jueces que suscriben la citada Opinión particular estimaron que no resultaba tan evidente que las reservas al artículo IX de la Convención contra el genocidio no fueran incompatibles con el objeto y fin del tratado, por lo que el Tribunal debería haber reconsiderado su jurisprudencia. Para estos jueces, resulta especialmente grave que a principios del siglo XXI los Estados puedan seguir eligiendo si son o no juzgados por delitos de genocidio.34 Más contundente se muestra la Opinión disidente del juez Koroma. Además de incidir sobre el carácter normativo de la Convención contra el crimen del genocidio ajena a las consideraciones de reciprocidad, declara que su artículo IX constituye un elemento crucial para alcanzar el objetivo y fin previstos en la misma puesto que es la única disposición de la Convención que permite la determinación del grado de 32 Cfr. párrs. 10-11 de la opinión conjunta. A estos efectos recuerdan que a la Convención del genocidio se ha presentado 28 reservas que han suscitado la objeción de 18 Estados; 54 reservas se han presentado a la Convención contra la discriminación racial que ha generado 26 objeciones; 75 reservas a la Convención sobre la eliminación de la discriminación contra la mujer con 18 Estados objetantes. 33 Cfr. apartado 11 de la Observación General Nº 24. 34 Acerca de la Opinión conjunta de los cinco jueces, véanse los comentarios de la Sentencia efectuados por WECKEL, Ph. y G. AREOU en la RGDIP 2006-2, pp. 491-492 y LATTY, F. AFDI, 2005, pp. 211-212. 84 Francisco Jiménez García responsabilidad de los Estados en la comisión de un crimen de genocidio (en cuya perpetración es difícil de imaginar la no intervención del aparato estatal), pues el resto de sus disposiciones se refieren a la responsabilidad penal de los individuos implicados. Para el juez de Sierra Leona, tal conclusión resulta conforme con lo establecido en la Directriz 3.1.13 del décimo Informe del Relator especial sobre las reservas a los tratados que dispone que las reservas a una disposición convencional relativa al arreglo de controversias o al control de la aplicación de un tratado será incompatible con el objeto y fin del tratado si tal disposición constituye la razón de ser del tratado.35 Denegar esta función a la Corte no solo le impide resolver las controversias resultantes de su aplicación e interpretación, sino principalmente las relativas a su ejecución así como deducir, en su caso, la responsabilidad de los Estados por actos de genocidio. Que el artículo IX de la Convención es una disposición nuclear para la determinación de la responsabilidad estatal por actos de genocidio, ha resultado posteriormente confirmada por la propia Corte Internacional de Justicia en su Sentencia de 26 de febrero de 2007 en el asunto de la Aplicación de la Convención para la prevención y sanción del crimen del genocidio (Bosnia-Herzegovina c. Serbia), en la que tal disposición convencional ha constituido el fundamento jurídico de base para que el Tribunal se pronunciase sobre la participación de Serbia en el genocidio de Srebrenica, precisando que tal Convención no sólo prevé la responsabilidad penal individual sino también la estatal.36 Esta situación de mero reconocimiento formal de las normas imperativas ha provocado que alguno de los jueces del Tribunal, el juez Kooijmans, haya mantenido en el plano doctrinal que la Corte Internacional de Justicia debe superar tal inmovilismo judicial, estéril y asfixiante, y que, sin caer en un activismo judicial destructivo, apueste por un método integrador e incidental de las disposiciones normativas –proactivo en palabras del juez– que permita al Tribunal avanzar de conformidad con los nuevos desarrollos del Derecho internacional.37 35 Cfr. párr. 11 de la Opinión disidente y párr. 99 del Informe, AG. Doc. A/CN.4/558/Add.1, p. 31. CIJ Reports 2007, párrs. 167-169. 37 P. KOOIJMANS asume que «I am certainly not in favour of judicial activism which may turn into a destructive trap. But neither am I in favour of a form of judicial restraint closes windows which need to be opened and thus becomes barren. Earlier I used the word ‘proactive’. In one of my dictionaries I found the following meaning of that term: ‘having orientation to the future, anticipating problems and taking affirmative steps to deal positively with them rather than reacting after a situation has already occurred». «The ICJ in the 21st Century, judicial restraint, judicial activism, or proactive judicial policy». ICQL 2007-4, p. 753. 36 Activismo judicial en la evolución del Derecho Internacional 85 3. Constitucionalismo sustantivo frente al constitucionalismo institucional El asunto Kadi38 ha generado un controvertido pero necesario debate39 en la era del reglamentarismo del Consejo de Seguridad sin que las estructuras de garantías de los derechos fundamentales, como parte integrante del estado de derecho, se hayan alterado en el ordenamiento internacional. El Derecho internacional se había caracterizado –y se sigue caracterizando en términos generales– por ser un ordenamiento esencialmente de cooperación, correspondiendo a los ordenamientos nacionales el desarrollo y la ejecución de la normativa internacional así como las garantías pertinentes para que tal normativa resultase conforme con los principios constitucionales del ordenamiento nacional sin cuestionar la primacía del Derecho internacional. No obstante, este planteamiento se ha visto profundamente alterado mediante la adopción de resoluciones por parte del Consejo de Seguridad que proyectan su normatividad no exclusivamente sobre los Estados sino directamente sobre el patrimonio jurídico (derechos, obligaciones y libertades) de los ciudadanos. A tal efecto surge la cuestión si determinados principios esenciales del estado de derecho (como los relativos al derecho a la defensa y la tutela judicial efectiva)40 como parte integrante del 38 Aunque nos vamos a centrar en el asunto Kadi, se ha de precisar que este no es más que uno de los asuntos llevados ante el Tribunal de Justicia de las Comunidades Europeas (TJCE) sobre esta materia. Véanse también los asuntos Yusuf/Al Barakaat International Foundation c. Consejo y Comisión, asunto T-306/01, Sentencia de 21 de septiembre de 2005; Chafiq Ayadi c. Consejo, Sentencia de 12 de julio de 2006, asunto T-253/02; Hassan c. Consejo y Comisión, asunto T-49/04, Sentencia de 12 de julio de 2006; Organización de los Modjahedines del Pueblo de Irán c. Consejo Asunto T-228/02, Sentencia del TPI (Sala Segunda), de 12 de diciembre de 2006. 39 Sobre este asunto véase: DE WET, E. «The role of European Courts in the development of a hierarchy of norms within international law: evidence of Constitutionalisation». European Constitutional Law Review, 2009-2, pp. 284-306; ZIEGLER, K. S. «Strengthening the rule of law, but fragmenting international law: the Kadi decision of the ECJ from the perspective of human rights». HRLR 2009-2, pp. 288-305; DE SENA, P. y M. Ch. VITUCCI. «The European Courts and the Security Council: between dédoublement fonctionnel and balancing of values«. EJIL 2009-1, pp.193-228 así como el debate suscitado en torno a este artículo por parte de DE BÚRCA, G. y A. NOLLKAEMPER en EJIL 2009-3, pp. 853-862 y 862-887 respectivamente; SIMMA, B. «Universality of international law from perspective of a practitioner». EJIL 2009-2, pp. 265-297, en particular, pp. 292-296; JIMÉNEZ GARCÍA, F. «Tutela judicial efectiva, Pilares intergubernamentales de la Unión Europea y Naciones Unidas o viceversa», l. cit.; SANTOS VARA, J. «El control de las sanciones contra Al-Qaeda y los talibanes en la Unión Europea: ¿un desafío a los poderes del Consejo de Seguridad?». RDCE, Nº 32, 2009, pp. 91-120; BORE EVENO, V. «Le contrôle juridictionnel des résoltions du Conseil de Securité: vers un constitutionnalisme international?». RGDIP 2006-4, pp. 827-860. 40 Conforme a la Observación General N° 32 del Comité de Derechos Humanos sobre el artículo 14 del Pacto de Derechos Civiles y Políticos relativo a los derechos de defensa y tutela judicial efectiva, «el artículo 14 establece garantías que los Estados Partes deben respetar, independientemente de su tradición jurídica y de su derecho interno. Si bien los Estados Partes deben informar sobre la interpretación que dan a estas garantías en sus respectivos ordenamientos jurídicos, el Comité observa que el contenido esencial de las garantías del Pacto no puede dejarse exclusivamente a la discreción del derecho interno». Además se recuerda que «si bien el artículo 14 no está incluido en la lista de derechos que no pueden suspenderse, que figuran en el párrafo 2 del artículo 4 del Pacto, los Estados que en circunstancias de emergencia pública decidan dejar en suspenso los procedimientos normales previstos en el artículo 14 deben asegurarse de que tal suspensión no vaya más allá de lo que exija estrictamente la situación. Las garantías procesales nunca podrán ser objeto de medidas 86 Francisco Jiménez García ius cogens conforman un orden constitucional de carácter internacional que vertebra e integra los sistemas jurídicos. En tal sentido y dada la naturaleza facultativa y el alcance limitado del sistema jurisdiccional internacional, no debería existir ningún problema en que tribunales sectoriales o regionales –incluso los nacionales desde el planteamiento del desdoblamiento funcional– controlaran dicha legalidad internacional con efectos jurídicos limitados en sus respectivos ordenamientos (aunque con una potencialidad jurídica superior), al margen de las consecuencias prácticas que esto pudiera suscitar y de la activación de los mecanismos internacionales oportunos para subsanar tales contradicciones o deficiencias (incluida la solicitud por parte del Consejo de Seguridad de una opinión consultiva a la CIJ sobre esta materia). No se ha de olvidar, en este sentido, que el propio Consejo de Seguridad ha reafirmado que la lucha antiterrorista ha de ser conforme con la Carta de las Naciones Unidas y el Derecho internacional. Igualmente, ha subrayado que los Estados deben asegurarse de que cualquier medida adoptada para luchar contra el terrorismo se ha de ajustar a las obligaciones que les incumben en virtud del Derecho internacional y que deben adoptar dichas medidas de conformidad con ese derecho, en particular, con el Derecho internacional relativo a los derechos humanos, el derecho relativo a los refugiados y el derecho humanitario.41 El origen del asunto Kadi proviene de la interacción entre las resoluciones dictadas por el Consejo de Seguridad en el marco del Capítulo VII de la Carta de las Naciones Unidas y aplicadas por el Comité de Sanciones 1267 creado al efecto, que imponen distintas sanciones económicas y financieras contra el régimen afgano de los talibanes y Usama bin Laden, así como contra las personas y entidades asociadas, incluyendo empresas, que se encontraran bajo su control directo o indirecto,42 y las medidas de ejecución adoptadas por la Unión Europea (UE) concretadas en distintas posiciones comunes aprobadas en el ámbito exclusivo de su Política Exterior y de Seguridad Común (PESC)43 que devinieron en reglamentos adoptados por el Con- derogatorias que soslayen la protección de derechos que no son susceptibles de suspensión. Cfr. CCPR/C/ GC/32 de 23 de agosto de 2007. 41 Cfr. párrafo 2 de la Resolución 1624 (2005) del Consejo de Seguridad que impone a los Estados la obligación de prohibir por ley la incitación a la comisión de un acto o actos de terrorismo. 42 Resoluciones 1267 (1999), 1333 (2000), 1390 (2002), 1452 (2003), en relación con las competencias ejecutivas del Comité de Sanciones, creado al efecto por el Consejo de Seguridad mediante la Resolución 1267 (1999), en la identificación y elaboración de las personas y entidades sancionadas, quien además, en virtud de la resolución 1452 (2003), aprueba las excepciones a la congelación de fondos y de recursos económicos que por razones humanitarias podían conceder los Estados. Este régimen de sanciones fue modificado por las Resoluciones 1526 (2004), 1617 (2005), 1735 (2006), 1822 (2008) y 1904 (2009) de modo que ahora las sanciones abarcan a las personas asociadas dondequiera que se encuentren. Junto a este Comité, operan otros dos, el Comité contra el Terrorismo y el Comité 1540. 43 Posiciones comunes 1999/727/PESC, 2001/154/PESC, 2002/402/PESC y 2003/140/PESC. Activismo judicial en la evolución del Derecho Internacional 87 sejo y la Comisión sobre la base jurídica de los artículos 60 y 301 del Tratado de la Comunidad Europea (TCE)44 Según los demandantes, la aplicación de tal normativa vulneraba directamente su derecho de defensa y tutela, pues la imposición de tales sanciones se había realizado en virtud de un procedimiento totalmente confidencial, sin que se les informara de las razones que las motivaron, ni se les comunicara las pruebas ni circunstancias invocadas en su contra, negándoseles todo oportunidad de defensa. Su inscripción en la lista del anexo I del Reglamento impugnado se debía únicamente al hecho de que figuraban en la lista que el Comité de Sanciones elaboró a partir de la información recibida de los Estados y de las organizaciones internacionales o regionales. Por estas razones alegan que, a su juicio, las resoluciones del Consejo de Seguridad no son directamente aplicables, sino que deben ser objeto de transposición al derecho interno de conformidad con las disposiciones constitucionales pertinentes así como con arreglo a los principios fundamentales del Derecho.45 Por su parte, el Consejo de la UE reconocía en sus alegatos que, cuando la Comunidad decide adoptar medidas unilaterales de coerción económica y financiera por iniciativa propia, el control jurisdiccional debe extenderse al examen de las pruebas utilizadas contra las personas sancionadas. En cambio, según el Reino Unido y el Consejo, cuando la Comunidad actúa sin ejercitar potestad discrecional alguna, basándose en una decisión adoptada por un órgano al que la comunidad internacional ha atribuido considerables facultades a fin de preservar la paz y seguridad internacionales, un control jurisdiccional completo entrañaría el riesgo de socavar el sistema de la ONU establecido en 1945, podría perjudicar gravemente a las relaciones internacionales de la Comunidad y de los Estados Miembros y entraría en conflicto con la obligación de la Comunidad de respetar el Derecho internacional. El Consejo arguyó que, en el presente asunto, el control jurisdiccional del juez comunitario no podía ir más allá del que existe en los Estados miembros en lo que respecta a la recepción, en su ordenamiento jurídico interno, de las decisiones adoptadas por los órganos de la comunidad internacional con el objetivo de preservar la paz y la seguridad internacionales. A este respecto, el Consejo invocó que, en varios Estados Miembros, los actos de aplicación de las resoluciones del Consejo de Seguridad son calificados de «actos de gobierno» y escapan por completo a la competencia del juez. En otros Estados Miembros, a su juicio, la amplitud de dicho control jurisdiccional está muy limitada.46 44 Reglamentos del Consejo: 337/2000, 467/2001, 881/2002, 561/2003 y Reglamentos de la Comisión, 2199/2001, 1580/2002 y 866/2003. 45 Cfr. apartados 137-152 de la Sentencia Kadi. 46 Cfr. ibíd., apartados 154-164 y párr. 225 de la Sentencia dictada en el asunto Yusuf. 88 Francisco Jiménez García Una vez afirmado que la UE se encuentra vinculada por las obligaciones dimanantes de Naciones Unidas y por las del Consejo de Seguridad, no directamente,47 sino en virtud de que «la Comunidad no puede violar las obligaciones que la Carta de las Naciones Unidas impone a sus Estados miembros ni obstaculizar el cumplimiento de las mismas y, por otra parte, que la Comunidad se encuentra obligada, en virtud del propio Tratado que la creó, a adoptar en el ejercicio de sus competencias todas las disposiciones necesarias para permitir que sus Estados miembros respeten tales obligaciones»,48 el Tribunal de Primera Instancia (TPI) asume que en este caso las instituciones actuaron en ejercicio de una competencia reglada de modo que no disponían de ningún margen de apreciación autónomo. En particular, no podían ni modificar directamente el contenido de las resoluciones de que se trata ni establecer un mecanismo que pudiera provocar tal modificación. Por otra parte, rechaza que pueda ejercerse un control jurisdiccional sobre tales actos, pues cualquier control de la legalidad interna del Reglamento impugnado efectuado por el Tribunal, en particular desde el punto de vista de las disposiciones o principios generales del Derecho comunitario relativos a la protección de los derechos fundamentales, exigiría por tanto un examen, de modo incidental, de la legalidad de las resoluciones del Consejo de Seguridad que impusieron las sanciones. De esta forma el Tribunal de Justicia da marchamo judicial a la producción en cascada de actos políticos o de gobierno que no dejan margen alguno al control jurisdiccional, en el ámbito de las Naciones Unidas porque no existen mecanismos jurisdiccionales con tal competencia, en los ámbitos internos de aplicación, como es el caso de la UE, porque se trata de actuaciones regladas que los órganos judiciales no pueden revisar ni articular mecanismos que promuevan tal revisión. La determinación y calificación política de que una situación constituye una amenaza para la paz y la seguridad internacionales ejercida por el Consejo de Seguridad en el ámbito del Capítulo VII de la Carta supone la creación de un «espacio político de actuación inmune a cualquier control jurisdiccional en defensa de los derechos fundamentales» ejercida por los tribunales nacionales o comunitarios. Las previsiones de los artículos 25, 48 y 103 de la Carta de las Naciones Unidas y del artículo 27 de la Convención de Viena sobre el derecho de los tratados avalan, según el TPI, tal afirmación. Para el Tribunal «habida cuenta en particular de las disposiciones del artículo 307 CE y del artículo 103 de la Carta de las Naciones Unidas, la alegación de que se han vulnerado, bien los derechos fundamentales protegidos por el ordenamiento jurídico comunitario, bien los principios de dicho ordenamiento jurídico, no puede afectar a la validez 47 48 Cfr. párrs. 93-203 del asunto Kadi. Véase ibíd., párr. 204. Activismo judicial en la evolución del Derecho Internacional 89 de una resolución del Consejo de Seguridad ni a su eficacia en el territorio de la Comunidad».49 Tal constatación, contraria a la propia esencia de la UE y de su ordenamiento jurídico así como a la jurisprudencia del TEDH, obliga al Tribunal de Primera Instancia a acudir a la noción paliativa del ius cogens (orden público internacional que se impone a todos los sujetos del Derecho internacional, incluidos los órganos de la ONU, y que no tolera excepción alguna50) para justificar su posición, abriendo a tal efecto una sedicente posibilidad de control en el caso de que tal actuación onusiana constituyera una violación grave de las obligaciones derivadas de las normas imperativas del Derecho internacional público, tal y como se desprende de los artículos 53 y 64 de la Convención de Viena sobre el derecho de los tratados. Solo en el hipotético caso de que la tutela judicial efectiva constituyera una norma superior del Derecho internacional integrante del ius cogens, esto es, un principio inviolable del Derecho internacional consuetudinario en términos de la Opinión consultiva de la Corte Internacional de Justicia en el asunto de la Licitud de la amenaza o del uso de armas nucleares, entonces el control jurisdiccional incidental ejercido por el TPI en virtud del recurso de anulación podría extenderse a los actos impugnados.51 Así pues, el TPI se reconoce con competencia universal para enjuiciar la legalidad internacional de las sanciones decretadas por el Consejo de Seguridad de las Naciones Unidas, aunque previamente se había declarado incompetente para enjuiciar los efectos territoriales en la Comunidad Europea de las propias disposiciones comunitarias dictadas en ejecución de las resoluciones del Consejo de Seguridad, ni siquiera para articular mecanismos comunitarios que pudieran provocar su revisión. No obstante, esta posibilidad no supone ningún avance en el razonamiento jurídico del Tribunal, sino que se erige en una premeditada válvula de escape sin consecuencia alguna, pues sobre la base de que el derecho a la defensa y a la tutela judicial efectiva no constituyen, según el Tribunal, un derecho absoluto –sin entrar en un mayor análisis sobre las consecuencias de la derogación absoluta de tal derecho en un ordenamiento que pivota sobre el mismo–, finalmente se reitera la incuestionable autoridad política del Consejo de Seguridad y el nulo margen de actuación tanto para las instancias políticas comunitarias, esto es el Consejo como la Comisión, como para la propia instancia judicial de la Unión Europea. Es más y en una pirueta de difícil comprensión por parte de un órgano judicial, la ausencia de este control 49 50 51 Véase ibíd., párrs. 214-224. Véase ibíd., párr. 226. Apartado 231. 90 Francisco Jiménez García jurisdiccional es suplida, según el TPI, por la existencia de controles políticos de carácter discrecional.52 La falta de conocimiento de los hechos y las pruebas sobre la vinculación terrorista de los afectados se proyecta sobre la fase jurisdiccional y sobre el ejercicio del derecho a la tutela judicial efectiva, situación que es justificada por el TPI mediante el reconocimiento de una nueva regla de ius cogens: la inmunidad de jurisdicción absoluta de los actos del Consejo de Seguridad cuando actúa en el marco del Capítulo VII, así como de la normativa regional de aplicación.53 Conforme al tenor literal de la Sentencia, Dicha limitación se justifica tanto por la naturaleza de las decisiones que el Consejo de Seguridad se ve obligado a adoptar en virtud del capítulo VII de la Carta de las Naciones Unidas como por el objetivo legítimo que se persigue. En las circunstancias del presente asunto, el interés de los demandantes en que el fondo del litigio sea examinado por un tribunal no tiene entidad suficiente para prevalecer sobre el interés general esencial en el mantenimiento de la paz y la seguridad internacionales frente a una amenaza claramente identificada por el Consejo de Seguridad, con arreglo a las disposiciones 52 La única posibilidad que acierta a proponer el TPI es la puesta en marcha de un sistema particular de protección diplomática que transciende al previsto en el Derecho internacional en vigor en cuanto viene determinado por el principio de jurisdicción, en lugar del principio de nacionalidad, y porque se entabla directamente ante un órgano político como es el Comité de Sanciones, aun cuando sea sobre un procedimiento bilateral entre Estados. Tal situación es la descrita en las Directrices del Comité de Sanciones, aprobadas por el 7 de noviembre y enmendadas el 10 de abril de 2003, así como la Resolución 1526 (2004), por las cuales se prevé la posibilidad de que un requirente (persona, grupo, empresa o entidad incluida en las lista consolidada del Comité de Sanciones) pueda pedir al Gobierno de su país de residencia o nacionalidad para que solicite una revisión del caso. Para tal efecto, se regula un procedimiento bilateral y discrecional entre el Gobierno requerido y el Gobierno o Gobiernos que propusieron originalmente la designación (el Gobierno o gobiernos proponentes) para reexaminar el mantenimiento o exclusión de la lista ante el Comité quien decide por consenso y, en caso de no hallarse este, el asunto se remitirá al Consejo de Seguridad quien decidirá en ultima instancia. Este procedimiento ha sido revisado el 12 de febrero de 2007 en virtud de las Resoluciones 1730 (2006) y 1735 (2006) del Consejo de Seguridad que destacan el carácter preventivo de estas medidas «que no se basan en criterios penales establecidos en el derecho interno», inciden en la necesidad de justificar la propuesta de inclusión en la lista, además de recordar el deber de los Estados de informar por escrito, en la medida de lo posible, a las personas y entidades incluidas en la Lista Consolidada sobre las medidas adoptadas, y mejoran el mecanismo de exclusión de la lista previendo la posibilidad de que el particular afectado pueda presentar directamente una solicitud de revisión del caso ante el punto focal, siendo éste el que tramite la solicitud sin que suponga reconocer la intervención directa del particular ante las instancias sancionadoras Cfr. Comité del Consejo de Seguridad establecido en virtud de la Resolución 1267 (1999) relativa a AlQaida y los talibanes y personas y entidades asociadas. Directrices del Comité para el desempeño de su labor (Aprobadas el 7 de noviembre de 2002, y modificadas el 10 de abril de 2003, el 21 de diciembre de 2005, el 29 de noviembre de 2006 y el 12 de febrero de 2007. Conforme a la Resolución 1730 (2006) del Consejo de Seguridad, un Estado puede decidir como norma general que sus nacionales o residentes presenten sus solicitudes directamente al punto focal. A tal efecto, el Estado hará una declaración dirigida al Presidente del Comité que se publicará en su sitio web. Por el momento, Francia, mediante carta de 30 de abril de 2007, ha formulado tal declaración. Las Directrices así como las Resoluciones del Consejo de Seguridad y demás documentación pertinente relativa a este Comité de Sanciones, incluyendo información sobre el punto focal, puede consultarse en la siguiente dirección: <http://www.un.org/spanish/sc/committees/1267/index.shtml>. 53 Cfr. párrs. 287-288. Activismo judicial en la evolución del Derecho Internacional 91 de la Carta de las Naciones Unidas. A este respecto, procede atribuir especial importancia al hecho de que las resoluciones que el Consejo de Seguridad ha ido aprobando sucesivamente no sólo no han establecido unas medidas de duración ilimitada o indeterminada, sino que han previsto siempre un mecanismo para revisar la oportunidad de mantener tales medidas tras un período de tiempo de 12 o 18 meses como máximo.54 Y se concluye que tal déficit de control jurisdiccional queda salvaguardado por el control político ejercido por el Comité de Sanciones: Por último, el Tribunal de Primera Instancia considera que, al no existir un tribunal internacional competente para controlar la legalidad de los actos del Consejo de Seguridad, la creación de un órgano tal como el Comité de Sanciones y la posibilidad, prevista en la normativa, de dirigirse a él en todo momento para obtener la revisión de cualquier caso individual, mediante un mecanismo formalizado en el que participan tanto «el Gobierno requerido» como «el Gobierno proponente» constituyen otra vía razonable para proteger adecuadamente los derechos fundamentales de los demandantes, tal como son reconocidos por el ius cogens.55 No obstante, tales conclusiones favorables a la indefensión absoluta fueron revisadas por la Gran Sala del TJCE en su Sentencia de 3 de septiembre de 200856. Tal y como había mantenido el Abogado General, Sr. Poiares Maduro en sus conclusiones,57 la Gran Sala niega la existencia de la pretendida inmunidad de los actos comunitarios ejecutivos de las resoluciones del Consejo de Seguridad, lo que lleva al Tribunal a declarar la nulidad del Reglamento por vulneración de los derechos de defensa y tutela judicial efectiva y, de forma colateral, el derecho de propiedad.58 Conforme a las conclusiones del Tribunal, con arreglo a las competencias que les confiere el Tratado, los tribunales comunitarios deben garantizar un control, en principio completo, de la legalidad de todos los actos comunitarios desde el punto de vista de los derechos fundamentales que forman parte integrante de los principios generales del Derecho comunitario –constitutivos a su vez del «bloque de legalidad constitucional» de la UE–, control que también se extiende a los actos comunitarios destinados a aplicar resoluciones aprobadas por el Consejo de Seguridad en virtud del capítulo VII de la Carta de las Naciones Unidas, como el Reglamento controvertido.59 Eso sí, el Tribunal de Luxemburgo declara que no le corresponde controlar la legalidad de las 54 Véase ibíd., apartado 289. Ibíd., apartado 290. 56 Véase TJCE, Sentencia de 3 de septiembre de 2008, asuntos acumulados C-402/05 P y C-415/05 P. En el mismo sentido la Sentencia de 3 de diciembre de 2009 dictada en el caso Hassan c. Consejo y Comisión, asuntos acumulados C-399/06 P y 403/06 P. 57 Conclusiones presentadas respectivamente el 16 de enero y 23 de enero de 2008 respecto a los asuntos C-402/05 P y C-415/05 P. 58 Véanse apartados 278-330. 59 Véase apartado 326. 55 92 Francisco Jiménez García resoluciones aprobadas por otros órganos internacionales, como es el caso del Consejo de Seguridad, ni siquiera limitando su control al examen de la compatibilidad de tales resoluciones con el ius cogens.60 Así pues, podemos afirmar que finalmente el TJCE reconoció que la UE, como comunidad de derecho, no puede aceptar ciegamente el «constitucionalismo puramente formal o extrínseco derivado de la institución de las Naciones Unidas» en virtud de sus artículos 25, 48 y 103 de su Carta sin un análisis de fondo de la norma que se ha de aplicar de acuerdo con los principios que conforman su «bloque de legalidad», lo que implica la exigencia de un control de constitucionalidad sustantivo o intrínseco. Ello no supone el reconocimiento de un poder de derogación internacional de la disposición onusiana cuestionada, pues carece de tal competencia, sino que sus efectos se limitan al ámbito normativo de la UE poniendo en atención del propio Consejo de Seguridad y de los Estados miembros (no se ha de olvidar que Francia y el Reino Unido son miembros permanentes del Consejo de Seguridad) las dificultades ínsitas en la normativa adoptada para que sean subsanadas. Tal «activismo judicial» ya ha tenido sus consecuencias en el ámbito sancionador de las Naciones Unidas. Así las Resoluciones del Consejo de Seguridad 1822 (2008) y 1904 (2009), relativas a la amenaza a la paz y la seguridad internacionales causadas por actos terroristas, toman nota de los problemas jurídicos y de otra índole surgidos como consecuencia de las medidas de aplicación adoptadas por los Estados miembros en ejecución de las resoluciones contra el terrorismo internacional así como de las mejoras que se han introducido en los procedimientos del Comité antiterrorista en cuestión y la calidad de la Lista consolidada.61 En concreto, la Resolución 1904 (2009) reafirma no solo que todos los Estados miembros han de tomar todas las medidas necesarias posibles, de conformidad con su legislación y prácticas nacionales, para notificar e informar oportunamente a la persona o entidad que se proponga incluir en la Lista y adjunten a esa notificación el resumen de los motivos para la inclusión, sino que prevé la instauración de una Oficina del Ombudsman donde se podrán remitir solicitudes de exclusión de la Lista consolidada sustituyendo en tal labor a los puntos focales establecidos mediante la Resolución 1730 (2006). A tal efecto, se regula un procedimiento contradictorio entre, por una parte, el peticionario y el Ombudsman que se hace cargo de su causa, y de otra, los Estados y los órganos competentes de Naciones Unidas: Equipo de Vigilancia y el Comité antiterrorista.62 60 Cfr. párr. 287. Véase Directrices del Comité 1267 para la realización de su labor aprobadas el 7 de noviembre de 2002 con las modificaciones introducidas el 12 de febrero de 2007 y el 9 de diciembre de 2008 <http://www.un.org/ spanish/sc/committees/1267/1267_guidelines_sp.shtml>. 62 Cfr. párrs. 19-21 de la Resolución así como su Anexo II. 61 Activismo judicial en la evolución del Derecho Internacional 93 4. Los Tribunales como garantes y responsables de la efectividad del Derecho Internacional. La persecución positiva de los crímenes internacionales y el control de convencionalidad El actual Derecho internacional ha consagrado como uno de sus principios estructurales el fin de la impunidad entendida ésta, tal y como se declara en el Acuerdo entre la ONU y el Gobierno de Guatemala relativo al establecimiento de una Comisión Internacional contra la Impunidad en Guatemala de 12 de diciembre de 2006,63 como la inexistencia de hecho o de derecho de responsabilidad penal, administrativa, disciplinaria o civil para los responsables que atentan gravemente contra los derechos humanos, eludiendo la investigación o la condena; todo lo cual conduce al debilitamiento del estado de derecho, impidiendo al Estado con su deber de garantizar a los ciudadanos afectados la protección de su vida, integridad física y el pleno acceso a la justicia, con la consecuente pérdida de la confianza de los ciudadanos en las instituciones democráticas del país. Tal principio se concibe además como uno de los presupuestos necesarios para alcanzar un verdadero estado de derecho internacional donde prime el imperio de las leyes frente al imperio de los Estados, garantice la generalidad de los derechos por él declarados así como la efectividad de su control y respeto mediante instancias jurisdiccionales,64 ya sean internacionales o nacionales en virtud de distintos criterios de atribución de competencia, asumiendo en el ámbito penal internacional el principio de complementariedad declarado en el Estatuto de Roma por el que se crea la Corte Penal Internacional. Por otra parte, el principio normativo del fin de la impunidad ante los crímenes internacionales ha adquirido una dimensión que va más allá de la garantía de la existencia de un recurso judicial efectivo (piedra angular, no obstante). En estos momentos tal derecho se concibe de una forma holística que comprende la búsqueda de la verdad y la obtención de una adecuada reparación tanto individual como de carácter social o colectivo. Tal concepción es la que preconizan los «Principios y directrices básicos sobre el derecho de las victimas de violaciones manifiestas de las normas internacionales de derechos humanos y de violaciones graves del derecho internacional humanitario a interponer recursos y obtener reparaciones», anexos a la Resolución 60/147 aprobada sin necesidad de votación por la Asamblea General de las Naciones Unidas de 16 de diciembre de 2005. Por otro lado, mientras que el fin de la impunidad y el deber de todo Estado de ejercer su jurisdicción penal contra los responsables de los crímenes internacionales se declaran en los Preámbulos 63 Acerca de este acuerdo y el mandato de esta Comisión véase: <http://cicig.org/index.php?page=mandato>. Cfr. CHESTERMAN, S. «An International Rule of Law?». New York University School of Law, April 11, 2008, Papers 70 <http://lsr.nellco.org/nyu/plltwp/papers/70>. 64 94 Francisco Jiménez García de la Convención Internacional para la protección de todas las personas contra las desapariciones forzadas, adoptada el 20 de diciembre de 2006, y del Estatuto de la Corte Penal Internacional de 17 de julio de 1998, la decisión de 13 de mayo de 2008 adoptada por la Sala de Cuestiones Preliminares de la CPI en el asunto Fiscal c. Germain Katanga and Mathieu Ngudjolo Chui, reconoció como derechos de las víctimas de los crímenes de la competencia de la Corte, el derecho a la verdad y el derecho a la justicia tal y como han sido interpretados por la CorteIDH y, en menor medida, por el TEDH65 y de conformidad con la doctrina sentada sobre esta cuestión por la denominada justicia transicional.66 Asimismo y en consecuencia, se afirma de manera contundente que el conjunto de derechos procesales de las víctimas no debe ser determinado de manera casuística sino mediante una aproximación sistemática. En tal sentido, la jueza Silvia Steiner concluye que a la hora de concretar el estatuto procesal de la víctima en el procedimiento ante la Corte, incluso en la fase preliminar, se ha de tener en cuenta su legítimo interés en la determinación veraz de los hechos, la identificación de los responsables de tales crímenes y la declaración de su responsabilidad como elementos integrantes de su derecho a la verdad. Además en el reconocimiento del derecho a la justicia, ha de presidir el objetivo de poner fin a la impunidad mediante el establecimiento judicial de las circunstancias en las cuales se consumaron las violaciones, la declaración de responsabilidad de los perpetradores de tales crímenes y la seguridad de que se impondrán sanciones de conformidad con la gravedad de los crímenes por los que hayan sido condenados.67 La consagración de este principio, sin embargo, se ha encontrado con importantes obstáculos en distintos ámbitos, desde las reglas de las inmunidades jurisdiccionales penales de los representantes internacionales de los Estados (asuntos Yerodia, Al-Adsani …), hasta las legislaciones nacionales relativas a las amnistías, indultos, prescripción o al principio de irretroactividad. No obstante, en el reconocimiento paulatino del principio del fin de la impunidad como principio estructural del Derecho internacional también cabe destacar el activismo de ciertos tribunales internacionales. En primer lugar, la Corte Interamericana de Derechos Humanos en el caso de la Masacre de Mapiripán v. Colombia reiteraba que: 65 Se cita la siguiente jurisprudencia de la Corte Interamericana de Derechos Humanos: Bàmaca- Velasquez c. Guatemala, Sentencia de 25 de noviembre de 2000, Serie C, nº 70, párr. 201, Barrios Altos c. Perú, Sentencia de 14 de marzo 2001, Series C, nº 75, par. 48, Masacre de Mapiripá c. Colombia, Sentencia de 15 de septiembre de 2005, Serie C, nº 134, párr. 297; Almohacid-Arellano y otros c. Chile, Sentencia de 26 de septiembre de 2006, Serie C, nº 154, párrs. 148 y ss. Respecto al TEDH, se cita el asunto Hugh Jordan c. Reino Unido, Sentencia de 4 de mayo de 2001, Demanda No. 24746/1994, [2001] ECHR 327, párr. 93. 66 Sobre este tema, véase J. Chinchón Álvarez, Derecho Internacional y Transiciones a la Democracia y la Paz. Madrid: Ediciones Parthenon, 2007. 67 Cfr. ICC-01/04-01/07, pars. 31-51 <http://www.icc-cpi.int/library/cases/ICC-01-04-01-07-474-ENG.pdf>. Activismo judicial en la evolución del Derecho Internacional 95 [...] ninguna ley ni disposición de derecho interno puede impedir a un Estado cumplir con la obligación de investigar y sancionar a los responsables de violaciones de derechos humanos. En particular, son inaceptables las disposiciones de amnistía, las reglas de prescripción y el establecimiento de excluyentes de responsabilidad que pretendan impedir la investigación y sanción de los responsables de las violaciones graves de los derechos humanos –como las del presente caso, ejecuciones y desapariciones. El Tribunal reitera que la obligación del Estado de investigar de manera adecuada y sancionar, en su caso, a los responsables, debe cumplirse diligentemente para evitar la impunidad y que este tipo de hechos vuelvan a repetirse.68 El principio de irretroactividad de las leyes penales es un principio general del Derecho internacional consustancial al principio de legalidad penal. Tanto el artículo 15 del Pacto Internacional de Derechos Civiles y Políticos de Naciones Unidas como los respectivos artículos 7 y 9 del Convenio Europeo de Derechos Humanos y de la Convención Americana sobre Derechos Humanos lo declaran. Según las disposiciones internacionales citadas, nadie será condenado por actos u omisiones que en el momento de su comisión no fueran delictivos según el derecho nacional o internacional, precisando que tal derecho no impide el juicio ni la condena de una persona por actos u omisiones que, en el momento de cometerse, fueran delictivos conforme a los principios generales del derecho reconocidos por la comunidad internacional. Esto es, que a la hora de concretar el alcance de este principio no sólo se ha de tener en cuenta la legalidad nacional sino también la legalidad internacional (incluida la consuetudinaria así como la derivada de los principios generales), por lo que estrictamente hablando la aplicación de la ley penal posterior a hechos anteriores que estaban condenados internacionalmente no supone una aplicación retroactiva de la ley penal nacional sino su aplicación retrospectiva en atención al cumplimiento de las obligaciones internacionales existentes para el Estado y para los individuos desde el momento en que se pueda afirmar la existencia de responsabilidad penal internacional sobre el crimen en cuestión. Como ha destacado el profesor Antonio Remiro Brotóns, «no hay razón alguna para hacer a los criminales beneficiarios de la morosidad de los legisladores estatales una vez que los tipos ya establecidos en el orden internacional son recibidos –miméticamente o por equivalencia– por los ordenamientos internos».69 68 Sentencia Corte IDH. de 15 de septiembre de 2005, Serie C. nº 134, párr. 304. En el mismo sentido y con especial referencia al artículo 20 del Estatuto de la Corte Penal Internacional, Sentencia de 26 de septiembre en el asunto Almonacid Arellano y otros c. Chile, CorteIDH Serie C nº 154 y Sentencia de 29 de noviembre de 2006 en el asunto La Cantuta c. Perú, CorteIDH Serie C nº 162. 69 Véase Remiro Brotóns, A. El caso Pinochet. Los límites de la impunidad. Madrid: Biblioteca Nueva, 1999, p. 60. 96 Francisco Jiménez García Así pues no resultan contrarios al principio de legalidad penal internacional aquellos procesos judiciales abiertos contra actuaciones delictivas que si bien en el momento de su comisión no estaban tipificadas en el código penal en vigor sin embargo eran consideradas criminales conforme al Derecho internacional, convencional o consuetudinario, vigente en ese momento y tales conductas fueron posteriormente incorporadas al respectivo código penal de aplicación por parte del tribunal enjuiciador. Por su parte, la Audiencia Nacional española mantuvo en su sentencia de 19 de abril de 2005 sobre el asunto Scilingo que debe negarse […] el argumento de que el legislador español, al introducir dicho precepto penal (el crimen de lesa humanidad en el artículo 607 bis CP) se estaba limitando a permitir en el ordenamiento español, pero sólo de cara al futuro y para el ámbito interno español, la persecución de este tipo de crímenes, a modo de dar formal cumplimiento a la exigencia derivada del principio de complementariedad del Estatuto de la Corte Penal Internacional o de la obligación de cooperar con dicho Tribunal, dejando sin previsión en derecho interno la norma penal internacional que ya desde hace décadas castigaba ese tipo de conductas. Ello significaría negar cualquier clase de internacionalidad a esta clase de delitos lo que representa una absoluta contradicción, además de no ajustarse siquiera a la ubicación sistemática dada a estos delitos.70 Las anteriores conclusiones no son meras aseveraciones o desiderata doctrinales, sino que cuentan con un importante soporte jurisprudencial. Además de las decisiones de la Corte Interamericana de Derechos Humanos ya indicadas, se ha de destacar la doctrina más reciente del Tribunal Europeo de Derechos Humanos que ha declarado la conformidad con el principio de legalidad penal internacional del artículo 7 del Convenio Europeo de Derechos Humanos de las legislaciones nacionales, así como de la jurisprudencia de ellas derivadas, que reconocen efectos retroactivos (o retrospectivos) a los crímenes contra la humanidad tipificados en sus respectivos códigos penales. Doctrina que viene a completar los desarrollos jurisprudenciales dictados en 1997 sobre el asunto Touvier c. Francia y en 2001 acerca de los asuntos Papon c. Francia y Streletz, Kesskest y Krentz c. Alemania.71 En los asuntos Kolk y Kislyiy c. Estonia, resuelto por el TEDH mediante decisión de admisibilidad dictada el 17 de enero de 2006 y Kononov c. Letonia, resuelto mediante sentencia del 24 de julio de 2008, se reconoce por parte del Tribunal de Estrasburgo que no resultan contrarias al artículo 7 del Convenio las decisiones judiciales nacionales que habían concluido que los crímenes contra la humanidad tipificados por el Estatuto Militar Internacional de Nuremberg en 1945, los Principios de dicho Tribunal declarados en 1950 por la Asamblea General de las Naciones Unidas e incluso en 70 71 Cfr. Fundamento Jurídico 1.4.3 de la sentencia. Las decisiones citadas pueden consultarse en la siguiente dirección: http://www.echr.coe.int/echr Activismo judicial en la evolución del Derecho Internacional 97 la Convención de la Haya de 1907, que reproduce casi literalmente el texto de la Convención de la Haya de 1989 y que asume la Cláusula Martens, son perseguibles independientemente de la fecha en que fueron cometidos y ello a pesar de que en el momento en que se produjeron los hechos impugnados no estuvieran tipificados como tales crímenes en los códigos penales entonces vigentes, sino que fueron códigos posteriores (de 1994 y 1993 respectivamente) los que introdujeron con efectos retroactivos y sin posibilidad de prescripción la persecución penal de tales crímenes. Es más, el TEDH salva las exigencias del principio de legalidad de las penas al mostrar su conformidad con el hecho de que la condena de los demandantes y la pena que les habían sido impuestas tienen su base jurídica en los respectivos códigos penales de la década de 1990. Con anterioridad en el asunto Jorgic c. Alemanía, el Tribunal de Estrasburgo había declarado compatible con los derechos a la libertad y seguridad (artículo 5) y a un proceso equitativo (artículo 6) la aplicación del principio de justicia universal en relación con la persecución del crimen del genocidio de conformidad con las previsiones de la Convención de 1948 sobre la prevención y sanción del crimen en cuestión. Para el TEDH, del artículo I de esta Convención deriva para las partes contratantes la obligación erga omnes de prevenir y sancionar el genocidio cuya prohibición forma parte del ius cogens internacional.72 Por su parte, en la sentencia en casación dictada el 17 de mayo de 2010 por la Gran Sala del TEDH sobre el asunto Kononov, se reitera que la garantía que consagra el artículo 7, elemento esencial de la preeminencia del derecho, ocupa un lugar primordial en el sistema de protección de la Convención, tal y como lo constata el hecho de que dicha disposición no pueda ser objeto de suspensión en caso de guerra o de otro peligro público. En cuanto a su contenido, recuerda que la previsión de legalidad penal hace referencia tanto a la existencia de una norma, escrita o no escrita, así como a los principios de accesibilidad y previsibilidad de tales disposiciones penales. A tal efecto insiste respecto al principio de previsibilidad que este comprende la interpretación judicial, pues está sólidamente establecido en la tradición jurídica de ciertos Estados Partes que la jurisprudencia, como fuente del derecho, contribuye necesariamente a la evolución pregresiva del derecho penal.73 En cuanto a los crímenes de guerra por los que fue el demandante condenado, acaecidos en 1944, se declara que tales crímenes habían sido codificados en las Reglas de la Haya de 1907, en las que se incluye la Cláusula Martens, además de en los Convenios de Ginebra de 1949 y en los Principios de Nuremberg. Es más, la prohibición de tales crímenes había sido declarada 72 73 TEDH, asunto Jorgic c. Alemanía, sentencia de 12 de julio de 2007, párr. 68. Cfr. párr. 185 de la Sentencia Kononov (2010). 98 Francisco Jiménez García por la jurisprudencia internacional como parte integrante de las «consideraciones elementales de la humanidad» así como de los «principios intransgredibles del derecho internacional consuetudinario».74 Por su parte el Tribunal Supremo español con ocasión del reiteradamente citado asunto Scilingo ha elaborado una interesante doctrina conforme a la cual se sancionan los hechos imputados de conformidad con los delitos tipificados en el momento de su comisión en los respectivos códigos penales de los Estados involucrados, salvando así los problemas de retroactividad, pero advirtiendo que tales conductas delictivas resultaban asimismo en ese momento sancionadas internacionalmente como crímenes contra la humanidad y que tal sanción era no solo plenamente conocida por los Estados implicados sino también por los autores de tales de crímenes, conjurando así los problemas relativos a la prescripción de los delitos o a la aplicación del principio de justicia universal. Se trata de la contextualización de delitos nacionales en categorías de crímenes sancionados por el ordenamiento internacional, incluyendo el derecho consuetudinario. Para el Tribunal Supremo, las referencias a estas conductas en el Derecho internacional penal con anterioridad a los hechos enjuiciados han sido generalmente previas a su constatación en normas de derecho interno. Desde la primera aparición escrita, en 1907 (Cláusula Martens), la evolución normativa ha permitido la consolidación internacional de un elemento de contexto identificable, añadido al hecho individual, que permite atribuir mayor cantidad de injusto a conductas que ya previamente eran constitutivas de delito. Las consecuencias de su apreciación se concretan en el reconocimiento de esa extraordinaria gravedad desde la perspectiva de la protección de los derechos humanos, y en la aceptación internacional de la necesidad de proceder a su persecución y castigo, haciendo efectivo un impulso internacional que supere las dificultades derivadas de su propia naturaleza.75 En atención a estos desarrollos jurisprudenciales y de forma complementaria a ellos, habría que destacar dos doctrinas fundamentales reconocidas por la Corte Interamericana de Derechos Humanos. En primer lugar, la relativa a las obligaciones positivas derivadas de las convenciones suscritas y del principio de diligencia debida que permiten declarar la responsabilidad del Estado por actuaciones delictivas cometidas por los particulares. En segundo lugar, la referente al control de la convencionalidad internacional por parte de los jueces nacionales de su derecho interno. Estas doctrinas jurisprudenciales han supuesto un importante avance respecto a la responsabilidad en el ámbito de la protección de los derechos humanos. Respecto a la teoría de la 74 75 Ibíd., párr. 215. Cfr. Fundamento Jurídico 6.3 de la sentencia. Activismo judicial en la evolución del Derecho Internacional 99 obligaciones positivas, se altera la estructura vertical del Derecho internacional de los derechos humanos (individuo v. Estado) introduciendo la dimensión horizontal (individuo v. individuo) que potencia la efectividad y plenitud de los derechos reconocidos por los Estados en los convenios internacionales que suscriben. A tal efecto, en el caso Campo Algodonero v. México, con importante referencia al acervo jurisprudencial del TEDH sobre la materia, se ha reconocido la responsabilidad del Estado por su falta de actuación respecto al asunto de los crímenes de mujeres de Ciudad Juárez. El Tribunal ha constatado en este caso que Las irregularidades en el manejo de evidencias, la alegada fabricación de culpables, el retraso en las investigaciones, la falta de líneas de investigación que tengan en cuenta el contexto de violencia contra la mujer en el que se desenvolvieron las ejecuciones de las tres víctimas y la inexistencia de investigaciones contra funcionarios públicos por su supuesta negligencia grave, vulneran el derecho de acceso a la justicia, a una protección judicial eficaz y el derecho de los familiares y de la sociedad a conocer la verdad de lo ocurrido. Además, denota un incumplimiento estatal de garantizar, a través de una investigación seria y adecuada, los derechos a la vida, integridad personal y libertad personal de las tres víctimas. Todo ello permite concluir que en el presente caso existe impunidad y que las medidas de derecho interno adoptadas han sido insuficientes para enfrentar las graves violaciones de derechos humanos ocurridas. El Estado no demostró haber adoptado normas o implementado las medidas necesarias, conforme al artículo 2 de la Convención Americana y al artículo 7.c de la Convención Belém do Pará, que permitieran a las autoridades ofrecer una investigación con debida diligencia. Esta ineficacia judicial frente a casos individuales de violencia contra las mujeres propicia un ambiente de impunidad que facilita y promueve la repetición de los hechos de violencia en general y envía un mensaje según el cual la violencia contra las mujeres puede ser tolerada y aceptada como parte del diario vivir.76 Ahora bien, y conforme a su propia jurisprudencia, la Corte interamericana deja claro que un Estado no puede ser responsable por cualquier violación de derechos humanos cometida entre particulares dentro de su jurisdicción. En efecto, las obligaciones convencionales de garantía a cargo de los Estados no implican una responsabilidad ilimitada de los Estados frente a cualquier acto o hecho de particulares, pues sus deberes de adoptar medidas de prevención y protección de los particulares en sus relaciones entre sí se encuentran condicionados al conocimiento de una situación de riesgo real e inmediato para un individuo o grupo de individuos determinado y a las posibilidades razonables de prevenir o evitar ese riesgo. Es decir, aunque un acto u omisión de un particular tenga como consecuencia jurídica la violación de determinados derechos humanos de otro particular, aquel no es automáticamente 76 Véase CorteIDH, caso González y otras («Campo Algodonero») v. México, Sentencia de 16 de noviembre de 2009, Serie C, nº 205, párr. 388. 100 Francisco Jiménez García atribuible al Estado, pues debe atenderse a las circunstancias particulares del caso y a la concreción de dichas obligaciones de garantía.77 En lo que respecta a la doctrina del control de convencionalidad, con ella se mueve el foco de la responsabilidad desde el Gobierno, principal valedor del cumplimiento de las obligaciones internacionales, hacía el poder judicial al que, en cuanto órgano del Estado, le corresponde un papel primordial y definitivo en el reconocimiento y garantía de los derechos asumidos por el ordenamiento internacional con independencia del sistema monista o dualista que impere en su ordenamiento nacional. Con este fin, el Tribunal de San José de Costa Rica ha establecido en su jurisprudencia que es consciente de que los jueces y tribunales internos están sujetos al imperio de la ley y, por ello, están obligados a aplicar las disposiciones vigentes en el ordenamiento jurídico. Pero cuando un Estado ha ratificado un tratado internacional como la Convención Americana, sus jueces, como parte del aparato del Estado, también están sometidos a ella, lo que les obliga a velar porque los efectos de las disposiciones de la Convención no se vean mermados por la aplicación de leyes contrarias a su objeto y fin, que desde un inicio carecen de efectos jurídicos. En otras palabras, el Poder Judicial debe ejercer un «control de convencionalidad» ex officio entre las normas internas y la Convención Americana, evidentemente en el marco de sus respectivas competencias y de las regulaciones procesales correspondientes. En esta tarea, el Poder Judicial debe tener en cuenta no solamente el tratado, sino también la interpretación que del mismo ha hecho la Corte Interamericana, intérprete última de la Convención Americana.78 No resulta ajena a esta doctrina cierta jurisprudencia nacional relativa a la persecución de los crímenes internacionales. A este respecto se puede destacar la Sentencia de la Corte Suprema de Perú en el asunto del ex presidente Alberto Fujimori de 7 de abril de 200979 en la que se incide sobre el valor interpretativo de la jurisprudencia de la Corte IDH,80 la virtualidad del derecho consuetudinario en la calificación de los actos criminales que justifica su perseguibilidad internacional, la improcedencia de la prescripción y la necesidad imperativa de su castigo,81 declarando la nulidad de toda 77 Cfr. ibíd., párr. 280. Cfr. Caso Almonacid Arellano y otros vs. Chile, Sentencia de 26 de septiembre de 2006 CorteIDH Serie C nº 154, párrs. 123 a 125 y caso La Cantuta vs. Perú, Sentencia de 29 de noviembre de 2006, CorteIDH Serie C nº 162, párrs. 173-189; Caso Boyce y otros vs. Barbados. Excepción Preliminar, Fondo, Reparaciones y Costas. Sentencia de 20 de noviembre de 2007. Serie C No. 169, párr. 78; asunto Radilla Pacheco vs. México, Corte IDH Serie C nº 209 Sentencia de 23 de noviembre de 2009, párr. 339 79 La sentencia puede consultarse en la siguiente dirección: <http://www.derechos.org/nizkor/peru/doc/fujimori.pdf>. 80 Cfr. párr. 107.1. 81 Cfr. párrs.710 y ss. 78 Activismo judicial en la evolución del Derecho Internacional 101 norma de amnistía que pudiera amparar la impunidad de los mismos.82 Asimismo, la Sentencia de la Corte Suprema argentina dictada el 13 de julio de 2007 en el asunto Mazzeo, Julio Lilo y otros que, reiterando su jurisprudencia anterior contraría las leyes de amnistía (Arancibia Clavel y Simon), declara la imposibilidad constitucional de indultar a autores y partícipes de crímenes internacionales, pues dicho acto de gobierno conllevaría de modo inescindible la renuncia a la verdad, a la investigación, a la comprobación de los hechos, a la identificación de sus autores y a la desarticulación de los medios y recursos eficaces para evitar la impunidad.83 En España destacan la Sentencia del Tribunal Constitucional 237/2005 de 26 de septiembre en el denominado asunto del genocidio maya en Guatemala, en la que se reconocía el carácter absoluto y pleno del principio de justicia universal tal y como entonces lo reconocía el artículo 23.4 de la Ley Orgánica del Poder Judicial (actualmente reformada y tal principio limitado), así como la Sentencia del Tribunal Supremo en el mencionado asunto Scilingo dictada en casación el 2 de septiembre de 2007.84 Según esta sentencia, los principios contenidos en el Derecho internacional penal deben ser tenidos en cuenta al proceder a la interpretación y aplicación del Derecho nacional, con mayor motivo cuando aquellos revisten naturaleza de ius cogens. Consiguientemente, tanto las normas de Derecho penal sustantivo como las de orden orgánico o procesal, deben ser interpretadas teleológicamente en coherencia con la necesidad de protección eficaz y con la efectividad de la prohibición de lesión de los derechos humanos conforme al contexto jurídico internacional existente en el momento de la comisión de la conducta criminal.85 5. Conclusiones Los asuntos analizados nos permiten apreciar la relevancia que el «activismo judicial» presenta actualmente en el panorama del Derecho internacional. No se trata de interpretaciones contra legem o apologéticas que pretendan movilizar la normativa internacional en la consecución de ciertos valores o principios meramente auspiciados por el ordenamiento internacional, sino de interpretaciones ajustadas a la normativa internacional vigente tendentes a la consecución de un equilibrio equitativo entre los intereses en presencia salvando la coherencia y la unidad del Derecho internacional. Si bien este ordenamiento se encuentra arraigado en principios 82 Cfr. párrs. 621 y ss. Cfr. párr. 29 de la Sentencia. La jurisprudencia de la Corte Suprema Argentina puede consultarse en <http://www.csjn.gov.ar/>. 84 Tribunal Supremo, Sala de lo Penal, sentencia 798/2007 de 1 de octubre de 2007. 85 Cfr. Fundamento Jurídico 6.3 de la sentencia. 83 102 Francisco Jiménez García estructurales de corte clásico incuestionables (soberanía, relativismo jurídico…), no puede obviar los progresos alcanzados en otros ámbitos que han de resultar necesariamente compatibles con los esquemas jurídicos anteriores. Se trata de significativos avances que quizá nos permitan hablar de la existencia de un incipiente orden público internacional concretado, en este caso, en el ámbito del Derecho internacional de los derechos humanos.
https://openalex.org/W3107799629	http://www.scielo.br/pdf/csp/v36n11/1678-4464-csp-36-11-e00194619.pdf	Portuguese	null	Determinantes do trabalho remunerado entre brasileiros mais velhos usando modelagem de equações estruturais: evidências do ELSI-Brasil	Cadernos de Saúde Pública	2,020	cc-by	9,255	Resumo Correspondência C. M. S. Castro Instituto René Rachou, Fundação Oswaldo Cruz, Belo Horizonte, Brasil. Av. Augusto de Lima 1715, Belo Horizonte, MG 30190-002, Brasil. camilamscastro@gmail.com O objetivo do estudo foi desenvolver um modelo conceitual e explorar asso ciações diretas e indiretas entre trabalho remunerado e fatores que operam ao longo da vida em amostra nacional representativa da população brasileira com 50 anos e mais. A análise foi baseada em 8.903 participantes da linha de base do Estudo Longitudinal da Saúde dos Idosos Brasileiros (ELSI- -Brasil). Os fatores de exposição foram sociodemográficos, de saúde, trabalho e interação social. Utilizando modelo de equações estruturais, observou-se que o trabalho remunerado apresentou associação total com a condição social ao longo da vida entre mulheres (coeficiente de padronização – CP = 0,489) e as sociação direta com a capacidade para o trabalho entre homens (CP = 0,527) Apenas para as mulheres, associação indireta e negativa foi observada com o esforço físico intenso no trabalho ao longo da vida, via aposentadoria (CP = -0,156). Homens com trabalho remunerado foram mais propensos à partici pação social, aferida pelo pertencimento a grupos ou associações (CP = 0,209) Entre as mulheres, essa participação ocorreu por meio de interações com fa mília e amigos (CP = 0,047), via capacidade para o trabalho. Tanto entre ho mens quanto entre mulheres, a condição de saúde ao longo da vida mostrou associação indireta e positiva (CP = 0,298 homens; CP = 0,142 mulheres) com o trabalho remunerado, via capacidade para o trabalho. Todos os fatores men cionados apresentaram associação significativa com o trabalho remunerado Os resultados do estudo mostraram que a participação de homens e mulheres mais velhos no mercado de trabalho ocorre por mecanismos diferentes, princi palmente ao considerar os fatores relacionados ao trabalho e interação Envelhecimento; Trabalho; Modelos Estatísticos; Situação Sociecônomica; Interação Social Este é um artigo publicado em acesso aberto (Open Access) sob a licença Creative Commons Attribution, que permite uso, distribuição e reprodu ção em qualquer meio, sem restrições, desde que o trabalho original seja corretamente citado. O objetivo do estudo foi desenvolver um modelo conceitual e explorar asso ciações diretas e indiretas entre trabalho remunerado e fatores que operam ao longo da vida em amostra nacional representativa da população brasileira com 50 anos e mais. A análise foi baseada em 8.903 participantes da linha de base do Estudo Longitudinal da Saúde dos Idosos Brasileiros (ELSI- -Brasil). ARTIGO ARTICLE Determinantes do trabalho remunerado entre brasileiros mais velhos usando modelagem de equações estruturais: evidências do ELSI-Brasil Determinants of paid work among older Brazilians using structural equation modeling: evidence from the ELSI-Brazil study Determinantes del trabajo remunerado entre adultos mayores brasileños usando modelos de ecuaciones estructurales: evidencias del ELSI-Brasil doi: 10.1590/0102-311X00194619 doi: 10.1590/0102-311X00194619 Envelhecimento; Trabalho; Modelos Estatísticos; Situação Sociecônomica; Interação Social Este é um artigo publicado em acesso aberto (Open Access) sob a licença Creative Commons Attribution, que permite uso, distribuição e reprodu ção em qualquer meio, sem restrições, desde que o trabalho original seja corretamente citado. Cad. Saúde Pública 2020; 36(11):e00194619 Introdução O envelhecimento populacional traz desafios significativos para o mercado de trabalho. Como alter nativa para minimizar esse desafio, diversos países têm considerado a inclusão das pessoas mais velhas nas atividades 1,2,3. Estudos sobre ocupação utilizam, frequentemente, idade de 45 ou 50 anos como critério base para o termo “trabalhadores mais velhos”. Esse ponto de vista oferece melhores possibilidades de medidas preventivas na saúde dos trabalhadores 4. Em nível mundial, a proporção da participação de adultos mais velhos no mercado de trabalho, em relação ao gênero, é maior entre os homens 5,6. Observa-se que, após um período de declínio constante, a participação dos homens mais velhos na força de trabalho aumentou recentemente na maioria dos países de alta renda da Organização para a Cooperação e Desenvolvimento Econômi co (OECD) 5. Concomitantemente, a participação das mulheres de todas as idades no mercado de trabalho vem aumentando tanto em países de alta quanto de média renda 5. No Brasil, verificou-se a predominância dos homens (56,1%) entre as pessoas ocupadas, o que foi confirmado em todas as regiões brasileiras 6. Em relação à análise por grupos de idade, o segmento dos trabalhadores mais velhos foi o que apresentou as maiores taxas de crescimento da ocupação. No terceiro trimestre de 2019, comparado com o mesmo período de 2017 e 2018, observou-se uma expansão da participação dos adultos mais velhos no mercado de trabalho. A participação das pessoas de 40-59 anos de idade passou de 39,9% para 40,4%, e a participação das pessoas com 60 anos e mais passou de 7,6% para 8,3%. Por outro lado, houve uma diminuição da participação no mercado de trabalho das pessoas entre 18-39 anos 6. A participação de adultos mais velhos no mercado de trabalho é um processo complexo, influen ciado por experiências passadas e atuais da vida. Pesquisas em países de alta renda têm mostrado que a participação das pessoas mais velhas na força de trabalho não é determinada por um único fator. Características individuais (sexo, idade, status socioeconômico) e da família interagem com os fatores relacionados à organização do trabalho e com os fatores contextuais, como mercado de trabalho e cobertura do sistema previdenciário 2,7. Outro exemplo de complexidade na participação dos mais velhos na força de trabalho é que muitos determinantes desta participação estão sujeitos a mudanças substanciais e, às vezes, temporalmente muito rápidas 7,8. Resumo Os fatores de exposição foram sociodemográficos, de saúde, trabalho e interação social. Utilizando modelo de equações estruturais, observou-se que o trabalho remunerado apresentou associação total com a condição social ao longo da vida entre mulheres (coeficiente de padronização – CP = 0,489) e as sociação direta com a capacidade para o trabalho entre homens (CP = 0,527). Apenas para as mulheres, associação indireta e negativa foi observada com o esforço físico intenso no trabalho ao longo da vida, via aposentadoria (CP = -0,156). Homens com trabalho remunerado foram mais propensos à partici pação social, aferida pelo pertencimento a grupos ou associações (CP = 0,209). Entre as mulheres, essa participação ocorreu por meio de interações com fa mília e amigos (CP = 0,047), via capacidade para o trabalho. Tanto entre ho mens quanto entre mulheres, a condição de saúde ao longo da vida mostrou associação indireta e positiva (CP = 0,298 homens; CP = 0,142 mulheres) com o trabalho remunerado, via capacidade para o trabalho. Todos os fatores men cionados apresentaram associação significativa com o trabalho remunerado. Os resultados do estudo mostraram que a participação de homens e mulheres mais velhos no mercado de trabalho ocorre por mecanismos diferentes, princi palmente ao considerar os fatores relacionados ao trabalho e interação 1 Instituto René Rachou, Fundação Oswaldo Cruz, Belo Horizonte, Brasil. 2 Programa de Pós-graduação em Saúde Púbica, Universidade Federal de Minas Gerais, Belo Horizonte, Brasil. 1 Instituto René Rachou, Fundação Oswaldo Cruz, Belo Horizonte, Brasil. 2 Programa de Pós-graduação em Saúde Púbica, Universidade Federal de Minas Gerais, Belo Horizonte, Brasil. Castro CMS et al. 2 Cad. Saúde Pública 2020; 36(11):e00194619 Variáveis do estudo A variável dependente do estudo foi participação no trabalho remunerado obtida pela pergunta: “O(A) Sr(a). trabalhou remuneradamente nos últimos 30 dias?”. Trabalho remunerado foi considerado como o exercício de qualquer atividade com remuneração em dinheiro, produtos, mercadorias ou benefícios. Essa variável incluiu assalariados, patrões, trabalhador autônomo, trabalho por produção e biscate, pessoas afastadas por motivo de férias, licença por doença, entre outros. Todas as outras condições, quais sejam aposentados, pedintes, estudantes, donas de casa que se dedicam exclusiva mente aos afazeres domésticos, pessoas que trabalham sem remuneração ou se dedicam a trabalho voluntário e aqueles que viviam exclusivamente de renda ou ajuda de parentes e/ou conhecidos foram incluídos na categoria não trabalho. As variáveis independentes foram definidas considerando-se os fatores de exposição que operam ao longo da vida e foram classificadas em fatores sociodemográficos e fatores relacionados à saúde, ao trabalho e à interação social. As variáveis sociodemográficas consideradas foram: (1) anos de escolari dade da mãe (0-4 anos vs. 5 anos ou mais); (2) local de residência aos 10 anos (rural vs. urbano); (3) idade em que começou a trabalhar (até 17 anos vs. 18 anos e mais); (4) anos de escolaridade do participante (0-7 anos vs. 8 anos ou mais); (5) idade na data da entrevista; (6) posição no domicílio (responsável pelo domicílio ou não); (7) situação conjugal (vive ou não com cônjuge ou companheiro(a); e (8) local de residência atual (rural ou urbano). Para a presente análise foi construída a variável latente condição social ao longo da vida, com base nas variáveis anos de escolaridade da mãe, local de residência aos 10 anos, idade em que começou a trabalhar e anos de escolaridade do participante. Essa variável foi baseada na análise de componentes principais e, posteriormente, foi dicotomizada em acima da média e abaixo deste valor. As variáveis relacionadas à saúde foram: (1) saúde até os 15 anos de idade, aferida pela pergunta: “O(a) Sr(a). diria que a sua saúde, desde o nascimento até os 15 anos de idade, era:” boa (boa, muito boa ou excelente) vs ruim (razoável ou ruim); (2) autoavaliação da saúde atual, definida pela resposta à pergunta: “Em geral, como o(a) Sr(a). avalia a sua saúde?”, categorizada como boa (boa ou muito boa) vs. ruim (regular, ruim ou muito ruim); e (3) número de doenças crônicas, categorizado em pelo menos uma vs duas ou mais. Fonte de dados e amostra Foram usados dados do inquérito da linha de base do Estudo Longitudinal da Saúde dos Idosos Brasileiros (ELSI-Brasil), cuja amostra foi delineada para representar a população brasileira não institucionali zada, com idade igual ou superior a 50 anos. A coleta de dados da linha de base da coorte foi condu zida entre 2015 e 2016. A amostragem utilizou um delineamento complexo, combinando estratifica ção por municípios, setores censitários e domicílios. Todos os residentes nos domicílios selecionados com idade igual ou superior a 50 anos foram elegíveis para entrevista e outros procedimentos. A amostra final foi estimada em 10 mil pessoas (9.412 participaram), residentes em 70 municípios das cinco regiões do país. Mais detalhes estão disponíveis na homepage do ELSI-Brasil (http://elsi.cpqrr. fiocruz.br/) e em publicação anterior 18. O ELSI-Brasil foi aprovado pelo Comitê de Ética em Pesquisa da Fundação Oswaldo Cruz, Minas Gerais (CAAE: 34649814.3.0000.5091). Todos os participantes assinaram o termo de consentimento livre e esclarecido separados para cada procedimento da pesquisa. Introdução Somando-se a isso, alguns estudos também mostraram que fatores biológicos, sociais e ambientais nas diferentes fases da vida podem acumu lar-se ao longo do tempo, impactando na tomada de decisão de permanecer trabalhando nas idades avançadas 9,10,11. Existem poucos estudos brasileiros examinando os determinantes da participação de adultos mais velhos no mercado de trabalho. Pesquisa conduzida em São Paulo, no ano 2000, entre idosos (60 anos e mais), mostrou que a idade e a escolaridade diminuem seu poder explicativo para o trabalho remunerado nas idades mais velhas quando são consideradas as condições de saúde 12. Um estudo mais recente, realizado na Região Metropolitana de Belo Horizonte, Minas Gerais, entre adultos mais velhos (50-69 anos de idade), mostrou que a associação entre autoavaliação de saúde e trabalho remunerado foi modificada pela escolaridade, principalmente entre as mulheres 13. Em comparação àqueles com boa autoavaliação de saúde e escolaridade igual ou superior a oito anos (grupo de refe rência), a participação no trabalho remunerado foi duas vezes mais baixa entre os homens com pior autoavaliação de saúde, independentemente do nível de escolaridade. Para as mulheres com pior autoavaliação de saúde, a participação no trabalho remunerado foi cerca de nove vezes menor entre aquelas com escolaridade mais baixa 13. Frente a essas evidências, é preciso aprofundar na compreensão dos mecanismos pelos quais homens e mulheres em idades avançadas permanecem trabalhando. O modelo de equação estrutural tem sido cada vez mais utilizado na área da saúde para medir e testar relações em fenômenos com plexos que exigem a análise de múltiplas relações simultaneamente 14,15. A vantagem da utilização do modelo de equação estrutural está no fato de que esta técnica estatística estima uma série de equações de regressão múltipla separadas, mas dependentes entre si, baseadas no modelo conceitual proposto pelo pesquisadores 16,17. O presente estudo baseou-se em evidências da literatura nacional e interna cional desenvolvidas em estudos longitudinais que investigaram especificamente os determinantes da participação na força de trabalho de pessoas em idades avançadas 2,8. Assim, o objetivo deste estudo foi Cad. Saúde Pública 2020; 36(11):e00194619 DETERMINANTES DO TRABALHO REMUNERADO ENTRE OS MAIS VELHOS 3 desenvolver um modelo conceitual e explorar as associações diretas e indiretas entre fatores sociode mográficos, de saúde, trabalho, interação social e a participação no trabalho remunerado de homens e mulheres em amostra nacional representativa da população com 50 anos e mais. Introdução desenvolver um modelo conceitual e explorar as associações diretas e indiretas entre fatores sociode mográficos, de saúde, trabalho, interação social e a participação no trabalho remunerado de homens e mulheres em amostra nacional representativa da população com 50 anos e mais. Variáveis do estudo As doenças foram definidas pela resposta à pergunta: “Algum médico já disse que o(a) Sr(a). tinha ‘tal doença’?”, sendo consideradas hipertensão, diabetes, colesterol, acidente vascular cerebral, asma, artrite ou reumatismo, osteoporose, problema crônico de coluna, câncer, doença do Cad. Saúde Pública 2020; 36(11):e00194619 Cad. Saúde Pública 2020; 36(11):e00194619 Castro CMS et al. 4 coração e depressão. Para a presente pesquisa, a variável latente condição de saúde ao longo da vida foi construída baseada na análise de componentes principais, com todas as variáveis descritas anterior mente. Em seguida, a variável foi dicotomizada em acima da média e abaixo deste valor. As variáveis relacionadas ao trabalho foram: (1) esforço físico intenso no trabalho ao longo da vida, definido pela informação: “Como o(a) Sr(a). descreveria as exigências físicas do trabalho que exerceu durante a maior parte da vida?”, categorizada como ausência de esforço físico intenso (nunca trabalhou ou ficava sentado a maior parte do tempo, ficava em pé/andando ou o trabalho exigia algum esforço físico) vs. presença de esforço físico intenso (o trabalho exigia esforço físico intenso a maior parte do tempo); (2) capacidade para o trabalho, definida pela pergunta: “Como o(a) Sr(a). avalia a sua capacidade atual para o trabalho?”, categorizada como boa (boa/muito boa) vs. ruim (razoável/ruim/ muito ruim); e (3) aposentadoria (sim/não), definido como aposentado quando o entrevistado decla rou receber salário ou pensão do Instituto Nacional do Seguro Social (INSS) ou governo. As variáveis relativas à interação social foram: (1) interação social primária, elaborada baseando-se na informação sobre a frequência com que a pessoa se encontrava ou conversava com filhos, parentes e/ou amigos, sem contar os que moravam com ele, categorizada como três ou mais vezes na semana, uma ou duas vezes por semana, uma ou duas vezes por mês, a cada dois ou três meses, uma ou duas vezes por ano e menos de uma vez por ano ou nunca; e (2) interação social secundária, elaborada com base na informação se o indivíduo tinha, nos últimos 12 meses, participado de atividades sociais organizadas ou associações civis, conselhos, lideranças comunitárias, cooperativas, partidos políticos etc, categorizada como não vs. sim. Para a interação social primária, as opções três ou mais vezes na semana e uma ou duas vezes por semana foram categorizadas em interação social elevada. Para a interação secundária, a categoria “sim” foi classificada em interação social elevada. Modelo conceitual O modelo proposto foi desenvolvido tendo como base dois modelos teóricos da literatura internacio nal, o lidA Cohort Study (German Cohort Study on Work, Age, Health and Work Participation) 2 e o Study on Transitions in Employment, Ability and Motivation 8, com o intuito de explorar as relações diretas e indiretas entre os fatores associados à participação no trabalho remunerado de homens e mulheres brasileiros com 50 anos e mais. A condição social ao longo da vida ocupou a posição mais distal do modelo; as variáveis intermediárias foram condição de saúde ao longo da vida, esforço físico inten so no trabalho ao longo da vida, aposentadoria e interação social e, por último, a capacidade para o trabalho foi considerada a variável proximal na associação com o trabalho remunerado. A idade na data da entrevista foi usada como variável de ajuste para todas as associações do modelo. As variáveis posição no domicílio, situação conjugal e local de residência atual foram consideradas como variáveis de ajuste para o desfecho (trabalho remunerado). Na Figura 1, as formas circulares ou elípticas repre sentam as variáveis latentes e os retângulos as variáveis observadas. As setas indicam as relações entre as variáveis, podendo ser diretas ou indiretas. O modelo proposto assume que as variáveis condição social ao longo da vida, condição de saúde ao longo da vida, esforço físico intenso no trabalho ao longo da vida, capacidade para o trabalho, aposen tadoria, interação social primária e interação social secundária estão associadas de forma direta com a participação no trabalho remunerado. Além disso, a condição social ao longo da vida está associada indiretamente ao trabalho remunerado, pelas vias da condição de saúde, trabalho e interação social. Variáveis do estudo No presente artigo usamos os termos “interação primária”, que é uma proxy para o conceito de “laço forte”, e “interação secundária”, que é uma proxy para o conceito de “laço fraco” proposto por Granovetter 19. Figura 1 Modelo conceitual desenvolvido para explorar as inter-relações entre as variáveis associadas à participação no trabalho remunerado de homens e mulheres com 50 anos ou mais. Estudo Longitudinal da Saúde dos Idosos Brasileiros (ELSI-Brasil), 2015-2016. Fonte: elaboração própria dos autores (2018). APOS: aposentadoria; CIVIL: situação conjugal; CS: condição social ao longo da vida (anos de escolaridade da mãe, local de residência aos 10 anos, idade em que começou a trabalhar, anos de escolaridade do participante); CT: capacidade para o trabalho; IDADE: idade na data da entrevista; OCUP: esforço físico intenso no trabalho ao longo da vida; PRIMÁRIA: interação social primária; SAÚDE: condição de saúde ao longo da vida (saúde até os 15 anos de idade, autovaliação da saúde atual, número e doenças crônicas); SECUNDÁRIA: interação social secundária; RESP: posição no domicílio; RESID: local de residência atual; TRABALHO: trabalho remunerado. Fonte: elaboração própria dos autores (2018). Fonte: elaboração própria dos autores (2018). APOS: aposentadoria; CIVIL: situação conjugal; CS: condição social ao longo da vida (anos de escolaridade da mãe, local de residência aos 10 anos, idade em que começou a trabalhar, anos de escolaridade do participante); CT: capacidade para o trabalho; IDADE: idade na data da entrevista; OCUP: esforço físico intenso no trabalho ao longo da vida; PRIMÁRIA: interação social primária; SAÚDE: condição de saúde ao longo da vida (saúde até os 15 anos de idade, autovaliação da saúde atual, número e doenças crônicas); SECUNDÁRIA: interação social secundária; RESP: posição no domicílio; RESID: local de residência atual; TRABALHO: trabalho remunerado. mensuradas 20. Nas análises das estimativas padronizadas para a construção das variáveis latentes deste estudo (condição social ao longo da vida e condição de saúde ao longo da vida) considerou-se a carga fatorial e os autovalores. Todas as variáveis latentes resultaram de fatores com autovalores maiores do que 1,00 e todos os itens dos fatores apresentaram cargas fatoriais maiores do que 0,70. mensuradas 20. Nas análises das estimativas padronizadas para a construção das variáveis latentes deste estudo (condição social ao longo da vida e condição de saúde ao longo da vida) considerou-se a carga fatorial e os autovalores. Todas as variáveis latentes resultaram de fatores com autovalores maiores do que 1,00 e todos os itens dos fatores apresentaram cargas fatoriais maiores do que 0,70. O modelo de equação estrutural também permite a estimação de múltiplas funções lineares, incluindo efeitos diretos e indiretos, característica não permitida pelos métodos tradicionais de regressão. Análise dos dados Na análise descritiva dos participantes do estudo foi usado o teste qui-quadrado de Pearson, para examinar diferenças entre homens e mulheres. O modelo de equação estrutural foi usado para ana lisar, simultaneamente, os fatores associados ao trabalho remunerado. Esse método permite testar a validade de um modelo conceitual baseado em um conjunto de variáveis observáveis e latentes. Uma variável latente ou constructo é aquela que não pode ser observada diretamente, com base nas unidades de análise em estudo, mas supostamente está relacionada a muitas variáveis que podem ser Cad. Saúde Pública 2020; 36(11):e00194619 DETERMINANTES DO TRABALHO REMUNERADO ENTRE OS MAIS VELHOS 5 ç p p APOS: aposentadoria; CIVIL: situação conjugal; CS: condição social ao longo da vida (anos de escolaridade da mãe, local de residência aos 10 anos, idade em que começou a trabalhar, anos de escolaridade do participante); CT: capacidade para o trabalho; IDADE: idade na data da entrevista; OCUP: esforço físico intenso no trabalho ao longo da vida; PRIMÁRIA: interação social primária; SAÚDE: condição de saúde ao longo da vida (saúde até os 15 anos de idade, autovaliação da saúde atual, número e doenças crônicas); SECUNDÁRIA: interação social secundária; RESP: posição no domicílio; RESID: local de residência atual; TRABALHO: trabalho remunerado. Resultados A amostra final deste estudo foi composta por 8.903 participantes do inquérito de base do ELSI-Bra sil, ou seja, aquelas pessoas que possuíam informações completas para todas as variáveis. Desses, 4.961 (56%) eram mulheres e 3.942 (44%) eram homens. O trabalho remunerado foi informado por 31,3% dos participantes, sendo maior para o sexo masculino (39,6% em relação a 24,2% para mulheres; p < 0,001). Na Tabela 1, são mostradas as características dos participantes do estudo, segundo o gênero. Foram observadas diferenças estatisticamente significantes entre homens e mulheres para residência urbana aos 10 anos, escolaridade do indivíduo, idade em que começou a trabalhar, idade na data da entrevista, posição no domicílio, situação conjugal, condição de saúde ao longo da vida, número de doenças crônicas, esforço físico intenso no trabalho ao longo da vida e interação social primária. Entre os homens, o trabalho remunerado apresentou associação significativa com condição social ao longo da vida, condição de saúde ao longo da vida, capacidade para o trabalho e interação social secundária. A situação conjugal e a idade na data da entrevista compuseram o modelo final como variáveis de ajuste. Para as mulheres, o trabalho remunerado apresentou associação significativa com condição social ao longo da vida, condição de saúde ao longo da vida, interação social primária, esfor ço físico intenso no trabalho ao longo da vida, capacidade para o trabalho e aposentadoria. A idade na data da entrevista compôs o modelo final como variável de ajuste. Os modelos conceituais finais das inter-relações entre os fatores associados à participação no trabalho remunerado, segundo o gênero, estão representados na Figura 2. Na Tabela 2, são apresentadas as OR (IC95%) e os coeficientes padronizados (CP) do modelo de equação estrutural das variáveis que compuseram o modelo final dos homens e das mulheres. Na Tabela 3, estão apresentadas as associações diretas, indiretas e totais das variáveis associadas ao trabalho remunerado para homens e mulheres. De modo geral, os resultados mostraram maior associação entre a participação no trabalho remunerado das mulheres com a condição social ao longo da vida, apresentando um coeficiente padronizado (CP) total de 0,489, e a participação dos homens registrou maior associação com a capacidade para o trabalho (CP = 0,527). Entre os homens, a con dição social ao longo da vida apresentou associação indireta e positiva (CP = 0,352) com o trabalho remunerado, principalmente via capacidade para o trabalho (CP = 0,186). Figura 1 O método combina aspectos de regressão múltipla e análise fatorial para estimar as relações entre cada variável dependente (endógena) e as variáveis explicativas (exógenas). Para esta análise, as variáveis consideradas como endógenas foram condição de saúde ao longo da vida, esforço físico intenso no trabalho ao longo da vida, aposentadoria, capacidade para o trabalho, interação social primária, interação social secundária e trabalho remunerado. Neste estudo, utilizou-se uma versão específica do modelo de equação estrutural para dados binários proposta por Kupek 15. Realizou-se inicialmente regressões logísticas com todas as variáveis do modelo conceitual. Num segundo momento, as variáveis com valor de p > 0,05 foram excluídas do modelo. Logo após essa etapa, realizou-se novamente as estimações das regressões logísticas do modelo somente com as variáveis que foram significativas. Na terceira etapa, os coeficientes do modelo de equação estrutural foram calculados com base nas odds ratio (OR) encontradas no segundo passo. Na quarta etapa, os coeficientes menores ou iguais a 0,18 foram excluídos do modelo. Após o desenvolvimento do modelo final foram calculados os coeficientes das associações indiretas e total, denominados coeficientes padronizados. Esse cálculo foi realizado baseando-se na transformação de Cad. Saúde Pública 2020; 36(11):e00194619 Cad. Saúde Pública 2020; 36(11):e00194619 Castro CMS et al. 6 Yule. Esse método aproxima a matriz dos coeficientes de correlação de Pearson das OR pela fórmula (OR – 1)/(OR + 1) 15. Quanto maior o valor do coeficiente padronizado, mais forte é a associação entre a variável explicativa e a variável dependente. Para determinar se o modelo apresentou bom ajuste foram considerados os seguintes critérios: (a) a qualidade de ajuste de cada equação de regressão logística, com base nos valores de p, nos R2 e na comparação dos qui-quadrados dos modelos; (b) foram incluídas nos modelos de equação estrutural finais apenas as variáveis com coeficientes maiores ou iguais a 0,18. Decidiu-se assumir uma postura mais conservadora para que o modelo fosse mais parcimonioso. A busca da parcimônia nesse modelo se justifica pelo fato de não ser possível o cálculo de medidas gerais de ajuste, pois o modelo utilizado proposto por Kupek 15 baseia-se na transformação de estimadores de regressão logística (especifica mente, as razões de chance) em um modelo de equações estruturais. Assim, só é possível ter indicado res de ajuste para cada equação isoladamente. Todas as análises foram realizadas separadamente para homens e mulheres. Figura 1 As estimativas foram realizadas usando-se os procedimentos para amostras complexas do programa Stata 14.1 (https:// www.stata.com), de forma a considerar o efeito do delineamento do estudo e os pesos dos indivíduos. Resultados Para esse grupo, a condição social ao longo da vida também registrou associação indireta e positiva com o trabalho remunerado pela via da condição de saúde ao longo da vida e capacidade para o trabalho (CP = 0,111). Entre as mulheres, a condição social ao longo da vida apresentou associação total (CP = 0,489) e direta (CP = 0,257) positivas com o trabalho remunerado. Além disso, apresentou associação indireta e positiva (CP = 0,232), principalmente via esforço físico intenso no trabalho ao longo da vida e aposentadoria (CP = 0,076). Entre homens e mulheres a condição de saúde ao longo da vida registrou associação indireta e positiva (CP = 0,298 entre homens; CP = 0,142 entre mulheres) com o trabalho remunerado, via Cad. Saúde Pública 2020; 36(11):e00194619 DETERMINANTES DO TRABALHO REMUNERADO ENTRE OS MAIS VELHOS 7 7 Tabela 1 Distribuição do trabalho remunerado e demais características da população com 50 anos ou mais, segundo o gênero. Estudo Longitudinal da Saúde dos Idosos Brasileiros (ELSI-Brasil), 2015-2016. Resultados Variáveis Total Homens Mulheres Valor de p ** % * IC95% % * IC95% % * IC95% Trabalho remunerado 31,3 28,2-34,5 39,6 36,0-43,4 24,2 21,7-26,8 < 0,001 Melhor condição social ao longo da vida 41,1 38,1-44,2 41,6 38,1-45,3 40,6 37,5-43,9 0,516 Anos de escolaridade da mãe (5+ anos) 7,1 6,1-8,4 7,9 6,4-9,7 6,5 5,4-7,8 0,117 Residência urbana aos 10 anos 39,7 36,6-42,8 38,1 34,9-41,4 41,0 37,5-44,6 0,043 Anos de escolaridade do participante (8+ anos) 35,7 32,9-38,7 37,4 34,0-40,9 34,3 31,4-37,3 0,033 Idade em que começou a trabalhar (18+ anos) 17,5 15,4-19,8 12,1 10,5-13,8 22,3 19,7-25,2 < 0,001 Idade na data da entrevista (65+ anos) 35,9 32,2-39,7 33,3 28,7-38,3 38,1 35,0-41,3 0,006 Responsável pelo domicílio 73,2 71,4-75,0 86,7 84,5-88,7 61,7 57,9-65,4 < 0,001 Vive com cônjuge/companheiro(a) 63,5 60,5-66,3 75,2 72,5-77,7 53,5 50,4-56,5 < 0,001 Residência atual urbana 84,7 79,4-88,8 84,8 79,2-89,1 84,6 79,2-88,8 0,855 Melhor condição de saúde ao longo da vida 49,3 46,9-51,8 52,6 49,6-55,4 46,6 43,9-49,3 < 0,001 Saúde boa até os 15 anos 78,0 76,4-79,6 78,8 76,9-80,7 77,3 75,4-79,1 0,139 Nenhuma ou uma doença crônica *** 39,1 36,9-41,3 49,7 47,2-52,1 30,1 28,0-32,3 < 0,001 Autoavaliação da saúde atual como boa 43,7 41,3-46,2 43,6 40,3-46,9 43,8 41,4-46,3 0,873 Esforço físico intenso no trabalho ao longo da vida 19,3 17,1-21,8 27,8 24,6-31,2 11,9 9,8-14,4 < 0,001 Capacidade para o trabalho boa 48,4 46,0-50,8 49,0 45,9-52,0 47,8 45,5-50,2 0,381 Aposentadoria 54,4 51,3-57,5 54,3 50,4-58,1 54,6 51,0-58,1 0,877 Interação social primária elevada 48,4 46,4-50,5 44,6 42,2-47,1 51,6 49,2-54,1 < 0,001 Interação social secundária elevada 50,3 47,2-53,5 49,0 45,2-52,8 51,5 48,3-54,6 0,087 Total 8.903 3.942 4.961 IC95%: intervalo de 95% de confiança. * Porcentagens ponderadas pelos parâmetros amostrais; Teste qui-quadrado de Pearson ou teste t de student com nível de significância ≤ 0,05; para diferenças entre gêneros; * Diagnóstico médico de hipertensão, diabetes, colesterol, acidente vascular cerebral, asma, artrite ou reumatismo, osteoporose, problema crônico de coluna, câncer, doença do coração e depressão. Distribuição do trabalho remunerado e demais características da população com 50 anos ou mais, segundo o gênero. Estudo Longitudinal da Saúde dos Idosos Brasileiros (ELSI-Brasil), 2015-2016. erado e demais características da população com 50 anos ou mais, segundo o gênero. Estudo Longitudinal da Saúde dos capacidade para o trabalho. Ressalta-se que a força da associação para o grupo dos homens foi o dobro da magnitude da associação do grupo das mulheres. capacidade para o trabalho. Ressalta-se que a força da associação para o grupo dos homens foi o dobro da magnitude da associação do grupo das mulheres. Em relação à interação social, a participação mas culina no trabalho remunerado esteve associada direta e positivamente à interação social secundária (CP = 0,209), já a participação feminina mostrou associação indireta e positiva com a interação social primária (CP = 0,047). Apenas para o grupo das mulheres, aposentadoria apresentou associação direta e negativa (CP = -0,439) com o trabalho remunerado. Além disso, o trabalho remunerado também esteve associado indireta e negativamente com esforço físico intenso no trabalho ao longo da vida, via aposentadoria (CP = -0,156). Resultados Em relação à interação social, a participação mas culina no trabalho remunerado esteve associada direta e positivamente à interação social secundária (CP = 0,209), já a participação feminina mostrou associação indireta e positiva com a interação social primária (CP = 0,047). Apenas para o grupo das mulheres, aposentadoria apresentou associação direta e negativa (CP = -0,439) com o trabalho remunerado. Além disso, o trabalho remunerado também esteve associado indireta e negativamente com esforço físico intenso no trabalho ao longo da vida, via aposentadoria (CP = -0,156). Cad. Saúde Pública 2020; 36(11):e00194619 Castro CMS et al. 8 Figura 2 Figura 2 Resultado do modelo de equação estrutural final com coeficientes padronizados dos fatores associados à participação no trabalho remunerado, segundo o gênero. Estudo Longitudinal da Saúde dos Idosos Brasileiros (ELSI-Brasil), 2015-2016. Fonte: elaboração própria dos autores (2018). APOS: aposentadoria; CIVIL: situação conjugal; CS: condição social ao longo da vida; CT: capacidade para o trabalho; IDADE: idade na data da entrevista; OCUP: esforço físico intenso no trabalho ao longo da vida; SAÚDE: condição de saúde ao longo da vida; PRIMÁRIA: interação social primária; SECUNDÁRIA: interação social secundária; TRABALHO: trabalho remunerado. Nota: N = 8.903 (3.942 homens e 4.961 mulheres). 2a) Homens 2b) Mulheres 2a) Homens 2a) Homens 2b) Mulheres 2b) Mulheres Fonte: elaboração própria dos autores (2018). Fonte: elaboração própria dos autores (2018). APOS: aposentadoria; CIVIL: situação conjugal; CS: condição social ao longo da vida; CT: capacidade para o trabalho; IDADE: idade na data da entrevista; OCUP: esforço físico intenso no trabalho ao longo da vida; SAÚDE: condição de saúde ao longo da vida; PRIMÁRIA: interação social primária; SECUNDÁRIA: interação social secundária; TRABALHO: trabalho remunerado. Nota: N = 8.903 (3.942 homens e 4.961 mulheres). Fonte: elaboração própria dos autores (2018). APOS: aposentadoria; CIVIL: situação conjugal; CS: condição social ao longo da vida; CT: capacidade para o trabalho; IDADE: idade na data da entrevista; OCUP: esforço físico intenso no trabalho ao longo da vida; SAÚDE: condição de saúde ao longo da vida; PRIMÁRIA: interação social primária; SECUNDÁRIA: interação social secundária; TRABALHO: trabalho remunerado. Nota: N = 8.903 (3.942 homens e 4.961 mulheres). Cad. Saúde Pública 2020; 36(11):e00194619 Resultados Resultados da regressão logística multivariada e respectivo coeficiente de padronização do modelo de equação estrutural para cada variável endógena, segundo o gênero. Estudo Longitudinal da Saúde dos Idosos Brasileiros (ELSI-Brasil), 2015-2016. ca multivariada e respectivo coeficiente de padronização do modelo de equação estrutural para cada variável endógena, tudinal da Saúde dos Idosos Brasileiros (ELSI-Brasil), 2015-2016. Resultados da regressão logística multivariada e respectivo coeficiente de padronização do modelo de equação estrutura segundo o gênero. Estudo Longitudinal da Saúde dos Idosos Brasileiros (ELSI-Brasil), 2015-2016. CP: coeficiente padronizado; IC95%: intervalo de 95% de confiança; OR: odds ratio. Nota: N = 8.903 (3.942 homens e 4.961 mulheres). Resultados ç p p APOS: aposentadoria; CIVIL: situação conjugal; CS: condição social ao longo da vida; CT: capacidade para o trabalho; IDADE: idade na data da entrevista; OCUP: esforço físico intenso no trabalho ao longo da vida; SAÚDE: condição de saúde ao longo da vida; PRIMÁRIA: interação social primária; SECUNDÁRIA: interação social secundária; TRABALHO: trabalho remunerado. h lh Cad. Saúde Pública 2020; 36(11):e00194619 DETERMINANTES DO TRABALHO REMUNERADO ENTRE OS MAIS VELHOS 9 Tabela 2 Resultados da regressão logística multivariada e respectivo coeficiente de padronização do modelo de equação estrutural para cada variável endógena, segundo o gênero. Estudo Longitudinal da Saúde dos Idosos Brasileiros (ELSI-Brasil), 2015-2016. Variáveis endógenas * Variáveis exógenas Homens Mulheres OR * IC95% CP # OR *** IC95% CP # Condição de saúde ao longo da vida Condição social ao longo da vida 2,19 1,86-2,58 0,37 1,92 1,65-2,24 0,32 Esforço físico intenso no trabalho ao longo da vida Condição social ao longo da vida - - - 0,24 0,17-0,33 -0,61 Aposentadoria Esforço físico intenso no trabalho ao longo da vida - - - 2,09 1,70-2,58 0,36 Idade na data da entrevista - - - 11,69 9,59- 14,25 0,84 Capacidade para o trabalho Condição social ao longo da vida 2,09 1,82-2,40 0,35 1,99 1,66-2,38 0,33 Condição de saúde ao longo da vida 3,61 3,06-4,26 0,57 3,89 3,23-4,56 0,59 Interação social primária - - - 1,49 1,26-1,77 0,20 Interação social primária Condição social ao longo da vida - - - 1,69 1,41-2,02 0,26 Interação social secundária Condição social ao longo da vida 1,71 1,42-2,05 0,26 - - Trabalho remunerado Condição social ao longo da vida - - - 1,69 1,40-2,03 0,26 Aposentadoria - - - 0,39 0,32-0,47 -0,44 Capacidade para o trabalho 3,23 2,71-3,86 0,53 1,63 1,37-1,95 0,24 Interação social secundária 1,53 1,30-1,81 0,21 - - - Vive com cônjuge/companheiro(a) 1,47 1,17-1,86 0,19 - - - Idade na data da entrevista 0,46 0,34-0,61 -0,37 0,34 0,27-0,42 -0,49 CP: coeficiente padronizado; IC95%: intervalo de 95% de confiança; OR: odds ratio. Nota: N = 8.903 (3.942 homens e 4.961 mulheres). * Variáveis endógenas: variáveis dependentes para cada regressão logística; Variáveis exógenas: variáveis explicativas para cada regressão logística; * OR ajustada por todas as variáveis listadas como explicativas pela regressão logística, com variância robusta; # CP = (OR – 1)/(OR + 1). Tabela 3 Resultados do modelo de equação estrutural com as associações diretas, indiretas e totais das variáveis associadas ao trabalho remunerado, segundo o gênero. Estudo Longitudinal da Saúde dos Idosos Brasileiros (ELSI-Brasil), 2015-2016. Associação das variáveis Homens Mulheres CP associação direta CP Associação indireta CP Associação total CP Associação direta CP Associação indireta CP Associação total * CS → TR - - 0,352 0,257 0,232 0,489 CS → CT → TR - 0,186 - - 0,079 - CS → OCUP → APOS → TR - - - - 0,076 - CS → SAÚDE → CT → TR - 0,111 - - 0,045 - CS → PRIMÁRIA → CT → TR - - - - 0,012 - CS → SECUNDÁRIA → TR - 0,055 - - - - SAÚDE → CT → TR - 0,298 - - 0,142 - CT → TR 0,527 - - 0,249 - - APOS → TR - - - -0,439 - - OCUP → APOS → TR - - - - -0,156 - PRIMÁRIA → CT → TR - - - - 0,047 - SECUNDÁRIA → TR 0,209 - - - - -i APOS: aposentadoria; CP: coeficiente padronizado ; CS: condição social ao longo da vida; CT: capacidade para o trabalho; APOS: aposentadoria; CP: coeficiente padronizado ; CS: condição social ao longo da vida; CT: capacidade para o trabalho; OCUP: esforço físico intenso no trabalho ao longo da vida; PRIMÁRIA: interação social primária; SAÚDE: condição de saúde ao longo da vida; SECUNDÁRIA: interação social secundária; TR: trabalho remunerado. Nota: N = 8.903 (3.942 homens e 4.961 mulheres). * Associação total = associação direta + associação indireta; ** CP = (OR – 1)/(OR + 1). * Associação total = associação direta + associação indireta; ** CP = (OR – 1)/(OR + 1). entre fatores relacionados ao trabalho (esforço físico intenso no trabalho ao longo da vida e aposen tadoria) e participação no trabalho remunerado. A condição social ao longo da vida parece afetar a participação no trabalho remunerado de homens e mulheres em idades avançadas 1. Uma pesquisa conduzida na Região Metropolitana de Belo Horizonte 21 mostrou que para homens e mulheres, tanto o rendimento, quanto o nível de esco laridade dos pais garante a educação dos filhos. Discussão Os resultados deste estudo mostraram que a participação no trabalho remunerado de homens e mulheres com 50 anos ou mais ocorre por mecanismos diferentes. A participação feminina foi influenciada diretamente pela condição social ao longo da vida, capacidade para o trabalho e aposen tadoria, e indiretamente pela condição de saúde ao longo da vida, esforço físico intenso no trabalho ao longo da vida e interação social primária. Por outro lado, a participação masculina no trabalho remu nerado foi influenciada diretamente pela capacidade para o trabalho e interação social secundária, e indiretamente pela condição social ao longo da vida e condição de saúde ao longo da vida. Segundo os resultados do modelo de equação estrutural, a variável que apresentou maior força de associação com a participação feminina no trabalho remunerado foi a condição social ao longo da vida, já que para os homens foi a capacidade para o trabalho. Para ambos os gêneros, a condição de saúde ao longo da vida apresentou associação indireta e positiva com o trabalho remunerado, mas a magnitude da força da associação para os homens foi duas vezes maior que para as mulheres. Os resultados do modelo de equação estrutural identificaram ainda diferenças de gênero em relação à interação social, com a participação dos homens no trabalho remunerado caracterizada pelo engajamento em atividades sociais organizadas ou associações civis, e a participação das mulheres caracterizada pela relação com parentes e amigos próximos. Além disso, somente entre as mulheres observou-se associação negativa Cad. Saúde Pública 2020; 36(11):e00194619 Cad. Saúde Pública 2020; 36(11):e00194619 Castro CMS et al. 10 Tabela 3 O que pode ser explicado pelo fato de que os homens tendem a exercer um trabalho mais desgastante fisicamente ao longo da vida, quando comparados às mulheres. Segundo um estudo baseado na teoria do capital social 26, o fato de um indivíduo pertencer a grupos ou associações (interação social secundária) afeta positivamente a probabilidade de ter uma maior participação no trabalho remunerado. Relacionamentos baseados em laços fracos (secundá rios), como aqueles encontrados entre os participantes de um grupo ou associação, são importantes pois conectam as pessoas a uma maior gama de informações e conhecimentos relativos a emprego. Apesar dos amigos próximos de um indivíduo serem mais motivados a ajudá-lo, os laços fracos estão estruturalmente localizados de tal modo a serem mais úteis no processo de busca por emprego 26. O presente estudo acrescentou a esses achados, uma vez que mostrou que a participação dos homens no trabalho remunerado está direta e positivamente associada à interação social secundária. Ao passo que, entre as mulheres, ter contato mais frequente com família e amigos (interação social primária) aumenta a probabilidade da participação feminina no trabalho remunerado, via capacidade para o trabalho 26. Parece que, para as mulheres, o contexto social formado pela família e amigos pode ser importante para a percepção da sua capacidade para o trabalho e consequente participação no traba lho remunerado. Isso, provavelmente, deve-se ao fato de que no modelo de família patriarcal, que é o predominante, o funcionamento da família é uma atribuição feminina. Uma interação mais forte das mulheres dentro de redes primárias pode, assim, garantir às mulheres de maior idade a segurança de que a funcionalidade familiar pode acontecer mesmo com sua ausência mais prolongada do ambiente doméstico. Nesse caso, não se pode descartar a possibilidade de causalidade reversa, uma vez que a interação social pode ser causa ou consequência da maior participação no trabalho remunerado. Somente entre as mulheres, a associação entre condição social ao longo da vida e trabalho remu nerado foi parcialmente mediada pelo esforço físico intenso no trabalho ao longo da vida e pela apo sentadoria. Mulheres que exerceram esforço físico intenso no trabalho ao longo da vida apresentaram maior probabilidade de estar aposentada e, consequentemente, menor probabilidade de participar do trabalho remunerado. Tabela 3 No entanto, a participação das mulheres no trabalho remunerado, em qualquer idade, depende mais do seu nível socioeconômico, em particular da esco laridade, em comparação ao nível socioeconômico dos pais (renda e escolaridade). O presente estudo mostrou que a condição social ao longo da vida apresentou associação direta com o trabalho remu nerado apenas entre as mulheres, além de registrar associação indireta via esforço físico intenso no trabalho ao longo da vida e aposentadoria. Esses resultados indicam a importância da condição social ao longo da vida para a participação feminina no trabalho remunerado. Independentemente de outros fatores, as mulheres com melhor condição social ao longo da vida apresentam maior probabilidade de participar do trabalho remunerado em idades avançadas. Além disso, os resultados evidenciam que mulheres com melhor condição social ao longo da vida apresentam menor probabilidade de exercer esforço físico intenso no trabalho ao longo da vida, levando à menor probabilidade de aposentadoria e, consequentemente, maior probabilidade de participar do trabalho remunerado. Em relação aos homens a condição social ao longo da vida apresentou somente associação indireta, via condição de saúde ao longo da vida e capacidade para o trabalho. Os resultados sugerem que homens com melhor condição social ao longo da vida registram melhor condição de saúde ao longo da vida, percepção da capacidade para o trabalho como boa e maior probabilidade de participar do trabalho remunerado. A saúde é considerada o principal fator determinante da capacidade para o trabalho 4,22, e um nível de saúde ruim aumenta a probabilidade de deterioração desta capacidade 23. Estudos mostram que independentemente de como a saúde é medida, seja objetiva ou subjetivamente 24, ela continua sendo um fator determinante da participação no trabalho remunerado em idades avançadas 25. Cad. Saúde Pública 2020; 36(11):e00194619 Cad. Saúde Pública 2020; 36(11):e00194619 DETERMINANTES DO TRABALHO REMUNERADO ENTRE OS MAIS VELHOS 11 Os resultados do presente estudo sugerem que, tanto homens quanto mulheres com melhores con dições de saúde ao longo da vida tendem a perceber sua capacidade para o trabalho como boa, apre sentando maior probabilidade de participar do trabalho remunerado. Os resultados do modelo de equação estrutural mostraram ainda que, para os homens, a força da associação entre condição de saúde ao longo da vida e participação no trabalho remunerado foi o dobro da magnitude da associação para as mulheres. Tabela 3 Há evidências de que os fatores psicossociais e físicos do trabalho, como alta demanda física e baixo controle, podem mediar a associação entre posição social de um indivíduo e o risco de aposentadoria por invalidez 2. Os resultados deste estudo reforçam a hipótese de que o traba lho remunerado pode representar uma sobrecarga para a saúde das mulheres quando a qualidade do emprego é baixa, e estar associada ao trabalho doméstico para a própria família 2,27. A principal vantagem deste estudo é a sua grande base populacional, representativa da população brasileira com 50 anos ou mais. Ao nosso conhecimento, este é o primeiro estudo baseado em amos tra nacional a analisar as relações entre os determinantes da participação no trabalho remunerado entre adultos mais velhos. Outra vantagem do estudo é o uso do modelo de equações estruturais, que permite analisar, simultaneamente, os fatores associados ao trabalho remunerado. Entre as limitações do estudo podemos citar o viés de informação e o viés de desejabilidade social, isto é, o desejo de transmitir uma imagem social e culturalmente aceitável. Outra limitação diz respeito à sua natureza transversal, que não permite estabelecer associações temporais para as variáveis relativas às condições atuais. Finalmente, uma terceira limitação do estudo é que a especificação do modelo final analisado não contou com uma medida geral de ajuste. Como recomendações finais, os resultados do modelo de equação estrutural mostraram a com plexidade da participação no trabalho remunerado de pessoas com 50 anos ou mais, principalmente em relação às diferenças de gênero. Para o grupo das mulheres, investimentos na condição social ao longo da vida, em particular na escolaridade, podem contribuir para aumentar a participação nas atividades econômicas. Além disso, estudos sobre a participação feminina no mercado de trabalho devem considerar o tipo de trabalho ao longo da vida e entender melhor o papel da aposentadoria na vida das mulheres. Por outro lado, a participação dos homens mais velhos no mercado de trabalho depende mais de melhorias nas condições de saúde ao longo da vida e na capacidade para o trabalho. Pesquisas que analisam a participação de adultos mais velhos no mercado de trabalho devem abordar Cad. Saúde Pública 2020; 36(11):e00194619 Cad. Saúde Pública 2020; 36(11):e00194619 Castro CMS et al. 12 Castro CMS et al. 12 uma perspectiva do curso de vida, considerando os fatores nos níveis micro, meso e macro. Colaboradores Todos os autores contribuíram igualmente para todas as fases do estudo e na elaboração e revisão do manuscrito. 1. Camarano AA, Kanso S, Fernandes D. Menos jovens e mais idosos no mercado de trabalho. In: Camarano AA, organizador. Novo regime demográfico: uma nova relação entre popula ção e desenvolvimento? Rio de Janeiro: Insti tuto de Pesquisa Econômica Aplicada; 2014. p. 377-406. Tabela 3 Além disso, devem verificar se as relações entre os fatores associados a essa participação se mantêm ao longo do tempo. Concluindo, apesar de diferenças importantes nos determinantes do trabalho remunerado entre homens e mulheres mais velhos, as condições de saúde ao longo da vida apresentaram associações indiretas e positivas em ambos os gêneros, via capacidade para o trabalho. Esses resultados sugerem que melhorias nas condições de saúde em todas as idades têm o potencial de aumentar a participação de adultos mais velhos brasileiros no mercado de trabalho. Informações adicionais 2. Hasselhorn HM, Apt W. Understanding em ployment participation of older workers: cre ating a knowledge base for future labour mar ket challenges. http://www.jp-demographic. eu/ (acessado em 10/Mai/2018). ORCID: Camila Menezes Sabino de Castro (0000- 0002-5383-9796); Maria Fernanda Lima-Costa (0000-0002-3474-2980); Jorge Alexandre Barbo sa Neves (0000-0003-4141-2282); Fabíola Bof de Andrade (0000-0002-3467-3989); Rosana Ferreira Sampaio (0000-0002-4775-9650). 3. 3. Nonato F, Pereira RHM, Nascimento PAMM, Araújo TC. O perfil da força de trabalho bra sileira: trajetórias e perspectivas. Boletim de mercado de trabalho – conjuntura e análise. Rio de Janeiro: Instituto de Pesquisa Econômi ca Aplicada; 2012. DETERMINANTES DO TRABALHO REMUNERADO ENTRE OS MAIS VELHOS 13 13 8. Ybema, JF, Geuskens GA, van den Heuvel S, Wind A, Leijten FRM, et al. Study on Transi tions in Employment, Ability and Motivation (STREAM): the design of a four-year longitu dinal cohort study among 15.118 persons aged 45 to 64 years. British Journal of Medicine & Medical Research 2014; 4:1383-99. 18. Lima-Costa MF, Andrade FB, Souza Jr. PRB, Neri AL, Duarte YAO, Castro-Costa E, et al. The Brazilian Longitudinal Study of Aging (ELSI-Brazil): objectives and design. Am J Epi demiol 2018; 187:1345-53. 19. Granovetter M. The strength of the weak ties. Am J Sociol 1973; 78:1360-80. Medical Research 2014; 4:1383-99. 20. Silva AAM, Vasconcelos AGG, Bettiol H, Bar bieri MA. Socioeconomic status, birth weight, maternal smoking during pregnancy and adiposity in early adult life: an analysis us ing structural equation modeling. Cad Saúde Pública 2010; 26:15-29. 9. Ploubidis GB, Silverwood RJ, DeStavola B, Grundy E. Life-course partnership status and biomarkers in midlife: evidence from the 1958 British Birth Cohort. Am J Public Health 2015; 105:1596-603. 10. World Health Organization. Active ageing: a policy framework, 2002. http://www.who. int/ageing/publications/en (acessado em 26/ Abr/2018). 21. Aguiar NF, Fernandes DC, Neves JAB. Mobi lidade social feminina. In: Aguiar NF, organi zador. Desigualdades sociais, redes de sociabi lidade e participação política. v. 1. Belo Hori zonte: Editora da UFMG; 2007. p. 165-80. 11. Bem-Shlomo Y, Kuh D. A life course approach to chronic disease epidemiology: conceptual models, empirical challenges and interdis ciplinar perspectives. Int J Epidemiol 2002; 31:285-93. 22. Martinez MC, Latorre MRDO. Saúde e capa cidade para o trabalho em trabalhadores de área administrativa. Rev Saúde Pública 2006; 40:851-8. 12. Pérez ER, Wajnman S, Oliveira AMHC. Análi se dos determinantes da participação no mer cado de trabalho dos idosos em São Paulo. Rev Bras Estud Popul 2006; 23:269-86. 23. Gould R, Ilmarinen J, Järvisalo J, Koskinen S, editors. Dimension of work ability: results of the health 2000 survey. Helsinki: Finnish Cen tre of Pensions/The Social Insurance Institu tion/National Public Health Institute/Finnish Institute of Occupational Health; 2008. 13. Castro CMS, Mambrini JVM, Sampaio RF, Macinko J, Lima-Costa MF. Aspectos socio demográficos e de saúde associados ao tra balho remunerado em adultos (50-69 anos) na Região Metropolitana de Belo Horizonte, Minas Gerais, Brasil. Cad Saúde Pública 2015; 31:1775-87. 24. Lima-Costa MF, Cesar CC, Chor D, Proietti FA. Agradecimentos M. F. F. L. Castro, J. A. B. Neves e R. F. Sampaio são bolsistas de produtividade em pesquisa do Con selho Nacional de Desenvolvimento Científico e Tecnológico, Ministério da Saúde (DECIT/SCTIE); COSAPI/DAPES/SAS; Ministério da Ciência, Tec nologia, Inovação e Comunicação. p 4. Ilmarinem JE. Aging workers. https://oem. bmj.com/content/58/8/546 (acessado em 29/ Jul/2019). 5. Department of Economic and Social Affairs, United Nations. World population ageing 2017. http://esa.un.org/wpp (acessado em 10/ Mai/2018). 6. Instituto Brasileiro de Geografia e Estatística. Pesquisa Nacional por Amostra de Domicílios Contínua. 3o trimestre de 2019. Rio de Janeiro: Instituto Brasileiro de Geografia e Estatística; 2019. 7. Bélanger A, Carrière Y, Sabourin P. Under standing employment participation o folder workers: the Canadian perspective. Can Public Policy 2016; 42:94-109. Cad. Saúde Pública 2020; 36(11):e00194619 Cad. Saúde Pública 2020; 36(11):e00194619 DETERMINANTES DO TRABALHO REMUNERADO ENTRE OS MAIS VELHOS 13 Cad. Saúde Pública 2020; 36(11):e00194619 Abstract This study aimed to develop a conceptual model and to explore direct and indirect associations be tween paid work and life-course factors in a repre sentative national sample of the Brazilian popula tion 50 years and older. The analysis was based on 8,903 participants in the baseline of the Brazilian Longitudinal Study of Aging (ELSI-Brazil). The exposure variables were sociodemographic, health, work, and social interaction variables. Based on a structural equation model, paid work showed total association with lifetime social status in women (standardized coefficient – SC = 0.489) and direct association with capacity for work in men (SC = 0.527). For women alone, an indirect and negative association was observed with life time intense physical effort at work, via retirement (SC = -0.156). Men with paid work were more prone to social participation, measured by belong ing to groups or associations (SC = 0.209). Among women, this participation was through interaction with family and friends (SC = 0.047), via capac ity for work. For both men and women, lifetime health status showed an indirect and positive asso ciation (SC = 0.298 men; SC = 0.142 women) with paid work, via capacity for work. All the above- mentioned factors showed a significant associa tion with paid work. The study’s results showed that participation in the work market occurs by different mechanisms for men and women, prin cipally considering the factors related to work and social interaction. El objetivo de este estudio fue desarrollar un mod elo conceptual y explorar asociaciones directas e indirectas entre trabajo remunerado y factores que operan a lo largo de la vida, en una muestra na cional representativa de población brasileña con 50 años y más. El análisis se basó en 8.903 partici pantes procedentes del Estudio Brasileño Longi tudinal del Envejecimiento (ELSI-Brasil). Los factores de exposición fueron: sociodemográficos, de salud, trabajo e interacción social. Utilizando un modelo de ecuaciones estructurales, se observó que el trabajo remunerado presentó una aso ciación total con la condición social a lo largo de la vida entre mujeres (coeficiente de estandarización- CP = 0,489) y asociación directa con la capacidad para el trabajo entre hombres (CP = 0,527). Sola mente para las mujeres, la asociación indirecta y negativa se observó con el esfuerzo físico intenso en el trabajo a lo largo de la vida, vía jubilación (CP = -0,156). DETERMINANTES DO TRABALHO REMUNERADO ENTRE OS MAIS VELHOS 13 Self-rated health compared with objective ly measured health status as a tool for mortal ity risk screening in older adults: 10-year fol low-up of the Bambuí Cohort Study of Aging. Am J Epidemiol 2012; 175:228-35. 14. Wind A, Geuskens GA, Ybema JF, Bongers PM, van der Beek AJ. The role of ability, motiva tion, and opportunity to work in the transition from work to early retirement – testing and optimizing the Early Retirement Model. Scand J Work Environ Health 2015; 41:24-35. 25. Castro CMS, Lima-Costa MF, César CC, Ne ves JAB, Bof de Andrade F, Souza-Junior PRB, et al. Curso da vida e capacidade para o traba lho entre adultos mais velhos: ELSI-Brasil. Rev Saúde Pública 2018; 52 Suppl 2:11s. 15. Kupek E. Beyond logistic regression: struc tural equations modelling for binary variables and its application to investigating unobserved confounders. BMC Med Res Methodol 2006; 6:13. 26. Neves JAB, Helal DH. Associativismo, capital social e mercado de trabalho. In: Aguiar NF, organizador. Desigualdades sociais, redes de sociabilidade e participação política. v. 1. Belo Horizonte: Editora da UFMG; 2007. p. 61-72. 16. Amorim LDAF, Fiaccone RL, Santos CAST, Santos TN, Moraes LTLP, Oliveira NF, et al. Structural equation modeling in epidemiology. Cad Saúde Pública 2010; 26:2251-62. 27. Barata RB. Relações de gênero e saúde: desi gualdade ou discriminação? In: Barata RB. Co mo e por que as desigualdades sociais fazem mal à saúde. Rio de Janeiro: Editora Fiocruz; 2009. p. 73-94. 17. Morais AODS, Simões VMF, Rodrigues LS, Batista RFL, Lamy ZC, Carvalho CA, et al. Sin tomas depressivos e de ansiedade maternos e prejuízos na relação mãe/filho em uma coorte pré-natal: uma abordagem com modelagem de equações estruturais. Cad Saúde Pública 2017; 33:e00032016. Cad. Saúde Pública 2020; 36(11):e00194619 Castro CMS et al. 14 Cad. Saúde Pública 2020; 36(11):e00194619 Recebido em 04/Out/2019 Versão final reapresentada em 14/Fev/2020 Aprovado em 01/Abr/2020 Envejecimiento; Trabajo; Modelos Estadísticos; Situación Socioeconómica; Interacción Social Abstract Hombres con trabajo remunerado fueron más propensos a la participación social, medida por la pertenencia a grupos o asociaciones (CP = 0,209). Entre mujeres, esta participación se produjo mediante interacciones con la familia y amigos (CP = 0,047), vía capacidad para el tra bajo. Tanto entre hombres como entre mujeres, la condición de salud a lo largo de la vida mostró una asociación indirecta y positiva (CP = 0,298 hombres; CP = 0,142 mujeres) con el trabajo re munerado, vía capacidad para el trabajo. Todos los factores mencionados presentaron una asociación significativa con el trabajo remunerado. Los resul tados del estudio mostraron que la participación en el mercado de trabajo de hombres y mujeres mayores se produce por mecanismos diferentes, principalmente al considerar los factores relacio nados con el trabajo e interacción social. Aging; Work; Statiscal Models; Socieconimic Status; Social Interaction Envejecimiento; Trabajo; Modelos Estadísticos; Situación Socioeconómica; Interacción Social Recebido em 04/Out/2019 Versão final reapresentada em 14/Fev/2020 Aprovado em 01/Abr/2020 Cad. Saúde Pública 2020; 36(11):e00194619
https://openalex.org/W4281257337	https://quantum-journal.org/papers/q-2023-09-21-1117/pdf/	English	null	Anonymous conference key agreement in linear quantum networks	arXiv (Cornell University)	2,022	cc-by	15,153	Anonymous conference key agreement in linear quantum networks Jarn de Jong1, Frederik Hahn1,2, Jens Eisert2, Nathan Walk2, and Anna Pappa1 1Electrical Engineering and Computer Science, Technische Universität Berlin, 10587 Berlin, Germany 2Dahlem Center for Complex Quantum Systems, Freie Universität Berlin, 14195 Berlin, Germany Sharing multi-partite quantum entanglement between parties allows for diverse secure communication tasks to be performed. Among them, conference key agreement (CKA) – an extension of key distribution to multiple parties – has received much attention recently. Interestingly, CKA can also be performed in a way that protects the identities of the participating parties, therefore pro- viding anonymity. In this work, we propose an anonymous CKA protocol for three parties that is implemented in a highly practical network setting. Specif- ically, a line of quantum nodes is used to build a linear cluster state among all nodes, which is then used to anonymously establish a secret key between any three of them. The nodes need only share maximally entangled pairs with their neighbours, therefore avoiding the necessity of a central server sharing entan- gled states. This linear chain setup makes our protocol an excellent candidate for implementation in future quantum networks. We explicitly prove that our protocol protects the identities of the participants from one another and per- form an analysis of the key rate in the finite regime, contributing to the quest of identifying feasible quantum communication tasks for network architectures beyond point-to-point. v:2205.09169v2 [quant-ph] 5 Sep 2023 arXiv:2205.09169v2 [quant-ph] 5 Sep arXiv:2205.09169v2 [quant-ph Jarn de Jong: dejong@tu-berlin.com Accepted in Quantum 2023-09-05, click title to verify. Published under CC-BY 4.0. 1 Introduction The goal of conference key agreement (CKA) protocols is to establish a shared key between multiple parties that do not use trusted means of communication. CKA has been explored in the quantum domain, with the aim of developing new schemes for cryptography and communication beyond bi-partite key distribution [1, 2, 3, 4]. Various quantum states have been proposed to achieve CKA, including both discrete-variable [5, 6, 7] and continuous- variable [8, 9, 10] states. In this work, we focus on the discrete-variable case. Particularly suitable for CKA protocols are quantum resource states that exhibit symmetric measure- ment outcome correlations for all participants of the respective communication protocol. Such states include GHZ states [11], which are ideal CKA resources due to their obvious correlation when measured in the computational basis [1, 5, 6]. Other useful symmetric resources are W states [12], although they only allow for the probabilistic generation of a conference key [7]. In recent years, the need for privacy and anonymity has led researchers to develop anonymous versions of protocols that implement important cryptographic primitives [13, 14, 15]. In the case of CKA, the goal would be to establish a key between a number of participants, while keeping their identities hidden from the other non-participating parties, Jarn de Jong: dejong@tu-berlin.com 1 Accepted in Quantum 2023-09-05, click title to verify. Published under CC-BY 4.0. and in some instances even from each other. Using shared GHZ states, anonymous proto- cols have been proposed [16, 17] and implemented [18] that also provide an advantage in keyrate compared to sharing bi-partite entanglement [17]. However, GHZ states are highly loss-prone and cannot be easily exchanged over long distances. Moreover, most previous approaches using these states require a central server to distribute the state. In this work, we address and answer the question of how to achieve anonymous CKA within a minimal and in several ways experimentally feasible nearest neighbour architec- ture of discrete variable quantum states. Specifically, we assume a linear chain of quantum nodes1, and study how to anonymously share a secret key between three of them. These three participants (also referred to as Alice, Bob and Charlie) use shared bi-partite entan- glement with their neighbours to establish a linear cluster state [19, 20, 21] that ‘connects’ the three of them and all the nodes in between. 1 Introduction This linear cluster state is further used to anonymously establish a three-partite maximally entangled state between Alice, Bob and Charlie, which is used to run a conference key agreement protocol and share a key with provable security. Our new protocol is one of the few cryptographic demonstrations where non-maximally entangled states are used for practical tasks. It highlights the flexibility that multi-partite entanglement provides for cryptographic tasks involving more than two parties and complements known results supporting this approach [16, 17, 22, 23, 24]. [ ] This work is structured as follows. In Subsection 1.1 we provide the necessary pre- liminaries and definitions to subsequently introduce the protocol in Section 2. Section 3 contains an analysis and explanation of the protocol and its performance, specifically how anonymity and security are achieved, as well as how the (finite) key rate is calculated. Section 4 contains a general discussion. Some details of the protocol, as well as the proofs of security and anonymity have been deferred to Appendices A, B and C, respectively. Accepted in Quantum 2023-09-05, click title to verify. Published under CC-BY 4.0. 1.1 Preliminaries and definitions Throughout this work, we denote with the ordered set N = {N1, N2, . . . , Nn−1, Nn} the collection of all nodes in a linear network (Fig. 1). We fix a set of three nodes P = {Na, Nb, Nc} ⊂N such that a < b < c and call them Alice, Bob and Charlie, respectively. The goal of these three parties is to establish a secret key between them, without revealing their identities to everyone else. We will refer to them as the participants and to all other nodes ¯P := N \ P as the non-participants of our anonymous conference key agreement protocol. N1 N2 · · · Na−1 Na Na+1 · · · Nb−1 Nb Nb+1 · · · Nc−1 Nc Nc+1 · · · Nn Alice Bob Charlie Figure 1: Participants P = {Na, Nb, Nc} and non-participants ¯P are connected by a linear network. The position of Alice, Bob and Charlie are not known by any other node in the network. Figure 1: Participants P = {Na, Nb, Nc} and non-participants ¯P are connected by a linear network. The position of Alice, Bob and Charlie are not known by any other node in the network. 1Note that “linear chain” setups often refer to quantum repeater setups involving quantum memories. Our protocol however does not require quantum memories. The feasibility of implementing this protocol in the short term, and the relevance of the photonics experiments discussed in Section 4, are due to the fact that the setup is a linear chain of source and measurement stations without quantum memories, rather than a traditional linear repeater chain. Accepted in Quantum 2023-09-05, click title to verify. Published under CC-BY 4.0. 2 Alice, Bob and Charlie know each other’s position within the network and can iden- tify the communication from each Ni with its index i. Moreover, they have access to a pre-shared secret key; note that our scheme is a key-expansion scheme, like all known quantum key distribution and CKA protocols. We assume that the participants are coop- erating: neither do they reveal each other’s identity nor do they reveal the created secret key. However, we allow the non-participants to be dishonest and to actively deviate from the protocol – as long as they do not collude with each other. 2 Protocol Our protocol is divided into three parts – the preparation of the required multi-partite states from the Bell pairs (2.1), the anonymous extraction of the GHZ3 states from the multi-partite states (2.2), and the subsequent key generation with post-processing (2.3). 1.1 Preliminaries and definitions This means that non- participants can perform arbitrary maps and measurements in deviating bases or disclose false measurement outcomes, but they cannot, jointly with other non-participants, perform a coordinated attack. Regarding initial resources, we assume that all nodes share one copy of an entangled (potentially noisy) Bell pair – i.e. (\|0, 0⟩+\|1, 1⟩)/ √ 2 – with each of their neighbours: node Ni therefore holds two qubits with labels τi and ωi. Qubit τi is entangled with qubit ωi+1 from node Ni+1 while qubit ωi is entangled with qubit τi−1 from Ni−1. Since nodes N1 and Nn both have a single neighbour, they only have a qubit τ1 and ωn, respectively. These Bell pairs are used to create three linear cluster states between the nodes. For a set of qubits {1, 2, . . . , n}, we define the n-qubit linear cluster state vector \|L1,...,n⟩:= n−1 Y i=1 CZi,i+1 \|+⟩⊗n , (1) (1) where \|+⟩⊗n := Nn i=1 \|+⟩i and CZi,i+1 is the controlled-Z quantum gate that is applied between neighbouring qubits labelled i and i + 1. Finally, from the linear cluster state, a three-partite GHZ state with state vector \|GHZ3⟩:= 1 √ 2 (\|0, 0, 0⟩+ \|1, 1, 1⟩) (2) (2) is extracted in an anonymous fashion. Fig. 2 depicts a visualization of our protocol. For clarity, throughout this work, we denote a unitary Pauli operation with a capital letter, where a superscript indicates a real power (e.g. Zb is a unitary Z operation for b = 1 and I for b = 0). To indicate measurement bases, we use the σ-notation, where a superscript indicates the qubit (i.e. σi x refers to a measurement of qubit i in the Hadamard basis). 2.1 Preparation of linear cluster states From the Bell resources, we create the network state vector \|N⟩:= \|Ll⟩⊗\|Lm⟩⊗\|Lr⟩, where \|Ll⟩, \|Lm⟩and \|Lr⟩– with l, m, r denoting left, middle and right – are linear cluster states on the qubits labelled {τ1, τ2, . . . , τa−1, ωa}, {τa, τa+1, . . . , τc−1, ωc} and {τc, τc+1, . . . , τn−1, ωn}, respectively (Fig. 2 top and middle), by performing Protocol 1 for State preparation. Protocol 1 is divided into three steps. Some nodes execute only a subset of the steps and a small selection of nodes execute a variant of Step 2a, listed as Step 2b. These considerations are reflected in Tab. 1, which indicates which nodes perform which steps. Accepted in Quantum 2023-09-05, click title to verify. Published under CC-BY 4.0. 3 3 · · · · · · · · · · · · · · · · · · · · · · · · y 1 · · · · · · · · · · · · · · · · · · · · · · · · y 2 · · · · · · · · · · · · N1 N2 . . . Na−1 Na Na+1 . . . Nb . . . Nc−1 Nc Nc+1 . . . Nn−1 Nn Figure 2: Top: Bell pairs shared between nodes Ni and Ni+1. Qubit τi (dark gray) of Ni and ωi+1 (light gray) of Ni+1 are entangled. The three qubits to be part of the GHZ state are colored green, blue and pink. Middle: In Protocol 1, the Bell resources are used to create three linear cluster states via Bell state projection. Alice and Charlie do not perform the projection. Bottom: In Protocol 2 the network states \|N⟩are transformed into \|GHZ3⟩states between Na, Nb and Nc. Figure 2: Top: Bell pairs shared between nodes Ni and Ni+1. Qubit τi (dark gray) of Ni and ωi+1 (light gray) of Ni+1 are entangled. The three qubits to be part of the GHZ state are colored green, blue and pink. Middle: In Protocol 1, the Bell resources are used to create three linear cluster states via Bell state projection. Alice and Charlie do not perform the projection. Bottom: In Protocol 2 the network states \|N⟩are transformed into \|GHZ3⟩states between Na, Nb and Nc. Protocol 1: State preparation Input. Bell pairs between nodes Ni and Ni+1. Goal. 2.1 Preparation of linear cluster states Prepare the network state vector \|N⟩. All Ni perform the following steps consecutively: 1. Receive oi−1. If oi−1 = 1 apply Z on ωi. 2a. Perform CZ between τi and ωi. Measure στi x and record measurement outcome bit as oi. 2b. Draw uniformly random bit oi. If oi = 1 apply Z on τi. Apply H on τi. 3. Send oi to Ni+1. Table 1: Protocol 1 Overview Not all steps of Protocol 1 are performed by everyone. This table indicates which steps are performed by whom. Note that since N1 does not perform 1. and Nn does not perform 3. there is neither o0 nor Nn+1. Node N1 Na Ni Nc Nn 1. 2a. 2b. 3. Table 1: Protocol 1 Overview Accepted in Quantum 2023-09-05, click title to verify. Published under CC-BY 4.0. Protocol 1: State preparation Not all steps of Protocol 1 are performed by everyone. This table indicates which steps are performed by whom. Note that since N1 does not perform 1. and Nn does not perform 3. there is neither o0 nor Nn+1. Node N1 Na Ni Nc Nn 1. 2a. 2b. 3. After discarding all measured qubits, each node but Alice and Charlie holds only one qubit. Therefore, we can rename τi →i for all Ni ∈N \ {Na, Nc, Nn} and ωn →n. For Alice and Charlie we rename τa and ωc to a and c as part of \|Lm⟩and ωa and τc to ˜a and ˜b as part of \|Ll⟩and \|Lr⟩, respectively. From the network state we will now show how to anonymously extract a GHZ3 state for Alice, Bob and Charlie. Protocol 1 and the above discussion is phrased under the assumption that neither Alice nor Charlie are at the ‘ends’ of N. If indeed Na = N1, Alice performs the steps of the column marked N1 in Tab. 1, and similarly for Charlie if Nc = Nn. Note that Alice and/or Charlie then also hold just one qubit. As a small example, consider a network with only five nodes, where P = {Na, Nb, Nc} = {N2, N4, N5} and thus ¯P = {N1, N3}. Note that Nn = Nc. All nodes except the first apply Z to their qubit ωi based on the outcome bit oi−1 they received. Any node other than Na, Accepted in Quantum 2023-09-05, click title to verify. Published under CC-BY 4.0. 4 4 Nc or the first or last node performs step 2a.: they apply a CZ gate and then measure στi x , sending the outcome oi to the next node. To disguise that they are not performing this step, N1 and Na apply Z to their τi based on randomly drawn bit oi and send it to the next node. Moreover, they apply H to τi. Since Nc is the last node, they only perform step 1. 2.2 Anonymous extraction of GHZ states After the n network nodes have created L network states as presented in Protocol 1, each of these states is used to anonymously establish a \|GHZ3⟩state between Alice, Bob and Charlie via Protocol 2 for GHZ extraction, as visualized in Fig. 2. Protocol 2: GHZ extraction Input. Network state vector \|N⟩from Protocol 1. Configuration corrections {Ci}i∈P calculated by Ni, as explained in App. A. Goal. Anonymous \|GHZ3⟩for Na, Nb, Nc. All Ni perform the following steps consecutively: 1. Receive bit βi−1 and compute βi = βi−1⊕1. 2a. Measure σi x or σi y if βi is 0 or 1, respectively. Record the measurement outcome bit mi. 2b. Draw a uniformly random bit mi. If i ∈P: apply Ci. 3. Communicate βi to node Ni+1. Table 2: Protocol 2 Overview N1 draws a uniformly ran- dom bit β1; Nn does not perform Step 3. Node N1 P ¯P Nn 1. ✗ ✓ ✓ ✓ 2a. ✗ ✗ ✓ ✗ 2b. ✓ ✓ ✗ ✓ 3. ✓ ✓ ✓ ✗ Protocol 2: GHZ extraction Accepted in Quantum 2023-09-05, click title to verify. Published under CC-BY 4.0. 2.3 Measurements and post-processing The participants now use a fraction p of the L GHZ3 states to check for eavesdropping and the rest to generate conference keys. Using L · h2(p) bits of the pre-shared key – where h2(p) := −p log2(p) −(1 −p) log2(1 −p) is the binary entropy of p – Alice, Bob, and Charlie coordinate their measurements to be in either the σz-basis (for KeyGen rounds) or σx-basis (for Verification rounds). During the latter, ⟨m⟩:= L · p states are measured to estimate the σx-basis error rate is the binary entropy of p – Alice, Bob, and Charlie coordinate their measurements to be in either the σz-basis (for KeyGen rounds) or σx-basis (for Verification rounds). During the latter, ⟨m⟩:= L · p states are measured to estimate the σx-basis error rate Qm X := 1 2 1 −⟨σa xσb xσc x⟩ , that is the relative number of erroneous (i.e. odd-parity) measurements. Every party announces a uniformly random bit after every KeyGen and every Verification round – with the exception of Bob and Charlie who announce their measurement outcome after each Verification round; this allows Alice to calculate Qm X. When Alice determines that Qm X is above a predetermined tolerance threshold Qtol, she sets her abort bit to 1 to abort the protocol. that is the relative number of erroneous (i.e. odd-parity) measurements. Every party announces a uniformly random bit after every KeyGen and every Verification round – with the exception of Bob and Charlie who announce their measurement outcome after each Verification round; this allows Alice to calculate Qm X. When Alice determines that Qm X is above a predetermined tolerance threshold Qtol, she sets her abort bit to 1 to abort the protocol. The other ⟨k⟩:= L −⟨m⟩= L · (1 −p) states are used to generate conference keys by Alice, Bob, and Charlie measuring in the σz-basis. This results in k bits of raw key for each participant which is then post-processed with error correction and privacy amplification. i To perform error correction, Alice applies a publicly known error-correcting code to her raw key and encrypts the resulting error syndrome with a one-time pad using a portion of the pre-shared key. Alice now announces the encrypted syndrome, while everyone else announces a string of random bits of the same length. Protocol 2: GHZ extraction Table 2: Protocol 2 Overview Input. Network state vector \|N⟩from Protocol 1. Configuration corrections {Ci}i∈P calculated by Ni, as explained in App. A. Goal. Anonymous \|GHZ3⟩for Na, Nb, Nc. N1 draws a uniformly ran- dom bit β1; Nn does not perform Step 3. All Ni perform the following steps consecutively: Node N1 P ¯P Nn 1. ✗ ✓ ✓ ✓ 2a. ✗ ✗ ✓ ✗ 2b. ✓ ✓ ✗ ✓ 3. ✓ ✓ ✓ ✗ Protocol 2 is divided into three steps as well; some nodes only execute a subset of the steps and for Step 2 there are again two different options; this is reflected in Tab. 2. After a given amount of time for everyone to measure their qubit, all nodes broadcast their mea- surement outcome mi. The participants P then perform local unitary corrections on their own qubits based on the number of nodes between Alice, Bob and Charlie as well as the collection of measurement outcomes {mi}i∈¯P, resulting in L GHZ3 states shared between them. These corrections can be found in App. A and consist of Clifford operations only. Importantly, the corrections invoked by the measurement outcomes can be accounted for in post-processing, so that all actions of the protocol can be carried out simultaneously; this ensures no quantum memories are necessary. The final steps that enable secure anonymous conference key agreement are explained in the next section. Similarly to the previous one, Protocol 2 is phrased under the assumption that neither Alice nor Charlie are at the ‘ends’ of N. If indeed Na = N1, Alice performs the steps of the column marked N1 in Tab. 1, and similarly for Charlie if Nc = Nn. Note that Alice and/or Charlie then also hold just one qubit. Consider again the example where P = {Na, Nb, Nc} = {N2, N4, N5}, ¯P = {N1, N3} and Nn = Nc. To start Protocol 2, N1 draws uniformly random bits β1 and m1. They send β1 to N2. Here, N2 is a participant and therefore does not measure their qubit after calculating β2 = β1 ⊕1. Instead they apply C2, draw the bit m2 uniformly and send β2 to 5 N3. The following node N3 is again not a participant. It flips the received bit, measures it, and records the result as m3 before sending β3 to N4. Protocol 2: GHZ extraction The node N4 now acts as the first participant N2: apply C4, compute β4, draw m4 and send β4 to N5. This last participant N5 is now in a special position at the end of the network, which allows them to skip the last step and just to apply C5, to compute β5 and to draw m5. Accepted in Quantum 2023-09-05, click title to verify. Published under CC-BY 4.0. 2Note that the CZ operation and the two subsequent measurements of each non-participant can be viewed as a measurement in the Bell-state basis, i.e. as a Bell-state projection, and therefore can be performed in one step. 3 Analysis of the protocol Our nearest neighbour state preparation ensures that no central server is needed to pro- vision resources. First, the parties create the linear cluster state by exchanging Bell pairs with their nearest neighbours, performing an entangling operation between their two qubits and subsequently measuring one. The non-participants then measure their remaining qubit in order to establish a GHZ3 state between the participants.2 The resulting corrections to obtain the GHZ3 are non-trivial only for Alice and Charlie: The correction for Alice de- pends only on the number of non-participants between Alice and Bob and the measurement outcomes {mi}b−1 i=a+1 of these nodes, while the correction for Charlie can be constructed by analogy. Importantly, the part of the correction that depends on the measurement results generates only Pauli corrections, so all these corrections can be considered as post- processing of KeyGen and Verification rounds of Alice and Charlie; this means that the participants do not have to wait for announcements from non-participants before perform- ing their measurements. 3.1 Anonymity of the protocol The definition of anonymity is taken from [17]. The protocol is defined to be εan-anonymous if, for any choice of participants, it is at most εan-close (in trace distance) to any state with a desired property. This property, adapted to our attack model, loosely states that the reduced state for any subset G ⊂N that does not contain the participants, is independent of the choice of participants P. For the rigorous definition and more details, see App. C. The alternating σx-σy measurement pattern of Step 2a in Protocol 2 only works when Alice and Charlie are at the respective ends of the linear cluster state. It is for this reason that we designed our extraction of the GHZ state to effectively only use the middle state \|Lm⟩. Since all non-participants perform steps that are independent of the position of Alice, Bob, and Charlie, the nodes {N1, . . . , Na−1} and {Nc+1, . . . , Nn} generate the linear cluster states \|Ll⟩and \|Lr⟩as a byproduct. Alice and Charlie not performing the Bell projection creates the tri-separable network state \|N⟩:= \|Ll⟩⊗\|Lm⟩⊗\|Lr⟩. While this could make their actions distinguishable from those of all other nodes, it is easy to see that the reduced quantum state of each node Ni is maximally mixed – even given all announced measurement results. In particular, this means that the reduced state of each node does not contain information about the identity of any other node in the network. The definition of anonymity is taken from [17]. The protocol is defined to be εan-anonymous if, for any choice of participants, it is at most εan-close (in trace distance) to any state with a desired property. This property, adapted to our attack model, loosely states that the reduced state for any subset G ⊂N that does not contain the participants, is independent of the choice of participants P. For the rigorous definition and more details, see App. C. i The alternating σx-σy measurement pattern of Step 2a in Protocol 2 only works when Alice and Charlie are at the respective ends of the linear cluster state. It is for this reason that we designed our extraction of the GHZ state to effectively only use the middle state \|Lm⟩. Since all non-participants perform steps that are independent of the position of Alice, Bob, and Charlie, the nodes {N1, . . . , Na−1} and {Nc+1, . . 2.3 Measurements and post-processing Bob and Charlie decrypt Alice’s error syndrome using their pre-shared key and then perform error correction on their respective keys using the publicly known error-correcting code. To verify the error correction, all participants apply a hash function hEC to their corrected key; Alice announces her outcome after encrypting it with a one-time pad using part of the pre-shared key, while everyone else announces a uniformly random bit string instead. Bob and Charlie now both decrypt Alice’s announced output and verify the error correction by comparing their output of hEC with Alice’s. If either Bob or Charlie find a discrepancy, they abort by setting their abort bit to 1; this ensures that the key they share is correct. To potentially abort the protocol, the participants each announce their abort bit, which is 1 if and only if they want to abort, while the non-participants all announce random bits. The participants encrypt again by one-time padding their bit each with a separate single bit of the pre-shared key, using another 3 bits in total. Finally, the participants perform privacy amplification to remove any correlation be- tween the key and a potential eavesdropper, thereby reducing the length of the key. To this end, they apply another hash function hPA; after subtracting the necessary key to replenish the pre-shared key for communication in subsequent rounds, this results results in a secure and correct key s of length ℓ(see App. B) shared by Alice, Bob, and Charlie if the protocol was not aborted. 6 3.1 Anonymity of the protocol . , Nn} generate the linear cluster states \|Ll⟩and \|Lr⟩as a byproduct. Alice and Charlie not performing the Bell projection creates the tri-separable network state \|N⟩:= \|Ll⟩⊗\|Lm⟩⊗\|Lr⟩. While this could make their actions distinguishable from those of all other nodes, it is easy to see that the reduced quantum state of each node Ni is maximally mixed – even given all announced measurement results. In particular, this means that the reduced state of each node does not contain information about the identity of any other node in the network. For noiseless scenarios, these announced measurement results {oi \| Ni ∈N\{Na, Nc, N1} are all uniformly random and uncorrelated – see App. C for a proof. To mask their iden- tity, the other three nodes therefore announce uniformly random bits oa, oc and o1. For noisy scenarios, the announced measurement bits remain uniformly random, if there is no noise bias in the σx or σy basis, as for e.g. depolarizing noise. If there were such a bias, it could however reveal some information about the participants’ identities, as the announced measurement results would be distinguishable from the uniformly random announcements of the participants. This can be avoided if the latter introduce some bias to their source of randomness, and hence mimic the non-uniformity of the announced measurement results of the non-participants; see Sec. 4 for more discussion. Furthermore, the neighbours to- the-right of the participants are not aware that the latter announce randomly chosen bits and will therefore perform Z corrections conditioned on them (i.e. Step 1 in Protocol 1); Accepted in Quantum 2023-09-05, click title to verify. Published under CC-BY 4.0. 7 hence Step 2b of the protocol works as “anti-correction” performed by Na, Nc and N1 on their part of the Bell pair. Similar to Protocol 1, during Protocol 2 all non-participants ¯P (except N1 and Nn) measure and announce their outcomes {mi \| Ni ∈¯P, i /∈{1, n}}, which are uniformly random and uncorrelated – see App. C for a proof. To hide their identity, the participants P as well as N1 and Nn announce a uniformly randomly drawn bit (in case of biased noise, the same considerations and solution as in Protocol 1 apply). In approaches where anonymity is of no concern, an error syndrome can be announced publicly. However, this syndrome might not be uniformly random and could potentially disclose Alice’s identity. 3.2 Security and performance of the protocol Following [1], we define the protocol to be εc-correct if the probability that the generated keys are the same for all participants is greater than 1−εc. Similarly, we define the protocol to be εs-secret if the output state is εs-close (in trace distance) to an ideal state where the key is uniformly random and uncorrelated. The protocol is then called (εc + εs)-secure. For more details and the rigorous definition, see App. B. In principle, all resulting GHZ3 states could be used for key generation by each par- ticipant measuring in the σz basis. However, to ensure secrecy of the key, it is of utmost importance that the states are verified, which is achieved by having all participants mea- sure their qubit in the σx basis instead. Crucially, the Verification rounds are selected such that potentially malicious non-participants do not learn of their selection. This is achieved by coordinating the Verification rounds in advance between Alice, Bob, and Charlie using secret communication. There are m = L · p Verification rounds and we therefore need L·h2(p) bits to coordinate their choices. As the k bits of the raw conference key resulting from the L·(1−p) KeyGen rounds are neither perfectly correlated nor secret, error correction and privacy amplification are required. Error correction. To make the key εc-correct they use a publicly known error-correcting code (e.g., a low-density parity-check code as in Ref. [25]) with an error syndrome of length lEC := k · h2(Qz). Here, Qz = maxi=B,C 1 2(1 −⟨σa zσi z⟩) is the maximum pairwise σz- basis error rate between Alice and Bob or Alice and Charlie; it is estimated in advance and thus regarded as a given parameter. Since the error syndrome is one-time padded, the participants use up lEC of the pre-shared key. To verify the error correction, the participants apply a hash function hEC, which is drawn from a family of two universal hash functions using a seed shEC. This seed is sourced from the pre-shared key3 and has length k −log2(εc) −1, which ensures an εc-correct key. The output of the hash function hEC is a bitstring of length log2( 2 εc ); the same amount is used from the pre-shared key to encrypt the outcome using one-time pad before Alice announces it. Privacy amplification. 3.1 Anonymity of the protocol Hence Alice one-time-pad encrypts the error syndrome so that her communication is indistinguishable from all other parties; the same reasoning applies to all other communication of the post-processing (i.e. the verification and abort), too. 3It should be noted that unlike the other uses of the pre-shared key, the same seed can be used in subsequent runs of the protocol and therefore it only needs to be determined once. 3.2 Security and performance of the protocol Privacy amplification works in a similar fashion: Alice, Bob and Charlie apply a hash function hPA drawn from a family of two-universal hash functions using a seed shPA of length k + lPA −1 sourced from the pre-shared key3, where lPA := k · 1 −h2(Qtol + µ εs−ε 2 ) +2−2 log2(ε) is the length of the output of the hash function. Accepted in Quantum 2023-09-05, click title to verify. Published under CC-BY 4.0. 8 Here, µ is a statistical correction and εs is the security parameter, while ε > 0 is a free parameter (see App. B for details). Note that in other approaches [6], privacy amplification also affects the lEC-parity bits that are transmitted during error correction. Since these have been encrypted with the pre-shared key in our approach, no leakage is possible and thus no privacy amplification is needed for these bits. Here, µ is a statistical correction and εs is the security parameter, while ε > 0 is a free parameter (see App. B for details). Note that in other approaches [6], privacy amplification also affects the lEC-parity bits that are transmitted during error correction. Since these have been encrypted with the pre-shared key in our approach, no leakage is possible and thus no privacy amplification is needed for these bits. Finally, the unencrypted announcement of the – not uniformly random – abort bits could reveal the identities of the participants. This is solved by all participants encrypting their abort bits with one-time pads; each participant uses a single bit of their pre-shared key to hide the correlation of the abort bits. In order to obtain an accurate key rate, we need to replenish the pre-shared key used as a one-time pad in the various communications: coordinating the verification rounds, the error correction’s syndrome and its verification, and the three bits for the abort communication, obtaining the secret key length ℓ(see App. B). Dividing by the number of rounds L, we obtain the key rate r := l/L with r = (1 −p) 1 −h2 Qtol + µ εs −ε 2 −h2(Qz) −h2(p) + 1 L log2 ε2εc −2 , where µ εs−ε 2 is a statistical correction (see App. B) and ε > 0 is a free parameter. 3.2 Security and performance of the protocol Note that for fixed εs > 0 and εc > 0 and given Qtol, Qz and L, one can optimise over p and ε. In the asymptotic limit (i.e. L →∞), not only the L-dependent terms of Eq. (3.2) vanish, but so do p and µ. We refer to Tab. 3 for a representation of the performance of the protocol in terms of the required number L of network states for a fixed key length ℓ, while Fig. 3 contains the achievable finite key rate r as a function of L. log10(ℓ) Qm x = 0.03 Qm x = 0.06 Qm x = 0.10 0 4.093e+04 1.277e+05 1.691e+06 3 4.652e+04 1.359e+05 1.709e+06 4 8.528e+04 1.992e+05 1.874e+06 5 3.420e+05 6.359e+05 3.222e+06 6 2.236e+06 3.731e+06 1.246e+07 Table 3: The number of network states L necessary to obtain vari- ous secret key lengths ℓ, for differ- ent σx-basis error rates Qm x (Qtol has been fixed at this value). Here, Qz is fixed at two thirds of Qm x to simulate white noise. The se- curity parameters εc and εs have both been fixed at 10−8 and the rates are optimised over p and ε. 4 Discussion In this work, we have investigated how to anonymously establish a secret key between three participants in a line of nearest-neighbour quantum nodes. We find that by sharing maximally entangled pairs that are then projected into linear cluster states, a secret key can be obtained without revealing the identity of the participants. This contrasts with previous approaches to anonymous conference key agreement [16, 17], which directly distribute large GHZ states that are not only error-prone but also rely on a central server. Although in this work we explicitly show how the three-partite GHZ state is generated using linear cluster states, this is in fact equivalent to performing Bell-state projections, as is for example common in quantum repeater schemes. There are other ways to extract Accepted in Quantum 2023-09-05, click title to verify. Published under CC-BY 4.0. 9 9 105 106 107 108 109 1010 Total number of rounds L −0.6 −0.4 −0.2 0.0 0.2 0.4 0.6 0.8 Finite key rate r Qm x = 0.120 Qm x = 0.105 Qm x = 0.090 Qm x = 0.075 Qm x = 0.060 Qm x = 0.045 Qm x = 0.030 Finite key rate r per total number of rounds L Figure 3: Finite key rate r as a function of the total number of network states L. Here, Qtol is fixed at various values of Qm x and Qz is fixed at two thirds of this value to simulate white noise. The blue dotted line is the asymptotic key rate for the minimum Qm x shown. Security parameters εc and εs have both been fixed at 10−8; the rates are optimised over p and ε. Finite key rate r per total number of rounds L Figure 3: Finite key rate r as a function of the total number of network states L. Here, Qtol is fixed at various values of Qm x and Qz is fixed at two thirds of this value to simulate white noise. The blue dotted line is the asymptotic key rate for the minimum Qm x shown. Security parameters εc and εs have both been fixed at 10−8; the rates are optimised over p and ε. smaller GHZ states from Bell pairs [26], however, these might not be as straightforward to perform anonymously. Accepted in Quantum 2023-09-05, click title to verify. Published under CC-BY 4.0. 4 Discussion After all measurements have taken place everyone can then announce their (potentially fabricated) bits. Since these secret measurements are in place of the actual measurements that need to be performed, the corresponding round cannot be used for either verification or key generation. As such, this approach would slightly diminish the keyrate. We now briefly discuss the prospects for implementation of these protocols via multi- partite photonic entanglement. The dominant cause of noise is typically presence of higher- order photon events for parametric down conversion (PDC) photon sources [18, 27, 25, 28, 29] and photon distinguishability for solid state sources [30, 31, 32], although other effects such as detector dark-counts and misalignment also contribute. Furthermore, for practical implementations it is important that the generated photons are at telecom wavelengths compatible with low loss transmission over optical fibre, which renders some multi-partite entanglement sources unsuitable. Regardless of the source of the noise, for the ACKA protocol the only quantity that ultimately matters are the measured QBER’s in the X and Z bases. In that sense our results in Fig. 3 already provide a detailed description of the tradeoff between the total tolerable noise and block size. An implementation of our ACKA protocol has already been carried out demonstrating positive asymptotic keyrates, albeit at non-telecom wavelengths [28]. Comparison with other experiment results shows that for moderate block-sizes the required noise thresholds are well within those that have already been demonstrated in state-of-the-art PDC sources deployed through optical fibre at telecom wavelengths for 4 and 6 photon entangled states [25, 29] showing that proof-of-principle demonstrations of this protocol are within the reach of present-day technology. Nevertheless, the observed rates are quite low due to losses, and these would only become more severe with increasing transmission distance and number of parties. This observation, coupled with previous work on the increasingly demanding noise thresholds for large scale multi-partite CKA [1, 5, 6, 7], suggest that a robust, large-scale implementation would require more sophisticated networks incorporating quantum repeaters or error-correction protocols [2, 3, 4]. We leave the generalization of our protocol to more than three participants as an open question: the extraction of larger GHZ states (i.e. more than three qubits) from linear cluster states is possible (see Ref. [33, 34]). However, the size of the larger GHZ state is bounded from above by roughly half of the number of nodes between Alice and Charlie [33]. 4 Discussion We also note that our method of generating the linear cluster state connecting the three participants, is not unique: in fact, any scheme that anonymously generates a linear cluster state from Alice to Charlie can be used, and from there, our proposed method for generating the conference key can be further applied. With respect to non-trusted participants, if they were allowed to collaborate with an adversary who controls the entanglement sources and does not care about protocol termina- tion, a trivial attack would be to distribute an eigenstate of a to-be-measured observable to one or more of the parties. Since participants and non-participants behave differently and the protocol contains announcements, albeit encrypted ones, the attacker would, at least probabilistically, learn the roles of the parties in the protocol. This attack can in principle be circumvented by first performing randomized verification of the shared entanglement as in Ref. [15, 16]; however, this would require access to sufficiently good quantum memories and/or would drastically decrease the keyrate. In the absence of such countermeasures, anonymity can only be guaranteed against the non-trusted participants assuming they cannot collaborate with an all powerful eavesdropper. However, we emphasise that the security, as opposed to the anonymity, does hold in a fully adversarial model where all non-trusted participants and the eavesdropper collaborate (see Appendix B). As mentioned in Section 3.1, the measurement outcomes that are announced throughout the protocol could, in the presence of noise, be distinguishable from the chosen (uniformly random) announced bits of the nodes that do not perform a measurement; this would be detrimental to their anonymity, even if the non-trusted participants are not collaborating with an eavesdropper, since they could potentially learn about noise characteristics in the network via measurements announced in the current (or previous) runs of the protocol. To mitigate this risk, these nodes can add a bias to their random announcements that is consistent with the noise of their detectors. If they want to mimic the exact bias that their detectors have, they could – in an adjusted protocol – postpone all announcements until 10 all L rounds of measurements have taken place. Such a protocol would be modified such that N1 and the participants can secretly estimate their bias, by performing additional measurements in the bases that they would have had to perform if it wasn’t for their special role. 4 Discussion In addition, the specific measurement bases used to extract the state may depend on the position of the participants, so particular care must be taken to prevent identity leakage in this way. Finally, closed-form expressions for post-processing corrections in this generalized form are not straightforward either – and obtaining them remains an open question. Our work contributes to the growing body of multi-partite quantum cryptographic schemes that live up to stringent security requirements in protocols that go beyond point-to-point protocols. It is the hope that this work further stimulates theoretical and experimental research towards understanding notions of secure quantum communication in multi-partite settings. Accepted in Quantum 2023-09-05, click title to verify. Published under CC-BY 4.0. Accepted in Quantum 2023-09-05, click title to verify. Published under CC-BY 4.0. col. We thank Federico Grasselli for carefully reading an early version of the manuscript and providing valuable feedback. J. de Jong and A. Pappa acknowledge support from the Emmy Noether DFG grant No. 418294583. F. H. acknowledges support from the Ger- man Academic Scholarship Foundation and J. E. from the BMBF (Q.Link.X and QR.X), J. E. and A. P. also acknowledge support from the Einstein Research Unit on Quantum Devices, for which this is an inter-node project. Appendices The appendix consists of three parts. Part A contains an explanation of the corrections that the participants have to perform on their qubits due to the network layout and the measurement results of the non-participants. Part B contains a restatement of the protocol, the definition of security, and the proof of security for the protocol. Finally, part C deals with the anonymity of the protocol, showing in particular that the announcements of the measurement do not reveal any information about the identity of the participants or non- participants. 5 Acknowledgements We thank Stefanie Barz, Jakob Budde, Lukas Rückle and Christopher Thalacker for fruitful discussions during our ongoing collaboration on experimentally implementing our proto- 11 Accepted in Quantum 2023-09-05, click title to verify. Published under CC-BY 4.0. col. We thank Federico Grasselli for carefully reading an early version of the manuscript and providing valuable feedback. J. de Jong and A. Pappa acknowledge support from the Emmy Noether DFG grant No. 418294583. F. H. acknowledges support from the Ger- man Academic Scholarship Foundation and J. E. from the BMBF (Q.Link.X and QR.X), J. E. and A. P. also acknowledge support from the Einstein Research Unit on Quantum Devices, for which this is an inter-node project. 12 A Corrections for Alice, Bob and Charlie during Protocol 2 Alice and Charlie need to perform a correction to obtain the GHZ3 state with Bob, whereas Bob never has to perform a non-trivial rotation. The corrections for Alice and Charlie are structurally similar; we first introduce those for Alice. In order to achieve this, we define the following quantities. • δab := b −a −1, the number of non-participants between Alice and Bob. • δab := b −a −1, the number of non-participants between Alice and Bob. • pab := δab mod 4, the mod-four value of δab • pab := δab mod 4, the mod-four value of δab • pab := δab mod 4, the mod-four value of δab • gab := δab−pab 4 , the integer number of groups of four that fit between Alice and Bob For Charlie, δcb, gcb and pcb are defined in a similar fashion. We refer to Fig. 4 for two potential configurations of the network that exemplifies these definitions. Na Na+1 Na+2 Na+3 Nb−4 Nb−3 Nb−2 Nb−1 Nb Nb+1 Nb+2 Nb+3 Nb+4 Nc−2 Nc−1 Nc Alice Bob Charlie pab = 3 gab = 1 pcb = 2 gcb = 1 Na Na+1 Na+2 Nb−8 Nb−7 Nb−6 Nb−5 Nb−4 Nb−3 Nb−2 Nb−1 Nb Nc−3 Nc−2 Nc−1 Nc Alice Bob Charlie pab = 2 gab = 2 pcb = 3 Figure 4: Two exemplary configurations. Top: δab = 7 (with pab = 3 and gab = 1) and δcb = 6 (with pcb = 2 and gcb = 1). Bottom: δab = 10 (with pab = 2 and gab = 2) and δcb = 3 (with pcb = 3 and gcb = 0). Figure 4: Two exemplary configurations. Top: δab = 7 (with pab = 3 and gab = 1) and δcb = 6 (with pcb = 2 and gcb = 1). Bottom: δab = 10 (with pab = 2 and gab = 2) and δcb = 3 (with pcb = 3 and gcb = 0). Alice now performs the following correction steps: Alice now performs the following correction steps: Alice now performs the following correction steps: 1. Apply a configuration correction Cab depending on pab and gab, as shown in Tab. 4, picking the left (brown, βa = 1) or right (green, βa = 0) table. 1. Apply a configuration correction Cab depending on pab and gab, as shown in Tab. Accepted in Quantum 2023-09-05, click title to verify. Published under CC-BY 4.0. A Corrections for Alice, Bob and Charlie during Protocol 2 The kx column contains the measurement outcomes that add to kx, which induce together with lx a correction Xkx⊕lx; similarly the kz column contains the measurement outcomes that create kz. δab mod 4 Cab kx kz 0 Zgab ✗ ✗ 1 ZgabH ✗ ma+1 2 ZgabPz ma+1 ma+1 ⊕ma+2 3 ZgabHPz ma+1 ⊕ma+2 ma+1 ⊕ma+3 δab mod 4 Cab kx kz 0 Xgab ✗ ✗ 1 XgabHPx ✗ ma+1 2 XgabPx ma+1 ma+2 3 XgabHX ma+2 ma+1 ⊕ ma+2 ⊕ma+3 Table 4: Local corrections that Alice needs to perform to obtain the GHZ state with Bob and Charlie after the non-participants measured their qubits. The left table shows the corrections if the non- participant a + 1 after Alice measured in the σx-basis (βa = 1), the right table the corrections if it was in the σy-basis (βa = 0). The Cab column contains the configuration correction which only depends on the number of non-participants δab between Alice and Bob – note that gab := ⌊δab/4⌋. The kx column contains the measurement outcomes that add to kx, which induce together with lx a correction Xkx⊕lx; similarly the kz column contains the measurement outcomes that create kz. Table 4: Local corrections that Alice needs to perform to obtain the GHZ state with Bob and Charlie after the non-participants measured their qubits. The left table shows the corrections if the non- participant a + 1 after Alice measured in the σx-basis (βa = 1), the right table the corrections if it was in the σy-basis (βa = 0). The Cab column contains the configuration correction which only depends on the number of non-participants δab between Alice and Bob – note that gab := ⌊δab/4⌋. The kx column contains the measurement outcomes that add to kx, which induce together with lx a correction Xkx⊕lx; similarly the kz column contains the measurement outcomes that create kz. βa = 1 βa = 0 lx 1st, 3rd 1st, 2nd, 3rd lz 2nd, 3rd, 4th 2nd, 4th βa = 1 βa = 0 lx 1st, 3rd 1st, 2nd, 3rd lz 2nd, 3rd, 4th 2nd, 4th Table 5: Selection of measure- ment outcomes out of every pair of four from the second set to calculate lx and lz, respectively. For example, when δab = 7 and βa = 1, lx = ma+4 ⊕ma+6 and lz = ma+5 ⊕ma+6 ⊕ma+7. Note that all three corrections (i.e. A Corrections for Alice, Bob and Charlie during Protocol 2 the configura- tion correction, the X correction and the Z correction) can be contracted into a single Clifford operation. How- ever, since the measurement-outcome dependent correc- tions are only Pauli operators, they will at most flip the measurement outcomes for Alice in the subsequent steps of the protocol – and need not be physically imple- mented. This also means that the participants can per- form their KeyGen or Verification measurements be- fore the measurement outcomes of the non-participants are announced. By having all nodes {Na+1, . . . , Nb−1} perform their measurements and Alice subsequently per- form the aforementioned corrections, the linear cluster state is contracted towards a \|La,b,b+1,...,c−1,c⟩linear clus- ter state. Hence, Charlie can perform the same steps (while using the measurement out- comes {mc−1, mc−2, . . . , mb+1}, δbc := c −b −1 and its redefined derivatives) to contract the state towards a three-partite linear cluster state \|La,b,c⟩. Two final H gates for Alice and Charlie result in the desired GHZ3 state between Alice, Bob and Charlie. Table 5: Selection of measure- ment outcomes out of every pair of four from the second set to calculate lx and lz, respectively. For example, when δab = 7 and βa = 1, lx = ma+4 ⊕ma+6 and lz = ma+5 ⊕ma+6 ⊕ma+7. Accepted in Quantum 2023-09-05, click title to verify. Published under CC-BY 4.0. A Corrections for Alice, Bob and Charlie during Protocol 2 4, picking the left (brown, βa = 1) or right (green, βa = 0) table. 2. Divide all the measurement outcomes {mi}b−1 a+1 into a set {mi}a+1+pab a+1 and a set {mi}b−1 a+2+pab – where it is to be understood that if pab = 0, the first set is empty. 2. Divide all the measurement outcomes {mi}b−1 a+1 into a set {mi}a+1+pab a+1 and a set {mi}b−1 a+2+pab – where it is to be understood that if pab = 0, the first set is empty. 3. From the outcomes in the first set, they calculate the bits kx and kz using Tab. 4. 3. From the outcomes in the first set, they calculate the bits kx and kz using Tab. 4. cepted in Quantum 2023-09-05, click title to verify. Published under CC-BY 4.0. 13 4. From the outcomes in the second set, out of every pair of four they select the mea- surement outcomes as described in Tab. 5 and add them all together to calculate lx and lz, respectively (e.g. if βa = 1, Alice selects every odd element of the second set to calculate lx, and every second, third and fourth out of four to calculate lz). 5. They apply an X operation on their qubit if and only if kx ⊕lx = 1. 6. They apply a Z operation on their qubit if and only if kz ⊕lz = 1. y pp y p q y z z δab mod 4 Cab kx kz 0 Zgab ✗ ✗ 1 ZgabH ✗ ma+1 2 ZgabPz ma+1 ma+1 ⊕ma+2 3 ZgabHPz ma+1 ⊕ma+2 ma+1 ⊕ma+3 δab mod 4 Cab kx kz 0 Xgab ✗ ✗ 1 XgabHPx ✗ ma+1 2 XgabPx ma+1 ma+2 3 XgabHX ma+2 ma+1 ⊕ ma+2 ⊕ma+3 Table 4: Local corrections that Alice needs to perform to obtain the GHZ state with Bob and Charlie after the non-participants measured their qubits. The left table shows the corrections if the non- participant a + 1 after Alice measured in the σx-basis (βa = 1), the right table the corrections if it was in the σy-basis (βa = 0). The Cab column contains the configuration correction which only depends on the number of non-participants δab between Alice and Bob – note that gab := ⌊δab/4⌋. A.1 Calculating the corrections Using the stabiliser formalism, it is straightforward to show that, starting from a linear cluster state \|La,a+1,...,c−1,c⟩, a measurement on node Na+1 in the σx- or σz-basis results in \|La,a+2,...,c−1,c⟩up to a local correction Ca+1 ab for Alice, where this correction depends on both the measurement basis βa+1 and outcome ma+1 as Ca+1 ab (ma+1, βa+1) = P (2ma+1+βa+1) z H = HP (2ma+1+βa+1) x , (3) (3) 14 where Pz := Rz π 2 is a half-rotation around the Z-axis and Px is defined similarly. Note that either identity (i.e. the Z- or X-based rotation) can be used. A series of multiple measurements then introduces a concatenation of these corrections, where the corrections are performed in order from Na+1 to Nb−1. They do not necessarily commute, but by using the X- and Z-based correction interchangeably (and thus cancelling out the H operations), and using the identity ZmiPx = PxXmiZmi (and likewise for Pz) one can group all the corrections that are not measurement outcome based together as the first corrections; this allows to partition the complete correction into a ‘configuration’ correction and an outcome-based correction. Specifically, for the alternating pattern of σx and σy measurements, each group of four consecutive measurements together introduces only Pauli corrections. For example, for any group of four consecutive nodes {N1, N2, N3, N4} (note that these labels resemble any set of four consecutive nodes) these corrections are X(m1+m3)Z(m2+m3+m4)X, (βa = 1) X(m1+m2+m3)Z(m2+m4)Z. (βa = 0) (βa = 0) Up to an irrelevant global phase, all these operators commute with each other. Therefore, starting from the last measured node (i.e. Nb−1) an integer multiple of four can be ‘stitched together’. Since there are gab := ⌊δab/4⌋of such groups, the correction becomes Up to an irrelevant global phase, all these operators commute with each other. Therefore, starting from the last measured node (i.e. Nb−1) an integer multiple of four can be ‘stitched together’. Accepted in Quantum 2023-09-05, click title to verify. Published under CC-BY 4.0. A.1 Calculating the corrections Since there are gab := ⌊δab/4⌋of such groups, the correction becomes gab−1 Y i=0 X(mb−4i−4⊕mb−4i−2)Z(mb−4i−3⊕mb−4i−2⊕mb−4i−1)X = XlxZlzXgab, (βa = 1) gab−1 Y i=0 X(mb−4i−4⊕mb−4i−3⊕mb−4i−2)Z(mb−4i−3⊕mb−4i−1)X = XlxZlzZgab, (βa = 0) where lx is defined as where lx is defined as lx := gab−1 M i=0 mb−4i−4 ⊕mb−4i−2, (βa = 1) lx := gab−1 M i=0 mb−4i−4 ⊕mb−4i−3 ⊕mb−4i−2, (βa = 0) and lz is defined as and lz is defined as lz := gab−1 M i=0 mb−4i−3 ⊕mb−4i−2 ⊕mb−4i−1, (βa = 1) lz := gab−1 M i=0 mb−4i−3 ⊕mb−4i−1. (βa = 0) (βa = 0) (βa = 0) The corrections for the measurements of the nodes Na+1, . . . , Na+pab (i.e. the first pab measurements) are then also grouped together; by splitting them into a measurement- outcome dependent and -independent part, they can be written as XkxZkzCab, where Cab, kx and kz can be read from Tab. 4. Note that the Xgab or Zgab in Tab. 4 is technically not part of the correction here, but that they will commute with XkxZkz and hence the total correction that Alice needs to perform becomes (where now Cab is as in Tab. 4): XlxZlzXkxZkzCab ˆ= Xkx⊕lxZkz⊕lzCab, (4) (4) 15 where ˆ= here indicates ‘up to an (irrelevant) global phase’. Since these corrections only consider nodes between Alice and Bob, and since there are actions that Bob needs to perform, the corrections for Charlie work in a similar fashion and can be seen separately from these. Accepted in Quantum 2023-09-05, click title to verify. Published under CC-BY 4.0. B Protocol statement and security proof We now state the protocol and proof the security of the generated key. Note that we have omitted the network state generation (i.e. Protocol 1) as it does not affect security. Input: Alice, Bob and Charlie then hash their string using the seed shEC. Alice encrypts her output using her copy of shEC. Using their copy, Bob and Charlie each decrypt Alice’s hash outcome and compare it to their own; if they do not align, they abort by setting their abort bit to 1. 6. Bob and Charlie use their copies of sEC to obtain lEC and correct their k σz mea- surement strings, i.e. their raw key. Alice, Bob and Charlie then hash their string using the seed shEC. Alice encrypts her output using her copy of shEC. Using their copy, Bob and Charlie each decrypt Alice’s hash outcome and compare it to their own; if they do not align, they abort by setting their abort bit to 1. 7. Alice, Bob and Charlie, using another three bits of the pre-shared key, encrypt their abort bit – which is equal to 1 if and only if they want to abort – and announce it, while all other parties announce uniformly random bits instead. If any participants announced a 1, everyone aborts (meaning they will not use the generated key). 7. Alice, Bob and Charlie, using another three bits of the pre-shared key, encrypt their abort bit – which is equal to 1 if and only if they want to abort – and announce it, while all other parties announce uniformly random bits instead. If any participants announced a 1, everyone aborts (meaning they will not use the generated key). 8. Finally, the participants hash their measurement results with the seed shPA to pro- duce the final key s of length 8. Finally, the participants hash their measurement results with the seed shPA to pro- duce the final key s of length lPA := k 1 −h2(Qtol + µ εs −ε 2 ) + 2 + 2 log2(ε) = ℓ+ ℓEC + ℓhEC + L · h2(p) + 3. However, to fairly evaluate performance the parties should replenish their stock of secret-shared key so as to be able to perform subsequent CKA protocols. Subtracting off the non-reusable shared randomness results in a string of length ℓthat is available for applications. Input: 16 (a) Alice and Charlie apply their configuration corrections Ca and Cc, respectively (cf. Tab. 4). (b) Alice (i = a) and Charlie (i = c) both calculate their parameters li x, ki x and li z, ki z from the measurement outcomes of the non-participants (cf. Tabs. 4 and 5) and each apply Xlx⊕kx and Zlz⊕kz to their qubit. (c) Alice and Charlie each apply a Hadamard operation H to their qubit to obtain the final desired GHZ state. (c) Alice and Charlie each apply a Hadamard operation H to their qubit to obtain the final desired GHZ state. 3. Using the pre-shared string sb, the participants coordinate their measurements of all L GHZ states into m Verification rounds (i.e. σx-basis) and k KeyGen rounds (i.e. σz- basis). Everyone announces after each measurement a random bit mi, except for Bob and Charlie, who announce their measurement outcomes for the Verification rounds. 3. Using the pre-shared string sb, the participants coordinate their measurements of all L GHZ states into m Verification rounds (i.e. σx-basis) and k KeyGen rounds (i.e. σz- basis). Everyone announces after each measurement a random bit mi, except for Bob and Charlie, who announce their measurement outcomes for the Verification rounds. 4. Alice, who can locate Bob’s and Charlie’s measurement outcomes from the Verifi- cation rounds, estimates the σx-basis error rate Qm X = 1 2(1 −⟨σa xσb xσc x⟩). If this is above the tolerance Qtol, she aborts by setting her abort bit to 1. 4. Alice, who can locate Bob’s and Charlie’s measurement outcomes from the Verifi- cation rounds, estimates the σx-basis error rate Qm X = 1 2(1 −⟨σa xσb xσc x⟩). If this is above the tolerance Qtol, she aborts by setting her abort bit to 1. 5. Alice computes the necessary information for error correction – the error syndrome of length ℓEC – and then one-time pad encrypts this information with the string sEC. All other players announce uniform random strings of length ℓEC. 5. Alice computes the necessary information for error correction – the error syndrome of length ℓEC – and then one-time pad encrypts this information with the string sEC. All other players announce uniform random strings of length ℓEC. 6. Bob and Charlie use their copies of sEC to obtain lEC and correct their k σz mea- surement strings, i.e. their raw key. Input: Input: • L network states \|N⟩connecting {Ni}n i=1, including Na, Nb and Nc. • Desired secrecy parameter εs > 0, which determines a correlation threshold Qtol, and correctness parameter εc > 0. • A random string sb of length L · h2(p) secretly pre-shared between the participants to randomly choose m out of the L cluster states to be measured in the σx-basis for parameter estimation where p = m/L, leaving k := L −m measurements in the σz-basis for key generation. • An estimate of the expected bit error rate Qz in the σz-basis between Alice and Bob and Alice and Charlie. The worst of these will be used to select an error-correcting code that requires an error syndrome of length ℓEC := k · h2(Qz) to be announced. • A pre-shared secret random string sEC of length ℓEC to be used to one-time pad the error reconciliation announcements, another pre-shared string shEC of length ℓhEC := log2(2/εc) to one-time pad the error correction verification announcements, and three bits of pre-shared key to communicate aborting by the participants. • Two pre-shared random strings, sh and shEC, of lengths k + ℓPA −1 and k + ℓhEC −1 respectively to be used as the seeds for hashing, where ℓPA is the output of the privacy amplification hashing as defined below. The string sh is used for privacy- amplification of the private key, while shEC is used to verify the error correction step has succeeded. Note that unlike the previous seeds, these can be used indefinitely and need not be replenished after each run of the protocol. utput: A key of length ℓshared anonymously between Alice, Bob and Charlie that is -secret and εc-correct. 1. For i = 1, . . . , n: (a) Node Ni receives bit βi−1 and computes βi = 1−βi−1, except for N1 who draws a random bit β0 instead. i. If Ni ∈¯P, they measure the operator σi x or σi y if βi = 0 or βi = 1, respec- tively. They broadcast the measurement outcome mi. ii. If Ni ∈P, they announce a uniform randomly drawn bit mi. (b) Node Ni sends bit βi to neighbour Ni+1, except for node Nn. 2. The participants perform corrections on their qubits to rotate the post-measurement state to the desired GHZ state. cepted in Quantum 2023-09-05, click title to verify. Published under CC-BY 4.0. Input: We now prove the security of our protocol in the scope of an even more general adversary model than the one introduced in the main text, so that we can resort to a powerful machinery that has been developed in the literature [35, 36] and we can build on the strategy of proof laid out in Ref. [6]; the security of our protocol within our adversary model then follows readily. However, there are some variations to the tools necessary to preserve the anonymity of the participants which is key to the present work. We briefly explain some critical quantities and definitions. Let ρSASBSCE′ be the joint classical-quantum state between the final keys of the participants and an eavesdropper conditioned on passing all 17 Accepted in Quantum 2023-09-05, click title to verify. Published under CC-BY 4.0. checks. Note that the eavesdroppers system, E′ = ER, is made up of a quantum system, E, that completely purifies the pre-measurement state ρABC (and is, therefore, assumed to include system of the non-participating player) and a classical register R that contains all of the information announced during the protocol. A protocol is called εrob-robust if it passes the correlation and the error correction checks with probability 1 −εrob. Defining a uniformly distributed state as ρU ≡ X s∈S 1 \|S\|\|s⟩⟨s\| (5) (5) with S the set of possible secret keys we have the following definition [6]. Definition 1 (Approximate robustness and secrecy) A CKA protocol that is εrob-robust is εc-correct if Definition 1 (Approximate robustness and secrecy) A CKA protocol that is εrob-robust is εc-correct if (1 −εrob) Pr [SA ̸= SB ∨SA ̸= SC] ⩽εc (6) (6) and εs-secret if (1 −εrob) 1 2 ∥ρSAE′ −ρU ⊗ρE′∥⩽εs (7) (7) is called (εs + εc)-secure if it is εc-correct and εc secret. Turning first to multi-partite error correction we have the following statement. Theorem 1 (Theorem 2 in Ref. [6]) Given a probability distribution PXA,B1,B2,...,BN , be- tween Alice and n other players there exists a one-way error-correction protocol for all n players that is: εc-correct, and 2(n −1)ε′ -robust on PXA,B1,B2,...,Bn, and has leakage ℓEC ⩽max i Hε′ 0 (XA\|Bi) + log2 2(n −1) εc . (8) (8) In terms of secrecy the critical results are leftover hashing against quantum side- information, an entropic uncertainty relation for smoothed min- and max-entropies, applied to our protocol, states the following. cepted in Quantum 2023-09-05, click title to verify. Published under CC-BY 4.0. Input: Lemma 1 (Leftover hashing against quantum side information in Refs. [37, 35]) Let ε′ ≥ 0 and ρZAE be a classical-quantum state where ZA is defined over a discrete-valued and finite alphabet, E is a quantum system and R is a register containing the classical infor- mation learnt by Eve during information reconciliation. If Alice applies a hash function, drawn at random from a family of two-universal hash functions that maps ρZAE to ρSAE and generates a string of length ℓ, then 1 2 ∥ρSAER −ρU ⊗ρER∥⩽2−1 2 (Hε′ min(ZA\|ER)−ℓ+2) + 2ε′, (9) (9) where Hε′ min (ZA\|ER) is the conditional smooth min-entropy of the raw measurement data given Eve’s quantum system and the leakage of the information reconciliation. leads to the following corollary. This leads to the following corollary. This leads to the following corollary. Accepted in Quantum 2023-09-05, click title to verify. Published under CC-BY 4.0. 18 Corollary 1 (Secret string extraction) For an εrob-robust protocol an εs-secret string of length Corollary 1 (Secret string extraction) For an εrob-robust protocol an εs-secret string of length ℓ= Hε′ min (ZA\|ER) + 2 −2 log2 1 ε (10) (10) ℓ Hmin (ZA\|ER) + 2 2 log2 ε (10) can be extracted for any εs, ε, ε′ ≥0 such that ′ can be extracted for any εs, ε, ε′ ≥0 such that can be extracted for any εs, ε, ε′ ≥0 such that can be extracted for any εs, ε, ε′ ≥0 such that can be extracted for any εs, ε, ε′ ≥0 such that (11) εs ≥ε + 2ε′ (11) where Hε′ min (ZA\|ER) is the conditional smooth min-entropy of the raw measurement data given Eve’s quantum system and the information reconciliation leakage conditioned on the protocol not aborting. Proof: Note that if we choose Proof: Note that if we choose ℓ= Hε′ min (ZA\|ER) + 2 −2 log2 (1 −εrob) ε , (12) (12) then the right hand side of (9) is equal to ε/(1 −εrob) + 2ε′. Comparing with (7) in Definition 1 we see we want this expression to satisfy ε/(1 −εrob) + 2ε′ ≤εs/(1 −εrob) so our security condition is satisfied for any εs ≥ε + 2(1 −εrob)ε′ which is true for any εs ≥ε+2ε′ where we used that (1−εrob) ≤1. Noting further that log2(1−εrob) ≤0 yields (10). This means that, provided the constraint in (11) is satisfied, the positive constant ε can be optimised over. Input: ε′ max(XA\|XBi) ≤kh2(d0 + µ(ε′(1 −εrob))), where µ(ε) := s m + k mk m + 1 m ln 1 ε. (14) Hε′ max(XA\|XBi) ≤kh2(d0 + µ(ε′(1 −εrob))), where µ(ε) := s m + k mk m + 1 m ln 1 ε. (14) (14) Putting all of these results together we can prove the following security statement. Putting all of these results together we can prove the following security statement. Theorem 2 (Security statement) If the anonymous CKA protocol defined above proceeds without aborting an (maxi∈{B,C} ℓi EC, εc) error correction protocol and a two-universal hash- ing are successfully applied then an (εs + εc)-secure key of length ℓ= k 1 −h2 Qtol + µ εs −ε 2 + 2 −2 log2 1 ε −ℓEC −ℓhEC −L · h2(p) −3 = L (1 −p) 1 −h2 Qtol + µ εs −ε 2 −h2 (Qz) −h2(p) + log2(ε2εc) −2 (15) (15) can be anonymously extracted. can be anonymously extracted. Proof: At the conclusion of the protocol we can immediately apply Corollary 1 to the k round classical-quantum state ρk ZAE = trBi(\|ΨABiE⟩⟨ΨABiE\|) to extract an εs-secret key of length ℓ= Hε′ min (ZA\|ER) + 2 −2 log2 1 ε (16) (16) for positive constants satisfying for positive constants satisfying εs ≥ε + 2(1 −εrob)ε′. (17) (17) Now, because all of the communication involved in error reconciliation is one-time padded to ensure anonymity we have that Hε′ min (ZA\|E, R) = Hε′ min (ZA\|E) by definition. This gives Now, because all of the communication involved in error reconciliation is one-time padded to ensure anonymity we have that Hε′ min (ZA\|E, R) = Hε′ min (ZA\|E) by definition. This gives ℓ = Hε′ min (ZA\|E) + 2 −2 log2 1 ε (13) ≥ k −Hε′ max (XA\|XBi) + 2 −2 log2 1 ε (17) ≥ k −H(εs−ε)/2/(1−εrob) max (XA\|XBi) + 2 −2 log2 1 ε (14) ≥ k −kh2 Qtol + µ εs −ε 2 + 2 −2 log2 1 ε, (18) (18) where in the third line we have also used that Hε1 max(X\|Y ) < Hε2 max(X\|Y ) for ε1 > ε2. This string is guaranteed to be εs-secret and, by Theorem 1, if the error correction process did not abort then the string is also εc-correct. Input: However, this is not a fair representation of the performance of the protocol, since we had to use up the reservoir of pre-shared key for the basis choices and for one-time padding the error reconciliation information. Thus, to get the length of useable key we need to calculate how much remains after we have replenished the pre-shared strings necessary for the next protocol implementation. Subtracting off the seed for basis choices, Lh2(p), and the length of the error correction information and verification, ℓEC and ℓhEC and the 3 bits for the abort step, gives (15). Accepted in Quantum 2023-09-05, click title to verify. Published under CC-BY 4.0. Input: Typically this makes little difference to the final performance and and they are commonly chosen as ε = εs/2. Now we see that the problem has condensed to determining Eve’s conditional smooth min-entropy for Zk A (in the following we will suppress the k superscript), the variable de- scribing the outcome of Alice’s σz measurements on the k key-generating qubits. To begin with, consider the situation before any information reconciliation is exchanged (there is no register R) so we simply have Hmin(ZA\|E). Since Eve’s state is taken to include that of all the non-participating players we can assume without loss of generality that there is an overall pure tripartite state between Alice, the remaining participants (which we denote Bi), and Eve. The required bound for this situation has been derived by applying an en- tropic uncertainty relation [37] for the smoothed min- and max-entropies specialised to the case of observables made up of the k-fold tensor product of either σz and σx measurements, (i.e. the observables ZA = σ1 z ⊗σ2 z ⊗· · · ⊗σk z and XA = σ1 x ⊗σ2 x ⊗· · · ⊗σk x) [35] Hε min(ZA\|E) + Hε max(XA\|Bi) ≥ k, ⇒Hε min(ZA\|E) ≥ k −Hε max(XA\|Bi), (13) (13) where we have used the data processing inequality, Hε max(XA\|XBi) ≥Hε max(XA\|XBi), in the second line. Naively, this bound cannot be evaluated since it is counterfactual, i.e. the k qubits are always measured in the σz-basis so we have no direct access to Hε max(XA\|XBi), which is the conditional max-entropy of the participants given their Pauli measurements if Alice had instead measured in the σx in these k rounds. However, since the parameter estimation and key generation rounds were selected at random then it has been shown that Serfling’s bound can be applied to statistically bound the σx correlation that would have been observed in the k key generation rounds based upon those that were actually observed in the parameter estimation rounds. This is expressed in the following result. Lemma 2 (Lemma 3 in Ref. [35]) Let k be the number of key generation rounds, m be the number of parameter estimation rounds, d0 a threshold on the number of errors that can be observed during parameter estimation without the protocol aborting and ε′ > 0. 19 Accepted in Quantum 2023-09-05, click title to verify. Published under CC-BY 4.0. C.1 Definition of anonymity We base our definition of anonymity on [17] (Eqs. B7 and B13) and adapt it to our setting. The definition is given in terms of the relation to an ideal output state of the protocol. Let σN\|abc be the ideal output state on the entire network, conditioned on a particular choice of participants P = {Na,Nb,Nc}. Let G ⊂¯P be a random subset of nodes that does not include the participants and define σG\|abc = trN\G[σN\|abc]. For all sets G and every other choice of participants P′ = {Na′, Nb′, Nc′}, σN\|abc should have the property: σG\|abc = σG\|a′b′c′. (19) (19) This property ensures that the reduced state of any set of non-participants is indepen- dent of the choice of participants.i We define a protocol with output state ρN\|abc to be εan-anonymous, if for every choice of participants P = {Na,Nb,Nc}, we have: ρ ¯P\|abc −σ ¯P\|abc ⩽εan, (20) (20) where σ ¯P\|abc is any state that fulfills property (19). where σ ¯P\|abc is any state that fulfills property (19). where σ ¯P\|abc is any state that fulfills property (19). C Anonymity in the protocol This section is concerned with the anonymity of the protocol. We first define anonymity according to the definition presented in [17] and adapt it to the setting of our protocol. Most importantly for our analysis, we need to show that all public communication – the announced measurement results – is independent of the choice of participants. We do this by showing that they are uniformly random and uncorrelated. 20 Accepted in Quantum 2023-09-05, click title to verify. Published under CC-BY 4.0. C.2.1 Honest-but curious setting Consider the stabilizer of the network state after all CZ operations have been performed in Step 2a of Protocol 1. It is generated by the following collection of operators: στ1 x σω2 z , στ1 z σω2 x στ2 z , {στi z σωi+1 z }a−2 i=2 , {σωi z στi x σωi+1 x στi+1 z }a−2 i=2 , σωa−1 z στa−1 x σωa x , στa−1 z σωa z . (21) (21) The measurement operator of all measurement outcomes together depends on β1 as M = ( σω2 y στ2 x σω3 x στ3 x σω4 y στ4 x σω5 x στ5 x σω6 y . . . στa−1 x , (β1 = 0) σω2 x στ2 x σω3 y στ3 x σω4 x στ4 x σω5 y στ5 x σω6 x . . . στa−1 x , (β1 = 1) (22) (22) where all (σx-)observables acting on {τi}a−1 i=2 are associated with the measurements of Protocol 1 (i.e. the outcomes {oi}) and all others are associated with Protocol 2 (i.e. the outcomes {mi}). It is now our goal to show that all these measurement outcomes are uniformly random, and that there are no correlations between the measurement outcomes associated with any subset S ⊂Q, where Q = {ω2, τ2, . . . , ωa−1, τa−1} is the set of qubits measured throughout both Protocol 1 and Protocol 2. Any such S has an associated observable MS = O i∈S σi b(i), (23) (23) where b(i) ∈{x, y} indicates the type of support on qubit i as shown in Eq. (22). If MS does not commute with at least one generator of the stabiliser (i.e. any operator from Eq. (21)), by Gottesman-Knill simulation, the measurement outcome for MS is uniformly random 0 or 1. If this holds for any S, there cannot be any correlations between any of the measurement outcomes. The uniform randomness of the individual measurement outcomes follows readily for the case when S contains only a single qubit. We now show that any MS indeed always anti-commutes with at least a single generator. Suppose that MS does commute with all generators but is non-trivial. If it has (non- trivial) support on τa−1, this is necessarily with σx. It will then not commute with στa−1 z σωa z (the last generator of Eq. (21)) and hence cannot have support on τa−1. Accepted in Quantum 2023-09-05, click title to verify. Published under CC-BY 4.0. C.2 Proof of anonymity In the proposed protocol, the output state ρ ¯P\|abc has several registers. The only non-trivial registers that need to be addressed are the ones containing the classical communication of all the measurement outcomes {oi} ∪{mi}. The reason is that the reduced quantum state of any dishonest party is the maximally mixed state, which is independent of the choice of participants, and therefore trivially fulfills (19). Moreover, all other parties do not hold a quantum register by the end of the protocol. In the remainder of this section we will show that there are no correlations between any of the announced measurement outcomes {oi} ∪{mi}, i.e. that the outcome distribution is indistinguishable from that of the uniformly drawn announcements of the nodes N1, Na and Nc during Protocols 1 and 2. We can then conclude that we have complete anonymity, i.e. our protocol is εan-anonymous for εan = 0. Since the state of the network always remains separable between the tri-partition of the nodes to the left of (and including) Alice, the nodes to the right of (and including) Charlie, and the nodes between (and including) Alice and Charlie, it suffices to show that there are no correlations within the measurement announcements associated with these three separate groups. We show this absence of correlations only for the left set, since the argument applies analogously to the other two sets. We first show this in the case of an honest-but-curious non-participant, followed by the case where a non-participant may actively deviate from the protocol. 21 C.2.1 Honest-but curious setting 2.1 Honest-but curious setting C.2.1 Honest-but curious setting C.2.2 Dishonest participant We are now allowing a single non-participant to deviate from the protocol in an arbitrary way. Let the index of this dishonest non-participant be i. To try to force any other node in the network to implicitly reveal their identity, Ni can actively perform a different measurement than described, where their outcomes would then be correlated with its (e.g.) direct neighbours. If these correlations then do not exist between their outcomes and the announced outcomes, then they can infer that these announced outcomes are artificial, and therefore that those who have announced them are in fact participants. Let this arbitrary measurement be represented by a 2-qubit POVM µi := {µj i}, where without loss of generality j ∈{1, 2, 3, 4}. Slightly abusing notation by combining POVM elements and observables, the measure- ment operator then becomes M = ( σω2 y στ2 x . . . σωi−1 x στi−1 x N µj i N σωi+1 x στi+1 x σωi+2 y . . . στa−1 x , (β1 = 0) σω2 x στ2 x . . . σωi−1 y στi−1 x N µj i N σωi+1 y στi+1 x σωi+2 x . . . στa−1 x , (β1 = 1). (24) (24) Without loss of generality, the underlying network state is still the same4 as in (21). Likewise, all of the single-qubit measurement operators in M for any node Nj̸=i do not commute with at least one of these generators, indicating that the individual measurement outcomes are uniformly random 0 or 1. Similar to before, the goal is to show that no choice of µi can create a measurement operator MS that shows correlations between the qubits of i and any subset S ⊂Q. It suffices to show that there is no MS with support on any of the qubits in Q \ {τi, ωi} that commutes with all generators. By the same analysis as in the previous section, MS cannot have any support on the qubits of any Nj\|j∈{a−1,...,i+1}. Moreover, we can make a similar inductive argument for nodes Nj\|j∈{2,...,i−1}. Independent of β1, if MS has support on ω2 it will not commute with the generator στ1 x σω2 z . Likewise, if MS has support on τ2, it will not commute with the generator στ1 z σω2 x στ2 z . 4Any non-trivial map that Ni may perform on their subsystem can be merged with the measurements {µi}. The other participants don’t deviate, or Ni is not aware of the deviation and therefore cannot exploit it. C.2.1 Honest-but curious setting Then, if MS has (non-trivial) support on ωa−1, with either a σx or σy, it will not commute with the generator σωa−1 z στa−1 x σωa x - thus it cannot have support on ωa−1 either. We can inductively go through the rest of the qubits in Q in reversed order, i.e. from right to left through the observable from Eq. (22). For j ∈{a −2, a −3, . . . , 3, 2}: Suppose MS has non-trivial support on τj, it is of type σx. Since MS has by con- struction no support on any qubit to the right of τj, it does not commute with the generator στj z σωj+1 z - hence MS cannot have support on τj. Suppose MS has non-trivial support on ωj, either of type σx or σy. Since MS has by construction no support on any qubit to the right of ωj, it does not commute with the generator σωj z στj x σωj+1 x στj+1 z – hence MS cannot have support on ωj. 22 We conclude that there is no MS with non-trivial support on at least a single qubit that does not anti-commute with at least one generator. From this, we can conclude that there are no correlations possible between any set of measurement outcomes from {oi} and {mi}, and that they are thus uniformly random and uncorrelated. Moreover, it stays uniformly random under any noise that does not add a bias in the used measurement bases (i.e. σx and σy). Accepted in Quantum 2023-09-05, click title to verify. Published under CC-BY 4.0. References [1] Gláucia Murta, Federico Grasselli, Hermann Kampermann, and Dagmar Bruß. “Quan- tum conference key agreement: A review”. Advanced Quantum Technologies 3, 2000025 (2020). [2] S. Pirandola, U. L. Andersen, L. Banchi, M. Berta, D. Bunandar, R. Colbeck, D. En- glund, T. Gehring, C. Lupo, C. Ottaviani, J. L. Pereira, M. Razavi, J. Shamsul Shaari, M. Tomamichel, V. C. Usenko, G. Vallone, P. Villoresi, and P. Wallden. “Advances in quantum cryptography”. Adv. Opt. Photon. 12, 1012–1236 (2020). [3] Antonio Acín, Immanuel Bloch, Harry Buhrman, Tommaso Calarco, Christopher Eichler, Jens Eisert, Daniel Esteve, Nicolas Gisin, Steffen J. Glaser, Fedor Jelezko, Stefan Kuhr, Maciej Lewenstein, Max F. Riedel, Piet O. Schmidt, Rob Thew, Andreas Wallraff, Ian Walmsley, and Frank K. Wilhelm. “The European quantum technologies roadmap”. New Journal of Physics 20, 080201 (2018). [4] Sumeet Khatri and Mark M. Wilde. “Principles of quantum communication theory: A modern approach” (2020). arXiv:2011.04672. [5] Michael Epping, Hermann Kampermann, Chiara Macchiavello, and Dagmar Bruß. “Multi-partite entanglement can speed up quantum key distribution in networks”. New Journal of Physics 19, 093012 (2017). [6] Federico Grasselli, Hermann Kampermann, and Dagmar Bruß. “Finite-key effects in multipartite quantum key distribution protocols”. New Journal of Physics 20, 113014 (2018). [7] Federico Grasselli, Hermann Kampermann, and Dagmar Bruß. “Conference key agree- ment with single-photon interference”. New Journal of Physics 21, 123002 (2019). [8] Yadong Wu, Jian Zhou, Xinbao Gong, Ying Guo, Zhi-Ming Zhang, and Guangqiang He. “Continuous-variable measurement-device-independent multipartite quantum communication”. Phys. Rev. A 93, 022325 (2016). [9] Carlo Ottaviani, Cosmo Lupo, Riccardo Laurenza, and Stefano Pirandola. “Modular network for high-rate quantum conferencing”. Communications Physics 2, 118 (2019). [10] Zhaoyuan Zhang, Ronghua Shi, and Ying Guo. “Multipartite continuous variable quantum conferencing network with entanglement in the middle”. Applied Sci- ences8 (2018). [11] Daniel M. Greenberger, Michael A. Horne, and Anton Zeilinger. “Going beyond bell’s theorem”. Pages 69–72. Springer Netherlands. Dordrecht (1989). [12] W. Dür, G. Vidal, and J. I. Cirac. “Three qubits can be entangled in two inequivalent ways”. Phys. Rev. A 62, 062314 (2000). [13] Zixin Huang, Siddarth Koduru Joshi, Djeylan Aktas, Cosmo Lupo, Armanda O. Quintavalle, Natarajan Venkatachalam, Sören Wengerowsky, Martin Lončarić, Sebas- tian Philipp Neumann, Bo Liu, Željko Samec, Laurent Kling, Mario Stipčević, Rupert Ursin, and John G. Rarity. “Experimental implementation of secure anonymous pro- tocols on an eight-user quantum key distribution network”. npj Quantum Information 8, 25 (2022). C.2.2 Dishonest participant We can inductively go through all qubits from the nodes Nj\|j∈{2,...,i−1} to show that there exists no MS that has non-trivial support on any qubit of the nodes {2, . . . , i −1, i + 1, . . . , a −1} and at the same time commutes with all the generators. We can conclude that, even for a dishonest node Ni, there are no correlations in the measurement outcomes announced by the other nodes. 23 References [14] Yu-Guang Yang, Xiao-Xiao Liu, Shang Gao, Yi-Hua Zhou, Wei-Min Shi, Jian Li, and Dan Li. “Towards practical anonymous quantum communication: A measurement- device-independent approach”. Physical Review A 104, 052415 (2021). Accepted in Quantum 2023-09-05, click title to verify. Published under CC-BY 4.0. 24 [15] Anupama Unnikrishnan, Ian J. MacFarlane, Richard Yi, Eleni Diamanti, Damian Markham, and Iordanis Kerenidis. “Anonymity for practical quantum networks”. Physical Review Letters 122, 240501 (2019). [16] Frederik Hahn, Jarn de Jong, and Anna Pappa. “Anonymous quantum conference key agreement”. PRX Quantum 1, 020325 (2020). [17] Federico Grasselli, Gláucia Murta, Jarn de Jong, Frederik Hahn, Dagmar Bruß, Her- mann Kampermann, and Anna Pappa. “Secure Anonymous Conferencing in Quantum Networks”. PRX Quantum 3, 040306 (2022). [18] Christopher Thalacker, Frederik Hahn, Jarn de Jong, Anna Pappa, and Stefanie Barz. “Anonymous and secret communication in quantum networks”. New Journal of Physics 23, 083026 (2021). [19] Hans J. Briegel and Robert Raussendorf. “Persistent entanglement in arrays of inter- acting particles”. Physical Review Letters 86, 910 (2001). [20] Robert Raussendorf and Hans J. Briegel. “A one-way quantum computer”. Physical Review Letters 86, 5188 (2001). [21] Marc Hein, Jens Eisert, and Hans J. Briegel. “Multi-particle entanglement in graph states”. Physical Review A 69, 062311 (2004). [22] Matthias Christandl and Stephanie Wehner. “Quantum anonymous transmissions”. In Bimal Roy, editor, Advances in Cryptology - ASIACRYPT 2005. Pages 217–235. Berlin, Heidelberg (2005). Springer Berlin Heidelberg. [23] Nathan Walk and Jens Eisert. “Sharing classical secrets with continuous-variable entanglement: Composable security and network coding advantage”. PRX Quantum 2, 040339 (2021). [24] Damian Markham and Barry C. Sanders. “Graph states for quantum secret sharing”. Phys. Rev. A 78, 042309 (2008). [25] Massimiliano Proietti, Joseph Ho, Federico Grasselli, Peter Bar- row, Mehul Malik, and Alessandro Fedrizzi. “Experimental quan- tum conference key agreement”. Science Advances 7, eabe0395 (2021). arXiv:https://www.science.org/doi/pdf/10.1126/sciadv.abe0395. [26] Clément Meignant, Damian Markham, and Frédéric Grosshans. “Distributing graph states over arbitrary quantum networks”. Physical Review A 100, 052333 (2019). [27] Xi-Lin Wang, Luo-Kan Chen, W. Li, H.-L. Huang, C. Liu, C. Chen, Y.-H. Luo, Z.-E. Su, D. Wu, Z.-D. Li, H. Lu, Y. Hu, X. Jiang, C.-Z. Peng, L. Li, N.-L. Liu, Yu-Ao Chen, Chao-Yang Lu, and Jian-Wei Pan. “Experimental Ten-Photon Entanglement”. Physical Review Letters 117, 210502 (2016). [28] Lukas Rückle, Jakob Budde, Jarn de Jong, Frederik Hahn, Anna Pappa, and Ste- fanie Barz. “Experimental anonymous conference key agreement using linear cluster states” (2022). arXiv:2207.09487. Accepted in Quantum 2023-09-05, click title to verify. Published under CC-BY 4.0. References [29] Alexander Pickston, Joseph Ho, Andrés Ulibarrena, Federico Grasselli, Massimiliano Proietti, Christopher L Morrison, Peter Barrow, Francesco Graffitti, and Alessan- dro Fedrizzi. “Experimental network advantage for quantum conference key agree- ment” (2022). arXiv:2207.01643. [30] Zi-Heng Xiang, Jan Huwer, Joanna Skiba-Szymanska, R. Mark Stevenson, David J. P. Ellis, Ian Farrer, Martin B. Ward, David A. Ritchie, and Andrew J. Shields. “A Accepted in Quantum 2023-09-05, click title to verify. Published under CC-BY 4.0. 25 tuneable telecom wavelength entangled light emitting diode deployed in an installed fibre network”. Communications Physics 3, 121 (2020). tuneable telecom wavelength entangled light emitting diode deployed in an installed fibre network”. Communications Physics 3, 121 (2020). [31] Chao-Wei Yang, Yong Yu, Jun Li, Bo Jing, Xiao-Hui Bao, and Jian-Wei Pan. “Se- quential generation of multiphoton entanglement with a Rydberg superatom”. Nature Photonics 16, 658–661 (2022). [32] Philip Thomas, Leonardo Ruscio, Olivier Morin, and Gerhard Rempe. “Efficient gen- eration of entangled multiphoton graph states from a single atom”. Nature 608, 677–681 (2022). [33] Jarn de Jong, Frederik Hahn, Nikolay Tcholtchev, Manfred Hauswirth, and Anna Pappa. “Extracting ghz states from linear cluster states” (2023). arXiv:2211.16758. [34] Frederik Hahn, Anna Pappa, and Jens Eisert. “Quantum network routing and local complementation”. npj Quantum Information 5, 1–7 (2019). [35] Marco Tomamichel, Charles Ci Wen Lim, Nicolas Gisin, and Renato Renner. “Tight finite-key analysis for quantum cryptography”. Nature Communications 3, 634 (2012). [36] Christopher Portmann and Renato Renner. “Security in quantum cryptography”. Rev. Mod. Phys. 94, 025008 (2022). [37] Marco Tomamichel, Christian Schaffner, Adam Smith, and Renato Renner. “Leftover Hashing Against Quantum Side Information”. IEEE Transactions on Information Theory 57, 5524–5535 (2011). 26
https://openalex.org/W3181944422	https://discovery.ucl.ac.uk/10130961/1/Tsoumpas_RSTA2021_Synergistic%20tomographic%20image%20reconstruction%20-%20part%202.pdf	English	null	Synergistic tomographic image reconstruction: part 2	Philosophical transactions - Royal Society. Mathematical, Physical and engineering sciences/Philosophical transactions - Royal Society. Mathematical, physical and engineering sciences	2,021	cc-by	3,542	Accepted: 17 May 2021 Accepted: 17 May 2021 Downloaded from https://royalsocietypublishing.org One contribution of 9 to a theme issue ‘Synergistic tomographic image reconstruction: part 2’. Subject Areas: computer modelling and simulation, image processing, medical physics, computational mathematics, biomedical engineering, artificial intelligence 8Institute of Nuclear Medicine, and 9Centre for Medical Image Computing, University College London, London, UK CT, 0000-0002-4971-2477; JSJ, 0000-0001-9114-754X; CK, 0000-0002-4355-8368; KT, 0000-0002-5514-199X Keywords: This special issue is the second part of a themed issue that focuses on synergistic tomographic image reconstruction and includes a range of contributions in multiple disciplines and application areas. The primary subject of study lies within inverse problems which are tackled with various methods including statistical and computational approaches. This volume covers algorithms and methods for a wide range of imaging techniques such as spectral X-ray computed tomography (CT), positron emission tomography combined with CT or magnetic resonance imaging, bioluminescence imaging and ﬂuorescence-mediated imaging as well as diffuse optical tomography combined with ultrasound. Some of the articles demonstrate their positron emission tomography, X-ray computed tomography, magnetic resonance imaging, diffuse optical tomography, tomography, open-source software Synergistic tomographic image reconstruction: part 2 royalsocietypublishing.org/journal/rsta Charalampos Tsoumpas1,2,3, Jakob Sauer Jørgensen4,5, Christoph Kolbitsch6,7 and Kris Thielemans8,9 Introduction 1Biomedical Imaging Science Department, University of Leeds, West Yorkshire, UK 2Biomedical Engineering and Imaging Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA 3Invicro, London, UK 4Department of Applied Mathematics and Computer Science, Technical University of Denmark, Kongens Lyngby, Denmark 5Department of Mathematics, The University of Manchester, Manchester, UK 6Physikalisch-Technische Bundesanstalt, Braunschweig and Berlin, Germany 7School of Biomedical Engineering and Imaging Sciences, King’s College London, London, UK 8Institute of Nuclear Medicine, and 9Centre for Medical Image Computing, University College London, London, UK CT, 0000-0002-4971-2477; JSJ, 0000-0001-9114-754X; CK, 0000-0002-4355-8368; KT, 0000-0002-5514-199X 1Biomedical Imaging Science Department, University of Leeds, West Yorkshire, UK 2Biomedical Engineering and Imaging Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA 3Invicro, London, UK 4Department of Applied Mathematics and Computer Science, Technical University of Denmark, Kongens Lyngby, Denmark 5Department of Mathematics, The University of Manchester, Manchester, UK 6Physikalisch-Technische Bundesanstalt, Braunschweig and Berlin, Germany 7School of Biomedical Engineering and Imaging Sciences, King’s College London, London, UK 8Institute of Nuclear Medicine, and 9Centre for Medical Image Computing, University College London, London, UK CT, 0000-0002-4971-2477; JSJ, 0000-0001-9114-754X; CK, 0000-0002-4355-8368; KT, 0000-0002-5514-199X Cite this article: Tsoumpas C, Sauer Jørgensen J, Kolbitsch C, Thielemans K. 2021 Synergistictomographicimage reconstruction: part 2. Phil.Trans.R.Soc.A 379: 20210111. https://doi.org/10.1098/rsta.2021.0111 lsocietypublishing.org/ on 05 July 2021 om https://royalsocietypublishing.org/ on 05 July 2021 1. Introduction Image reconstruction describes the process from acquired data to the corresponding image information. Often research in this area is guided by the individual modalities (e.g. magnetic resonance imaging (MRI), X-ray computed tomography (CT) and positron emission tomography (PET) for medical imaging) or by different ﬁelds (e.g. tomographic imaging in medicine, biology or industry). Although each modality has its own unique challenges, the underlying image reconstruction problems also have a lot in common and the mathematical, computational and software aspects are not necessarily so different. Downloaded from https://royalsocietypublishing.org/ on 05 July 2021 lsocietypublishing.org/ on 05 July 2021 Each of these areas uses innovative solutions to optimize the reconstructed images. However, there is a lot of synergy between different ﬁelds and modalities. The special issue focuses on ideas or methodologies that could be translated across domain areas and hence beneﬁt many different applications. Thus, we hope that the special issue will help accelerate future research across different scientiﬁc ﬁelds. The common element of the research and review articles is the synergy of different measurements to provide substantially improved information on the unknown quantity/quantities being reconstructed. The approach to synthesizing and combining information intelligently is of great relevance in various research domains beyond the scope of this special issue and we envisage that techniques and ideas presented in this issue may attract interest in completely different application domains. Downloaded from https://royalsocietypublishing.o In order to bring together scientists and academics across different research domains, we organized in November 2019 a symposium on Synergistic Image Reconstruction (http://www.ccpsynerbi.ac.uk/symposium2019) and this special issue includes work presented at this event. The meeting was sponsored by two EPSRC-sponsored networks: the collaborative computational project (CCP) in synergistic PET-MR image reconstruction (CCP-PET-MR, http://www.ccppetmr.ac.uk) and the collaborative computational project in tomographic imaging (CCPi, https://www.ccpi.ac.uk/). Both these networks, which will stay active at least until 2025, with the former expanded to synergistic reconstruction in biomedical imaging (CCP SyneRBI, https://www.ccpsynerbi.ac.uk/), welcome national and international collaborations to better support their scientiﬁc communities. The special issue entitled ‘Synergistic Tomographic Reconstruction‘ is separated in two different parts. The ﬁrst issue (Part 1) included three review articles and ﬁve research investigations [1]. 1. Introduction This is the second part of the issue, and focuses on novel concepts as well as methods and software tools that can be useful for multi-spectral and multi-modality imaging such as spectral X-ray CT, MRI, PET-MRI, PET/CT, bioluminescence imaging and ﬂuorescence- mediated imaging as well as diffuse optical tomography with ultrasound. Many of these techniques have been developed only recently and several potential synergies still need to be explored [2]. Author for correspondence: Charalampos Tsoumpas e-mail: Tsoumpas@ieee.org 2021 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/ by/4.0/, which permits unrestricted use, provided the original author and source are credited. 2 utility on real-world challenges, either medical applications (e.g. motion compensation for imaging patients) or applications in material sciences (e.g. material decomposition and characterization). One of the desired outcomes of the special issues is to bring together different scientiﬁc communities which do not usually interact as they do not share the same platforms such as journals and conferences. utility on real-world challenges, either medical applications (e.g. motion compensation for imaging patients) or applications in material sciences (e.g. material decomposition and characterization). One of the desired outcomes of the special issues is to bring together different scientiﬁc communities which do not usually interact as they do not share the same platforms such as journals and conferences. royalsocietypublishing.org/journal/rsta Phil. Trans. R.Soc. A 379: 20210111 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . j This article is part of the theme issue ‘Synergistic tomographic image reconstruction: part 2’. 3 royalsocietypublishing.org/journal/rsta Phil. Trans. R.Soc. A 379: 20210111 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . artefacts in the images. Synergy can be achieved on various levels including motion tracking, estimation and correction. The review presents successful examples and discusses software and data available to the scientiﬁc community for validation and further use in clinical and preclinical scanners. 3 The next contribution—in two parts—presents the Core Imaging Library (CIL), a new open- source Python framework for processing and reconstruction of tomographic imaging data. Both articles provide complete open-access code and data to fully reproduce results and ﬁgures. Part I by Jørgensen et al. [4] gives an overview of CIL and its capabilities to handle both conventional but also challenging datasets such as those with high noise, incomplete data, non-standard scan geometries or obtained with new modalities such as dynamic and spectral CT. A modular object-oriented design and a ﬂexible convex (smooth and non-smooth) optimization framework enable users to experiment with existing reconstruction algorithms or easily implement new ones. The capabilities are demonstrated in four case studies covering synchrotron parallel-beam X-ray CT, laboratory cone-beam laminography, golden-angle neutron tomography and positron emission tomography. Downloaded from https://royalsocietypublishing.org/ on 05 July 2021 Part II by Papoutsellis et al. [5] focuses on the capabilities of CIL for multi-channel cases such as dynamic (time-resolved) and spectral (energy-resolved) tomography. CIL natively supports multi-channel data and provides a number of dedicated regularization methods, such as spatial, spectral and spatio-spectral variants of Total Variation (TV). CIL handles general linear inverse problems, as demonstrated by the ﬁrst case study of colour denoising and inpainting. The second case study covers fast dynamic CT and compares a range of regularized reconstruction methods including directional TV in terms of compensating for highly undersampled data and exploiting the dynamic dimension. The third and ﬁnal case study considers K-edge imaging in hyperspectral tomography. The use of CIL algorithms and building blocks demonstrate how cross-channel regularization can produce substantial improvements in reconstruction quality, and the use of a stochastic algorithm to accelerate computations is further discussed (ﬁgure 1). Downloaded from https://royalsocietypublishing.o Motion-correction for PET-MRI is the subject of the next paper by Brown et al. [6]. It describes recent additions to the open-source Synergistic Image Reconstruction Framework (SIRF) [7], enabling researchers to develop and evaluate new methods of motion correction and image reconstruction for either PET, MRI or (synergistic) PET-MRI. The article describes the basic functionality and illustrates the software in the context of respiratory motion. 2. Contributions in the second issue The issue commences with a review article by Polycarpou et al. [3] which summarizes various recently proposed motion compensation methods in PET-MRI. Physiological motion of the subject during scanning can affect the diagnostic value of PET-MRI substantially. The synergistic nature of integrated PET-MRI scanners can help combine information and potentially correct for motion 3 royalsocietypublishing.org/journal/rsta Phil. Trans. R.Soc. A 379: 20210111 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In the example, motion estimation is performed on cardiac MRI data. This is then followed by motion- compensated image reconstruction for both MRI and PET, combining functionality of SIRF and CIL to obtain fully corrected PET-MRI images that are spatially matched. A ﬁnal software-related contribution by Peter [8] describes a new framework for multi- modality simulation and reconstruction called Musiré. This provides a bash script that allows easy simulation for CT, MR, PET but also bioluminescence imaging (BLI) and ﬂuorescence- mediated imaging (FMI), starting from a single object description. The tool is an interface to several standard open-source packages and handles data format conversions. Although currently, all reconstructions are single-modality, the package simpliﬁes data generation for studying synergistic reconstruction methods. Li et al. [9] describe the development of a promising technique that exploits PET/CT data to perform material decomposition (in two components) without the need for dual-energy X-ray CT, as attenuation for high-energy (511 keV) is estimated from time-of-ﬂight PET data [10]. The paper describes a novel method to reconstruct both activity and high energy attenuation from the PET data by using the X-ray CT for guided reconstruction. The method uses a kernel reconstruction with kernels estimated by an autoencoder convolutional neural network (CNN) with the X-ray CT image as input. The article provides proof-of-concept results in a simulation context. Open-source software implementing the method is also made available. The article by Cueva et al. [11] proposes a new variant of directional TV regularization for spectral CT. Speciﬁcally, a spectral CT setup with three energy channels is considered and the three energy channel datasets are initially summed and reconstructed using TV-regularization Figure1. Examplefrom[5]ofhyperspectralX-raytomographicimaginginthreedimensionsofgoldandothermineraldeposits using synergistic reconstruction methods. Localizing and distinguishing inclusions with similar densities is not possible using conventionalX-rayCT.Newenergy-sensitiveX-raydetectorscan,inprinciple,distinguishinclusionsthroughK-edgeabsorption profiles which act as elemental fingerprints. However, the hyperspectral data is very noisy, causing simple reconstruction of energy channels independently to fail: channelwise SIRT reconstruction (left column) of energy channel below (top row) and above(bottomrow)goldandgalenaK-edgesarenoisyandinclusionspoorlydefined.Usingasynergisticreconstructionmethod described in [5] employing both spectral and spatial regularization (right column, same energy channels as before in top and bottom rows) the imaging of mineral phases and small inclusions is strongly improved. Inclusions can be distinguished due to different responses at different energies. For example, unlike the many smaller inclusions (gold and galena) the two large inclusionsinthetoprightonlyshowahighresponseatthelowerenergy,whichhelpstodistinguishtheseaschalcopyrite.Image credit: Laura Murgatroyd, STFC, UKRI. 3. Conclusion 5 The contributions in this second part of the special issue provide an overview of the different scientiﬁc ﬁelds as well as several software-oriented papers. The latter describe open-source software packages enabling researchers to explore synergistic image reconstruction methods. Where possible, authors have ensured that part of their data has been made publicly available via open data platforms (e.g. Zenodo). We believe that open datasets play an important role in accelerating research outcomes and are equally important to open source code. One of the issues we have noted is the lack of access to clinical datasets primarily due to ethical considerations. Another challenge is that extracting raw data (rather than images) is much harder and usually requires the support by the scanner manufacturer. Having a common protocol or ﬁle format for making raw data accessible would strongly help the scientiﬁc community, especially scientists from less economically advanced countries who otherwise cannot effectively contribute their scientiﬁc ideas to this ﬁeld. It is our hope that the open-access software described in this issue points towards a more inclusive and open-access research environment to engage more scientists in state-of-the-art research projects, potentially supported by strong international collaborations. d from https://royalsocietypublishing.org/ on 05 July 2021 Downloaded from https://royalsocietypublishing.org/ on 05 July 2021 Data accessibility. This article has no additional data. Data accessibility. This article has no additional data. Data accessibility. This article has no additional data. Authors’contributions. C.T. drafted the manuscript; J.S.J., C.K. and K.T. wrote sections and revised the manuscript; all authors read and approved the manuscript. tributions. C.T. drafted the manuscript; J.S.J., C.K. and K.T. wrote sections and revised the manuscript; rs read and approved the manuscript. Downloaded from https://royalsocietypublishing.org Competing interests. C.T. serves as an executive member of the advisory board of Positrigo AG, Zürich, Switzerland. K.T. is the Director of Algorithms and Software Consulting Ltd, London, UK. The other authors report no conﬂicts of interest. Funding. This work was funded by the UK EPSRC grants ‘Computational Collaborative Project in Synergistic PET/MR Reconstruction’ (CCP PETMR) EP/M022587/1 and its associated Software Flagship project EP/P022200/1; the ‘Computational Collaborative Project in Synergistic Reconstruction for Biomedical Imaging’ (CCP SyneRBI) EP/T026693/1; ‘A Reconstruction Toolkit for Multichannel CT’ EP/P02226X/1 and ‘Collaborative Computational Project in tomographic imaging’ (CCPi) EP/M022498/1 and EP/T026677/1. C.T. is sponsored by a Royal Society Industry Fellowship (IF170011). J.S.J. was partially supported by The Villum Foundation (grant no. 25893). Downloaded from https://royalsocie Acknowledgements. 4 royalsocietypublishing.org/journal/rsta Phil. Trans. R.Soc. A 379: 20210111 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 hil. Trans. R.Soc. A 379: 20210111 Downloaded from https://royalsocietypublishing.o Figure1. Examplefrom[5]ofhyperspectralX-raytomographicimaginginthreedimensionsofgoldandothermineraldeposits using synergistic reconstruction methods. Localizing and distinguishing inclusions with similar densities is not possible using conventionalX-rayCT.Newenergy-sensitiveX-raydetectorscan,inprinciple,distinguishinclusionsthroughK-edgeabsorption profiles which act as elemental fingerprints. However, the hyperspectral data is very noisy, causing simple reconstruction of energy channels independently to fail: channelwise SIRT reconstruction (left column) of energy channel below (top row) and above(bottomrow)goldandgalenaK-edgesarenoisyandinclusionspoorlydefined.Usingasynergisticreconstructionmethod described in [5] employing both spectral and spatial regularization (right column, same energy channels as before in top and bottom rows) the imaging of mineral phases and small inclusions is strongly improved. Inclusions can be distinguished due to different responses at different energies. For example, unlike the many smaller inclusions (gold and galena) the two large inclusionsinthetoprightonlyshowahighresponseatthelowerenergy,whichhelpstodistinguishtheseaschalcopyrite.Image credit: Laura Murgatroyd, STFC, UKRI. to obtain a side information image. Each energy channel is then reconstructed separately using directional TV regularization with the side information as a reference from which to propagate edge information. Forward–backward splitting and linearized Bregman iterations are compared for solving the resulting optimization problem. Experiments with simulated and real data demonstrate that the proposed method compares favourably with TV-regularization. Di Sciacca et al. [12] present a method for Diffuse Optical Tomographic (DOT) reconstruction using concurrent ultrasound information as guidance. The inverse problem in DOT has a nonlinear forward model and is highly ill-posed. The paper suggests a practical way to guide the three-dimensional DOT reconstruction using a structural prior from a two-dimensional ultrasound acquisition which is segmented and extended to three-dimensional. The paper compares the proposed approach with non-guided Tikhonov regularization on simulated data using the geometry of the proposed system [13]. Open-source software and datasets are made available. 3. Conclusion We wish to thank Ms Alice Power who, as the commissioning editor of the journal, has provided great support in the timely and appropriate completion of both volumes of this special issue. Furthermore, we wish to thank all authors and reviewers of the articles for their work that made the second volume of the special issue possible even during a pandemic. Downloaded from https://r References 1. Tsoumpas C, Jørgensen JS, Kolbitsch C, Thielemans K. 2021 Synergistic tomographic image reconstruction: part 1. Phil. Trans. R. Soc. A 379, 20200189. (doi:10.1098/rsta.2020.0189) 2. Arridge SR, Ehrhardt MJ, Thielemans K. 2021 (An overview of) Synergistic reconstruction for multimodality/multichannel imaging methods. Phil. Trans. R. Soc. A 379, 20200205. (doi:10.1098/rsta.2020.0205) 2. Arridge SR, Ehrhardt MJ, Thielemans K. 2021 (An overview of) Synergistic reconstruction for multimodality/multichannel imaging methods. Phil. Trans. R. Soc. A 379, 20200205. (doi:10.1098/rsta.2020.0205) 3. Polycarpou I, Soultanidis G, Tsoumpas C. 2021 Synergistic motion compensation strategies for positron emission tomography when acquired simultaneously with magnetic resonance imaging. Phil. Trans. R. Soc. A 379, 20200207. (doi:10.1098/rsta.2020.0207) 3. Polycarpou I, Soultanidis G, Tsoumpas C. 2021 Synergistic motion compensation strategies for positron emission tomography when acquired simultaneously with magnetic resonance imaging. Phil. Trans. R. Soc. A 379, 20200207. (doi:10.1098/rsta.2020.0207) g g ( ) 4. Jørgensen JS et al. 2021 Core Imaging Library part I: a versatile Python framework for tomographic imaging. Phil. Trans. R. Soc. A 379, 20200192. (doi:10.1098/rsta.2020.0192) g g 4. Jørgensen JS et al. 2021 Core Imaging Library part I: a versatile Python framework for tomographic imaging. Phil. Trans. R. Soc. A 379, 20200192. (doi:10.1098/rsta.2020.0192) g p g g 5. Papoutsellis E et al. 2021 Core Imaging Library – Part II: multichannel reconstruction for dynamic and spectral tomography. Phil. Trans. R. Soc. A 379, 20200193. (doi:10.1098/rsta.2020.0193) 5. Papoutsellis E et al. 2021 Core Imaging Library – Part II: multichannel reconstruction for dynamic and spectral tomography. Phil. Trans. R. Soc. A 379, 20200193. (doi:10.1098/rsta.2020.0193) ( ) 6. Brown R et al. 2021 Motion estimation and correction for simultaneous PET/MR using SIRF and CIL. Phil. Trans. R. Soc. A 379, 20200208. (doi:10.1098/rsta.2020.0208) 6. Brown R et al. 2021 Motion estimation and correction for simultaneous PET/MR using SIRF and CIL. Phil. Trans. R. Soc. A 379, 20200208. (doi:10.1098/rsta.2020.0208) ( ) 7. Ovtchinnikov E et al. 2020 SIRF: synergistic image reconstruction framework. Comput. Phys. Commun. 249, 107087. (doi:10.1016/j.cpc.2019.107087) 7. Ovtchinnikov E et al. 2020 SIRF: synergistic image reconstruction framework. Comput. Phys. Commun. 249, 107087. (doi:10.1016/j.cpc.2019.107087) 8. Peter J. 2021 Musiré: multimodal simulation and reconstruction framework for the radiological imaging sciences. Phil. Trans. R. Soc. A 379, 20200190. (doi:10.1098/ rsta.2020.0190) 6 royalsocietypublishing.org/journal/rsta Phil. Trans. R.Soc. A 379: 20210111 . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 royalsocietypublishing.org/journal/rsta Phil. Trans. R.Soc. A 379: 20210111 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9. Li S, Wang G. 2021 Modiﬁed kernel MLAA using autoencoder for PET-enabled dual-energy CT. Phil. Trans. R. Soc. A 379, 20200204. (doi:10.1098/rsta.2020.0204) 10. Wang G. 2020 PET-enabled dual-energy CT: image reconstruction and a proof-of-concept computer simulation study. Phys. Med. Biol. 65, 245028. (doi:10.1088/1361-6560/abc5ca) 11. Cueva E, Meaney A, Siltanen S, Ehrhardt MJ. 2021 Synergistic multi-spectral CT reconstruction with directional total variation. Phil. Trans. R. Soc. A 379, 20200198. (doi:10.1098/rsta.2020.0198) ( ) 12. Di Sciacca G et al. 2021 Enhanced diffuse optical tomographic reconstruction using concurrent ultrasound information. Phil. Trans. R. Soc. A 379, 20200195. (doi:10.1098/rsta.2020.0195) 13. Pifferi A et al. 2021 SOLUS: an innovative multimodal imaging system to improve breast cancer diagnosis through diffuse optics and ultrasounds. In Optical Tomography and Spectroscopy of Tissue XIV, vol. 11639, p. 116390C. International Society for Optics and Photonics. Downloaded from https://royalsocietypublishing.org/ on 05 July 2021 lsocietypublishing.org/ on 05 July 2021 om https://royalsocietypublishing.org/ on 05 July 2021
https://openalex.org/W3045675383	https://journal.nurulfikri.ac.id/index.php/jtt/article/download/109/92	Indonesian	null	Penerapan Fasilitas TWAIN Untuk Digitalisasi Kearsipan Pada PT. PLN Area Flores Bagian Barat Berbasis Client-Server	Jurnal teknologi terpadu	2,018	cc-by-sa	3,811	Penerapan Fasilitas TWAIN Untuk Digitalisasi Kearsipan Pada PT. PLN Area Flores Bagian Barat Berbasis Client-Server Meliana Oktavia Meo Program Studi Teknik Informatika, STIKOM Uyelindo Kupang E-mail: meliana.oktavia.g@gmail.com ABSTRACT PLN Area Flores bagian barat masih mengalami banyak kendala, seperti dalam sistem penyimpanan arsipnya saat ini masih bersifat konvensional yaitu menggunakan media kertas sehingga tidak menerapkan sistem pengindeksan yang berakibat pencarian arsip dalam bentuk fisik menjadi sulit dan lambat, rusaknya kertas arsip surat dikarenakan penumpukan dan masih besarnya tingkat kehilangan arsip surat karena dokumen fisik yang berceceran dan tidak tertata dengan baik, serta kesulitan untuk memindahkan arsip surat yang berdasarkan klasifikasi kode arsip ISSN 2477-0043 e-ISSN 2460-7908 ISSN 2477-0043 e-ISSN 2460-7908 ISSN 2477-0043 e-ISSN 2460-7908 Jurnal Teknologi Terpadu Vol. 4, No. 1, Juli 2018 Jurnal Teknologi Terpadu Vol. 4, No. 1, Juli 2018 Jurnal Teknologi Terpadu Vol. 4, No. 1, Juli 2018 ABSTRACT Arsip adalah warkat, gambar atau dalam bentuk yang lain yang disimpan sebagai bahan informasi pada saat diperlukan. Arsip memiliki peranan yang sangat penting bagi suatu instansi atau suatu perusahaan. Arsip yang tidak dikelola dengan baik akan menganggu kelancaran aktivitas suatu organisasi karena arsip yang disimpan merupakan sumber data dan informasi yang berguna bagi suatu organisasi. Oleh karena itu, perlu adanya suatu sistem pengelolaan arsip yang baik dan benar dengan memanfaatkan teknologi komputer. PT. PLN Area Flores Barat adalah salah satu Badan Usaha Milik Negara (BUMN) yang tidak terlepas dari kegiatan surat menyurat dan kearsipan lainnya. Permasalahan yang dialami oleh PT. PLN Area Flores Barat adalah proses penyimpanan arsip surat masih bersifat konvensional dan belum menerapkan sistem pengindeksan serta pemanfaatan teknologi komputer. Hal ini menyebabkan pencarian arsip dalam bentuk fisik menjadi sulit dan lambat, selain itu rusaknya kertas arsip surat dikarenakan penumpukan. Penelitian ini bertujuan untuk membuat sebuah aplikasi bantu yang dapat mempermudah proses pengarsipan dokumen berbasis client-server pada PT. PLN area Flores bagian barat secara digital dengan menerapkan fasilitas TWAIN. Metode yang digunakan dalam penelitian ini adalah metode Waterfall dengan Bahasa pemrograman Microsoft Visual Basic. Hasil penelitian ini dapat membantu dan mempermudah proses pengolahan data kearsipan pada PT. PLN Area Flores Bagian Barat sehingga diharapkan dapat meminimalisasi kehilangan dan kerusakan data serta dapat memberikan informasi yang efektif dan efisien dalam pendukung pengambilan keputusan oleh pimpinan Kata Kunci : Digitalisasi, Kearsipan, Client-Server, TWAIN Flores bagian Barat yang tentunya tidak terlepas dari kegiatan surat menyurat dan kearsipan lainnya namun dalam pelaksanaan kegiatan administrasi, proses penyimpanan arsip surat pada PT. PLN Area Flores bagian barat masih mengalami banyak kendala, seperti dalam sistem penyimpanan arsipnya saat ini masih bersifat konvensional yaitu menggunakan media kertas sehingga tidak menerapkan sistem pengindeksan yang berakibat pencarian arsip dalam bentuk fisik menjadi sulit dan lambat, rusaknya kertas arsip surat dikarenakan penumpukan dan masih besarnya tingkat kehilangan arsip surat karena dokumen fisik yang berceceran dan tidak tertata dengan baik, serta kesulitan untuk memindahkan arsip surat yang berdasarkan klasifikasi kode arsip Flores bagian Barat yang tentunya tidak terlepas dari kegiatan surat menyurat dan kearsipan lainnya namun dalam pelaksanaan kegiatan administrasi, proses penyimpanan arsip surat pada PT. Kata Kunci : Digitalisasi, Kearsipan, Client-Server, TWAIN 2.4 Sistem Penyimpanan Arsip Sistem penyimpanan adalah sistem yang dipergunakan pada penyimpanan warkat agar kemudahan kerja penyimpanan dapat diciptakan dan penemuan warkat yang sudah disimpan dapat diketemukan dengan cepat bilamana warkat tersebut sewaktu-waktu diperlukan. Jurnal Teknologi Terpadu Vol. 4, No. 1, Juli 2018 Jurnal Teknologi Terpadu Vol. 4, No. 1, Juli 2018 benar menerapkan sistem kearsipan berbasis komputer secara optimal. Arsip dibedakan menurut fungsinya menjadi dua golongan [3], yaitu : Pemanfaatan perangkat komputer dalam PT. PLN Area Flores Bagian Barat untuk kearsipan hanya digunakan untuk mengetik surat yang dilakukan pada paket aplikasi Microsoft Office yaitu Microsoft Word dan file-file hasil kerjapun tidak tertata dengan baik yang akhirnya hilang maupun rusak 1. Arsip Dinamis Adalah arsip yang dipergunakan secara langsung dalam perencanaan, pelaksanaan, penyelenggaraan kehidupan kebangsaan pada umumnya atau dipergunakan secara langsung dalam penyelenggaraan administrasi. Oleh karena itu, dibutuhkan pengembangan dalam proses penyimpanan arsip berbasis database dengan memanfaatkan teknologi komputer dan alat scanner untuk melakukan dokumentasi terhadap arsip yang kemudian disimpan ke dalam database sehingga menjadi sistem informasi yang lebih baik. Arsip dinamis aktif dibedakan menjadi dua yaitu : a. Arsip dinamis aktif Adalah arsip dinamis yang masih sering dipergunakan bagi kelangsungan pekerjaan dilingkungan satuan kerja (unit pengolah) pada suatu organisasi. b. Arsip dinamis inaktif b. Arsip dinamis inaktif Adalah arsip dinamis yang frekuensi kegunaannya oleh unit pengolah sudah jarang dan hanya dipergunakan sebagai referensi bagi satu organisasi. g 2. Arsip Statis g 2. Arsip Statis Arsip berasal dari bahasa asing, orang Yunani mengatakan “Archivum” yang artinya tempat untuk menyimpan. Sering pula kata tersebut ditulis “Archeon” yang berarti Balai Kota (tempat untuk menyimpan dokumen) tentang masalah pemerintahan. Adalah arsip yang tidak dipergunakan secara langsung untuk perencanaan, penyelenggaraan kehidupan kebangsaan pada umumnya maupun pada penyelenggaraan sehari-hari administrasi negara. Menurut Bahasa Belanda yang dikatakan dengan “Archief” mempunyai arti [1]. Menurut Bahasa Belanda yang dikatakan dengan “Archief” mempunyai arti [1]. 2.3 Jenis Arsip Sesuai dengan perkembangan teknologi dan kemajuan peralatan data dan informasi yang sudah sampai kepada era komputerisasi, maka arsip masa kini dapat terekam pada kertas, kertas film, dan media komputer. Karena itu sekarang terdapat dua jenis arsip ditinjau dari sudut hukum dan perundang- undangan, yaitu menurut Undang Undang No 7 Tahun 1971 adalah : g f p y 1. Tempat untuk menyimpan catatan-catatan dan bukti-bukti kegiatan yang lain. 2. Kumpulan catatan atau bukti kegiatan yang berjudul tulisan, gambar, grafik dan sebagainya. 3. Bahan-bahan yang akan disimpulkan sebagai bahan pengingat. Menurut Undang Undang No 7 Tahun 1971, arsip adalah : a. Naskah-naskah yang dibuat dan diterima oleh lembaga-lembaga dan bahan-bahan pemerintahan dalam bentuk apapun, baik dalam keadaan tunggal maupun berkelompok dalam rangka pelaksanaan kegiatan pemerintah. 1. Arsip Otentik Yaitu arsip yang diantaranya terdapat tanda tangan asli dengan tinta (bukan fotokopi atau film) sebagai tanda keabsahan dari isi arsip bersangkutan. 2. Arsip Tidak Otentik 2. Arsip Tidak Otentik Yaitu arsip diatasnya tidak terdapat tanda tangan asli dengan tinta. b. Naskah-naskah yang dibuat dan diterima oleh badan-badan swasta atau perorangan, dalam bentuk corak apapun, baik dalam keadaan tunggal maupun berkelompok dalam rangka pelaksanaan kehidupan kebangsaan 1. PENDAHULUAN Seiring dengan berkembangnya teknologi yang semakin canggih di era globalisasi ini maka kegiatan administrasi saat ini juga menuntut akan sistem kearsipan yang terkomputerisasi. Salah satu kegiatan yang erat hubungannya dengan administrasi adalah pengelolaan arsip. Semua lembaga baik swasta maupun pemerintah dalam pelaksanaan kegiatannya tidak terlepas dari arsip surat. Ketepatan dalam pengelolaan arsip akan berpengaruh besar dan dapat memberikan nilai tambah bagi suatu kemajuan aktivitas kerja perusahaan di masa yang akan datang. Dengan memanfaatkan teknologi komputer untuk kelancaran aktivitas suatu organisasi karena arsip yang disimpan selain sebagai alat pengingat juga merupakan sumber data dan informasi yang berguna dalam proses pengambilan keputusan dan sebagai alat bukti dari pelaksanaan kegiatan organisasi. Kendala seperti ini bukan karena pada PT. PLN Flores Barat tidak tersedianya fasilitas listrik dan komputer tetapi pemahaman akan pentingnya sistem informasi kearsipan yang masih sangat minim dan juga kurangnya jumlah pegawai kearsipan yang benar- PT. PLN Area Flores Barat adalah salah satu Badan Usaha Milik Negara (BUMN) dengan area 1 ISSN 2477-0043 e-ISSN 2460-7908 5. Sistem tanggal Adalah sistem penyimpanan arsip menurut urut- urutan tanggal yang tertera pada tiap arsip itu. Sistem ini dapat dipakai bagi arsip yang harus memperhatikan sesutu jangka waktu tertentu, misalnya surat- surat tagihan. 1. Sistem abjad standar untuk mengakomodasi teknologi masa depan [5] standar untuk mengakomodasi teknologi masa depan [5] Adalah sistem penyimpanan arsip yang disimpan menurut abjad dari nama-nama orang/organisasi utama yang tertera dalam tiap-tiap arsip itu. Dengan sistem menurut urut-urutan abjad ini, sepucuk surat yang berhubungan dengan seseorang langsung dapat diketemukan kembali dengan lebih cepat daripada kalau semua surat dicampur adukkan. 4. Sistem nomor c. Crystal Reports sebagai dirancang untuk membuat laporan Adalah sistem penyimpanan arsip yang didasarkan pada kode nomor sebagai pengganti dari nama orang/nama badan atau pokok masalah. Pada sistem ini nomor yang diberikan akan selamanya tetap sama dan tidak pernah berubah. 2.2 Fungsi Arsip Yang dimaksud dengan arsip adalah warkat, gambar atau dalam bentuk yang lain yang disimpan sebagai bahan informasi pada saat diperlukan. Jadi warkat yang disimpan itu suatu ketika dikeluarkan dari tempat penyimpanan dan digunakan sebagai sumber informasi untuk keperluan kegiatan yang sedang dilaksanakan [2] Penyimpanan arsip merupakan aktivitas pokok dalam bidang kearsipan [4]. Sistem penyimpanan arsip dapat di bedakan menjadi lima macam yaitu : 2 ISSN 2477-0043 e-ISSN 2460-7908 Jurnal Teknologi Terpadu Vol. 4, No. 1, Juli 2018 Jurnal Teknologi Terpadu Vol. 4, No. 1, Juli 2018 3.3.1 Usecase Diagram Usecase menggambarkan interaksi yang terjadi dalam sistem, yang memberi gambaran user atau actor yang berhubungan dengan sistem dan hal-hal yang berhubungan dengan user di dalam sistem. Pengklasifikasian sistem penyimpanan ini dilakukan agar pegawai kearsipan dapat dengan mudah dan cepat dalam melakukan pekerjaan penyimpanan arsip dan juga dalam penemuan kembali arsip yang akan diperlukan. p 2. Sistem pokok soal 2. Sistem pokok soal Adalah sistem penyimpanan arsip yang disimpan menurut urutan yang dimuat, dalam tiap- tiap arsip bersangkutan. Isi arsip sering juga disebut sebagai perihal, pokok masalah, permasalahan dan pokok surat atau subjek. Misal, surat yang mengenai iklan dikumpulkan menjadi satu dibawah judul “iklan”. 3.2 Antarmuka Perangkat Lunak 3. Sistem geografis Dalam penelitian ini, adapun spesifikasi perangkat lunak yang digunakan: Adalah sistem penyimpanan arsip yang didasarkan pada pengelompokan menurut nama tempat (wilayah). Sistem ini sering disebut juga sistem lokasi. a. Microsoft Visual Basic 6.0 sebagai Bahasa pemrograman. p g b. StarUML sebagai media perancangan system 4. Sistem nomor 3.1 Jenis Penelitian Menurut penggunaannya, penelitian yang digunakan saat ini adalah penelitian terapan (applied research). Penelitian terapan ialah setiap penelitian yang bertujuan untuk meningkatkan pengetahuan ilmiah dengan suatu tujuan praktis. Hasil dari penelitian diharapkan dapat segera dipakai untuk keperluan praktis yaitu menerapkan teknologi TWAIN dalam bentuk aplikasi kearsipan secara digital pada PT. PLN Area Flores Bagian Barat. 3.3 Perancangan dan Konstruksi Sistem Tahapan dalam pembuatan perangkat lunak ini terdiri atas beberapa bagian utama sebagai berikut: 5. Sistem tanggal 2.5 Fasilitas TWAIN Usecase Diagram System Mengelola Data Bagian Mengelola Data Jabatan Mengelola Data Jenis Mengelola Data Sifat Mengelola Data Arsip Mengelola Data User Mengelola Data Scan Arsip Mengelola Data Instansi Mengelola Data Pegawai Mengelola Data Pemohon Mengelola Data Pelanggan Mengelola Data Meteran Mengelola Data Migrasi Mengelola Data Surat Masuk Petuga Mencetak Laporan Pegawai Mencetak Laporan Pemohon Mencetak Laporan Pelanggan Mencetak Laporan Meteran Mencetak Laporan Migrasi Mencetak Laporan Surat Masuk Mencetak Laporan Disposisi Surat Mencetak Laporan Surat Keluar 3.3.3 Activity Diagram Mengelola Data Jabatan Gambar 3. Activity Diagram Mengelola Data Jabatan Petugas User Interface System Database Initialize User Request Sent Request Execute Request Sent Response Accept Response Tambah Refresh Cari Data Tambah Refresh Hapus Ubah Cari Keyword Initialize ID Get ID Checking ID Sent ID Cek Validasi Data Submit Proses Execute Proses Display Data Jabatan Pilih Jabatan Request Data jabatan .3 Activity Diagram Mengelola Data Jabatan Gambar 3. Activity Diagram Mengelola Data Jabatan 3.3.4 Activity Diagram Mengelola Data Instansi Gambar 4. Activity Diagram Mengelola Data Instansi 3.3.5 Perancangan Antarmuka Bagian ini akan merupakan implementasi atau kontruksi tampilan dari program yang akan dibuat a. Perancangan Form Bagian Petugas User Interface System Database Initialize User Request Sent Request Execute Request Sent Response Accept Response Tambah Refresh Cari Data Tambah Refresh Hapus Ubah Cari Keyword Initialize ID Get ID Checking ID Sent ID Cek Validasi Data Submit Proses Execute Proses Request Data Instansi Display Data Instansi Pilih Instansi 3.3.4 Activity Diagram Mengelola Data Instansi Gambar 4. Activity Diagram Mengelola Data Instansi Petugas User Interface System Database Initialize User Request Sent Request Execute Request Sent Response Accept Response Tambah Refresh Cari Data Tambah Refresh Hapus Ubah Cari Keyword Initialize ID Get ID Checking ID Sent ID Cek Validasi Data Submit Proses Execute Proses Request Data Instansi Display Data Instansi Pilih Instansi 3.3.4 Activity Diagram Mengelola Data Instansi Gambar 1. Usecase Diagram 3.3.2 Activity Diagram Mengelola Data Bagian Gambar 2. Activity Diagram Mengelola Data Bagian Petugas User Interface System Database Initialize User Request Sent Request Execute Request Sent Response Accept Response Tambah Pilih Jabatan Refresh Cari Data Tambah Refresh Hapus Ubah Cari Keyword Initialize ID Get ID Checking ID Sent ID Cek Validasi Data Submit Proses Execute Proses Request Data Bagian Display Data Bagian 3.3.2 Activity Diagram Mengelola Data Bagian 3.2 Activity Diagram Mengelola Data Bagian Gambar 4. 2.5 Fasilitas TWAIN TWAIN (Technology Without An Interesting Name) adalah sebuah standar API (Application Programming Interface) untuk pengambilan gambar pada sistem operasi Microsoft Windows dan Machintos. Diperkenalkan pada 1992, dan saat ini diratifikasi pada versi 1.9 (per Januari 2000). Standar dikelola oleh TWAIN Working Group, sebuah organisasi nirlaba yang tidak mewakili industri pencitraan. Grup TWAIN adalah organisasi nirlaba yang mewakili industri pencitraan. Tujuan TWAIN adalah untuk menyediakan dan mengembangkan standar umum universal yang menghubungkan aplikasi dan perangkat akuisisi citra. Misi yang sedang berlangsung dari organisasi ini adalah untuk terus meningkatkan 3 ISSN 2477-0043 Jurnal Teknologi Terpadu e-ISSN 2460-7908 Vol. 4, No. 1, Juli 2018 Gambar 1. Usecase Diagram 3.3.2 Activity Diagram Mengelola Data Bagian 3.3.3 Activity Diagram Mengelola Data Jabatan Gambar 3. Activity Diagram Mengelola Data Jabatan 3.3.4 Activity Diagram Mengelola Data Instansi Gambar 4. Activity Diagram Mengelola Data Instansi 3.3.5 Perancangan Antarmuka Bagian ini akan merupakan implementasi atau kontruksi tampilan dari program yang akan dibuat a. Perancangan Form Bagian System Mengelola Data Bagian Mengelola Data Jabatan Mengelola Data Jenis Mengelola Data Sifat Mengelola Data Arsip Mengelola Data User Mengelola Data Scan Arsip Mengelola Data Instansi Mengelola Data Pegawai Mengelola Data Pemohon Mengelola Data Pelanggan Mengelola Data Meteran Mengelola Data Migrasi Mengelola Data Surat Masuk Petugas Mencetak Laporan Pegawai Mencetak Laporan Pemohon Mencetak Laporan Pelanggan Mencetak Laporan Meteran Mencetak Laporan Migrasi Mencetak Laporan Surat Masuk Mencetak Laporan Disposisi Surat Mencetak Laporan Surat Keluar Petugas User Interface System Database Initialize User Request Sent Request Execute Request Sent Response Accept Response Tambah Pilih Jabatan Refresh Cari Data Tambah Refresh Hapus Ubah Cari Keyword Initialize ID Get ID Checking ID Sent ID Cek Validasi Data Submit Proses Execute Proses Request Data Bagian Display Data Bagian Petugas User Interface System Database Initialize User Request Sent Request Execute Request Sent Response Accept Response Tambah Refresh Cari Data Tambah Refresh Hapus Ubah Cari Keyword Initialize ID Get ID Checking ID Sent ID Cek Validasi Data Submit Proses Execute Proses Display Data Jabatan Pilih Jabatan Request Data jabatan Petugas User Interface System Database Initialize User Request Sent Request Execute Request Sent Response Accept Response Tambah Refresh Cari Data Tambah Refresh Hapus Ubah Cari Keyword Initialize ID Get ID Checking ID Sent ID Cek Validasi Data Submit Proses Execute Proses Request Data Instansi Display Data Instansi Pilih Instansi ISSN 2477-0043 e-ISSN 2460-7908 Jurnal Teknologi Terpadu Vol. 4, No. 1, Juli 2018 3.3.3 Activity Diagram Mengelola Data Jabatan Gambar 3. 2.5 Fasilitas TWAIN Activity Diagram Mengelola Data Jabatan 3.3.4 Activity Diagram Mengelola Data Instansi Gambar 4. Activity Diagram Mengelola Data Instansi 3.3.5 Perancangan Antarmuka Bagian ini akan merupakan implementasi atau kontruksi tampilan dari program yang akan dibuat a. Perancangan Form Bagian Petugas User Interface System Database Initialize User Request Sent Request Execute Request Sent Response Accept Response Tambah Refresh Cari Data Tambah Refresh Hapus Ubah Cari Keyword Initialize ID Get ID Checking ID Sent ID Cek Validasi Data Submit Proses Execute Proses Display Data Jabatan Pilih Jabatan Request Data jabatan Petugas User Interface System Database Initialize User Request Sent Request Execute Request Sent Response Accept Response Tambah Refresh Cari Data Tambah Refresh Hapus Ubah Cari Keyword Initialize ID Get ID Checking ID Sent ID Cek Validasi Data Submit Proses Execute Proses Request Data Instansi Display Data Instansi Pilih Instansi Gambar 1. Usecase Diagram 3.3.2 Activity Diagram Mengelola Data Bagian Gambar 2. Activity Diagram Mengelola Data Bagian System Mengelola Data Bagian Mengelola Data Jabatan Mengelola Data Jenis Mengelola Data Sifat Mengelola Data Arsip Mengelola Data User Mengelola Data Scan Arsip Mengelola Data Instansi Mengelola Data Pegawai Mengelola Data Pemohon Mengelola Data Pelanggan Mengelola Data Meteran Mengelola Data Migrasi Mengelola Data Surat Masuk Petugas Mencetak Laporan Pegawai Mencetak Laporan Pemohon Mencetak Laporan Pelanggan Mencetak Laporan Meteran Mencetak Laporan Migrasi Mencetak Laporan Surat Masuk Mencetak Laporan Disposisi Surat Mencetak Laporan Surat Keluar Petugas User Interface System Database Initialize User Request Sent Request Execute Request Sent Response Accept Response Tambah Pilih Jabatan Refresh Cari Data Tambah Refresh Hapus Ubah Cari Keyword Initialize ID Get ID Checking ID Sent ID Cek Validasi Data Submit Proses Execute Proses Request Data Bagian Display Data Bagian Gambar 1. Usecase Diagram System Mengelola Data Bagian Mengelola Data Jabatan Mengelola Data Jenis Mengelola Data Sifat Mengelola Data Arsip Mengelola Data User Mengelola Data Scan Arsip Mengelola Data Instansi Mengelola Data Pegawai Mengelola Data Pemohon Mengelola Data Pelanggan Mengelola Data Meteran Mengelola Data Migrasi Mengelola Data Surat Masuk Petug Mencetak Laporan Pegawai Mencetak Laporan Pemohon Mencetak Laporan Pelanggan Mencetak Laporan Meteran Mencetak Laporan Migrasi Mencetak Laporan Surat Masuk Mencetak Laporan Disposisi Surat Mencetak Laporan Surat Keluar Gambar 1. 2.5 Fasilitas TWAIN Activity Diagram Mengelola Data Instansi 3.3.5 Perancangan Antarmuka Bagian ini akan merupakan implementasi atau kontruksi tampilan dari program yang akan dibuat a. Perancangan Form Bagian 3.3.5 Perancangan Antarmuka 3.3.5 Perancangan Antarmuka Bagian ini akan merupakan implementasi atau kontruksi tampilan dari program yang akan dibuat a. Perancangan Form Bagian Gambar 2. Activity Diagram Mengelola Data Bagian 4 ISSN 2477-0043 e-ISSN 2460-7908 Jurnal Teknologi Terpadu Vol. 4, No. 1, Juli 2018 d. Perancangan Form Jenis Form Jenis 1 Kode Nama Kode Nama Jenis Tambah Ubah Hapus Refresh Cari Tutup Halaman x/x Gambar 8. Perancangan Form Jenis e. Perancangan Form Scan Arsip Form Pilih Device Scan Arsip Simpan Gambar Tutup Gambar 9. Perancangan Form Scan Arsip f. Perancangan Form Pelanggan Form Pelanggan Kode Nama Pelanggan Alamat No. Telp Keterangan d. Perancangan Form Jenis d. Perancangan Form Jenis Form Bagian 1 Kode Nama Bagian Bagian Kode Nama Tambah Ubah Hapus Refresh Cari Tutup Halaman x/x Gambar 5. Perancangan Form Bagian d. Perancangan Form Jenis Form Jenis 1 Kode Nama Kode Nama Jenis Tambah Ubah Hapus Refresh Cari Tutup Halaman x/x Gambar 8. Perancangan Form Jenis Gambar 8. Perancangan Form Jenis Gambar 5. Perancangan Form Bagian Gambar 5. Perancangan Form Bagian e. Perancangan Form Scan Arsip e. Perancangan Form Scan Arsip e. Perancangan Form Scan Arsip Form Pilih Device Scan Arsip Simpan Gambar Tutup Gambar 9. Perancangan Form Scan Arsip e. e. Perancangan Form Scan Arsip Form Pilih Device Scan Arsip Simpan Gambar Tutup Gambar 9. Perancangan Form Scan Arsip b. Perancangan Form Jabatan b. Perancangan Form Jabatan b. Perancangan Form Jabatan Form Jabatan 1 Kode Nama Kode Nama Jabatan Tambah Ubah Hapus Refresh Cari Tutup Halaman x/x Gambar 6. Perancangan Form Jabatan b. Perancangan Form Jabatan Form Jabatan 1 Kode Nama Kode Nama Jabatan Tambah Ubah Hapus Refresh Cari Tutup Halaman x/x Gambar 6. Perancangan Form Jabatan Gambar 6. Perancangan Form Jabatan Gambar 6. Perancangan Form Jabatan f. Perancangan Form Pelanggan f. Perancangan Form Pelanggan Form Pelanggan 1 Kode Nama Pelanggan Kode Nama Tambah Ubah Hapus Refresh Cari Tutup Halaman x/x Alamat No. Telp Keterangan Gambar 10. Perancangan Form Pelanggan c. Perancangan Form Instansi c. Perancangan Form Instansi Form Bagian Instansi 1 Kode Nama Bagian Instansi Kode Nama Tambah Ubah Hapus Refresh Cari Tutup Halaman x/x Gambar 7. Perancangan Form Instansi Gambar 10. Perancangan Form Pelanggan Gambar 7. Perancangan Form Instansi 5 ISSN 2477-0043 e-ISSN 2460-7908 ISSN 2477-0043 e-ISSN 2460-7908 ISSN 2477-0043 e-ISSN 2460-7908 Jurnal Teknologi Terpadu Vol. 4, No. 1, Juli 2018 c. Tampilan Form Data Jenis surat Tabel 1. File pendukung implementasi program Tabel 1. File pendukung implementasi program Gambar 13. Tampilan Form Data Jenis Surat d. Tampilan Form Scan Dokumen Gambar 14 Tampilan form Scan Dokumen d. Tampilan Form Scan Dokumen d. 4.1 Implementasi Sistem 4.1 Implementasi Sistem Aplikasi dapat berjalan dengan baik apabila memiliki file-file pendukung untuk menunjang proses yang akan dijalankan. c. Tampilan Form Data Jenis surat Gambar 13. Tampilan Form Data Jenis Surat d. Tampilan Form Scan Dokumen Gambar 14 Tampilan form Scan Dokumen e. Tampilan Form Scan Dokumen Berikut adalah beberapa file pendukung dalam implementasi program ini yaitu: Berikut adalah beberapa file pendukung dalam implementasi program ini yaitu: Tabel 1. File pendukung implementasi program 4.2 Antarmuka Sistem a. Tampilan Form Utama Gambar 11. Tampilan form utama b. Tampilan Data Bagian Instansi Gambar 12. Tampilan Form Data Bagian Instansi c. Tampilan Form Data Jenis surat Gambar 13. Tampilan Form Data Jenis Surat d. Tampilan Form Scan Dokumen Gambar 14 Tampilan form Scan Dokumen e. Tampilan Form Scan Dokumen pa file pendukung dalam itu: lementasi program pilan form utama Instansi c. Tampilan Form Data Jenis surat Gambar 13. Tampilan Form Data Jenis Surat d. Tampilan Form Scan Dokumen Gambar 14 Tampilan form Scan Dokumen e. Tampilan Form Scan Dokumen c. Tampilan Form Data Jenis surat c. Tampilan Form Data Jenis surat c. Tampilan Form Data Jenis surat 5. KESIMPULAN Dari penelitian yang dilakukan dapat disimpukan bahwa aplikasi yang dibuat dapat berjalan dengan baik dan dapat diterima dengan baik. Dengan adanya aplikasi digitalisasi kearsipan pada PT. PLN Area Flores Bagian Barat berbasis client-server dengan menggunakan fasilitas TWAIN maka akan membantu dan mengolah data kearsipan yang lebih efektif dan efisien. Gambar 15. Tampilan form laporan pegawai Gambar 15. Tampilan form laporan pegawai 4.3 Deskripsi Sistem Sistem yang ditawarkan merupakan sistem baru dimana berbasis komputerisasi dalam penanganan data kearsipan berbasis clint-server. Selain itu, di dalam sistem ini juga dilengkapi dengan tool TWAIN untuk mempermudah proses scan dokumen sehingga langsung disimpan pada database dengan demikian demikian, data kearsipan selain disimpan dalam bentuk dokumen fisik juga dapat disimpan dalam bentuk data digital yaitu setiap file gambar yang discan akan tersimpan di dalam database (bukan di dalam folder tertentu) sehingga mengurangi kehilangan atau kekurangan data data Sistem yang dirancang juga menggunakan level otentifikasi untuk membedakan pengguna sistem berdasarkan bagian tertentu dan dengan level akses tertentu sehingga dilengkapi dengan password administrator yang berarti hanya user yang memiliki hak akses yang boleh menggunakan sistem yang dibuat 4.2 Antarmuka Sistem 4.2 Antarmuka Sistem a. Tampilan Form Utama Gambar 11. Tampilan form utama Gambar 11. Tampilan form utama b. Tampilan Data Bagian Instansi Gambar 12. Tampilan Form Data Bagian Instansi b. Tampilan Data Bagian Instansi Gambar 12. Tampilan Form Data Bagian Instansi b. Tampilan Data Bagian Instansi Gambar 14 Tampilan form Scan Dokumen Gambar 14 Tampilan form Scan Dokumen e. Tampilan Form Scan Dokumen e. Tampilan Form Scan Dokumen Gambar 12. Tampilan Form Data Bagian Instansi 6 ISSN 2477-0043 e-ISSN 2460-7908 ISSN 2477-0043 e-ISSN 2460-7908 Jurnal Teknologi Terpadu Vol. 4, No. 1, Juli 2018 Jurnal Teknologi Terpadu Vol. 4, No. 1, Juli 2018 Jurnal Teknologi Terpadu Vol. 4, No. 1, Juli 2018 DAFTAR PUSTAKA [1] Sedarmayanti. 2003. Tata Kearsipan dengan Memanfaatkan Teknologi Modern. Bandung : Mandar Maju j [2] Mulyono, Sularso. 2003. Manajemen Kearsipan. Semarang. UNNES [2] Mulyono, Sularso. 2003. Manajemen Kearsipan. Semarang. UNNES [3] Republik Indonesia. 1971. Undang-Undang Nomor 7 Tahun 1971 tentang Ketentuan- ketentuan Pokok Kearsipan. Lembaran Negara RI Tahun 1971 Nomor 32. Sekretariat Negara. Jakarta [4] The Liang Gie. 2000. Administrasi Perkantoran Moderen. Yogyakarta : Liberty [4] The Liang Gie. 2000. Administrasi Perkantoran Moderen. Yogyakarta : Liberty [5] TWAIN. 2017. Twain Initiative. http://www.twain.org (diakses tanggal 7 Maret 2017) [5] TWAIN. 2017. Twain Initiative. http://www.twain.org (diakses tanggal 7 Maret 2017) 7 7
https://openalex.org/W3214191682	https://scindeks-clanci.ceon.rs/data/pdf/1821-2077/2019/1821-20771902069P.pdf	Bosnian	null	The analysis of the market position and marketing potentials of crossfit	Sport - nauka i praksa	2,019	cc-by	6,453	1  nesaperic@gmail.com UDK 005.346:796.015.14 659.11:796 Nenad Perić1 Univerzitet Metropolitan i Univerzitet Union-Nikola Tesla, Beograd, Srbija Apstrakt: CrossFit je prepoznat kao jedan od najbrže rastućih programa trenin- ga na svetu. Usled svoje nezahtevnosti, širine obuhvata i suštinski jednostavne primenljivosti u prethodnih desetak godina je registrovan veliki broj vežbača i klubova koji ga praktikuju širom sveta. Njegov jednostavan i razumljiv koncept je omogućio stvaranje velikog socijalnog kapitala - velikog broja vežbača, instruk- tora i klubova koji su, takođe, vid promocije programa. Pored toga što je postao kategorija za sebe, CrossFit je izrastao i u posebnu kulturu, a temelji ovog progra- ma su funkcionalnost i potreba ljudi za vežbanjem i zdravim životom. Usled broja ljudi koji praktikuju ovaj program, petocifrenog broja CrossFit klubova širom sveta i njegove velike i kontinuirane medijske prisutnosti, može se reći da je CrossFit po- stao globalni sportski pokret koji se konstantno razvija i ima ogroman marketinški potencijal.CrossFit je jedan od najboljih primera da su jednostavne stvari i ideje obično i najdelotvornije u bilo kojoj grani, pa tako i sportskoj industriji. Ključne reči: CrossFit, vežbanje, vežbač, program, potencijal, jednostavnost Ključne reči: CrossFit, vežbanje, vežbač, program, potencijal, jednostavnost SPORT - Nauka i Praksa, Vol. 9, №2, 2019, str. 69-76. SPORT - Nauka i Praksa, Vol. 9, №2, 2019, str. 69-76. Stručni rad Stručni rad UVOD U svetu, pa i kod nas je u prethodnih desetak godina zabeležena veli- ka ekspanzija CrossFita.CrossFit je prepoznat kao jedan od najbrže rastućih programa treninga (visokog intenziteta) na svetu (Claudino i sar., 2018). Pa ipak, kod nas se ni jedan naučni rad ne bavi istim. Otuda i potiče motivacija za pisanje rada koji će predstaviti ovaj program, te analizirati njegovu tržišnu poziciju i marketinški potencijal. Pored toga, samo nekolicina naučnih radova 6  69 69  SPORT - Nauka i Praksa, Vol. 9, No 2, 2019. inostranih autora ovaj program u manjem obimu analizira kao marketinški fenomen, gotovo isključivo se radi o radovima koji izučavaju fiziološke efekte praktikovanja istog. CrossFit Inc. je kompanija koju je osnovao Greg Glassman 2000. godine. 2005. godine pod imenom CrossFit delovala je mreža od 13 teretana. Danas, ta mreža broji preko 13000 filijala u oko 150 zemalja širom sveta (https://map.cross- fit.com/?_ga=2.95823068.1142892516.1574639395-1739441689.1574639395). Godine 2007. osnovane su The CrossFit Games koje se od tada redovno održa- vaju svakog leta i iz godine u godinu privlače sve veći broj takmičara. Vreme- nom su postale toliko popularne da je nagradni fond drastično povećan, tako da prvoplasirani takmičari u muškoj i ženskoj konkurenciji osvajaju po 300.000 američkih dolara, a novčanu nagradu dobijaju (u opadajućim iznosima) svi tak- mičari na prvih dvadeset pozicija, što pokazuje finansijsku snagu ovog progra- ma i organizacije.Takođe, CrossFit svojom medijskom i internet zastupljenošću doprinosi stvaranju on-line zajednice, u kojoj se razmenjuju iskustva, stilovi, rezultati, koji unapređuju CrossFit kao veštinu vežbanja. CrossFit se bazira na dinamičnom programu vežbanja čiji je cilj pove- ćanje opšte spremnosti vežbača, njegove snage i kondicije, ali i jačanje psiho- loške strane, zajedništva i timskog rada. Trening se obično sastoji od kratkog zagrevanja, dvadesetominutnog napornog i vrlo zahtevnog treninga, i dugo- trajnog istezanja. CrossFit se bazira na 10 krucijalnih tačaka: kardiovasku- larna/respiratorna izdržljivost, snaga, kondicija, fleksibilnost, brzina, gipkost, koordinacija, agilnost, ravnoteža i preciznost (Benjamin, 2012).Obuhvatajući više olimpijskih disciplina (olimpijsko dizanje tegova, plivanje, gimnastiku, atletiku…), on teži stvaranju polivalentnih sportista. Sa visokim intenzitetom treninga i velikim brojem ponavljanja stavlja se naglasak na specifičan broj ponavljanja, specifične težine i specifične pokrete za određeno vreme. Ovim se omogućava jasno merenje rezultata u fizičkoj spremi. Takođe, CrossFit podrazumeva da vežbači mogu praviti svoje programe prema individualnim zahtevima. Ova vrsta treninga je popularna u vojsci SAD-a i koriste je policije SAD i Velike Britanije. UVOD Međutim, CrossFit zbog intenziteta i veoma eksplo- zivne akcije mišića ima mnogo pozitivnih strana i za ljude koji su prosečni kad je u pitanju vežbanje (Meyer, Morrison, Zuniga, 2017). Vežbe mogu biti raznolike (trčanje, vijača, veslanje, zgibovi, sklekovi, propadanja, mrtva di- zanja, trzaj, izbačaj…), a izvode se u bezbroj kombinacija, od kojih se jedna izabere kao VD-vežba dana (eng. WOD-workout of the day), tako da ni jedan trening nije isti. VD obično trener ispiše na tabli i onda svako na njoj upisuje svoj rezultat. Takvim načinom pristupa neguje se sportski takmičarski duh, ali i zajedništvo, međusobno pomaganje i savetovanje. Svaki trener mora po- sedovati sertifikat-licencu koju dobija pohađanjem kursa na kome obavezno učestvuje i u izvođenju vežbi, a ne samo u teoretskoj nastavi. Pored osnovnog 70  Nenad Perić: ANALIZA POZICIJE I MARKETINŠKIH POTENCIJALA CROSSFIT PROGRAMA ANALIZA POZICIJE I MARKETINŠKIH POTENCIJALA CROSSFIT PROGRAMA kursa, radi se i na kursevima koji se odnose na pripreme sportskih timova, raznih olimpijaca, trudnica, dece itd. kursa, radi se i na kursevima koji se odnose na pripreme sportskih timova, raznih olimpijaca, trudnica, dece itd. CrossFit i tržišno okruženje Kada je CrossFit u pitanju, možemo reći da je najznačajnije sagledati trendove u oblasti fitnesa i velnesa uopšte, kao i potrošače, odnosno ciljnu grupu. i i p p j g p Ako posmatramo fitnes iz tržišnog aspekta, mogli bi da ga okarakte- rišemo kao vrstu usluge iz oblasti rekreacije, a koja treba da zadovolji kori- snikovu potrebu za zdravljem, lepotom, kretanjem, zabavom i ublažavanjem negativnih efekata savremenog načina života. Iz te perspektive, fitnes (eng. fitness) predstavlja industriju koja prodaje mnoštvo usluga, proizvoda i fit- nes filozofiju življenja. Iz toga sledi da fitnes predstavlja svojevrsnu privrednu granu, pa unutar njega, važe isti principi kao i u svim ostalim delatnostima. Danas je fitnes mnogo više u odnosu na nekadašnji pristup rekreaciji i vezan je za naplative kategorije, kao što su: fitnes objekti, programi, oprema, pre- parati i stručno i specijalizovano osoblje. U fitnesu se sve više javljaju novi programi vežbanja koji se poslednjih godina jednim imenom nazivaju “grupni vođeni programi”. Pod tim imenom podrazumevaju se programi vežbanja koji su različiti po svom sadržaju, nameni, upotrebi rekvizita i sprava, ali isti po aerobnom režimu rada i grupnom vođenju treninga, najčešće uz muziku kao stimulišući medij. Praktikuju se sledeći oblici vežbanja: step aerobik, aqua aerobik, body sculpting ili body styling, body pump, kick boxing, tae-bo, spi- ning, latino, funky, hip-hop, salsa, afro, etno aerobik; jazzersize; low impact i mnogi drugi Za sve ove programe, možemo kod nas koristiti termin aerobik ili fitnes programi (Stojiljković, 2005). Trend velnesa (eng. wellness) kao načina života i razvoj velnes turi- zma u Severnoj Americi i Evropi razvija se u poslednje tri decenije, dok u našoj zemlji postaje popularan od početka tekuće. Koncept velnesa razvio je američki lekar Halbert Dun (Halbert Dunn) 1959. godine, kada prvi put piše o ,,specifičnom stanju zdravlja koje obuhvata sveopšti osećaj blagostanja, posmatrajući čoveka kao celinu sačinjenu od tela, duha i uma i koji je zavisan od okoline u kojoj se nalazi” (Dunn, 1957). Hansruedi i Kaufman pokušavaju da udruže razumevanje pojma velnesa u Severnoj Americi i Evropi, ističući da on predstavlja stanje zdravlja koje karakteriše harmonija tela, uma i duha, odgovornost prema samom sebi, fizička aktivnost, briga o lepoti, zdrava is- hrana, relaksiranje, meditacija, mentalna aktivnost, obrazovanje, osećanje za okolinu i društvene veze, kao osnovni elementi velnesa (Hansruedi, Kauf- man, 2000). CrossFit i tržišno okruženje Termin okruženje u smislu marketinga se različito koristi. Kotler, na primer, u definiciji marketinškog okruženja uključuje sve faktore izvan ak- tivnosti menadžmenta marketinga u preduzeću, što podrazumeva unutrašnje i spoljne uticaje. Razlike između kategorija okruženja ilustruje konceptom slojeva okruženja, koje se nadovezuju jedan na drugi. Za primer uzima sa- mo faktore koje smatra ključnim u odnosu na svaki pojedinačni nivo (Kotler, 1997). Interno okruženje. Pod ovim nazivom Russ i Kirpatrick podrazumeva- ju interaktivne odnose međuzavisnosti između marketinga i ostalih aktivnosti menadžmenta. Na primer, u nadmetanju oko obezbeđenja sredstava za svoje aktivnosti ili oko međusobne saradnje u donošenju odluka (Russ i Kirpatrick, 1982). Mikrosredina. Ovaj drugi nivo obuhvata faktore koji utiču na me- nadžment marketinga, a koji su izvan organizacionih granica preduzeća. Tu spadaju: konkurencija, distribucija, dobavljači, potrošači itd. i javnost. Strateg marketinga treba posebno da sagledava značaj javnosti i obezbedi efikasan odnos sa njenim važnim elementima. Makrosredina. Na faktore ovog nivoa pojedinačna organizacija gotovo da nema nikakav uticaj. To su: socio-kulturološka sredina, političko-zakon- ska, ekonomska, tehnološka i fizička. Svaki od njih utiče na strategiju marke- tinga i zato se moraju sistematski pratiti njihove promene i trendovi. Ključni problem u proceni okruženja koje obuhvata sve događaje i trendove izvan granica marketinške aktivnosti je upravo identifikovanje tren- dova koji su najznačajniji za analizu, jer ne postoje čvrsta i brza pravila za njihovo razlikovanje. Treba naglasiti da identifikovanje najvažnijih trendova zahteva znatnu kreativnost i sposobnost predviđanja. Ponekad na izgled nebi- tan trend ili promena može biti presudna u dugoročnom smislu. Najvažniji razlog zbog koga se procena okruženja i vrši je procena najverovatnijih uticaja na organizaciju. Planerima tržišne strategije, Kotler (1997) za ovu procenu sugeriše korišćenje matrice mogućnosti i opasnosti. Matrica mogućnosti služi planeru da proceni relativnu atraktivnost koju pru- ža okruženje, kao i šanse za uspeh ako se deluje u određenom pravcu. Uslov za to je procena kvaliteta i slabosti kompanije. Isto tako, planer treba da pro- ceni i mogući uticaj eventualnih opasnosti iz okruženja, kao i stepen njihove ozbiljnosti u odnosu na aktivnosti organizacije. Naravno, ovo su pojednosta- vljeni primeri. Prava analiza procene okruženja uzima u obzir nekoliko tren- dova odjednom i procenjuje njihov pojedinačni i unakrsni uticaj. Problemi u 71  SPORT - Nauka i Praksa, Vol. 9, No 2, 2019. definisanju trendova i teškoće u preciznom predviđanju posledica ne umanju- ju suštinski značaj postupaka procene za spremnije dočekivanje budućnosti. CROSSFIT I NJEGOV (TRŽIŠNI) OBUHVAT I POTENCIJAL CrossFit može da trenira svako, muškarci i žene svih uzrasta. Vežbač sam sebi određuje intenzitet treninga prevashodno uz nadzor instruktora, ia- ko to nije obavezno, CrossFit je „slobodna kategorija“. Prva faza ulaska u program je upoznavanje sa svim vežbama i gimnastičarskim elementima koje se koriste i usavršavanje izvođenja istih sa svojom telesnom težinom. Nakon uspešnog savladavanja takvih vežbi i postizanja dobre kondicije, koordina- cije, gipkosti, izdržljivosti prelazi se u drugu fazu. U njoj se unose u vežbe, bučice, tegovi i olimpijsko dizanje, ergometri. Tehnika izvođenja samih vežbi se prvo usavršava sa manjim kilažama, a većim brojem ponavljanja, nakon čega se postepeno kilaže povećavaju. Cilj drugog nivoa je povećanje snage i zadržavanje i povećavanje stečene kondicije.Treća faza je najozbiljnija jer u njoj počinje trka vežbača sa samim sobom u cilju postizanja ličnih rekorda. Ovde se ne koriste sprave na koje inače nailazite u teretanama. Koriste se girije, olimpijske šipke, tegovi, karike, vratila, traktorske gume, medicinske lopte, ergometri, mornarski kanapi, ramovi za vuču itd. Dakle, prema osnovnoj postavci, CrossFit programi su osmišljeni tako da ih može koristiti svako bez obzira na dotadašnje iskustvo i spremnost jer pružaju mogućnost prilagođavanja težine i načina treninga konkretnoj osobi i njenim ciljevima. Zahteva neznatnu investiciju kluba ili pojedinca što se tiče opreme potrebne za vežbanje. Takođe, jedan isti program može biti upotre- bljen za pripremu osoba koje se bave borilačkim veštinama kao i osoba koje imaju neke srčane probleme. Programi ostaju isti, ono što se menja jeste op- terećenje i intenzitet programa vežbanja. Stoga je potencijalni tržišni obuhvat ovog programa ogroman, kako u smislu pojedinaca koje može privući, tako i u smislu sportskih klubova koji ga mogu praktikovati i uspostaviti saradnju sa CrossFit mrežom koja je doživela ogroman rast u proteklih desetak godina. Temelji ovog sporta su, funkcionalnost i potreba svih ljudi za vežba- njem i zdravim životom. Filozofija CrossFita je jednostavna, to je da svaki prosečan vežbač treba da dostigne svoj maksimum u onim fitnes ciljevima koji su njemu najpotrebniji. Dodatna motivacija svim CrossFit vežbačima je to što je svaki trening baziran na kombinaciji različitih vežbi. Stoga i ne čudi što su CrossFit trenere zvali i karatisti i bejzbol igrači, plivači, bokseri, ne bi li usavršili neke segmente svoje forme. CrossFit koristi gimnastički izraz „progresije“ za definisanje procesa tranzicije za savladavanje svakog pokreta. Pristup progresiji osigurava samoefikasnost stvaranjem dostižnih koraka ka težim izazovima. CrossFit i tržišno okruženje Kao što vidimo, za razliku od fitnesa, velnes veći akcenat stavlja na ono što čini organizmu dobro, jer je fizička aktivnost samo jedna od stavki pobrojanih u gornjm nabrajanju. Dakle, velnes može uključivati fitnes, dok fitnes često proširuje svoje usluge onima iz oblasti fitnesa, a i jedno i drugo se, kao i CrossFit, bore za ideju dobrobiti osobe koja ih praktikuje. U tome su marketinški veoma razrađeni jer predtavljaju ozbiljne tržišne kategorije koje privlače sve više ljudi, pa samim tim i sredstava. 72  Nenad Perić: Nenad Perić: ANALIZA POZICIJE I MARKETINŠKIH POTENCIJALA CROSSFIT PROGRAMA CROSSFIT I NJEGOV (TRŽIŠNI) OBUHVAT I POTENCIJAL Posvećenost većem cilju će ojačati i potaknuti motivaciju članova tokom procesa (Meier, Becker, & Vandenberghe, 2004). Motivacija se takođe pojačava posmatranjem ostalih članova i interakcijom sa njima. Že- ljeni rezultati opažanja bili bi porast samoefikasnosti, uverenje da pojedinac 73  SPORT - Nauka i Praksa, Vol. 9, No 2, 2019. može izvršiti zadatak i očekivano trajanje ishoda, percepcija da će ispunjenje zadatka doneti pozitivan krajnji rezultat (Gibson, 2004). Članovi rade zajed- no na poboljšanju svojih veština i napretku u poteškoćama, čime se razvija kolektivan duh na zdravoj i jakoj, ali neinvazivnoj osnovi, ne postoji moranje da se trening ili određena vežba uradi do kraja, već članovi grupe podržavaju jedni druge da budu što bolji u vežbanju. Međuzavisnost je potrebna u razme- ni znanja kad god jedna strana ne može ostvariti svoj cilj bez pomoći druge (McNeish & Mann, 2010). Međutim, postoji drugi faktor koji održava grupu, jednom kada se for- mira: kako članovi napreduju, tako napreduje i njihov status unutar grupe (Holmes i Meierhoff, 1999). Ovo stvara strateški sistem nagrađivanja ugra- đen u model napredovanja. Sistemi nagrađivanja pomažu da se podstaknu članovi da zajedno rastu i razvijaju se kroz strukturu kolektivne zajednice (Martin-Perez, Martin-Cruz i Estrada-Vakuero, 2010). Kako se novi članovi razvijaju u iskusne članove, sistem nagrađivanja podstiče ih da podrže nove članove njihovim napredovanjem. Ovim se stvara odlična baza potencijalnih vežbača i imidž grupa koje vežbaju CrossFit kako u užoj javnosti (fitnes, vel- nes i sl), tako i široj javnosti, potencijalnim vežbačima. ZAKLJUČAK Svet je pun trendova u fitnesu, a novi trendovi počinju svakog dana. Većina njih, kao i novih brendova, nikada ne doživi pun razvoj. „Globaliza- cija današnjeg tržišta je široko prisutna u svim sferama društva i kao takva neizostavan je deo problematike tržišta sporta. Pozicioniranje i stvaranje pre- poznatljivog imena, odnosno dobrog imidža na globalnom tržištu, predstavlja zahtevan posao koji podrazumeva dugoročne kontinuirane napore, i to mnogo veće od onih koje definiše lokalna konkurencija“ (Ratković, Dašić, 2018). Ra- zlog ovome je prezasićenost tržišta različitim ponudama, kao i prezasićenost glava potrošača i auditorijuma tim istim ponudama i drugim marketinškim promocijama (Perić, Krasulja, Radojević, 2011). Razlika između mnogih drugih programa i CrossFita je korišćenje na- gona društvenog kapitala da utiče na promene (Durlauf i Fafchamps, 2004). CrossFit je započeo jedan čovek, sportski entuzijasta i od male grupe ljudi koja je razvila zajednički jezik, članstvo i napredovanja proširio je na sve kon- tinente. Glavni razlog uspeha ovog programa i njegova svetska popularnost se mogu naći u jednostavnosti njegovog koncepta: ispunjava potrebu mnogih vezanu za fizičko zdravlje, spremu i uzročno posledično fizički izgled; svako ga može praktikovati u meri u kojoj mu odgovara; podstiče koheziju grupa vežbača i želju za napretkom uz razvijanje zdravog takmičarskog duha, pre- vashodno na individualnom, pa potom na grupnom nivou; zahteva minimalnu 74  Nenad Perić: Nenad Perić: ANALIZA POZICIJE I MARKETINŠKIH POTENCIJALA CROSSFIT PROGRAMA ANALIZA POZICIJE I MARKETINŠKIH POTENCIJALA CROSSFIT PROGRAMA investiciju i pojedinca i kluba koji ga praktikuju; ima jednostavan i razumljiv koncept. Taj jednostavan i razumljiv koncept je omogućio stvaranje socijal- nog kapitala, velikog broja vežbača koji su, takođe, vid promocije programa (marketing od usta od usta je u većini slučajeva najdelotvorniji marketing). Pored toga, omogućio je organizaciju masovnog takmičenja koje zbog pri- rode sporta nije zahtevalo naročite tehničke preduslove za njegovo izvođe- nje, a sprovođenje organizacije istog se najvećim delom zasniva na ljudima koja praktikuju CrossFit i instruktorima istog. CrossFit je jedan od najboljih primera da su jednostavne stvari i ideje obično i najdelotvornije u bilo kojoj oblasti, pa i sportskoj industriji. Pored toga što je postao kategorija za sebe, CrossFit je izrastao i u posebnu kulturu, a temelji ovog programa su funkcionalnost i potreba svih ljudi za vežbanjem i zdravim životom. ZAKLJUČAK Usled broja ljudi koji praktikuju ovaj program, petocifrenog broja CrossFit klubova širom sveta i njegove velike i kontinuirane medijske prisutnosti, može se reći da je CrossFit postao globalni sportski pokret koji se konstantno razvija i ima ogroman marketinški poten- cijal. UDK 005.346:796.015.14 659.11:796 Nenad Perić1 Metropolitan University and University Union - Nikola Tesla, Belgrade, Serbia Nenad Perić1 Abstract: CrossFit is recognized as one of the fastest growing training programs in the World. Due to its width of scope and essentially simple applicability, it has registered a large number of practitioners and clubs practicing it worldwide in the last ten years. Its simple and understandable concept has made it possible to create a large social capital - a large number of practitioners, instructors and clubs, which are also a part of the program promotion (word-of-mouth). In addition to becoming a category in itself, CrossFit has grown into a distinct culture, and the foundations of this program are its functionality and people’s need to exercise and live a healthy life. Due to the number of people practicing this program, the 5-digit number of CrossFit clubs worldwide and its large and continuous media presence, it can be said that CrossFit has become a constantly evolving global sports movement with a huge marketing potential. CrossFit is one of the best examples that simple things and ideas are usually the most effective in any branch, including the sports industry. Key words: CrossFit, exercise, exerciser, program, potential, simplicity 1  nesaperic@gmail.com LITERATURA 1. Benjamin A. S., (2012). Using Sport Education to Implement a CrossFit Unit, Journal of Physical Education, Recreation & Dance Volume 83, 2012 - Issue 8. 2. Claudino, J.G., Gabbett, T.J., Bourgeois, F. et al. (2018). CrossFit Overvi- ew: Systematic Review and Meta-analysis, Sports Med - Open (2018) 4: 11. https://doi.org/10.1186/s40798-018-0124-5 3. Dunn, H., (1959). High level wellness for man and society, American Journal of Public Health, 49(6): 786–792. 4. Durlauf, S. N., Fafchamps, M. (2004). Social capital, NBER Working Pa- per Series, 1-89. (http://www.nber.org/papers/w10485) 5. Gibson, S. (2004). Social learning (cognitive) theory and implications for human Resource, Advances in Developing Human Resources, 6(2), 193-210.f 6. Holmes, J., Meyerhoff, M. (1999). The community of practice: Theories and methodologies in language and gender research, Language in socie- ty, 28(02), 173-183. 7. Jain, C., (1984). Environmental scanning: how the best companies do it, Journal of Long Rang Planning, Vol. 17. 8. McNeish, J., Mann, I. (2010). Knowledge sharing and trust in organizati- ons. IUP Journal of Knowledge Management, 8(1/2), 18-38. 9. Kotler, P. (1997). Marketing management: analisys, planning, implemen- tation and control, Prentice-Hall, New Jersey. 75  SPORT - Nauka i Praksa, Vol. 9, No 2, 2019. 10. Martín-Pérez, V., Martín-Cruz, N., Estrada-Vaquero, I. (2012). The in- fluence of organizational design on knowledge transfer. Journal of Kno- wledge Management, 16(3), 418-434.i 11. Meyer, J., Morrison, J. Zuniga, J. (2017). The Benefits and Risks of CrossFit: A Systematic Review, Workplace Health & Safety 2017, 612- 618. https://doi.org/10.1177/2165079916685568 12. Meyer, J., Becker, T., Vandenberghe, C. (2004). Employee commitment and motivation: a conceptual analysis and integrative model, Journal of applied psychology, 89(6).991-1007. 13. Mueller, H., Kaufmann, E. L., Wellness Tourism: Market Analysis of a special health tourism segment and implications for the hotel industry, Journal of Vacation Marketing, 2000, 2-3. 14. Perić, N., Krasulja, N., Radojević, I. (2011). Medijske, PR i brend tenden- cije, drugo, prošireno izdanje, Synopsis. 15. Ratković, M., Dašić, D. (2018). Marketing u sportu sa elementima indu- strije sporta, Visoka škola modernog biznisa 16. Russ, A., Kirkpatrick, C., Marketing, Little, Browne & Co, Boston, 1982.i 16. Russ, A., Kirkpatrick, C., Marketing, Little, Browne & Co, Boston, 1982. 17. Stojiljković, S., Fitness, Fakultet sporta i fizičkog vaspitanja, Beograd, 2005. 17. Stojiljković, S., Fitness, Fakultet sporta i fizičkog vaspitanja, Beograd, 2005. 18. https://map.crossfit.com/?_ga=2.95823068.1142892516.1574639395- 1739441689.1574639395 76  SPORT - Science & Practice, Vol. 9, №2, 2019, pp. 69-77. INTRODUCTION In the past ten years there has been a significant expansion of CrossFit. CrossFit is recognized as one of the fastest growing high-intensity functional training modes in the world (Claudino et. al., 2018). Yet, no scientific papers in Serbia deal with this topic. That served as a motivation for writing this paper, which will briefly introduce this program, analyse its marketing potential and market positioning in the field of sports and healthy living. In addition, only a 69  SPORT - Science & Practice, Vol. 9, No 2, 2019. few scientific papers analyse this program as a marketing phenomenon, to an extent; almost exclusively, papers concerning CrossFit deal with the physiolo- gical effects of practicing this program. CrossFit Inc. is a company founded by Greg Glassman in 2000. In 2005, the network of collaborating gyms under the name CrossFit numbered 13 venu- es. Today, the network counts over 13,000 branches in about 150 countries worl- dwide (https://map.crossfit.com/?_ga=2.95823068.1142892516.1574639395- 1739441689.1574639395). In 2007, The CrossFit Games was founded and since then it has been regularly held every summer and it attracts more and more competitors every year. Over time, they have become so popular that the prize pool has drastically increased, with first-place competitors in the men's and women's competitions winning $ 300,000 each, and prizes (in decreasing amounts) being awarded to all competitors in the top twenty positions, which shows the financial power of this program and organizations. Also, CrossFit contributes to the creation of an online community through its online presen- ce, in which experiences, styles, workout results are exchanged between the people participating in this programme. This enhances CrossFit as an exercise skill. CrossFit is based on a dynamic exercise program that aims at increasing the overall fitness of the body, strength and shape, as well strengthening the mental state of the exerciser, unity and teamwork. Training usually consists of short warm-ups, twenty minutes of strenuous and very demanding training, and prolonged stretching. CrossFit is based on 10 crucial points: cardiovascu- lar / respiratory endurance, strength, fitness, flexibility, speed, suppleness, coordination, agility, balance and precision (Benjamin, 2012). Encompassing multiple Olympic disciplines (Olympic weightlifting, swimming, gymnastics, athletics…), it seeks to create polyvalent athletes. This is done with high-in- tensity training, a high number of repetitions, specific weights and specific movements for a certain amount of time. This allows for a clear measurement of the results in terms of physical fitness. INTRODUCTION CrossFit also implies that exercisers can make their own programs according to individual requirements. This type of exercise is quite popular with the US military and used by the US and UK police. However, because of the intensity and very explosive action of the muscle, CrossFit has many positive effects on people of average fitness (Meyer, Morrison, Zuniga, 2017). Exercises can be varied (running, rowing, hinges, joints, decay, deadlifts, etc.), and they are performed in a myriad of combinations, one of which is selected as the WOD-workout of the day. The WOD is usually written by a trainer on a board and then everyone writes down their score. This approach fosters a competitive sporting spirit as well as fellowship, mutual assistance and counselling. Each trainer must have a certificate that he / she obtains by attending a specific course in which he / she is obliged to take practical training, and not just theoretical instruction. In 70  Nenad Perić:THE ANALYSIS OF THE MARKET POSITION AND MARKETING POTENTIALS OF CROSSFIT addition to the basic course, there are also courses related to the preparation of sports teams, various Olympic categories, pregnant women, children, etc. addition to the basic course, there are also courses related to the preparation of sports teams, various Olympic categories, pregnant women, children, etc. CROSSFIT FUNCTION AND EFFECTS A true environmental assessment analysis considers several trends at once and assesses their individual and unified impact. The problems in defi- ning trends and the difficulty in accurately predicting consequences do not diminish the essential importance of assessment procedures for better prepa- redness for the future. When it comes to CrossFit, we can say that it is crucial to look at trends in the field of fitness and wellness in general, as well as con- sumers and the target group.i If we look at fitness from a market point of view, we could characterize it as a type of service for recreation, which should satisfy the user's need for health, beauty, movement, fun and minimize the negative effects of modern lifestyle. From this perspective, fitness is an industry that sells a multitude of services, products and a fitness philosophy of living. It follows that fitness is a kind of an industry, and within it, the same principles apply as in all other activities. Today, fitness goes way beyond the former approach to recreation and is linked to payable categories, such as: fitness facilities, programs, equ- ipment, preparations and professional and specialized staff. There are more and more new fitness programs in fitness, especially group guided programs in recent years. These programs are different in content, purpose, use of props and weights, but the same in aerobic mode and group training, usually accom- panied by music as a stimulating medium. The following forms of exercise are practiced: step aerobics, aqua aerobics, body sculpting or body styling, body pumping, kick boxing, tae-bo, spinning, Latin, funky, hip-hop, salsa, afro, ethno aerobics; jazzersize; low impact and many more For all these programs, we can use the term aerobics or fitness programs (Stojiljković, 2005). The wellness trend as a lifestyle and the development of wellness tou- rism in North America and Europe has been ongoing over the last three deca- des, while it has become popular in Serbia since the beginning of the current decade. The concept of wellness was developed by an American physician Halbert Dunn in 1959, when he first wrote about „a specific state of health that encompasses a universal sense of well-being, viewing man as a whole made up of body, spirit and mind and dependent on the environment in which he/she is located” (Dunn, 1957). CROSSFIT FUNCTION AND EFFECTS The term environment is used differently in marketing terms. Kotler's definition of a marketing environment includes all factors beyond the activi- ties of marketing management within the company, including both intrinsic and extrinsic influences. The differences between environment categories are illustrated by the concept of the layers of environment, which overlap one another. He takes as an example only the factors he considers crucial in rela- tion to each individual level (Kotler, 1997). Internal environment. Russ and Kirpatrick imply interactive relations- hips of interdependence between marketing and other management activities. For example, when competing for the provision of resources for their activities or for mutual cooperation in decision making (Russ and Kirpatrick, 1982).f Microenvironment. The second level covers factors that affect marke- ting management beyond the organizational boundaries of a business. This includes: competition, distribution, suppliers, consumers, etc. and the public. A marketing strategist should especially consider the importance of the public and ensure an effective relationship with its important elements. Macro environment. Individual organization practically does not in- fluence factors at this level. These are: socio-cultural environment, political and legal, economic, technological and physical environment. Each of them influences the marketing strategy and therefore their changes and trends must be systematically monitored. The key problem in assessing the environment, which encompasses all events and trends beyond the boundaries of marketing activity is the identifi- cation of trends that are most relevant for analysis. Especially since there are no exact rules to distinguish them. It should be emphasized that identifying the most important ones requires considerable creativity and foresight. Espe- cially since sometimes a seemingly irrelevant trend or change can be crucial in the long run. The most important reason for assessing the environment is to evalu- ate the most likely impacts on an organization. For market strategy planners, Kotler (1997) suggests using an opportunity and threats matrix for this as- sessment. The opportunity matrix serves the planner to evaluate the relative attractiveness that the environment provides, as well as the chances of success if it works in a particular direction. The condition for this is to evaluate the quality and weakness of the company. The planner should also evaluate the potential impact and potential hazards of the environment, as well as their 71  SPORT - Science & Practice, Vol. 9, No 2, 2019. degree in relation to the organization's activities. Of course, this is just the basics. CROSSFIT AND ITS (MARKET) SCOPE AND POTENTIAL CrossFit can be practiced by anyone, men and women of all ages. The exerciser determines the intensity of the training primarily under the super- vision of their instructor, although this is not mandatory - CrossFit is a “free category”. The first phase of entering the program is getting acquainted with all the exercises and gymnastics elements used and perfecting their execution using one's own body weight. After successfully mastering such exercises and achieving good fitness, coordination, flexibility, endurance, the second stage comes in. It includes exercises with dumbbells, weights and Olympic lifting, ergometers. The technique of executing the exercises is first perfected with smaller weights and more repetitions, after which the weights gradually increase. The goal of the second level is to increase strength and maintain and boost the acquired fitness. The third phase is the most serious because it initi- ates a competition with oneself in order to achieve personal records. This does not imply the use of the devices you might normally find in gyms. It involves weights, Olympic bars, links, shafts, tractor tires, medical balls, ergometers, navy ropes, tow frames, etc. So, by default, CrossFit programs are designed to be practiced by anyo- ne, regardless of experience and willingness, as they provide the ability to ta- ilor the difficulty and way of training to a specific person and their goals. The equipment needed to practice CrossFit requires little investment by a club or an individual. Also, the same program with its varieties can be used to prepare martial arts practitioners as well as people with heart conditions. The basics of the program remain the same, what is changing is the workload and the intensity of the exercise programs. Therefore, the potential market coverage of this program is enormous, both in terms of the individuals it can attract and in terms of sports clubs that can implement it and establish collaboration with the CrossFit Network, which has seen a huge expansion in the past ten years. The foundations of the program are the functionality and the need of people to exercise and live a healthy life. CrossFit's philosophy is simple: eve- ry individual should reach their maximum in the fitness goals they needs the most. An additional motivation for all CrossFit workouts is that each workout is based on a combination of different exercises. CROSSFIT FUNCTION AND EFFECTS Hansruedi and Kaufman try to bring together the understanding of the wellness concept in North America and Europe, emp- hasizing that it represents a state of health characterized by harmony of body, mind and spirit, self-responsibility, physical activity, beauty care, healthy ea- ting, relaxation, meditation, mental activity, education, sense of environment and social connections, as the basic elements of wellness (Hansruedi, Kauf- man, 2000). As we can see, unlike fitness, wellness places greater emphasis on the benefits to the body, because physical activity is just one of the items listed above. So, wellness can include fitness, while fitness often extends its services to those in the fitness field, and both, like CrossFit, are struggling 72  Nenad Perić:THE ANALYSIS OF THE MARKET POSITION AND MARKETING POTENTIALS OF CROSSFIT with the idea of the well-being of the person who practices them. They are ve- ry sophisticated in terms of marketing because they represent serious market categories that are attracting more and more people, and therefore incomes. CROSSFIT AND ITS (MARKET) SCOPE AND POTENTIAL Therefore, it is not surprising that CrossFit trainers were also hired by karate practitioners, baseball players, swimmers, boxers and other athletes, in order to perfect certain segments of 73  SPORT - Science & Practice, Vol. 9, No 2, 2019. their fitness. CrossFit uses a gymnastics term progression to define the tran- sition process to master each movement. An approach to progression ensures self-efficacy by creating achievable steps toward more difficult challenges. Commitment to a larger goal will strengthen and encourage members' motiva- tion during the process (Meier, Becker, & Vandenberghe, 2004). Motivation is also reinforced through the observation of other members. The desired results of the observations would be an increase in self-efficacy, belief an individual can accomplish a task and the expected outcome, the perception that the com- pletion of the task will produce a positive end result (Gibson, 2004). Members work together to improve their skills and progress, but on a non-invasive basis - there is no need to go through with training or a specific exercise, but rather, group members are there to support each other to be as good as possible at exercising. Interdependence is needed in knowledge sharing whenever one party cannot accomplish their goal without the help of the other (McNeish & Mann, 2010). However, there is another factor that sustains the group, once formed: as members advance, so does their status within the group (Holmes and Me- ierhoff, 1999). These reward systems help encourage members to continue to grow and develop together through the collective community structure (Mar- tin-Perez, Martin-Cruz, and Estrada-Vaquero, 2010). As new members deve- lop into experienced members, the reward system encourages them to support newer members through their progression. This creates an excellent database of potential practitioners and image groups that practice CrossFit both in more narrow terms (fitness, wellness, etc.), as well as the wider public - potential exercisers. CONCLUSION The world is full of fitness trends, and new trends are starting every day. Most of them, like new brands, never fully develop. The globalization of today's market is widespread in all spheres of the society and it is an indi- spensable part of the sports market. Positioning and creating a recognizable brand, that is, a good image in the global market, is a demanding job that involves long-term continuous efforts, far greater than those defined by local competition (Ratković, Dašić, 2018). The reason for this is the overload of the market with different offers, as well as the overload of the minds of consumers and audiences with numerous offers and other marketing promotions (Perić, Krasulja, Radojević, 2011).f The difference between many other programs and CrossFit is the use of social capital to influence change (Durlauf and Fafchamps, 2004). CrossFit was started by a single man, a sports enthusiast, who spread a small group of 74  74  Nenad Perić:THE ANALYSIS OF THE MARKET POSITION AND MARKETING POTENTIALS OF CROSSFIT people that developed a common language, membership and advancements to all continents. The main reason for the success of this program and its worl- dwide popularity can be found in the simplicity of its concept: it fulfils the need of many people related to physical health, fitness and, consequently, their physical appearance; anyone can practice it to an adequate extent; it encoura- ges cohesion of training groups and a desire for progress while developing a healthy competitive spirit - primarily at the individual and then at the group level; it requires minimal investment both from exercisers and sport clubs; it has a simple and understandable concept. This simple and understandable concept made it possible to create social capital - a large number of exercisers who are also a form of program promotion (word-of-mouth marketing is in most cases the most effective marketing). In addition, it enabled the organiza- tion of a mass competition that, due to the nature of the sport, did not requi- re special technical prerequisites, and its implementation is largely based on CrossFit exercisers and instructors. CrossFit is one of the best examples that simple things and ideas are usually the most effective in any field, including the sports industry. CONCLUSION In addition to becoming a category for itself, CrossFit has grown into a distinct culture, and the foundations of this program are the functionality and need of all people to exercise and live a healthy life. Due to the number of pe- ople practicing this program, the 5-digit number of CrossFit clubs worldwide and its large and continuous media presence, it can be said that CrossFit has become a constantly evolving global sports movement with a huge marketing potential. 75  SPORT - Science & Practice, Vol. 9, No 2, 2019. 18. https://map.crossfit.com/?_ga=2.95823068.1142892516.1574639395- 1739441689.1574639395 Nenad Perić:THE ANALYSIS OF THE MARKET POSITION AND MARKETING POTENTIALS OF CROSSFIT 17. Stojiljković, S., Fitness, Fakultet sporta i fizičkog vaspitanja, Beograd, 2005. 17. Stojiljković, S., Fitness, Fakultet sporta i fizičkog vaspitanja, Beograd, 2005. 18. https://map.crossfit.com/? ga=2.95823068.1142892516.1574639395- REFERENCES 1. Benjamin A. S., (2012). Using Sport Education to Implement a CrossFit Unit, Journal of Physical Education, Recreation & Dance Volume 83, 2012 - Issue 8. 2. Claudino, J.G., Gabbett, T.J., Bourgeois, F. et al. (2018). CrossFit Overvi- ew: Systematic Review and Meta-analysis, Sports Med - Open (2018) 4: 11. https://doi.org/10.1186/s40798-018-0124-5 3. Dunn, H., (1959). High level wellness for man and society, American Journal of Public Health, 49(6): 786–792. 4. Durlauf, S. N., Fafchamps, M. (2004). Social capital, NBER Working Pa- per Series, 1-89. (http://www.nber.org/papers/w10485) 5. Gibson, S. (2004). Social learning (cognitive) theory and implications for human Resource, Advances in Developing Human Resources, 6(2), 193-210. 6. Holmes, J., Meyerhoff, M. (1999). The community of practice: Theories and methodologies in language and gender research, Language in socie- ty, 28(02), 173-183. 7. Jain, C., (1984). Environmental scanning: how the best companies do it, Journal of Long Rang Planning, Vol. 17. 8. McNeish, J., Mann, I. (2010). Knowledge sharing and trust in organizati- ons. IUP Journal of Knowledge Management, 8(1/2), 18-38. 9. Kotler, P. (1997). Marketing management: analisys, planning, implemen- tation and control, Prentice-Hall, New Jersey. 10. Martín-Pérez, V., Martín-Cruz, N., Estrada-Vaquero, I. (2012). The in- fluence of organizational design on knowledge transfer. Journal of Kno- wledge Management, 16(3), 418-434.i 11. Meyer, J., Morrison, J. Zuniga, J. (2017). The Benefits and Risks of CrossFit: A Systematic Review, Workplace Health & Safety 2017, 612- 618. https://doi.org/10.1177/2165079916685568 12. Meyer, J., Becker, T., Vandenberghe, C. (2004). Employee commitment and motivation: a conceptual analysis and integrative model, Journal of applied psychology, 89(6).991-1007. 13. Mueller, H., Kaufmann, E. L., Wellness Tourism: Market Analysis of a special health tourism segment and implications for the hotel industry, Journal of Vacation Marketing, 2000, 2-3. 14. Perić, N., Krasulja, N., Radojević, I. (2011). Medijske, PR i brend tenden- cije, drugo, prošireno izdanje, Synopsis. 15. Ratković, M., Dašić, D. (2018). Marketing u sportu sa elementima indu- strije sporta, Visoka škola modernog biznisa 16. Russ, A., Kirkpatrick, C., Marketing, Little, Browne & Co, Boston, 1982. 76  Nenad Perić:THE ANALYSIS OF THE MARKET POSITION AND MARKETING POTENTIALS OF CROSSFIT 77 
https://openalex.org/W4386479207	https://www.researchsquare.com/article/rs-3062037/latest.pdf	English	null	Determining Malware Attacks in Iot Health Care System Using Self Attention Based Depth Poly O (Optimized)-network	Research Square (Research Square)	2,023	cc-by	12,605	Research Article Keywords: Internet of Things, Healthcare, malware attacks, Min-max Hat normalization, wrapper discriminant component analysis, Hybrid cuckoo Adam optimization algorithm, self-attention based depth poly O (optimized)-Network Posted Date: September 6th, 2023 Abstract— Abstract— just some of the fields that have been affected by the Internet of Things. Patient information is typically stored and sent over an IoT-based healthcare system, making data security a top priority. All of a hospital's most critical patients are brought to the intensive care unit (ICU) to get the best care available. ICU staff are on call around the clock to care for patients who have suffered life-threatening illnesses or injuries. The life and health of the patient depend on the ICU's instruments and machinery. If a patient can't reach their doctors, it might have catastrophic effects for their health. In light of this, it is critical to develop a healthcare system based on the Internet of Things that is secure against cyberattacks. This highlights the need of ensuring secure communication between medical sensors and actuators in an IoT-based healthcare system. Many initiatives have been taken so far to automate patient monitoring and let medical practitioners remotely analyze a patient's health state using remote monitoring and analysis technologies. To prevent identifying data from leaving the hospital, several techniques employ fog computing to do certain calculations at the edge. IoT healthcare systems, however, have been a prime target for hackers in recent years. According to some estimates, hackers have successfully into 90% or more of hospital networks. Intel from the White House in 2017 suggests that the healthcare sector is increasingly being targeted by hackers. Note that cyber assaults increased dramatically during the COVID-19-time period. As of late, IoT healthcare services and infrastructure have been a prime target for hackers. Security and confidentiality of patient information are of paramount importance in IoT healthcare environments. The health of patients is seriously threatened when sensitive information is stolen or tampered with in healthcare systems. To the same extent, a patient's life might be jeopardized by unauthorized changes to his or her medical file. Furthermore, cyber-attacks on hospital IoT systems might significantly disrupt patient care. For example, a hacker may get access to a patient's infusion pump and change the settings in such a way that the pump releases an unsafe amount of insulin, putting the patient at risk of possibly fatal hypoglycemia. Now picture a similar situation in which a hacker gains access to a person's pacemaker and alters the device to cause the heart rate to decrease or increase. Abstract— A deadly bradycardia or tachycardia might arise suddenly in such a situation. In light of this, ensuring the security of healthcare networks is of critical significance. As a result of their small memory capacities, slower processors, and shorter battery lives, IoT devices have resource restrictions. For this reason, traditional security solutions fail to effectively identify malicious attacks on IoT devices. As a result, designing a The Internet of Things (IoT) is becoming more important in numerous sectors, including healthcare, industry, the military, and education. The framework successfully safeguards the privacy, authenticity, and accessibility of data in a networked setting. Health care is only one of many sectors that may benefit from the given solutions, which are essential for protecting the personal information of patients and maintaining the integrity of their medical records. The privacy, security, and reliability of the whole health care system online are still at danger owing to a broad variety of intermediary assaults and infiltration activities, despite the fact that the Internet of Things (IoT) offers trustworthy mechanisms for keeping data secure. In this research, we employ a hyper-tuned optimal classifier based on deep learning to overcome these issues. Here the real time patients sensor data are retrieved and it can be preprocessed for removing the error by using the Minmax Hat normalization. Then the features can be selected using wrapper discriminant component analysis. Then the proposed classifier can be optimized using the Hybrid CUADA (Cuckoo Adam) optimization algorithm. After classification parameter optimization, the collected features are fed into the newly- introduced self-attention based depth poly O (optimized)- Network to detect malware intrusions and monitor patient records. The system's effectiveness has been assessed based on experimental findings and subsequent discussions. Index Terms—Internet of Things, Healthcare, malware attacks, Min-max Hat normalization, wrapper discriminant component analysis, Hybrid cuckoo Adam optimization algorithm, self- attention based depth poly O (optimized)-Network DOI: https://doi.org/10.21203/rs.3.rs-3062037/v1 DOI: https://doi.org/10.21203/rs.3.rs-3062037/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. R d F ll Li ATTENTION BASED DEPTH POLY O (OPTIMIZED)-NETWORK Paul Rodrigues1 Harun Bangali2Syed Asif Basha3 T Gopalakrishnan4 Pandimurugan V5 Rajasoundaran S6,SVN Santhosh Kumar7 1 Professor, Department of Computer Engineering, College of Computer Science, King Khalid University, Al-Faraa, Saudi Arabia 2,3 Lecturer, Department of Computer Engineering, College of Computer Science, King Khalid University, Al-Faraa, Saudi Arabia 4Associate Professor, Department of Information Technology, Manipal Institute of Technology Bengaluru, Manipal Academy of Higher Education, Manipal, India g , p , 5,6 Assistant Professor, School of Computing, Networking and communications, SRMIST, Kattankulathur, Chennai, India. 7School of Information Technology and Engineering, Vellore Institute of Technology, Vellore,India nt Professor, School of Computing, Networking and communications, SRMIST, Kattankulathur, Chennai, Indi f Information Technology and Engineering, Vellore Institute of Technology, Vellore,India 5,6 Assistant Professor, School of Computing, Networking and communications, SRMIST, Kattankulathu 7School of Information Technology and Engineering, Vellore Institute of Technology, Vellore,India I. INTRODUCTION Currently, only the basic minimum in IDS technology [[6, 7]] is available for IoT systems, despite the need for more advanced options. Insufficient data storage and analysis capabilities of IoT nodes have been cited as a major cause of issues for host IDS [[8, 3]]. As different protocols raise different security risks and need for different implementations of intrusion detection systems [7], it is crucial that the communication protocols used by IoT devices (such as CoAP, MQTT, etc.) are not utilized in the traditional network. In order to effectively adapt IDS for an IoT-based critical care unit, it is important to amass a large and reliable IoT network traffic dataset. A breach in security in such a setting might potentially endanger the patient's life [9]. There are several state-of-the-art datasets accessible for the purpose of training and assessing IDS and IPS for both legacy and IoT networks. It's interesting to see how many of the older suggestions for measuring network traffic are still in use today. This data was generated either in real-time using real-time systems or in a simulated environment using simulation tools. The DARPA dataset, the KDD-99 dataset, the NSL-KDD dataset, the DEFCON dataset, the LBNL dataset, the CAIDA dataset, the UNIBS dataset, the foolproof system of protection for Internet of Things gadgets is an extremely difficult undertaking. Security mechanisms that function across the network are thus ideal for IoT devices. In this study, we show how the suggested AI-based architecture might protect IoT healthcare systems from cyberattacks. In particular, the proposed system contributes by, , ❖ “To classify IoT attacks and continuously learn to improve the accuracy of deep learning. ❖ We first use wrapper discriminant component analysis to analyze the source code and to isolate the faulty features ❖ Using the Hybrid CUADA (Cuckoo Adam) optimization technique, the suggested classifier may be fine-tuned for use in the classification phase. The retrieved characteristics are fed into the newly-introduced self-attention based depth poly O (optimal)-Network for detecting malware attacks once the classification parameters have been tuned.” The suggested architecture will aid in building context-aware IoT security solutions, notably in the sensitive healthcare setting. The remaining sections of the paper are organized as follows. Current IDS approach in an IoT setting is presented in Section 2. The suggested framework for creating context-aware security solutions for the Internet of Things is outlined in Section 3. I. INTRODUCTION "Internet of things" (IoT) refers to a possible future in which all of our ordinary objects will be internet-connected and able to communicate with one another and learn from each other. The IoT is a revolutionary transformation in how networks are constructed, allowing previously incompatible gadgets to communicate and share data. Maintaining human civilization will depend on this form of communication. Both the technological landscape and people's day-to-day lives have been profoundly altered by the advent of the Internet of Things. The number of gadgets that can connect to the Internet has exploded in recent years. New research suggests that in 2018, there were around 23.14 billion IoT devices connected online throughout the world. By 2025, it is expected that there will be 75.44 billion connected things in operation. Construction, farming, transportation, the electrical grid, and healthcare are were identified using ELM, we applied Fuzzy C-Means to fix several mislabeled instances in the dataset. See [4], which offered a feature engineering technique for efficiently identifying the irregularities, for a similar approach to boosting the performance of traditional IDSs. Health care IoT fog environment risks were also described and explored in [5]. Researchers used an online sequential ELM ensemble to prevent man-in-the-middle and distributed denial-of-service attacks on a fog-based smart home with a remote patient monitoring system. Since privacy and trust were first key concerns with the Internet of Things, security has emerged as the most pressing of those concerns. Protecting networks and devices against malicious software requires the use of firewalls and intrusion detection systems (IDS/IPS). Although this capability is available in many firewalls, static rules are often utilized to identify trusted and unsafe traffic. By contrast, AI- powered intrusion detection and prevention systems (IDS/IPS) have shown to be more reliable and successful than the static rules that came before them. In training and validating the IDS and IPS, both malicious and benign network traffic is utilized. Either real systems that create malicious and benign network traffic, or artificially generated traffic, may be used to construct such datasets. A simulated network environment may be created with the use of traffic generators. Training and testing IDS and IPS using data from live networks is essential, but it may be time-consuming and costly. Nevertheless, this may be avoided if a traffic generator were used in the creation of the dataset. I. INTRODUCTION The outcomes of the suggested framework were explored in Section 4, and the article is wrapped up in Section 5. II. RELATED WORKS They use a method of cognitively evaluating characteristics by analyzing the feature space. The first layer's input neurons were subjected to a pruning procedure to exclude those that were associated with less important properties. By the implementation of our technique, we were able to increase accuracy from 97.84% to 99.3% while decreasing the number of characteristics from 154 to only 6. Using an ensemble learning strategy, [19] proposes a system for intrusion detection in WLANs. They were able to accomplish this remarkable feat of feature reduction by excluding any characteristics that were either identical throughout the whole dataset or had more than half of their values missing. A total of 36 characteristics were generated using this method. Ensemble Learning's classification results for impersonation, flooding, injection, and normal showed an accuracy of 95.88% when all the assaults were combined into a single category. According to [20], machine learning may be used to construct an intrusion detection system tailored to the IoT with little resources and an emphasis on impersonation threats. This study categorizes Internet of Things devices into two distinct categories: semi-distributed and distributed architectures. A stacked auto encoder technique was employed for feature extraction. For each of the dataset subsets, the best 7 features were chosen from the original features using a variety of feature selection techniques. The classification was performed using a classifier called a multi-layer perceptron (MLP). The experimental findings demonstrated that the semi- distributed model outperformed the distributed models trained with 21 characteristics by a margin of 99.97%. This study [21] develops a three-stage framework for detecting impersonation attacks in wireless networks. The Mutual Information (MI) and C4.8 (C4.8) algorithms were employed to complete feature extraction and classification, respectively. They claimed a 98.22% success rate while employing only five factors. An intrusion detection system based on a deep learning model (stacked auto-encoder, or SAE) is provided in [22] to distinguish between impersonation, flooding, injection, and normal traffic. Both writers suggested adding an additional layer of complexity into their respective models. Without feature selection, two hidden layers were able to achieve greater accuracy (98.67%) than three. Several deep learning-based IDS course of seven weeks to facilitate the evaluation of IDS. Several protocols, including Simple Mail Transfer Protocol (SMTP), Internet Message Protocol (IMAP4), Internet Group Chat (ICQ), and Simple Network Management Protocol (SNMP), are used to bring the data in. II. RELATED WORKS Many simulated attacks, including DoS, R2L, and U2R, were tried on both Windows and UNIX platforms. Nevertheless, this dataset has several peculiarities, such as the absence of false positives and the exclusion of real-time traffic. [10]. New research [11] proposes using neural networks for intrusion detection, with a focus on how training might improve performance and efficiency. For WSNs, the authors of [12] recommend using an SVM classifier in conjunction with the binary grey wolf optimizer. In terms of feature count, accuracy, false alarm rate, and detection rate on the NSL KDD'99 dataset, the proposed IDS outperformed state- of-the-art approaches. The IDS system's feature extraction and selection process was given a new spin by combining the Aquila optimizer with the CNN DL (Deep Learning) technique [13]. The novel IDS approach was tested using the public datasets CIC2017, BoT-IoT, NSL KDD, and KDD99. The test results show that the devised approach works as intended. An IDS method based on Fruitfly optimization and deep Auto encoder is presented in [14]. They employed a deep Auto encoder to determine the most important aspects of the assaults, a back propagation neural network to categorize the data, and a fuzzy C-Means clustering approach to manage the gaps in the data. In addition, the Fruitfly algorithm was used to adjust the Auto encoder’s concealed layers. Testing on the NSL-KDD and UNSWNB15 datasets confirmed the efficacy of the proposed method. In light of the fact that the AWID dataset [15] was used to assess the proposed IDS system, we highlight some relevant work that was also evaluated on this dataset. The influence of feature selection on detection accuracy is a central topic of the selected research. Suitable contexts for this work may be found in the following fields linked to machine learning and deep learning. Apply Machine Learning to an Existing Issue With the goal of discovering the best efficient classifier for detecting intrusions, the AWID dataset [15] was created utilizing a wide range of traditional ML techniques (AdaBoost, OneR, J48, Naive Bayes, Random Forest, ZeroR, and Random Tree). Using all 154 features with J48 as the classifier, they got an accuracy of 96.20 percent, while using just 20 features, they got an accuracy of 96.26 percent. The feature selection strategies employed by the IDS given in [16] include information gain and Chi-squared statistics. II. RELATED WORKS The novel IDS approach was tested using the public datasets CIC2017, BoT-IoT, NSL KDD, and KDD99. The test results show that the devised approach works as intended. An IDS method based on Fruitfly optimization and deep Auto encoder is presented in [14]. They employed a deep Auto encoder to determine the most important aspects of the assaults, a back propagation neural network to categorize the data, and a fuzzy C-Means clustering approach to manage the gaps in the data. In addition, the Fruitfly algorithm was used to adjust the Auto encoder’s concealed layers. Testing on the NSL-KDD and UNSWNB15 datasets confirmed the efficacy of the proposed method. In light of the fact that the AWID dataset [15] was used to assess the proposed IDS system, we highlight some relevant work that was also evaluated on this dataset. The influence of feature selection on detection accuracy is a central topic of the selected research. Suitable contexts for this work may be found in the following fields linked to machine learning and deep learning. Apply Machine Learning to an Existing Issue With the goal of discovering the best efficient classifier for detecting intrusions, the AWID dataset [15] was created utilizing a wide range of traditional ML techniques (AdaBoost, OneR, J48, Naive Bayes, Random Forest, ZeroR, and Random Tree). Using all 154 features with J48 as the classifier, they got an accuracy of 96.20 percent, while using just 20 features, they got an accuracy of 96.26 percent. The feature selection strategies employed by the IDS given in [16] include information gain and Chi-squared statistics. Thereafter, they conducted tests using a variety of feature-size vectors (111, 41, and 10 features). The evaluation revealed that when trained with 41 features, Random Tree's classification accuracy improved from 92.17 to 95.12. In [17], a method is proposed for detecting impersonation attacks on wireless networks. There were three distinct machine learning (ML) techniques utilized for classification: an ANN, a decision tree and an SVM After extensive testing it was discovered that the SVM method, which employs 11 carefully selected features for training, had the highest overall performance (99.86%). They debated the first blow but disregarded the two that followed (injection and flooding attacks). Using a cognitive feature ranking algorithm and an ANN classifier, the authors of [18] present an intrusion detection system. They use this technique to determine whether an occurrence is benign or harmful. II. RELATED WORKS Many aspects of our everyday lives have been altered by the rapidly developing Internet of Things. With the advent of IoT, more advanced methods of health monitoring have become possible. Several proposals for IoT-based intelligent health monitoring systems have surfaced in recent years. Yet, healthcare IoT security is only getting started. Traditional network security measures are impractical for IoT-based healthcare systems because of the limited resources of IoT devices and the unpredictable security needs of these systems. [1]. Keep in mind that the types of data generated by IoT devices in an IoT ecosystem shift significantly from one use case to the next. Traditional security protocols need to be adjusted to suit the IoT's unique characteristics. [2]. In addition, there is an immediate need to secure the IoT network that links constrained medical devices. Many investigations have led to the development of several techniques for preventing cyberattacks on healthcare IoT devices. A solution is provided in [2] to help detect and prevent replay attacks on healthcare IoT devices that run on batteries. Their proposed solution looks at the device's unique identifier, timestamps, and power consumption patterns to detect and prevent replay attacks on battery-operated IoT healthcare equipment. According to [3], semi-supervised Fuzzy C-Means based on extreme learning machines may be used to identify cyberattacks in fog-based Internet of Things scenarios (ELM). As soon as cyberattacks course of seven weeks to facilitate the evaluation of IDS. Several protocols, including Simple Mail Transfer Protocol (SMTP), Internet Message Protocol (IMAP4), Internet Group Chat (ICQ), and Simple Network Management Protocol (SNMP), are used to bring the data in. Many simulated attacks, including DoS, R2L, and U2R, were tried on both Windows and UNIX platforms. Nevertheless, this dataset has several peculiarities, such as the absence of false positives and the exclusion of real-time traffic. [10]. New research [11] proposes using neural networks for intrusion detection, with a focus on how training might improve performance and efficiency. For WSNs, the authors of [12] recommend using an SVM classifier in conjunction with the binary grey wolf optimizer. In terms of feature count, accuracy, false alarm rate, and detection rate on the NSL KDD'99 dataset, the proposed IDS outperformed state- of-the-art approaches. The IDS system's feature extraction and selection process was given a new spin by combining the Aquila optimizer with the CNN DL (Deep Learning) technique [13]. II. RELATED WORKS [26] offers a framework for creating context-aware security solutions, which may be used to detect malicious communications in IoT deployment settings. Their strategy is built on the innovative new open-source IoT data producing tool IoT Flock. The IoT-Flock tool allows researchers to simulate both safe and insecure IoT environments. An open- source tool is included into the proposed architecture to centralize the IoT-Flock traffic records. They developed a healthcare IoT dataset including both safe and harmful IoT communications to evaluate their approach. Finally, they used several machine learning strategies to the newly minted data in an effort to spot assaults on the healthcare system. A real-time Improved Healthcare Monitoring System (EHMS) has been designed and deployed [33] to track patient vitals and gather data on network use. The system then employs a variety of machine learning techniques to both reinforce and weaken the dataset. They were able to construct a trustworthy intrusion detection system by gathering data in real time. They found that the SVM technique outperformed the other algorithms by a factor of 10, with a success rate of 92.44%. In [34], researchers used knowledge from many different areas to create a data fusion system for detecting intrusions. They suggest using a decision-based fusion model that makes use of steps like initialization, preprocessing, Feature Selection, and multimodal classification to effectively detect intruders. The suggested model achieves its best results with a detection rate of 99.21%, a precision of 98.93%, and an accuracy of 99.21%. Read [35] to learn about the long-term effects of intrusion detection in the Security of Things (SoT) paradigm on smart healthcare. Classifiers such as radial basis functions (RFs), neural networks (NBs), and logistic regressions were all used to the NSL-KDD dataset by the research community. Using a weighted genetic approach in conjunction with Random Forest, we were able to improve detection while simultaneously reducing false positives and negatives. They used a combination of a genetic algorithm and RF models and had a 98.81% success rate. After recognizing and assessing these assaults, the authors of [28] conducted a scoping study, where they discovered 18 solutions that may be categorized into the following three classes: intrusion detection and prevention (IDP), communication tunneling (CT), and hardware defenses (HWP). Authors at [29] suggested using wireless body area networks as part of an intrusion detection system for the IoMT (WBAN). II. RELATED WORKS Thereafter, they conducted tests using a variety of feature-size vectors (111, 41, and 10 features). The evaluation revealed that when trained with 41 features, Random Tree's classification accuracy improved from 92.17 to 95.12. In models performed far worse. Thus, a Smart E-Healthcare System might be supported by this architecture, boosting patients' online safety. Internet of Medical Things security and privacy issues continue to persist, despite ongoing technological developments aimed towards resolving them (IMoT). Hence, [31] built a trustworthy IDS using a DNN for better detection and prevention of intrusions. Some hyper parameter selection feature engineering was also included. The proposed DNN model improves on the baseline NSL-KDD results achieved using conventional machine learning approaches by 15%, reaching an overall accuracy of 99.7%. In [32], an anomaly detection approach for cardiac rhythm data is presented, focusing on the latter. A total of five techniques were used, with two being unsupervised and the other three supervised, all with the goal of spotting irregularities in the data. When used to characterize such systems, they found that random Forest and ensemble approaches achieved an accuracy of 99% or higher. removed. During the phase of classification and detection, it was recommended to use either a deep neural network with three hidden layers or a deep neural network with seven hidden layers. The 7-hidden-layer variant showed a 0.2 point increase in accuracy. Impersonation risks in wireless communications were identified by [24] using unsupervised clustering and a deep learning approach (stacked auto-encoder). The proposed method is split into two distinct phases: feature extraction using a two-layer stacked auto-encoder, and K-means-based unsupervised clustering. After calculating over all 50 characteristics, the total success rate was 94.81%. On top of that, [25] proposed a wireless IDS that using semi-supervised deep learning to classify network traffic as either benign, malicious (such as an impersonation attack), or malicious (such as a flooding assault or injection attack). They used a stacked auto-encoder, which uses a ladder network strategy by stacking a clean decoder unit on top of a noisy encoder unit and a decoder unit. The introduction of a focus loss function widened the application domain of the proposed method. Classification accuracies for "normal," "impersonation," "flooding," and "injection" were 99.77%, 89.32%, 73.41%, and 82.79%, respectively. Overall, this method had a 98.54 percent success rate. II. RELATED WORKS A hospital network architecture model was used to conduct extensive testing on many subsets of the IoMT, such as wireless body area networks and other types of connected medical equipment. Maximum intrusion detection accuracy at the network level is 99.6 percent, and maximum detection accuracy at the device level is 98.2 percent. Using a comprehensive set of criteria, the authors of [36] rate and assess several machine learning algorithms and data sources. Furthermore, they have evaluated the approaches' capabilities for avoiding false positives. In-depth research reveals obstacles to putting the algorithms into practice and suggests solutions. K-Nearest Neighbors seems to be widely used in the current literature for healthcare intrusion systems, as shown by their analysis. The authors of [30] present an intrusion detection system (IDS) that makes use of many Machine Learning methods and some complicated attack data. Bagging ensemble and Random Forest both achieved 97.67% accuracy, whereas other classifier [37] For the purpose of identifying and blocking known harmful, phishing, spammy, and malware-based domains, the K-Nearest Neighbors method for analyzing DNS traffic has been proposed. Their F1 score was 94.8 and 99.4%, respectively, using the gender- and skew-balanced (60/50) CIC Bell DNS 2021 dataset. The authors of [38] came up with a unique approach to the problem of assault identification by assessing the detection abilities of various base classifiers and then creating an ensemble to use in recognizing certain types of attack. They conducted rigorous experiments across a variety of methods on the CIC-IDS 2018 dataset, provided a thorough analysis of the class wise performance metrics, and arrived at the conclusion that neural networks reach 96.65% accuracy. Researchers [39] evaluate their ability to spot common zero-day threats by using open-source intrusion detection datasets. Their case study compares and contrasts the datasets (KDD99, NSL- KDD, and CIC-IDS-2018) utilized to develop their predictors. For the NSL-KDD datasets, an accuracy of about 99.97% was attained. On the other hand, [40] presented RCNN, a more complicated Intrusion detection system based on CNN and LSTM (Long Short Term Memory), which was evaluated on the KDD Cup 99 and CIC-IDS 2018 datasets (Recurrent Convolutional Neural Networks). Around 94% accuracy was attained using the same model across both datasets. rank's correlation. While designing our own neural network, we relied on the activation function MISH [45], also designed by the same author. When we spoke about how extensive it was, we realized this. II. RELATED WORKS The average accuracy of Mish on YOLOv4 utilizing a CSP-DarkNet-53 base is higher than that of alternative activation functions, such as Leaky ReLU. The LightGBM ensemble approach has been shown to be an effective tool for Intrusion Detection since its introduction in [46]. Results from the research demonstrated the superiority of their method over both conventional machine learning algorithms and other ensemble methodologies (98.37 percent). An auto encoder-based bi-level anomaly detector was published in [47], which achieved a maximum detection rate of 99.2 percent on the CIC IDS 2017 and 2018 datasets by training an artificial neural network to reconstitute its vectorized input. For what seems to be a problem with few variables, a complex algorithm was created. Using several datasets, the authors of propose a Convolutional auto-encoder (Conv-AE) for intrusion detection in [48]. It is shown that the Conv-AE algorithm is able to identify hostile assaults in the context of both anomaly- and misused-based ID systems by effectively integrating the benefits of conventional state-of-the-art techniques. They achieved a 98.2 percent success rate by training their algorithms with the help of a distributed and big-data-friendly processing pipeline. [49] demonstrates how combining anomaly detection with abuse detection helped overcome their respective constraints. They implemented K-Nearest Neighbors on the KDD-Cup99 dataset. They had an accuracy of over 98%, but a poor Area Under the Curve (AUC) of 0.58. In [50], we provide a concise overview of the CIC-IDS-2017 and CIC-IDS-2018 datasets, discussing how we evaluated each dataset's characteristics and iterated them over several Machine Learning and Deep Learning models. Several have stated that the more varied assaults, data collection methods, and attack infrastructure used by CIC IDS datasets make them superior to those of NSL KDD. For buildings to function at their full potential, socioeconomic differences must be taken into consideration at every stage of the design process. The authors of [41] analyzed online assaults with random undersampling ratios to investigate class balance in the context of different ensemble learning approaches. Accuracy of around 94.01% was attained using RCNN on the CIC IDS 2018 dataset, however the authors did find that undersampling at particular ratios may have a significant influence on the model's performance. An alternative perspective on the importance of network traffic and its hierarchical structure is provided by the packet byte-based convolutional neural network (PBCNN), which was initially introduced in [42]. II. RELATED WORKS For both the CIC IDS 2017 and CIC IDS 2018 datasets, PBCNN achieved an accuracy of 99.99%. In [43], a symmetrical method is used to evaluate the CIC-IDS- 2018 data set. It was also investigated whether or not there were any biases or imbalances in the classes represented in the CIC- IDS-2018 dataset. It has been shown that signature-based intrusion detection approaches are not as effective as other methods. [51] claims that an adaptive and ensemble system is superior to the anomaly-based method. Combining the KNN, DNN, and random forest algorithms, they developed an adaptive voting system for use on the KDD Cup99 dataset. Based on their findings, the Adaboost model outperformed the competition with a 99.99 percent accuracy rate. If you want to come up with creative and useful solutions, you need to master the dataset. Recurrent Neural Networks, such as Gated Recurrent Units, outperformed regular LSTM networks while trying to create a trustworthy anomaly-based intrusion detection system, as reported in [52]. It is possible that convolutions and recurrent algorithms are computationally demanding and time consuming compared to other approaches, yet they are effective for processing non-sequential tabular data. Almost 81% accuracy has been achieved in the ADFA Intrusion Detection dataset. About the drawbacks of anomaly-based detection, [53] The majority of the studies on IDS methods have been carried out in an ensemble learning framework. In order to determine the most effective basic classifiers for ensemble learning, the authors of [44] analyzed seven distinct options and compared the accuracy metrics of the evaluated architectures. They used just 23 features and yet achieved 98.8% accuracy on the CIC IDS 2018 dataset because to the excellent feature selection processes they used, including filter approaches and Spearman against network, software, and data breaches. In Figure 1, the whole process of the suggested method is shown. presented a hybrid solution that uses SVMs and decision trees. They get 97.881 and 99.982 percent accuracy on the CIC IDS 2018 and KDD Cup 99 datasets, respectively. In order to detect DoS assaults, Wenkhede and Kshirsagar created a system that makes use of ML and NN. Multi-Layer Perceptron and Random Forest are used by [54] for the CIC IDS 2017 dataset. The experimental findings showed that the Random Forest algorithm outperformed the Multi-Layer Perceptron method by a wide margin, achieving an accuracy of almost 99% on the CIC-IDS 2017 datasets. IoT security has two main objectives: Our research is based on the assumption that the setting is an indoor hospital, either an intensive or general care unit, with the patient confined to a single room. A reliable outside source is keeping tabs on the patient's vitals from a safe distance. Our IoT-based ICU use case includes two beds, each of which is outfitted with nine patient monitoring devices (sensors), and a single control unit known as the Bedx-Control-Unit. The IoT- Flock architecture serves as the basis for all of these devices. In this piece, we will discuss the transmission of ECG data, namely HRV. The suggested patch integrates with a current sensor network to get ECG data. Light-emitting diode (LED) or Bluetooth Low Energy (BLE) modifications are made to the 1.Ensuring the safety of data throughout its collection, storage, processing, and transmission 2. In order to achieve these objectives, an unique approach was created to detect vulnerabilities in IoT components. III. PROBLEM STATEMENT The Internet of Things is perhaps one of the most fascinating technological breakthroughs of recent times. IoT Insights predicts that by 2025 there will be more than 27 billion Internet- connected devices in use worldwide. Fears about software vulnerabilities and assaults are increasing, which might discourage many customers from embracing IoT devices. Companies in the healthcare, financial services, industrial, logistics, and retail sectors, among others, are particularly vulnerable to Internet of Things security threats because of their widespread use. Figure 1 Schematic representation of the suggested methodology II. RELATED WORKS Figure 1 Schematic representation of the suggested methodology We discover the foundation for an Internet of Things-based medical data preservation system in [55], where an unique block chain-based method to encryption of medical data is combined with an optimization algorithm. The Stochastic Gradient Descent with Warm Restarts method, introduced in [56], was developed using Cosine Annealing to schedule learning rates. We were able to do more precise studies as a result of this. In [57], a new method of weights initialization for neural networks is presented to improve model convergence and performance. Using Decision Trees and the KDD Cup 99 dataset, the authors of Article [58] suggest a recursive feature reduction technique for feature selection, claiming an accuracy of more than 99% when used to DOS assaults and other attacks. The authors of [59] offer a correlation-based feature selection strategy based on their findings; by using a neural network, they increase accuracy by 0.98 percentage points on the NSL-KDD dataset. [60] a. Data source IoT security has two main objectives: b. Preprocessing 𝐻(𝑘) = −∫ 𝜇𝑌(𝑘)log 𝜇𝑌(𝑘)𝑑𝑘 (5) (5) We shall describe the Min max_ Hat normalization approach we used in this section of the article. As a rule, raw data has a wider range of values than analyzed information. Data parameters with a large range of values will have a significant impact on the calculated distance. This is why it's important to normalize (or scale) all characteristics so that their values fall within the same general range. The conditional entropy of a set is the measure of the uncertainty associated with a set, such as k, for which we have indirect knowledge by virtue of our knowledge of X and j: The conditional entropy of a set is the measure of the uncertainty associated with a set, such as k, for which we have indirect knowledge by virtue of our knowledge of X and j: 𝐻(𝑘/𝑗) = −∫ 𝜇𝑋(𝑗) ∫ 𝜇𝑌(𝑘/𝑗= 𝑥)log 𝜇𝑌(𝑘/= 𝑥)𝑑𝑘𝑑𝑗(6) The joint entropy, defined as, is the collective degree of doubt about the (X,Y) pair: The simplest approach, known as min-max Hat normalization scaling, works by rescaling the range of feature values to the interval [0,1] or [-1,1]. When deciding on a target range, it's important to consider the data's context. In general, for [0,1], the formula for a min-max is: 𝐻(𝑗, 𝑘) = −∫ 𝜇𝑗,𝑘(𝑗, 𝑘)log 𝜇𝑋,𝑌(𝑗, 𝑘)𝑑𝑗𝑑𝑘 (7) (7) The degree to which j is informed by k is quantified by the mutual information between j and k. (or conversely on the amount of knowledge on j provided by k . Thus, it may be described as: 𝑙′ = 𝑙−n(𝑙) m (𝑙)−n(𝑙), (1) (1) 𝑀(𝑗; 𝑘) = 𝐻(𝑘) −𝐻(𝑘/𝑗) (8) where x is the unaltered value and x' is the new value. We may use this formula to rescale the range [a, b]: (8) (8) Which is precisely what happening to k's uncertainty when z is known and the latter is reduced. If k is the target of the prediction, then j's relevance to the model for k may be evaluated using the mutual information. Rewriting the mutual information in terms of the entropy's attributes results in: 𝑙′ = 𝑎+ (𝑙−n(𝑙))(𝑏−𝑎) n(𝑙)−n(𝑙) , (2) (2) a range of numbers between a minimum of a and a maximum of b. c. Feature selection Here, we employed discriminant component analysis to finish up the feature selection process. So, we'll pretend j and k are two independent variables (they can have real or vector values). It's how we indicate 𝜇𝑗,𝑘 the distribution of probabilities for both z and k together. The marginal density functions are, as we all know, defined by : 𝜇𝑋(𝑗) = ∫ 𝜇𝑗,𝑘(𝑗, 𝑘)𝑑𝑦 𝜇𝑌(𝑘) = ∫ 𝜇𝑗,𝑘(𝑗, 𝑘)𝑑𝑥 (4) (4) Its entropy, defined as, provides the measure of our uncertainty about k.: Its entropy, defined as, provides the measure of our uncertainty about k.: Figure 2 Process of dataset retrieval IV. PROPOSED WORK This research proposes a framework for detecting attacks on IoT devices. This research utilizes an optimized self-attention based depth poly O (optimal)-Network to safeguard IoT devices hat matrix model grows more accurate as the real value approaches 1, signal before transmission. LEDs are the source of information for stationary security cameras. At the end, data is sent to an authorized observer through an electronic healthcare gateway. 𝑙′ = 𝑎+ (𝑙−n(𝑙))(𝑏−𝑎) n(𝑙)−n(𝑙) . (3) 𝑙′ = 𝑎+ (𝑙−n(𝑙))(𝑏−𝑎) n(𝑙)−n(𝑙) . (3) Figure 2 Process of dataset retrieval When we standardized the raw data set, we got a value of 0.976. However, when we applied the normalized (scaled) data, we obtained a normalized value of 0.786. As such, we dissect the proposed algorithm as follows: The outcome of the ranking technique is a rating of the variables based on the mutual information between them. Thus, the approach multiplies each input variable by n before arriving at the final output value 𝐼(𝑗𝑖; 𝑘), 𝑖= 1 … 𝑛, Then, the variables are ranked according to this value, and the least significant ones are eliminated. Purpose of the Cuckoo Species is(𝑞); 𝑞= (𝑞1, … … 𝑞𝑣)𝑀, and the initial number of individuals inside the k host nest is shown as 𝑃𝑗(𝑧= 1,2, … , 𝑘). The following formula offers fresh information on how to help cuckoo birds. 𝑞𝑗 𝑢+2 = 𝑞𝑗 𝑢+ 𝛽⊕Levy (𝛾) (13) (13) The lowest redundancy - maximum relevance criteria consists of picking the variable 𝑗𝑖 from the features 𝑗−𝑠 that maximizes 𝑢𝑖-𝑗𝑖, where 𝑢𝑖 is a relevance term and 𝑗𝑖 is a redundancy term. 𝑗𝑖 is the mean redundancy of 𝑗𝑖 to each variable 𝑗𝑖∈z already selected. “where P(u+1) stand for the current answer, and z for the cuckoo, ⊕ represents the entry-wise multiplications, Levy 𝛾 𝛽 > 0 stands for the levy rise and for a potential increment in magnitude. “where P(u+1) stand for the current answer, and z for the cuckoo, ⊕ represents the entry-wise multiplications, Levy 𝛾 𝛽 > 0 stands for the levy rise and for a potential increment in magnitude. 𝑢𝑖= 𝐼(𝑗𝑖; 𝑘) 𝑗𝑖= 1 𝑑∑ 𝑧𝑗∈𝑧𝑆 𝐼(𝑗𝑖; j) 𝑋𝑀𝑅𝑀𝑅= arg max 𝑧∈𝑧−𝑆 (𝑢𝑖−𝑧𝑖) (12) When a cuckoo's egg looks like the host egg, you know that one of those birds has set an egg there. Whatever fitness solution has the least amount of error is the best. (12) 𝑅𝑗= 1 𝑔∑ 𝑔 𝑤=1 (𝑒𝑤−𝑞𝑡) (14) (14) This approach picks the variable with the optimal relevance- redundancy balance at each phase. This criterion for selection is quick and effective. At forward search step d, the search algorithm calculates n-d evaluations. where, 𝑒𝑤 𝑖𝑠 the predicted result of the classification, and the 𝑞𝑡 𝑖𝑠 𝑡ℎ𝑒 actual result of the classification. The update equation for the Adam optimization method is as follows, taking into consideration the step size 𝛽. b. Preprocessing 𝑀(𝑗; 𝑘) = 𝐻(𝑘) + 𝐻(𝑘) −𝐻(𝑗, 𝑘) (9) (9) Then, we built the Hat matrix for feature extraction, whereby xi, a matrix of features, is utilized to predict zi, a target variable, from the training data. In other words, using the previously remembered definitions, into: Through using the following formula, we were able to determine the extent to which the model was inaccurate. The 𝐼(𝑗; 𝑘) = −∫ 𝜇𝑋,𝑌(𝑗, 𝑘)log 𝜇𝑗,𝑘(𝑧,𝑘) 𝜇𝑧(𝑗)𝜇𝑘(𝑘) 𝑑𝑥𝑑𝑦 (10) (10) The definition of the conditional component data is as follows: The definition of the conditional component data is as follows: species will hatch before eggs of the host species, which is just cruel. After the first cuckoo chick hatches, the parents will shift the host eggs slightly out of the nest to make room for the new arrival and guarantee that all of the cuckoo chicks have enough to eat. It seems that most wild animals forage in a very chaotic fashion. Also, the quirks of levy flying are employed to determine the levy behavior of the animals. Below are some guidelines based on the cuckoo search paradigm that best explain the proposed approach. Each cuckoo female will deposit an egg in a nest of her own choosing. The nest's better solution is good for the progeny of the future (egg quality). When the host species discovers that alien eggs have been laid in its nest, it abandons the nest, destroys it by eating the eggs, and then starts over elsewhere. 𝐼(𝑗1; 𝑘/𝑘2) = 𝐻(𝑗1/𝑘) −𝐻(𝑗1/𝑘, 𝑧2) = 𝐼(j/𝑘, 𝑧2) −𝐼(𝑗1/𝑘) (11) (11) Calculates the proportion of 𝑗2's value that is included in j1, assuming that 𝑗1's value is known. A another way to look at it is as the difference between the amount of information needed to describe 𝑗1 given 𝑗2 and the amount of information needed to describe 𝑋1 given 𝑗2 and k. The conditional mutual information is 0 if the information contained in k and 𝑗2 regarding 𝑋1 is identical. For the contrary case, if both k and 𝑧2 provide information, and where that information is complimentary, the gap widens significantly. d. Attack detection 𝜑𝑢= 𝜑𝑢−1 −𝛽⋅ 𝑓ˆ𝑢 (√𝑠ˆ𝑢+𝐾) (15) (15) (15) In order to improve classification accuracy, the suggested method combines the hybrid cuckoo Adam algorithm with the self-attention based depth poly O (optimized)-Network. It's possible that the classifier's initial parameters would be optimized. Accuracy of classification is enhanced by using the cuckoo's nesting patterns along with the step size parameter. With a tunable learning rate parameter, one may generate a sparse gradient that provides a precise moment estimate. While adequate results may be obtained by using just one optimization technique for data classification, the accuracy of the classification performance is enhanced by combining the breeding parameter of the CS algorithm with the gradient function. This means that the suggested method not only speeds up the classification process but also increases accuracy. where, 𝑓ˆ𝑢 stands for the mean gradient over a certain time period, su for the square of the gradient, and for the increment size. Hence, for the time being, let's pretend that u=u+2 and u- 1=u. The values for u and u-1 may be found by solving the following equation. 𝛽⋅𝑓ˆ𝑢+2 √𝑠ˆ𝑢+2+𝐾 = 𝜙𝑢−𝜙𝑢+2 𝛽 = (𝜙𝑢−𝜙𝑢+2)(√𝑠ˆ𝑢+2+𝐾) 𝑓ˆ𝑢+2 (16) (16) Under the assumption that 𝜑 =P, the modified version of Eq. (16) is as follows, Levy's Flight: Some animals also have impeccable timing when it comes to laying eggs. Parasitic cuckoos choose the nest where the eggs of the host species were laid. Eggs of the cuckoo 𝑞𝑗 𝑢+2 = 𝑞𝑧 𝑢+ (𝑃𝑧𝑢−𝑃𝑧𝑢+2)(√𝑠ˆ𝑢+2+𝐾) 𝑓ˆ𝑢+2 ⊕Levy (𝛾) 𝑃𝑧 𝑢+2 = 𝑞𝑧 𝑢+ 𝑞𝑧𝑢(√𝑠ˆ𝑢+2+𝐾) 𝑓ˆ𝑢+2 − 𝑞𝑧𝑢+2(√𝑠ˆ𝑢+2+𝐾) 𝑓ˆ𝑢+2 ⊕Levy (𝛾) 𝑞𝑧 𝑢+2 + 𝑞𝑧𝑢+2(√𝑠ˆ𝑢+2+𝐾) 𝑓ˆ𝑢+2 = 𝑞𝑧 𝑢( 𝑓ˆ𝑢+2+(√𝑠ˆ𝑢+2+𝐾) 𝑓ˆ𝑢+2 ) ⊕Levy (𝛾) 𝑞𝑧 𝑢+2 ( 𝑓ˆ𝑢+2+(√𝑠ˆ𝑢+2+𝐾) 𝑓ˆ𝑢+2 ) = 𝑃𝑧 𝑢( 𝑓ˆ𝑢+2+(√𝑠ˆ𝑢+2+𝐾) 𝑓ˆ𝑢+2 ) ⊕Levy (𝛾) 𝑃𝑧 𝑢+2 = 𝑓ˆ𝑢+2 𝑓ˆ𝑢+2+(√𝑠ˆ𝑢+2+𝐾) × {𝑃𝑧 𝑢( 𝑓ˆ𝑢+2+(√𝑠ˆ𝑢+2+𝐾) 𝑓ˆ𝑢+2 ) ⊕Levy (𝛾)} (17) 𝑏1 = {𝑏1 1, 𝑏2 1, K, 𝑏𝑚 1 , K, 𝑏𝑙 1}; 1 ≤𝑚≤𝑙 𝑐1 = {𝑐1 1, 𝑐2 1, K, 𝑐𝑛 1, K, 𝑐𝑓 1}; 1 ≤𝑛≤𝑓 (19) (19) “ where f represents hidden neurons and represents the mth visible neuron in layer 1 𝑏𝑚 1 , and the nth unseen neuron in layer 1 is represented by 𝑐𝑛 1. Every single neuron in both the overt and covert levels is biased. Let x and y stand for the visible and invisible layers' biases, respectively. d. Attack detection With this, we can see the second hidden layer, which is expressed as,𝑐2 = {𝑐1 2, 𝑐2 2, … , 𝑐𝑛 2, … , 𝑐𝑓 2}; 1 ≤𝑛≤𝑓 (25) 𝑆2 = {𝑆𝑛𝑛 2 }; 1 ≤𝑛≤𝑓 (26) (26) In x2 and y2 Eq. (25) and (26) the parameters for the bias of the hidden and visible layers are the same (26). This vector form is used in layer 2 for weighting. where, 𝑆𝑛𝑛 2 is an abbreviation for the f-by-f layer 2 weight vector, where n"th" stands for the n"th" exposed neuron and n"th" concealed neuron. Hence, it is possible to determine the output of the nth secret neuron by d. Attack detection Seeing the biased behavior of the neurons in layers 1 and 2 is necessary for this purpose. 𝑞𝑧 𝑢+2 + 𝑞𝑧 (√𝑢+2 ) 𝑓ˆ𝑢+2 = 𝑞𝑧 𝑢( 𝑓ˆ𝑢+2+(√𝑠ˆ𝑢+2+𝐾) 𝑓ˆ𝑢+2 ) ⊕Levy (𝛾) 𝑞𝑧 𝑢+2 ( 𝑓ˆ𝑢+2+(√𝑠ˆ𝑢+2+𝐾) 𝑓ˆ𝑢+2 ) = 𝑃𝑧 𝑢( 𝑓ˆ𝑢+2+(√𝑠ˆ𝑢+2+𝐾) 𝑓ˆ𝑢+2 ) ⊕Levy (𝛾) 𝑃𝑢+2 (17) (17) 𝑥1 = {𝑥1 1, 𝑥2 1, K, 𝑥𝑚 1 , K, 𝑥𝑙 1} 𝑦1 = {𝑦1 1, 𝑦2 1, K, 𝑦𝑛 1, K, 𝑦𝑓 1} (20) (20) where 𝑦𝑛 1 signifies the hidden neuron bias and 𝑥𝑚 1 with the mth visible neuron. The weight for the first layer of the classifier is defined as,, = 𝑓ˆ𝑢+2 𝑓ˆ𝑢+2+(√𝑠ˆ𝑢+2+𝐾) × {𝑃𝑧 𝑢( 𝑓ˆ𝑢+2+(√𝑠ˆ𝑢+2+𝐾) 𝑓ˆ𝑢+2 ) ⊕Levy (𝛾)} 𝑆1 = {𝑆𝑚𝑛 1 }; 1 ≤𝑚≤𝑙; 1 ≤𝑛≤𝑓 (21) (21) where, 𝑆𝑚𝑛 1 provides, in units, the length of the weight vector between the mth visible and nth hidden item. To that end, the output of the first hidden layer is based on the weights and biases of the neurons included inside that layer. In this case 𝑓𝑢 and 𝑠𝑢 are calculated as, In this case 𝑓𝑢 and 𝑠𝑢 are calculated as, In this case 𝑓𝑢 and 𝑠𝑢 are calculated as, 𝑓𝑢+2 = 𝜅1𝑓𝑢+ (1 −𝜅1)ℎ𝑢 𝑠𝑢+2 = 𝜅2 ⋅𝑠𝑢−1 + (1 −𝜅2)ℎ𝑢 2 (18) (18) 𝑐𝑛 1 = 𝜆[𝑦𝑛 1 + ∑ 𝑚 𝑏𝑚 1 𝑆𝑚𝑛 1 ] (22) (22) “where, 𝐾= 10−8, and 𝑓𝑢 displays the squared gradient, while s u stands for the moving average gradient.”. where, 𝜆 represents the action that activates something. Moreover, layer 1's output is shown as 𝑐1 = {𝑐𝑛 1}; 1 ≤𝑛≤𝑓 (23) “Hyper parameters Ranges various batch sizes {252,512} variety of network sizes {small, normal} Functions of activation {ReLu, Swish} Dropout layer Learning rate Inrange[0.1,0.2] Optimization method {HC Adam} {3,11}” (23) Layer 2 classifiers are also trained with the use of the hidden layer's output. The visible layer of layer 2 receives information from layer 1 per the equation (24). This proves that the layer 2 neurons' representation is identical to that of the layer 1 neurons'. 𝑏2 = {𝑏1 2, 𝑏2 2, … , 𝑏𝑓 2} = {𝑐𝑛 1}; 1 ≤𝑛≤𝑓 (24) (24) (24) where, {𝑐𝑛 1} the layer 1 output vector should be indicated. V. PERFORMANCE ANALYSIS (28) In this research, we implement and assess a new attack detection system based on optimization. In this research, we provide a hybrid cuckoo Adam algorithm-based self-attention based depth poly O (optimized)-Network. Experiments were conducted in a MATLAB environment. We have a dataset for testing with 200,960 examples from the regular class and 15,682 examples from the assault class. Here we provide the outcomes of the experiments and talk about them. Once their output is processed, the f-labeled neurons in the input layer send their data on to the classifier layer. The information is received by this layer as, In this research, we implement and assess a new attack detection system based on optimization. In this research, we provide a hybrid cuckoo Adam algorithm-based self-attention based depth poly O (optimized)-Network. Experiments were conducted in a MATLAB environment. We have a dataset for testing with 200,960 examples from the regular class and 15,682 examples from the assault class. Here we provide the outcomes of the experiments and talk about them. 𝑝= {𝑝1, 𝑝2, K, 𝑝𝑛, K, 𝑝𝑓} = {𝑐𝑛 2}; 1 ≤𝑛≤𝑓 (29) (29) Neurons in the input layer are given a f label, and their output is passed to the classifier layer. This layer takes in data in the form 0) Take here be ) es in well When are ) ffers yer's Figure 4(a)Sample input 𝑑= {𝑑1, 𝑑2, K, 𝑑𝐴, K, 𝑑𝐶}; 1 ≤𝜔≤𝑣 (30) (30) where C is the total amount of brain cells we can't see. Take into account the unobserved neuron's D A bias A, where A=1,2,, K,C. The results of using poly layers might be represented as, 𝑒= {𝑒1, 𝑒2, K, 𝑒𝜔, K, 𝑒𝑣}; 1 ≤𝜔≤𝑣 (31) (31) Here in which v refers as the total number of hidden nodes in the network. Each layer, both the input and hidden ones, as well as the hidden and output ones, has its own weight vector. When weight vectors between the input and hidden layers are multiplied together, the result is the scalar product (SE). Here in which v refers as the total number of hidden nodes in the network. Each layer, both the input and hidden ones, as well as the hidden and output ones, has its own weight vector. When weight vectors between the input and hidden layers are multiplied together, the result is the scalar product (SE). V. PERFORMANCE ANALYSIS 𝑆𝐸= {𝑆𝑛𝐴 𝐸}; 1 ≤𝑛≤𝑓; 1 ≤𝜔≤𝑣 (32) (32) “where, 𝑆𝑛𝐴 𝐸 we may deduce that the nth input neuron differs from a hidden neuron of size SE by [fC]. The hidden layer's output may be expressed as, where and are the neurons' weights and biases, respectively. indicate the difference between an n- th input neuron and a hidden n-th neuron of size SE, where SE is the size of the input neuron's hidden input neuron. The output of the hidden layer may be written as where and are the weights and biases of the individual neurons. “where, 𝑆𝑛𝐴 𝐸 we may deduce that the nth input neuron differs from a hidden neuron of size SE by [fC]. The hidden layer's output may be expressed as, where and are the neurons' weights and biases, respectively. indicate the difference between an n- th input neuron and a hidden n-th neuron of size SE, where SE is the size of the input neuron's hidden input neuron. The output of the hidden layer may be written as where and are the weights and biases of the individual neurons. Figure 4(a)Sample input 𝑑𝐴= [∑ 𝑓 𝑛=1 𝑆𝑛𝐴 𝐸∗𝑝𝑛]𝐷𝐴 ∀𝑝𝑛= 𝑐𝑛 2 (33) (33) where 𝐷𝐴 is the hidden neuron's bias set to equal𝑝𝑛= 𝑐𝑛 2, Using Layer 2's Output as Layer 1's Input. Moreover, SG stands in for the mass of the covert layer, and its formula for its weight is, 𝑆𝐺= {𝑆𝐴𝜔 𝐺}; 1 ≤𝐴≤𝐶; 1 ≤𝜔≤𝑣 (34) (34) Figure 4 (b)Simulated output For the final output vector, we get by multiplying the hidden layer's output by the weight matrix SG. For the final output vector, we get by multiplying the hidden layer's output by the weight matrix SG. The amount of data from Internet of Things devices was increased to 1 GB, and the number of attack threads was raised from 1 to 50. (Bytes Per Second). Input examples and simulated results for predicting malicious traffic are shown in Figure 4. The amount of data from Internet of Things devices was increased to 1 GB, and the number of attack threads was raised from 1 to 50. (Bytes Per Second). Input examples and simulated results for predicting malicious traffic are shown in Figure 4. Figure 3 Process of hyper tuning Figure 3 Process of hyper tuning In a self-attention based depth poly O (optimized)-Network, the feature vector F is fed into the top layer, as shown in the following formula. 𝑐𝑛 2 = 𝜆[𝑦𝑛 2 + ∑ 𝑚 𝑏𝑚 2 𝑆𝑛𝑛 2 ], ∀𝑏𝑚 2 = 𝑐𝑛 1 (27) (27) Finally the attack can be classified precisely. Finally the attack can be classified precisely. where, 𝑦𝑛 2 finds the nth hidden neuron's associated bias. Hence, the hidden layer's output might be phrased as, 𝑐2 = {𝑐𝑛 2}; 1 ≤𝑛≤𝑓 (28) V. PERFORMANCE ANALYSIS 𝑒𝜔= ∑ 𝐶 𝐴=1 𝑆𝐴𝜔 𝐺∗𝑑𝐴 (35) (35) “where, 𝑆𝐴𝜔 𝐺 stands for the relative importance of A"th" concealed and 𝜔th neuronal output, and 𝑑𝐴 named the output of the hidden layer”. Figure 5 Validation accuracy and loss analysis The loss function, which is calculated for each data item in an epoch, may be used to quantitatively analyze the quantity of data lost over the course of an epoch. An iterative curve is Figure 6(b) True response Vs. Predicted response Figure 5 Validation accuracy and loss analysis Figure 6(b) True response Vs. Predicted response Figure 6(b) True response Vs. Predicted response Figure 5 Validation accuracy and loss analysis The loss function, which is calculated for each data item in an epoch, may be used to quantitatively analyze the quantity of data lost over the course of an epoch. An iterative curve is generated by repeatedly discarding a small percentage of the data. The produced curve shows that the training and testing losses for the suggested classifiers are closer to the bottom of the range. Figure 6(b) True response Vs. Predicted response Here, in Figure 6, we provide the confusion matrix for the multiclassification case where the optimum permutation does exist. As can be seen from this, the optimized network identifies all DDoS and DoS assaults properly, with the exception of the HTTP DoS attack, which it mistakes for a Scanning attack and an Xbash attack in two separate instances. Scanning assaults are also detected almost precisely, with just around of sample flows being incorrectly categorized, mostly as Mirai and regular traffic. About of misclassified flows include regular traffic and Xbash assaults, with errors in the categorization mostly focusing on malware attacks and normal traffic, respectively. In conclusion, misclassification occurs in 3% of situations even with the optimal hyperparameter setting for Mirai flows, with HTTP DoS assaults being the most confused type. The confusion matrix not only confirms the danger of Mirai attacks, but also shows that the vast majority of misclassifications concern either regular traffic or highly frequent types of traffic, such as HTTP flows. Here, in Figure 6, we provide the confusion matrix for the multiclassification case where the optimum permutation does exist. Figure 7 Precision rate analysis We were able to categorize the irregular data flow from the characteristics data we gathered. Figure 7 shows that the proposed technique achieves a high degree of precision (99.8%), which is exceptional in comparison to other current processes. V. PERFORMANCE ANALYSIS As before, the proposed technique had the greatest recall (99.8%) of all the approaches tested. Figure 9 Accuracy rate analysis Th SABDPON d l ll f i d i f “Rate of success in predicting instances, expressed as a fraction of the total number of cases .” “rate of Precision = 𝐶𝑂 𝐶𝑂+𝐷𝑂” “F measure or F1 score” “Only two measurements are needed to determine the F-measure (precision and recall) .” “F measure = 2∗ (𝑃𝑟𝑒𝑐𝑖𝑠𝑖𝑜𝑛 ∗𝑅𝑒𝑐𝑎𝑙𝑙) (𝑃𝑟𝑒𝑐𝑖𝑠𝑖𝑜𝑛+𝑅𝑒𝑐𝑎𝑙𝑙)” “Rate of success in predicting instances, expressed as a fraction of the total number of cases .” “rate of Precision = 𝐶𝑂 𝐶𝑂+𝐷𝑂” “F measure or F1 score” “Only two measurements are needed to determine the F-measure (precision and recall) .” “F measure = 2∗ (𝑃𝑟𝑒𝑐𝑖𝑠𝑖𝑜𝑛 ∗𝑅𝑒𝑐𝑎𝑙𝑙) (𝑃𝑟𝑒𝑐𝑖𝑠𝑖𝑜𝑛+𝑅𝑒𝑐𝑎𝑙𝑙)” “Rate of success in predicting instances, expressed as a fraction of the total number of cases .” “rate of Precision = 𝐶𝑂 𝐶𝑂+𝐷𝑂” “F measure or F1 score” “Only two measurements are needed to determine the F-measure (precision and recall) .” “F measure = 2∗ (𝑃𝑟𝑒𝑐𝑖𝑠𝑖𝑜𝑛 ∗𝑅𝑒𝑐𝑎𝑙𝑙) (𝑃𝑟𝑒𝑐𝑖𝑠𝑖𝑜𝑛+𝑅𝑒𝑐𝑎𝑙𝑙)” “Rate of success in predicting instances, expressed as a fraction of the total number of cases .” “rate of Precision = 𝐶𝑂 𝐶𝑂+𝐷𝑂” “F measure or F1 score” “Only two measurements are needed to determine the F-measure (precision and recall) .” “F measure = 2∗ (𝑃𝑟𝑒𝑐𝑖𝑠𝑖𝑜𝑛 ∗𝑅𝑒𝑐𝑎𝑙𝑙) (𝑃𝑟𝑒𝑐𝑖𝑠𝑖𝑜𝑛+𝑅𝑒𝑐𝑎𝑙𝑙)” Fig re 7 Precision rate anal sis V. PERFORMANCE ANALYSIS As can be seen from this, the optimized network identifies all DDoS and DoS assaults properly, with the exception of the HTTP DoS attack, which it mistakes for a Scanning attack and an Xbash attack in two separate instances. Scanning assaults are also detected almost precisely, with just around of sample flows being incorrectly categorized, mostly as Mirai and regular traffic. About of misclassified flows include regular traffic and Xbash assaults, with errors in the categorization mostly focusing on malware attacks and normal traffic, respectively. In conclusion, misclassification occurs in 3% of situations even with the optimal hyperparameter setting for Mirai flows, with HTTP DoS assaults being the most confused type. The confusion matrix not only confirms the danger of Mirai attacks, but also shows that the vast majority of misclassifications concern either regular traffic or highly frequent types of traffic, such as HTTP flows. Figure 6(a) Actual class Vs. Predicted class Some performance measures are shown below that may be used to compare the proposed technique with current methods [26] and demonstrate its efficacy. Table 1 Evaluation metrics “Evolution matrix” “Description” “Formula” “Accuracy” “The rate at which predictions were correct over the whole sample. .” “Accuracy = 𝐶𝑂+𝑅𝐵 𝐶𝑂+𝐶𝑂+𝑅𝐵+𝐷𝐵” “Recall” “The rate at which events are anticipated correctly as a percentage of all events.” “rate of Recall = 𝐶𝑂 𝐶𝑂+𝐷𝐵" “Precision” Figure 6(a) Actual class Vs. Predicted class Table 1 Evaluation metrics “Evolution matrix” “Description” “Formula” “Accuracy” “The rate at which predictions were correct over the whole sample. .” “Accuracy = 𝐶𝑂+𝑅𝐵 𝐶𝑂+𝐶𝑂+𝑅𝐵+𝐷𝐵” “Recall” “The rate at which events are anticipated correctly as a percentage of all events.” “rate of Recall = 𝐶𝑂 𝐶𝑂+𝐷𝐵" “Precision” Table 1 Evaluation metrics Table 1 Evaluation metrics “Rate of success in predicting instances, expressed as a fraction of the total number of cases .” “rate of Precision = 𝐶𝑂 𝐶𝑂+𝐷𝑂” “F measure or F1 score” “Only two measurements are needed to determine the F-measure (precision and recall) .” “F measure = 2∗ (𝑃𝑟𝑒𝑐𝑖𝑠𝑖𝑜𝑛 ∗𝑅𝑒𝑐𝑎𝑙𝑙) (𝑃𝑟𝑒𝑐𝑖𝑠𝑖𝑜𝑛+𝑅𝑒𝑐𝑎𝑙𝑙)” Figure 7 Precision rate analysis We were able to categorize the irregular data flow from the characteristics data we gathered. Figure 7 shows that the proposed technique achieves a high degree of precision (99.8%), which is exceptional in comparison to other current processes. Figure 8 Recall rate analysis The recall results for identifying fraudulent data traffic in real- time datasets are shown in Figure 8. Figure 8 Recall rate analysis The recall results for identifying fraudulent data traffic in real- time datasets are shown in Figure 8. As before, the proposed technique had the greatest recall (99.8%) of all the approaches tested. Figure 7 Precision rate analysis Figure 9 Accuracy rate analysis “Reference Extracted amount of the feature corrected data discrepancy Accuracy obttained” “[27] 11.0 ☑ 99.860% [27] 111, 41,10.0 ❌ 95.120% for 41.0 features [27] 6.0 ❌ 99.30% [27] 154.0 ❌ 98.670% [27] 154, 20.0 ❌ 96.260% [27] 5.0 ☑ 98.220% [27] 95.0 ☑ 98.540% for multi-class 99.520% for binary [27] 50.0 ☑ 94.810% [27] 71.0 ☑ 92.490% [27] 36.0 ❌ 95.880% for multi-class [27] 21.0 ☑ 99.970% semi-distributed, 97.800% for distributed [27] 76,13,8.0 ☑ 990% Proposed 78500.0 ☑ 99.990%” Table 2 briefly compare and contrast the existing literature with our planned study.. catastrophic. In this research, we aimed to develop context- aware security solutions for IoT medical contexts by proposing a paradigm for identifying malicious communications. In order to implement the proposed method, identifying malicious traffic is prioritized. To get meaningful information from the generated traffic, it may be collected and then processed using a MATLAB script. To identify potentially harmful traffic in an IoT healthcare environment, we construct a dataset and use it to train and evaluate the suggested classifier. The next phase involves field-testing the trained classifier. With an F1-score of 99.8%, 99.8% precision, 99.8% recall, and 99.9% accuracy, the SABDPON classifier is the most effective. The proposed architecture has been experimentally validated, suggesting its potential application in developing innovative IoT security solutions. The resultant dataset and proposed methodology may be used by researchers to enhance the security and situational awareness of IoT healthcare systems. Quickly simulating several IoT use cases is now possible thanks to the proposed framework, opening the path for the development of AI-based security solutions for a wider variety of use cases. Further work It has been experimentally shown that the proposed architecture is successful in detecting cyberattacks, leading to a safer and more secure IoT healthcare setting. By following the provided architecture and the major steps shown in Figure 1, it is possible to swiftly create AI-based security solutions for any novel IoT use case. The results of these studies might help in the development of more effective context-aware IoT security solutions, especially in the IoT healthcare industry. The dataset generated during this study will be made publicly accessible for use in future studies if and when requested by the scientific community. Figure 9 Accuracy rate analysis The SABDPON model allows for very precise detection of the kind of attack that has occurred. The proposed approach has more accuracy than the existing best practices (99.99 percent). From the figure 10 it will be shown that the suggested methodology have high F score (99.8%) range when compared to other existing mechanisms. Figure 10 Analysis of F1 score Figure 10 Analysis of F1 score Figure 11 AUC Calculation For each given categorization threshold, the AUC remains constant. Regardless of the chosen categorization threshold, it checks how well the model predicts. A larger area under the curve indicates a more effective classifier. In addition, the curve features a single node that stands for the classifier's ideal range in operation. In this figure 11, we provide the ROC curves for all evaluated data sets. We can see that the area under each curve is extremely large, which is evidence that the categorization using the SABDPON method is effective. Figure 11 AUC Calculation in operation. In this figure 11, we provide the ROC curves for all evaluated data sets. We can see that the area under each curve is extremely large, which is evidence that the categorization using the SABDPON method is effective. For each given categorization threshold, the AUC remains constant. Regardless of the chosen categorization threshold, it checks how well the model predicts. A larger area under the curve indicates a more effective classifier. In addition, the curve features a single node that stands for the classifier's ideal range For each given categorization threshold, the AUC remains constant. Regardless of the chosen categorization threshold, it checks how well the model predicts. A larger area under the curve indicates a more effective classifier. In addition, the curve features a single node that stands for the classifier's ideal range Table 2 briefly compare and contrast the existing literature with our planned study.. REFERENCES 1. S. Pundir, M. Wazid, D. P. Singh, A. K. Das, J. J. Rodrigues, and Y. Park, "Intrusion detection protocols in wireless sensor networks integrated to Internet of Things deployment: Survey and future challenges," IEEE Access, vol. 8, pp. 3343-3363, 2019. 14. R. Sekhar, K. Sasirekha, P. Raja, and K. Thangavel, "A novel GPU based intrusion detection system using deep autoencoder with Fruitfly optimization," SN Applied Sciences, vol. 3, p. 594, 2021. 15. C. Kolias, G. Kambourakis, A. Stavrou, and S. Gritzalis, "Intrusion detection in 802.11 networks: Empirical evaluation of threats and a public dataset," IEEE Communications Surveys & Tutorials, vol. 18, pp. 184-208, 2015. 2. P. Rughoobur and L. Nagowah, "A lightweight replay attack detection framework for battery depended IoT devices designed for healthcare," in 2017 International Conference on Infocom Technologies and Unmanned Systems (Trends and Future Directions)(ICTUS), 2017, pp. 811-817. 16. U. S. K. P. M. Thanthrige, J. Samarabandu, and X. Wang, "Machine learning techniques for intrusion detection on public dataset," in 2016 IEEE Canadian conference on electrical and computer engineering (CCECE), 2016, pp. 1-4. 3. S. Rathore and J. H. Park, "Semi-supervised learning based distributed attack detection framework for IoT," Applied Soft Computing, vol. 72, pp. 79-89, 2018. 4. S. Carta, A. S. Podda, D. R. Recupero, and R. Saia, "A local feature engineering strategy to improve network anomaly detection," Future Internet, vol. 12, p. 177, 2020. 17. M. E. Aminanto, H. Tanuwidjaja, P. D. Yoo, and K. Kim, "Weighted feature selection techniques for detecting impersonation attack in Wi-Fi networks," in Proc. Symp. Cryptogr. Inf. Secur.(SCIS), 2017, pp. 1- 8. 5. I. Alrashdi, A. Alqazzaz, R. Alharthi, E. Aloufi, M. A. Zohdy, and H. Ming, "FBAD: Fog-based attack detection for IoT healthcare in smart cities," in 2019 IEEE 10th Annual Ubiquitous Computing, Electronics & Mobile Communication Conference (UEMCON), 2019, pp. 0515-0522. 18. D. Kaleem and K. Ferens, "A cognitive multi-agent model to detect malicious threats," in Proceedings of the 2017 International Conference on Applied Cognitive Computing (ACC’17), 2017. 19. F. D. Vaca and Q. Niyaz, "An ensemble learning based wi-fi network intrusion detection system (wnids)," in 2018 IEEE 17th international symposium on network Computing and Applications (NCA), 2018, pp. 1-5. 6. L. Santos, C. Rabadao, and R. Gonçalves, "Intrusion detection systems in Internet of Things: A literature review," in 2018 13th Iberian conference on information systems and technologies (CISTI), 2018, pp. 1-7. 20. M. A. Rahman, A. VI. CONCLUSION Academics and engineers have developed an interest in the design of IoT healthcare systems as a result of the fast development of IoT technology. There have been several plans for IoT healthcare systems developed over the last few years, but they all have a common flaw: a lack of adequate security. The consequences of a security breach or cyberattack on healthcare systems connected to the Internet of Things might be 9. A. H. Seh, M. Zarour, M. Alenezi, A. K. Sarkar, A. Agrawal, R. Kumar, et al., "Healthcare data breaches: insights and implications," in Healthcare, 2020, p. 133. on our feature selection method will include undertaking extensive testing with a variety of DL network topologies and, more importantly, a focus on finding the most essential qualities themselves rather than simply their frequency. 10. I. Sharafaldin, A. H. Lashkari, and A. A. Ghorbani, "Toward generating a new intrusion detection dataset and intrusion traffic characterization," ICISSp, vol. 1, pp. 108-116, 2018. Compliance with Ethical Standards Disclosure of potential conflicts of Interest : There is no potential conflicts of interest among the authors. 11. T. Batiha and P. Krömer, "Design and analysis of efficient neural intrusion detection for wireless sensor networks," Concurrency and Computation: Practice and Experience, vol. 33, p. e6152, 2021. Research involving human participants and/or animals: This article does not contain any studies with human participants or animals performed by any of the authors. Funding : This work was done under grant No 420/year 2023 of Scientific Research of King Khalid University, Al-Faraa, Saudi Arabia.Competing Interests : There is no competing interests among the authors 12. M. Safaldin, M. Otair, and L. Abualigah, "Improved binary gray wolf optimizer and SVM for intrusion detection system in wireless sensor networks," Journal of ambient intelligence and humanized computing, vol. 12, pp. 1559-1576, 2021. Author Contributions: All the authors have contributed in equal manner. Author Contributions: All the authors have contributed in equal manner. Data Availability : Data is available based on personal request 13. A. Fatani, A. Dahou, M. A. Al-Qaness, S. Lu, and M. Abd Elaziz, "Advanced feature extraction and selection approach using deep learning and Aquila optimizer for IoT intrusion detection system," Sensors, vol. 22, p. 140, 2022. REFERENCES T. Asyhari, L. Leong, G. Satrya, M. H. Tao, and M. Zolkipli, "Scalable machine learning- based intrusion detection system for IoT-enabled smart cities," Sustainable Cities and Society, vol. 61, p. 102324, 2020. 7. B. B. Zarpelão, R. S. Miani, C. T. Kawakani, and S. C. de Alvarenga, "A survey of intrusion detection in Internet of Things," Journal of Network and Computer Applications, vol. 84, pp. 25-37, 2017. 8. S. Ghazanfar, F. Hussain, A. U. Rehman, U. U. Fayyaz, F. Shahzad, and G. A. Shah, "Iot-flock: An open-source framework for iot traffic generation," in 2020 International Conference on Emerging Trends in Smart Technologies (ICETST), 2020, pp. 1-6. 21. S. J. Lee, P. D. Yoo, A. T. Asyhari, Y. Jhi, L. Chermak, C. Y. Yeun, et al., "IMPACT: Impersonation attack detection via edge computing using deep autoencoder and feature abstraction," IEEE Access, vol. 8, pp. 65520-65529, 2020. IEEE Access. (2020) 8:106576–84. doi: 10.1109/ACCESS.2020.3000421 IEEE Access. (2020) 8:106576–84. doi: 10.1109/ACCESS.2020.3000421 22. V. L. Thing, "IEEE 802.11 network anomaly detection and attack classification: A deep learning approach," in 2017 IEEE Wireless Communications and Networking Conference (WCNC), 2017, pp. 1-6. doi: 34. Nguyen PT, Huynh VD, Vo KD, Phan PT, Elhoseny M, Le DN. Deep learning based optimal multimodal fusion framework for intrusion detection systems for healthcare data. CMC. (2021) 66:2555–71. doi: 10.32604/cmc.2021.012941 23. S. Wang, B. Li, M. Yang, and Z. Yan, "Intrusion detection for WiFi network: A deep learning approach," in Wireless Internet: 11th EAI International Conference, WiCON 2018, Taipei, Taiwan, October 15-16, 2018, Proceedings, 2019, pp. 95-104. 35. Iwendi C, Anajemba JH, Biamba C, Ngabo D. Security of things intrusion detection system for smart healthcare. Electronics. (2021) 10:1375. doi: 10.3390/electronics10121375 24. K. Kim, M. E. Aminanto, and H. C. Tanuwidjaja, Network intrusion detection using deep learning: a feature learning approach: Springer, 2018. 25. J. Ran, Y. Ji, and B. Tang, "A semi-supervised learning approach to ieee 802.11 network anomaly detection," in 2019 IEEE 89th vehicular technology conference (VTC2019-Spring), 2019, pp. 1-5. 36. Yeng PK, Nweke LO, Woldaregay AZ, Yang B, Snekkenes EA. Data-driven and artificial intelligence (AI) approach for modelling and analyzing healthcare security practice: a systematic review. In: Arai K, Kapoor S, Bhatia R, editors. Intelligent Systems and Applications. Cham: Springer (2021). doi: 10.1007/978-3-030-55180-3_1 26. F. Hussain, S. G. Abbas, G. A. Shah, I. M. Pires, U. U. Fayyaz, F. REFERENCES Shahzad, et al., "A framework for malicious traffic detection in IoT healthcare environment," Sensors, vol. 21, p. 3025, 2021. 27. T. Gaber, A. El-Ghamry, and A. E. Hassanien, "Injection attack detection using machine learning for smart IoT applications," Physical Communication, vol. 52, p. 101685, 2022. 37. Mahdavifar S, Maleki N, Lashkari AH, Broda M, Razavi AH. Classifying malicious domains using DNS traffic analysis. In: The 19th IEEE International Conference on Dependable, Autonomic, and Secure Computing (DASC). Calgary, AB (2021). 28. Tervoort T, De Oliveira MT, Pieters W, Van Gelder P, Olabarriaga SD, Marquering H. Solutions for mitigating cybersecurity risks caused by legacy software in medical devices: a scoping review. IEEE Access. (2020) 8:84352–61. doi: 10.1109/ACCESS.2020.2984376 38. Seth S, Chahal KK, Singh G. A novel ensemble framework for an intelligent intrusion detection system. IEEE Access. (2021) 9:138451–67. doi: 10.1109/ACCESS.2021.3116219 39. Serinelli BM, Collen A, Nijdam NA. On the analysis of open source datasets: validating IDS implementation for well-known and zero day attack detection. Proc Comp Sci. (2021) 191:192–9. doi: 10.1016/j.procs.2021.07.024 29. Thamilarasu G, Odesile A, Hoang A. An intrusion detection system for internet of medical things. IEEE Access. (2020) 8:181560–76. doi: 10.1109/ACCESS.2020.3026260 30. Subasi A, Algebsani S, Alghamdi W, Kremic E, Almaasrani J, Abdulaziz N. Intrusion detection in smart healthcare using bagging ensemble classifier. In: International Conference on Medical and Biological Engineering. Cham: Springer (2021) p. 164–71. doi: 10.1007/978-3-030-73909-6_18 40. Thilagam T, Aruna R. Intrusion detection for network based cloud computing by custom RC-NN and optimization. ICT Express. (2021) 7:512–20. doi: 10.1016/j.icte.2021.04.006 41. Zuech R, Hancock J, Khoshgoftaar TM. Detecting web attacks using random undersampling and ensemble learners. J Big Data. (2021) 8:1–20. doi: 10.1186/s40537-021-00460-8 31. Sarna Priya RM, Maddikunta PK, Parimala M, Koppu S, Gadekallu TR, Chowdhary CL, et al. An effective feature engineering for DNN using hybrid PCA-GWO for intrusion detection in IoMT architecture. Comp Commun. (2020) 160:139–49. doi: 10.1016/j.comcom.2020.05.048 42. Yu L, Dong J, Chen L, Li M, Xu B, Li Z, et al. PBCNN: packet bytes-based convolutional neural network for network intrusion detection. Comp Netw. (2021) 194:108117. doi: 10.1016/j.comnet.2021.108117 32. Šabić E, Keeley D, Henderson B, Nannemann S. Healthcare and anomaly detection: using machine learning to predict anomalies in heart rate data. AI Soc. (2021) 36:149–58. doi: 10.1007/s00146-020-00985-1 43. Gopalan SS, Ravikumar D, Linekar D, Raza A, Hasib M. Balancing approaches towards ML for IDS: a survey for the CSE-CIC IDS dataset. (ICCUBEA). Pune: IEEE (2018). doi: 10.1109/ICCUBEA.2018.8697702 doi: 44. Fitni QR, Ramli K. Implementation of ensemble learning and feature selection for performance improvements in anomaly-based intrusion detection systems. In: 2020 IEEE International Conference on Industry 4.0, Artificial Intelligence, and Communications Technology (IAICT). IEEE (2020) p. 118–24. doi: 10.1109/IAICT50021.2020.9172014 55. Mubashar A, Asghar K, Javed AR, Rizwan M, Srivastava G, Gadekallu TR, et al. Storage and proximity management for centralized personal health records using an ipfs-based optimization algorithm. J Circ Syst Comp. (2021) 15:2250010 56. Loshchilov I, Hutter F. Sgdr: Stochastic gradient descent with warm restarts. arXiv Preprint. (2016) arXiv:1608.03983. 45. Misra D. Mish: a self regularized non-monotonic neural activation function. arXiv Preprint. (2019) arXiv:1908.08681. 57. Glorot X, Bengio Y. Understanding the difficulty of training deep feedforward neural networks. In: Proceedings of the Thirteenth International Conference on Artificial Intelligence and Statistics. Sardinia: JMLR Workshop and Conference Proceedings (2010). p. 249–56. 46. Hua Y. An efficient traffic classification scheme using embedded feature selection and lightgbm. In: 2020 Information Communication Technologies Conference (ICTC). Nanjing: IEEE (2020). doi: 10.1109/ICTC49638.2020.9123302 58. Sharma NV, Yadav NS. An optimal intrusion detection system using recursive feature elimination and ensemble of classifiers. Microproc Microsyst. (2021) 23:104293. doi: 10.1016/j.micpro.2021.104293 47. Catillo M, Rak M, Villano U. 2L-ZED-IDS: a two- level anomaly detector for multiple attack classes. In: AINA Workshops. Caserta: Springer (2020). p. 687– 96. doi: 10.1007/978-3-030-44038-1_63 48. Khan MA, Kim J. Toward developing efficient Conv- AE-based intrusion detection system using heterogeneous dataset. Electronics. (2020) 9:1771. doi: 10.3390/electronics9111771 59. Sumaiya Thaseen I, Saira Banu J, Lavanya K, Rukunuddin Ghalib M, Abhishek K. An integrated intrusion detection system using correlation-based attribute selection and artificial neural network. Trans Emerg Telecommun Technol. (2021) 32:e4014. doi: 10.1002/ett.4014 49. Meryem A, Ouahidi BE. Hybrid intrusion detection system using machine learning. Netw Sec. (2020) 2020:8–19. doi: 10.1016/S1353-4858(20)30056-8 60. He K, Zhang X, Ren S, Sun J. Deep residual learning for image recognition. In: Proceedings of the IEEE Conference on Computer Vision and Pattern Recognition. Las Vegas, LA: IEEE (2016). p. 770–8. doi: 10.1109/CVPR.2016.90 50. Thakkar A, Lohiya R. A review of the advancement in intrusion detection datasets. Proc Comp Sci. (2020) 167:636–45. doi: 10.1016/j.procs.2020.03.330 51. Gao X, Shan C, Hu C, Niu Z, Liu Z. An adaptive ensemble machine learning model for intrusion detection. IEEE Access. (2019) 7:82512–21. doi: 10.1109/ACCESS.2019.2923640 52. Chawla A, Lee B, Fallon S, Jacob P. Host based intrusion detection system with combined CNN/RNN model. REFERENCES In: 2020 International Conference on Communications, Signal Processing, and Their Applications (ICCSPA). 33. Hady AA, Ghubaish A, Salman T, Unal D, Jain R. Intrusion detection system for healthcare systems using medical and network data: a comparison study. (ICCUBEA). Pune: IEEE (2018). doi: 10.1109/ICCUBEA.2018.8697702 Sharjah: IEEE (2021). doi: 10.1109/ICCSPA49915.2021.9385742 (ICCUBEA). Pune: IEEE (2018). doi: 10.1109/ICCUBEA.2018.8697702 (ICCUBEA). Pune: IEEE (2018). doi: 10.1109/ICCUBEA.2018.8697702 In: Joint European Conference on Machine Learning and Knowledge Discovery in Databases. Cham: Springer (2018). p. 149–58. doi: 10.1007/978- 3-030-13453-2_12 53. Freas CB, Harrison RW, Long Y. High performance attack estimation in large-scale network flows. In: 2018 IEEE International Conference on Big Data (Big Data). IEEE (2018). p. 5014–20. doi: 10.1109/BigData.2018.8622125 54. Wankhede S, Kshirsagar D. DoS attack detection using machine learning and neural network. In: 2018 Fourth International Conference on Computing Communication Control and Automation
W2747337326.txt	http://www.scielo.br/pdf/bjft/v20/1981-6723-bjft-20-e2016149.pdf	pt		Morangos produzidos no semiárido de Minas Gerais: qualidade do fruto e da polpa congelados	Brazilian journal of food technology/Brazilian Journal of Food Technology	2,017	cc-by	4,678	Original Article Campinas, v. 20, e2016149, 2017 http://dx.doi.org/10.1590/1981-6723.14916 ISSN 1981-6723 on-line version Morangos produzidos no semiárido de Minas Gerais: qualidade do fruto e da polpa congelados Strawberries produced in the semi-arid region of Minas Gerais, Brazil: quality of the frozen fruit and pulp Ariane Castricini1, Mário Sérgio Carvalho Dias1, Ramilo Nogueira Martins1, Leandra Oliveira Santos1 1 Empresa de Pesquisa Agropecuária de Minas Gerais (EPAMIG Norte), Nova Porteirinha/MG - Brasil Corresponding Author Ariane Castricini, Empresa de Pesquisa Agropecuária de Minas Gerais (EPAMIG Norte), Campo Experimental do Gorutuba, Rodovia MGT 122, km 155, Caixa Postal: 12, CEP: 39525-000, Nova Porteirinha/MG - Brasil, e-mail: castriciniariane08@gmail.com Cite as: Strawberries produced in the semi-arid region of Minas Gerais, Brazil: quality of the frozen fruit and pulp. Braz. J. Food Technol., v. 20, e20161493, 2017. Received: Dec. 01, 2016; Approved: June 13, 2017 Resumo O congelamento de morangos inteiros ou em polpa pode fornecer matéria-prima para sua utilização em iogurtes, recheios, coberturas, sorvetes e sucos, fora da safra. Entretanto, ainda que o consumo não seja in natura, a qualidade do produto final deve ser satisfatória. Objetivou-se avaliar a qualidade de morangos produzidos no Semiárido de Minas Gerais, congelados inteiros e em polpa, durante o armazenamento. Frutos das cultivares Tudla, Oso Grande, Albion e Portola foram produzidos em Mocambinho, distrito de Jaíba-MG, após a colheita, foram lavados, embalados (inteiros ou polpa) a vácuo e congelados por até 180 dias. Os tratamentos foram constituídos por cultivares (c) e dias de armazenamento (d), constituindo fatorial duplo c × d, em que se utilizaram três repetições de 10 frutos por parcela ou três polpas embaladas, para as avaliações de frutos congelados inteiros e em polpa, respectivamente. A intensidade da cor vermelha dos morangos inteiros ou em polpa reduziu-se durante o armazenamento (redução de °hue); quando inteiros, ‘Tudla’, ‘Oso Grande’ e ‘Portola’ foram vermelhos mais claros que ‘Albion’, mas, em polpa, não ocorreu diferença entre ‘Tudla’ e ‘Albion’. Morangos ‘Oso Grande’ armazenados inteiros ou em polpa tiveram maior teor de sólidos solúveis em relação àqueles das demais cultivares, e ‘Portola’, o menor teor e a menor acidez titulável (quando inteiros). ‘Tudla’ apresentou frutos e polpa mais ácidos. Durante o armazenamento de morangos congelados inteiros ou em polpa, produzidos no Semiárido de Minas Gerais, ocorreram modificações nos parâmetros cor, sólidos solúveis e acidez titulável. Palavras-chave: Fragaria × ananassa Duch; Pós-colheita; Armazenamento. Abstract The freezing of whole strawberries or pulp can provide raw material for the out of season use of strawberries in yogurts, fillings, toppings, ice cream and juices. However, although the consumption is not in natura, the quality of the final product should be satisfactory. This study aimed to evaluate the quality of strawberries produced in the semi-arid region of Minas Gerais, Brazil, frozen either whole or in the form of pulp, during storage. Fruits of the cultivars Tudla, Oso Grande, Albion and Portola were produced in the semi-arid region of Minas Gerais, and after harvesting, were washed, vacuum packed (whole or pulp) and frozen for up to 180 days. The treatments consisted of cultivars (c) and days of storage (d), constituting a double factorial c × d design, using three repetitions of 10 fruits per batch or three pulp-packs, respectively, for the frozen whole fruit assessments. The intensity of the red colour of the whole strawberries or strawberry pulp reduced during storage (reduction of hue). When whole, the cultivars ‹Tudla›, ‹Oso Grande› and ‹Portola’ were a lighter red than› Albion ‹, but in the form of pulp, there was no difference between ‹Tudla› and ‹Albion›. The ‹Oso Grande› strawberries, stored whole or in pulp, showed higher soluble solids contents than those of the other cultivars, and ‹Portola› showed the lowest content and also the lowest titratable acidity (when whole). The fruits and pulp of the ‘Tudla’ cultivar were more acidic. During the storage of frozen whole or pulped strawberries produced in the semi-arid region of Minas Gerais, changes occurred in the colour, soluble solids and titratable acidity. Keywords: Fragaria × ananassa Duch; Postharvest; Storage. Este é um artigo publicado em acesso aberto (Open Access) sob a licença Creative Commons Attribution, que permite uso, distribuição e reprodução em qualquer meio, sem restrições desde que o trabalho original seja corretamente citado. http://bjft.ital.sp.gov.br Morangos produzidos no semiárido de Minas Gerais: qualidade do fruto e da polpa congelados Castricini, A. et al. 1 Introdução O morango (Fragaria × ananassa) pode ser usado como ingrediente em diversas preparações, tais como caldas, iogurtes, bebidas, biscoitos e sorvetes, entre outras. No entanto, além de ter uma produção sazonal, é uma fruta muito delicada, com alta taxa respiratória, sendo, por isso, altamente perecível (ROCHA et al., 2008). Segundo Cunha Junior et al. (2012), a comercialização de morangos a grandes distâncias é dificultada devido à sua perecibilidade, decorrente principalmente da suscetibilidade ao desenvolvimento de agentes patogênicos. De acordo com Brackmann et al. (2011), após a colheita, os frutos devem ser refrigerados rapidamente, a fim de minimizar a deterioração, pois morangos com a epiderme brilhante, sem sinais de desidratação ou deterioração, são desejáveis. O armazenamento congelado do morango inteiro ou em polpa é uma alternativa para seu armazenamento durante maior período, em relação ao fruto in natura ou fresco. No entanto, devido às perdas das características do fruto in natura, como formato, textura etc., a destinação do produto pós-congelamento será para preparações industriais. A qualidade de polpas de fruta no Brasil é regulamentada pela Instrução Normativa n.1, de 07 de janeiro de 2000, que determina os Padrões de Identidade e Qualidade (PIQ) (BRASIL, 2000). Estes padrões incluem características e composição às quais a polpa ou purê do determinado fruto deve atender, como cor, sabor, teores de sólidos solúveis e acidez. A polpa de morango é o produto obtido da parte comestível do fruto por meio de processo tecnológico adequado e deve obedecer às seguintes características e composição: sólidos solúveis (a 20 °C) 7,5 °Brix, pH = 3,30, acidez titulável (ácido cítrico) 0,80 g 100 g-1 e 56 mg 100 g-1 de ácido ascórbico (BRASIL, 2016). O clima mais favorável para o cultivo do morangueiro é o temperado; no entanto, no início da década de 2000, a Empresa de Pesquisa Agropecuária de Minas Gerais (Epamig) iniciou pesquisas para avaliar o comportamento da cultura no clima semiárido. Os pesquisadores partiram do pressuposto de que, em consequência do clima quente e seco predominante na região, as doenças não se desenvolveriam, sendo possível, assim, praticar uma agricultura sem a utilização de agrotóxicos (DIAS et al., 2014). Os primeiros trabalhos concluídos apresentaram resultados satisfatórios de produtividade e qualidade dos frutos. Neste sentido, objetivou-se avaliar a qualidade de morangos produzidos no Semiárido de Minas Gerais, congelados inteiros e em polpa, durante o armazenamento. 2 Material e métodos Morangos das cultivares Tudla, Oso Grande, Albion e Portola foram produzidos no Semiárido de Minas Gerais, em Mocambinho, distrito de Jaíba-MG, no Campo Braz. J. Food Technol., Campinas, v. 20, e2016149, 2017 Experimental de Mocambinho, pertencente à Epamig. Para a implantação do experimento, inicialmente retirou-se uma amostra de solo para a realização da análise em laboratório. Posteriormente, o solo foi preparado através de uma aração e uma gradagem. Em seguida, foram preparados os canteiros com dimensões de 1,20 m × 50 m, com três linhas de plantio. A adubação mineral consistiu na aplicação de Superfosfato simples, Sulfato de amônio, Cloreto de potássio, Sulfato de magnésio e FTE BR10, de acordo com o resultado da análise do solo. A adubação orgânica foi realizada com a utilização de esterco curtido de curral na proporção de 20 t ha-1. Foi adotado o sistema de irrigação por microaspersão, sendo este instalado antes do transplantio das mudas, que foi realizado quinze dias após a adubação organomineral da área. A irrigação visou atender a demanda de 5 mm dia-1, que são exigidos pela cultura, geralmente, nas condições locais. O espaçamento adotado entre plantas e entre linhas foi de 0,40 m × 0,40 m, respectivamente. As adubações de cobertura iniciaram-se aos trinta dias após o transplantio, sendo estas realizadas quinzenalmente até o final do ciclo da cultura, seguindo as recomendações de análises de solo. O mulching utilizado foi a lona plástica dupla face branco e preta, colocada sobre os canteiros trinta dias após o transplantio. O excesso de folhas e estolhos foi eliminado periodicamente, visando melhorar o arejamento entre as plantas, tornando, assim, o ambiente menos propício à manifestação de fungos secundários e favorecendo também a ação de agentes polinizadores na área, os quais contribuem para a boa formação dos frutos. Para o controle de ácaros e manchas foliares, utilizaram-se produtos biológicos, objetivando um menor impacto ambiental, com uma produção de morangos isenta de resíduos de agrotóxicos. As pulverizações eram realizadas com extrato de folhas de Nim (espécie) (10%) e aplicação de Calda Bordalesa. Os frutos foram colhidos com 75% da superfície com coloração vermelha. Após a colheita e antes do processamento, da embalagem e do congelamento, os frutos foram caracterizados quanto cor, através dos parâmetros luminosidade (L), croma (C) e °hue (ângulo de cor), ao teor de sólidos solúveis e à acidez titulável. Os valores obtidos nesta avaliação constituíram o tempo zero de armazenamento, sendo as demais avaliações feitas aos 30, 60, 90, 150 e 180 dias de armazenamento. A coloração do fruto foi determinada por Colorímetro Minolta, modelo Chroma meter CR 400, sistema L* C* Hue. A luminosidade (L) varia entre zero (mais escuro) e 100 (mais claro); para a cromaticidade ou pureza da cor (C), valores relativamente inferiores representam cores impuras (menor saturação de pigmentos) e os superiores, as cores puras (maior saturação de pigmentos). O ângulo de tonalidade ou cor verdadeira (°Hue) varia entre 0° e 360°, sendo que o ângulo 0° corresponde à cor vermelha; 90° à cor amarela; 180° ou -90°, a cor verde, e 270° ou -180°, 2 http://bjft.ital.sp.gov.br Morangos produzidos no semiárido de Minas Gerais: qualidade do fruto e da polpa congelados Castricini, A. et al. a cor azul. A avaliação de sólidos solúveis foi feita por leitura direta em refratômetro manual/digital, com resultados expressos em °Brix. A acidez titulável, determinada por titulação, foi expressa em grama de ácido cítrico. 100 g-1 de polpa (IAL, 1987). teste Scott-Knott a 5% ou análise de regressão, de acordo com a variável independente ‒ cultivar ou armazenamento, respectivamente. Após a caracterização inicial, os frutos inteiros ou na forma de polpa foram armazenados em freezer (-16 °C). Tanto para o armazenamento de frutos inteiros quanto em polpa, utilizou-se saco plástico (0,20 × 0,30 m) de polietileno de baixa densidade (70 µm), selado a vácuo, com seladora a vácuo, modelo Selovac 200. Os sacos apresentam certa permeabilidade (não informada pelo fabricante), pois, de acordo com Bauer (2009), todo material polimérico, devido à própria estrutura molecular, mesmo quando revestido com materiais de alta barreira, ainda demonstra certo grau de permeabilidade, quando comparado ao vidro ou material metálico. Para obtenção da polpa, os frutos foram processados em liquidificador industrial, sem adição de água. 3.1 Qualidade de frutos congelados O armazenamento da polpa e dos frutos inteiros deu-se até 180 dias após o processamento e a embalagem, ou seja, até seis meses. Durante este período, foram retiradas amostras do freezer e, após o descongelamento, foram feitas as avaliações de cor (L, C e °hue), sólidos solúveis e acidez titulável, de acordo com as metodologias descritas anteriormente. Foi utilizado o delineamento inteiramente casualizado, com três repetições de 10 frutos por parcela, para as avaliações de frutos congelados inteiros, e três polpas embaladas, para as análises da polpa. A análise de variância foi feita em esquema fatorial 4 × 6, sendo quatro cultivares (Tudla, Oso Grande, Albion e Portola) avaliadas, em seis tempos de armazenamento (0, 30, 60, 90, 150 e 180 dias), quando armazenadas em frutos inteiros, e em sete tempos (0, 30, 60, 90, 120, 150 e 180 dias), quando em polpa. Os resultados foram submetidos à análise estatística utilizando-se o Software Saeg 9.1 (SAEG, 2007). A análise de variância foi feita depois de comprovada a distribuição normal ou homogênea dos dados, através do Teste de Lilliefors e do Teste Cochran e Bartlett, respectivamente. Uma vez observada significância pelo Teste F, foi utilizado o 3 Resultados e discussão Houve interação significativa entre o tempo de armazenamento e as cultivares, para a cromaticidade da cor, os sólidos solúveis e a acidez titulável. A luminosidade da cor da casca variou em função dos efeitos isolados dos tratamentos, enquanto a tonalidade da cor (°hue), em função dos dias de armazenamento (Tabela 1). Durante o armazenamento, ocorreu intensificação da cor vermelha dos frutos, evidenciada pela redução dos valores do °hue (Figura 1A). De acordo com Borges et al. (2013), no sistema C.I.E Lab, (padrão utilizado no colorímetro Minolta), o 0° corresponde à cor vermelha e o 90°, à amarela. Assim, quanto maior o valor, mais amarelo é o fruto, e, quanto menor, mais vermelho. Até 90 dias de armazenamento, verificou-se também redução de L (Figura 1B), que expressa o grau de luminosidade da cor medida (L* = 100 = branco; L* = 0 = preto); neste caso, do vermelho. Entretanto, a partir deste dia de armazenamento, ocorreu aumento de L, indicando que os frutos congelados tenderam ao vermelho mais claro. Segundo Ávila et al. (2012), menores valores relativos de °hue indicam coloração mais escura; no entanto, de acordo com Flores-Cantillano et al. (2008), o escurecimento acentuado da epiderme do morango durante o armazenamento é um atributo indesejável, tornando o produto pouco aceitável no mercado. Morangos ‘Tudla’, ‘Oso Grande’ e ‘Portola’ tiveram maior luminosidade da cor da epiderme que ‘Albion’ (Tabela 2), ou seja, esses cultivares tem frutos relativamente mais claros que ‘Albion’. A cromaticidade da cor dos frutos durante o armazenamento está apresentada na Tabela 3A. A cultivar Tudla apresentou redução deste componente da cor a partir de 30 dias de congelamento; para ‘Oso Grande’ e ambas as cultivares ‘Albion’ e ‘Portola’, esta redução ocorreu a partir de 60 dias e do tempo zero, respectivamente. A redução dos valores de croma indica Tabela 1. Análise de variância para luminosidade (L), croma (C), °hue, sólidos solúveis (ss) e acidez titulável (at) de cultivares de morangos inteiros, durante o armazenamento. Quadrado Médio Sólidos Acidez Fontes de Variação GL L C* °hue solúveis titulável Armazenamento (A) 5 156,3* 118,6* 77,1* 2,4* 0,2* Cultivares (C) 3 23,5* 73,6* 0,9ns 4,0* 0,3* ns ns A×C 15 5,6 19,3* 8,7 1,1* 0,3* Resíduo 48 4,4 9,1 6,0 0,2 0,9 C.V. (%) 6,2 10,7 8,2 6,0 12,7 Significativo; nsNão Significativo, a 5% de probabilidade pelo teste F. Braz. J. Food Technol., Campinas, v. 20, e2016149, 2017 3 http://bjft.ital.sp.gov.br Morangos produzidos no semiárido de Minas Gerais: qualidade do fruto e da polpa congelados Castricini, A. et al. redução da saturação de pigmentos da determinada cor; neste caso, da cor vermelha, tornando-se menos viva. Assim, verificou-se perda de brilho na epiderme dos frutos em tempos distintos para cada cultivar; esta resposta pode estar relacionada a maior ou menor resistência dos tecidos à mudança de cor (escurecimento), em função do frio. Entretanto, a diferença de cor entre cultivares é uma característica genética, influenciada por fatores climáticos e culturais (FLORES-CANTILLANO et al., 2008). A cultivar ‘Tudla’ apresentou maior valor de croma em relação às demais, em todos os dias de armazenamento, indicando frutos com coloração mais pigmentada, ou seja, vermelho mais “vivo” (Tabela 3). Menores valores para croma ocorreram para ‘Oso Grande’, em todos os dias de armazenamento, indicando que esta cultivar possui frutos mais opacos que as demais. Figura 1. Cor de morangos, durante o armazenamento. (A) Tonalidade; (B) Luminosidade. Tabela 2. Luminosidade da cor de morangos congelados inteiros. Cultivar Luminosidade da cor Tudla 34,07A Oso Grande 35,01A Albion 32,26B Portola 33,81A Médias seguidas de mesma letra não diferem estatisticamente pelo teste Scott-Knott a 5%. O teor de sólidos solúveis reduziu-se em todas as cultivares durante o armazenamento; entretanto, morangos ‘Tudla’ aos 180 dias não diferiram daqueles avaliados no dia zero (Tabela 3B). Andrade Júnior et al. (2016) observaram que, em média, houve diminuição dos sólidos solúveis do início ao fim do período de armazenamento (5,8 para 5,1°Brix). Entre as cultivares, Oso Grande apresentou maior teor de sólidos solúveis em todos os tempos de armazenamento e Portola, o menor teor relativo. Morangos de cultivares com maiores teores e manutenção destes, durante o armazenamento, são mais apreciados. Segundo Portela et al. (2012), o teor de sólidos solúveis é um dos atributos de Tabela 3. Croma da cor, sólidos solúveis e acidez titulável de morangos congelados inteiros, por 180 dias. A - Croma (C) Armazenamento (dias) Tudla Oso Grande Albion 0 34,12Aa 21,31Da 29,05Ba Aa Ca 30 37,03 30,52 32,35Bb Ab Da 60 30,82 27,65 30,47Bb 90 31,18Ab 26,70Db 29,38Bb 150 26,45Ab 20,02Dc 24,79Bd Ab Dc 180 29,17 22,94 26,92Bc B - Sólidos Solúveis (°Brix) 0 8,70Ba 8,80Aa 8,80Aa Ca Ab 30 7,37 8,23 7,80Bb Cb Ab 60 6,70 7,63 7,07Bc 90 6,97Cb 7,60Ac 7,10Bc Cb Ab 150 6,70 8,13 6,87Bc Ca Ab 180 7,53 8,23 7,73Bb -1 C - Acidez titulável (g de ácido cítrico.100 g de polpa) 0 0,28Aa 0,21Ba 0,21Ba Aa Bb 30 0,25 0,23 0,22Cd 60 0,20Ab 0,19Bc 0,18Cd 90 0,28Aa 0,28Ba 0,27Cb Aa Bb 150 0,38 0,29 0,28Cc Aa Bb 180 0,28 0,27 0,24Cc Portola 27,19Ca 32,04Bb 29,06Cb 28,03Cb 24,11Cc 24,49Cc 6,10Ca 7,17Dc 6,70Dd 6,57De 6,40Dd 6,97Db 0,21Ca 0,21Dc 0,16Dd 0,26Db 0,27Dc 0,20Db Médias seguidas de mesma letra maiúscula na linha e minúscula na coluna não diferem estatisticamente pelo teste Scott-Knott a 5%. Braz. J. Food Technol., Campinas, v. 20, e2016149, 2017 4 http://bjft.ital.sp.gov.br Morangos produzidos no semiárido de Minas Gerais: qualidade do fruto e da polpa congelados Castricini, A. et al. qualidade quanto ao sabor, pois indica a quantidade de açúcares existentes no fruto. Antunes et al. (2014) também verificaram menor teor de sólidos solúveis em morangos ‘Portola’, em relação às cultivares Camino Real, Palomar, Albion, Monterey e San Andreas, em dois ciclos de produção. Cultivares e clones de morangos estudados por Brackmann et al. (2011) tiveram, em média, menor teor de sólidos solúveis que das cultivares estudadas neste trabalho. A acidez titulável de morangos das quatro cultivares foi menor no 60º dia de armazenamento (Tabela 3C). Para ‘Portola’, não houve diferença significativa entre os demais dias de armazenamento, sendo estatisticamente superior a acidez dos frutos no 60º dia. Morangos ‘Albion’ e ‘Portola’ tiveram redução na acidez em relação ao dia zero de armazenamento; a cultivar Oso Grande também reduziu a acidez durante o armazenamento, apesar de não ter havido diferença entre o dia zero e o 90º. Tendência semelhante de alterações na acidez titulável de morangos com o armazenamento foi observada por Andrade Júnior et al. (2016) e Ávila et al. (2012). Os valores de acidez verificados neste trabalho foram inferiores àqueles encontrados por Brackmann et al. (2011), em diferentes cultivares e clones de morangos, antes e depois do armazenamento refrigerado. A luminosidade da cor está apresentada na Figura 2, na qual se verificou ligeiro aumento durante os dias de armazenamento. O aumentou dos valores de L* indica que a cor está tendendo ao clareamento; assim, a polpa de morangos tornou-se mais clara com o armazenamento. Entre as cultivares (Tabela 5), Oso Grande e Portola tiveram polpa com maior luminosidade que Tudla e Albion. Cunha Junior et al. (2012) também verificaram tendência de aumento nos valores de L* em morangos submetidos à atmosfera controlada e ao armazenamento refrigerado. De acordo com Kader (2010), o valor de Luminosidade (L) é um indicador de escurecimento ao longo do armazenamento, o qual pode ser ocasionado tanto por reações oxidativas quanto pelo aumento da concentração de pigmentos. A tonalidade da cor vermelha e as variações do croma estão apresentadas na Tabela 6A e B. Entre as cultivares, a polpa de morangos ‘Albion’ apresentou maiores valores de °hue, indicando tonalidade vermelha mais intensa, porém não tão “viva” quanto de ‘Tudla’, em todos os dias de armazenamento estudados. Inversamente, a polpa de ‘Oso Grande’ permaneceu vermelha menos intensa e opaca, em comparação às demais. Em relação Segundo Figueiredo et al. (2010), a acidez é importante atributo químico na definição da finalidade de uso das variedades de morango, visto que o desenvolvimento de cultivares de dupla aptidão é dificultado pelo fato de as exigências para uso industrial e para consumo in natura serem opostas, ou seja, maior e menor acidez, respectivamente. 3.2 Qualidade da polpa congelada Com exceção da luminosidade da cor, todas as variáveis de qualidade de polpa avaliadas sofreram efeito interativo dos fatores ‘cultivar’ e ‘dias de armazenamento’. A luminosidade variou em função dos efeitos isolados dos fatores estudados (Tabela 4). Figura 2. Luminosidade da polpa congelada de morangos, durante o armazenamento. Tabela 4. Análise de variância para luminosidade (L), croma (C), °hue, sólidos solúveis (ss) e acidez titulável (at) de polpa de morangos durante o armazenamento. Quadrado Médio Sólidos Acidez Fontes de Variação GL L C* °hue solúveis titulável Armazenamento (A) 6 147,9* 55,6* 51,4* 0,4* 0,3* Cultivares (C) 3 18,3* 195,8* 37,3* 5,3* 0,8* ns A×C 18 2,9 20,9* 14,2* 0,7* 0,5* Resíduo 56 3,3 4,9 1,9 0,1 0,7 C.V. (%) 4,8 9,0 4,7 4,2 8,8 Significativo; nsNão Significativo, a 5% de probabilidade pelo teste F. Braz. J. Food Technol., Campinas, v. 20, e2016149, 2017 5 http://bjft.ital.sp.gov.br Morangos produzidos no semiárido de Minas Gerais: qualidade do fruto e da polpa congelados Castricini, A. et al. aos dias de armazenamento, verificou-se que as polpas de ‘Tudla’ e ‘Portola’ permaneceram com tonalidade mais intensa até o 30º dia e ‘Oso Grande’ e ‘Albion’ tiveram maiores °hue somente no primeiro dia de avaliação. A polpa armazenada de morangos ‘Oso Grande’ foi aquela com maior teor de sólidos solúveis, em relação às demais cultivares, e o menor teor ocorreu em ‘Portola’ (Tabela 6C). Durante os dias de armazenamento, ocorreu redução dos teores de sólidos solúveis em todas as polpas. Em relação à acidez titulável (Tabela 6D) e a exemplo dos frutos congelados inteiros, a polpa de morangos ‘Tudla’ foi a mais ácida e a de ‘Oso Grande’ apresentou-se com menor acidez; os morangos ‘Portola’ congelados inteiros tiveram menos acidez. Segundo Figueiredo et al. (2010), a acidez revelada pelo pH de polpa são atributos físico-químicos importantes na definição da finalidade de uso das variedades. A acidez de todas as polpas, no presente trabalho, foi inferior àquela encontrada por Batista et al. (2013). Tabela 5. Luminosidade da polpa de morangos congelada. Cultivar Tudla Oso Grande Albion Portola Luminosidade da cor 35,31B 37,16A 35,89B 37,17A Médias seguidas de mesma letra não diferem estatisticamente pelo teste Scott-Knott a 5%. Tabela 6. Croma e tonalidade da cor, sólidos solúveis e acidez titulável da polpa de morangos congelada por 180 dias. A - Croma (C) Armazenamento (dias) Tudla Oso Grande Albion Portola 0 30 60 90 120 150 180 34,12Aa 32,41Aa 24,77Ad 24,57Ad 27,11Ac 30,34Ab 25,33Ac 0 30 60 90 120 150 180 33,60 32,11Ba 28,99Bb 28,43Bb 31,59Bb 30,98Bb 29,69Bb 0 30 60 90 120 150 180 8,70Ba 7,57Bb 7,43Cb 7,57Cb 7,40Cb 7,20Cb 7,40Cb 21,31Da 20,22Db 19,93Db 18,36Cb 19,98Db 18,79Db 20,98Db 29,05Ba 27,93Bb 22,36Be 22,58Bd 25,90Bc 28,70Bb 25,12Bd 27,19Ca 23,90Cb 22,15Ce 22,50Be 22,26Cd 25,46Cc 24,49Ce 34,83Aa 32,59Ab 29,15Ad 29,42Ad 31,76Ac 32,80Ae 30,54Ad 32,36Ca 31,35Ca 27,77Cd 27,62Cc 29,76Cb 28,87Ce 29,65Cc 8,80Aa 7,60Bb 7,58Bc 7,63Bb 7,93Bb 7,60Bb 7,50Bc 6,10Ca 7,50Cc 7,25De 7,30Db 7,13Dd 7,17Db 7,30Dd B - Tonalidade da Cor (°Hue) 30,21Da 27,91Db 22,56Dd 22,81Dc 27,93Db 27,47Dd 25,32Dc Ba C - Sólidos solúveis (°Brix) 8,80Aa 8,37Ac 8,40Ad 8,43Ac 8,47Ac 8,07Ab 8,20Ad D - Acidez titulável (g de ácido cítrico.100 g-1 de polpa) 0 30 60 90 120 150 180 0,28Ac 0,40Aa 0,34Ac 0,32Ac 0,32Ab 0,40Aa 0,43Aa 0,21Bb 0,29Cb 0,24Dc 0,26Db 0,27Db 0,26Ca 0,34Da 0,21Bd 0,31Bc 0,26Ce 0,27Cd 0,29Cc 0,36Bb 0,37Ca 0,21Bc 0,31Bb 0,28Be 0,28Bd 0,30Bb 0,40Aa 0,39Ba Médias seguidas de mesma letra maiúscula na linha e minúscula na coluna não diferem estatisticamente pelo teste Scott-Knott a 5%. Braz. J. Food Technol., Campinas, v. 20, e2016149, 2017 6 http://bjft.ital.sp.gov.br Morangos produzidos no semiárido de Minas Gerais: qualidade do fruto e da polpa congelados Castricini, A. et al. 4 Conclusões Morangos inteiros congelados por 180 dias apresentaram escurecimento da epiderme e redução do teor de sólidos solúveis. A polpa de morangos tornou-se mais clara com o armazenamento, sendo ‘Oso Grande’ e ‘Portola’, as mais claras. A cultivar Oso Grande tem maior teor de sólidos solúveis e Portola, menor teor, armazenados inteiros ou em polpa. A polpa e os frutos armazenados de ‘Tudla’ são mais ácidos. Morangos ‘Portola’ congelados inteiros são menos ácidos, assim como ‘Oso Grande’ em polpa. Durante o armazenamento de morangos congelados inteiros ou em polpa, produzidos no Semiárido de Minas Gerais, ocorreram modificações nos parâmetros cor, sólidos solúveis e acidez titulável. Agradecimentos Ao Banco do Nordeste pelo financiamento do projeto de pesquisa. À FAPEMIG pelas Bolsas de Incentivo à Pesquisa. Referências ANDRADE JÚNIOR, V. C.; GUIMARÃES, A. G.; AZEVEDO, A. M.; PINTO, N. A. V. D. FERREIRA, M. A. M. Conservação pós-colheita de frutos de morangueiro em diferentes condições de armazenamento. Horticultura Brasileira, v. 34, n. 3, p. 405-4112016. ANTUNES, M. C.; CUQUEL, F. L.; ZAWADNEAK, M. A. C.; MOGOR, A. F.; RESENDE, J. T. V. Postharvest quality of strawberry produced during two consecutive seasons. Horticultura Brasileira, v. 32, n. 2, p. 168-173, 2014. ÁVILA, J. M. M.; TORALLES, R. P.; CANTILLANO, R. F. F.; PERALBA, M. C. R.; PIZZOLATO, T. M. Influência do sistema de produção e do armazenamento refrigerado nas características físico-químicas e no desenvolvimento de compostos voláteis em morangos. Ciência Rural, v. 42, n. 12, p. 2265-2271, 2012. BATISTA, A. G.; OLIVEIRA, B. D’Á.; OLIVEIRA, M. A.; GUEDES, T. J.; SILVA, D. F.; PINTO, N. A. V. D. Parâmetros de qualidade de polpas de frutas congeladas: uma abordagem para produção do agronegócio familiar no Alto Vale do Jequitinhonha. Revista Tecnologia & Ciência Agropecuária, v. 7, n. 4, p. 49-54, 2013. BAUER, E. J. Pharmaceutical packaging handbook. New York: Informa Healthcare; 2009. 585 p. BORGES, C. D.; MENDONÇA, C. R. B.; ZAMBIAZI, R. C.; NOGUEIRA, D.; PINTO, E. M.; PAIVA, F. F. Conservação de morangos com revestimentos à base de goma xantana e óleo Braz. J. Food Technol., Campinas, v. 20, e2016149, 2017 essencial de sálvia. Bioscience Journal, v. 29, n. 5, p. 10711083, 2013. BRACKMANN, A.; PAVANELLO, E. P.; BOTH, V.; JANISCH, D. I.; SCHMITT, O. J.; GIMÉNEZ, G. Avaliação de genótipos de morangueiro quanto à qualidade e potencial de armazenamento. Revista Ceres, v. 58, n. 5, p. 542-547, 2011. BRASIL. Ministério da Agricultura do Abastecimento. Instrução Normativa nº 01/00, de 07/01/00. Regulamento técnico geral para fixação dos padrões de identidade e qualidade para polpa de fruta. Diário Oficial [da] República Federativa do Brasil, Brasília, DF, 2000. p. 54-58. BRASIL. Ministério da Agricultura do Abastecimento. Regulamento Técnico para fixação dos padrões de identidade e qualidade para polpa de morango. Diário Oficial [da] República Federativa do Brasil, Brasília, DF, 2016. p. 6. Disponível em: <http://www. agricultura.gov.br/acesso-a-informacao/participacao-social/ consultas-publicas/documentos/01_09-secao-1-portaria-86. pdf>. Acesso em: 18 jul. 2017. CUNHA JUNIOR, L. C.; JACOMINO, A. P.; OGASSAVARA, F. O.; TREVISAN, M. J.PARISI, M. C. M. Armazenamento refrigerado de morango submetido a altas concentrações de CO2. Horticultura Brasileira, v. 30, n. 4, p. 688-694, 2012. DIAS, M. S. C.; REIS, J. B. R. S.; JESUS, A. M. Morango no semiárido. Informe Agropecuário - morango: tecnologias de produção ambientalmente corretas, v. 35, n. 279, p. 92-98, 2014. FIGUEIREDO, F. C.; BOTREL, P. P.; TEIXEIRA, C. P.; PETRAZZINI, L. L.; LOCARNO, M.; CARVALHO, J. G. Pulverização foliar e fertirrigação com silício nos atributos físico-químicos de qualidade e índices de coloração do morango. Ciência e Agrotecnologia, v. 34, n. 5, p. 1306-1311, 2010. FLORES-CANTILLANO, R. F. F.; CASTAÑEDA, L. M. F.; TREPTOW, R. O.; SCHUNEMANN, A. P. P. Qualidade físico-química e sensorial de cultivares de morango durante o armazenamento refrigerado. Embrapa Clima TemperadoBoletim de Pesquisa e Desenvolvimento, v. 75, p. 1-31, 2008. INSTITUTO ADOLFO LUTZ - IAL. Métodos químicos e físicos de análise de alimentos. 3. ed. São Paulo: IAL, 1987. KADER, A. A. Future of modified atmosphere research. Acta Horticulturae, n. 857, p. 212-217, 2010. PORTELA, I. P.; PEIL, R. N.; RODRIGUES, S.; CARINI, F. Densidade de plantio, crescimento, produtividade e qualidade das frutas de morangueiro “Camino Real” em hidroponia. Revista Brasileira de Fruticultura, v. 34, n. 3, p. 792-798, 2012. SAEG. Sistema para análises estatísticas. versão 9. Viçosa: Fundação Arthur Bernardes, 2007. 7
https://openalex.org/W4284678262	https://orca.cardiff.ac.uk/id/eprint/155932/1/engproc-19-00001%20%281%29.pdf	English	null	TLBO Tuned a Novel Robust Fuzzy Control Structure for LFC of a Hybrid Power System with Photovoltaic Source	null	2,022	cc-by	6,447	Citation: Shouran, M.; Anayi, F. TLBO Tuned a Novel Robust Fuzzy Control Structure for LFC of a Hybrid Power System with Photovoltaic Source. Eng. Proc. 2022, 19, 1. https://doi.org/10.3390/ ECP2022-12684 Citation: Shouran, M.; Anayi, F. TLBO Tuned a Novel Robust Fuzzy Control Structure for LFC of a Hybrid Power System with Photovoltaic Source. Eng. Proc. 2022, 19, 1. https://doi.org/10.3390/ ECP2022-12684 Proceeding Paper TLBO Tuned a Novel Robust Fuzzy Control Structure for LFC of a Hybrid Power System with Photovoltaic Source † Mokhtar Shouran 1,2,* and Fatih Anayi 1 Mokhtar Shouran 1,2,* and Fatih Anayi 1 1 Wolfson Centre for Magnetics, School of Engineering, Cardiff University, Cardiff CF24 3AA, UK; anayi@cardiff.ac.uk 1 Wolfson Centre for Magnetics, School of Engineering, Cardiff University, Cardiff CF24 3AA, UK; anayi@cardiff.ac.uk 2 Department of Control Engineering, College of Electronics Technology, Bani Walid 90045, Libya * Correspondence: shouranma@cardiff.ac.uk; Tel.: +44-7424491429 † Presented at the 1st International Electronic Conference on Processes: Processes System Innovation, 17–31 May 2022; Available online: https://ecp2022.sciforum.net. 2 Department of Control Engineering, College of Electronics Technology, Bani Walid 90045, Libya * Correspondence: shouranma@cardiff.ac.uk; Tel.: +44-7424491429 † Presented at the 1st International Electronic Conference on Processes: Processes System Innovation, 17–31 May 2022; Available online: https://ecp2022.sciforum.net. Correspondence: shouranma@cardiff.ac.uk; Tel.: +44 7424491429 † Presented at the 1st International Electronic Conference on Processes: Processes System Innovation, 17–31 May 2022; Available online: https://ecp2022.sciforum.net. † Presented at the 1st International Electronic Conference on Processes: Processes System Innova 17–31 May 2022; Available online: https://ecp2022.sciforum.net. Abstract: This study proposes a new fuzzy logic control (FLC) design-based I controller plus a Fuzzy Cascade FOPI-FOPD (I + F C FOPI-FOPD) for load frequency control (LFC) in power systems. The structure of this design offers a satisfactory level of reliability as well as excellent robustness performance. The proposed fuzzy design is employed in a hybrid dual area power system based on a photovoltaic renewable energy plant in area one and a thermal generation unit in area two. In order to achieve the best possible dynamic performance of the proposed structure, the teaching learning-based optimization (TLBO) algorithm is suggested to optimally tune the scaling factor gains of the proposed fuzzy conﬁguration. The superiority of the suggested fuzzy control design is investigated by conducting a comparative study between this design and a previously applied PI-based ﬁreﬂy algorithm. Simulation results revealed that the fuzzy logic controller introduced in this study is reliable and superior, and appropriately handled the problem of frequency variation. Keywords: fuzzy logic control (FLC); load frequency control (LFC); dual area power system; teaching learning-based optimization (TLBO) 1. Introduction Modern power systems are becoming more reliant on renewable energy resources (RERs). The dependence on RERs is mainly due to the continuous increments in the prices of fossil fuels in addition to avoiding the emission of greenhouses gases and its obvious link to the global warming dilemma. Moreover, the fact of the increasing power demand in today’s world means there is a requirement for a huge number of different generating units to interconnect through existing transmission lines called tie-lines to meet the increasing power consumption; this also leads to the adoption of hybrid power systems where electrical networks rely on RERs along with the traditional power resources. The RERs used in interconnected power systems may be photovoltaic (PV), wind turbine (WT), energy storage system, fuel cell (FC) with electrolyser and battery. Solar and wind energy resources have been considered the most commonly used in power systems among the various RERs. However, many modern power systems remain reliant on thermal generation to meet the demands of end users. This gives an obvious idea of the vital role that thermal units play in power systems in addition to the continued increase in RERs reliance [1]. Published: 7 June 2022 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. g its play in power systems in addition to the continued increase in RERs reliance [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/). From a control engineering point of view, ensuring the stable and reliable operation of modern power systems is one of the most challenging processes. This is due to the continuous increase in their size, which in turn leads to more complexity in addition to having to consider the external/internal disturbances that power systems unpredictably experience. p Furthermore, load demand in most power systems is continually changing and this change leads to consequences of a deviation in frequency and tie-line power ﬂow and, in https://www.mdpi.com/journal/engproc Eng. Proc. 2022, 19, 1. https://doi.org/10.3390/ECP2022-12684 Eng. Proc. 2022, 19, 1 2 of 7 turn, requires a similar change in the generation side. 1. Introduction Accordingly, the mismatch between the demanded power and the generated power is the reason for the frequency and tie-line power deviations throughout the whole power system. Importantly, in stable, secure and reliable power systems, the frequency and tie-line power ﬂow are maintained in their pre- speciﬁed limits in normal operating conditions as well as in case of a sudden disturbance occurrence, which is achieved by balancing the active power output of the generated power and the requested demand plus the possible losses. This control mechanism is called load frequency control (LFC). As a result of installing this loop-LFC in power systems, frequency and tie-line power deviations are guaranteed to be always zero or within acceptable limits. Based on this, an improper LFC system design may undermine the overall behaviour of the controlled system causing undesired ﬂuctuations in frequency and tie-line ﬂow, which in turn may lead to system instability [2]. Due to the above-mentioned issues, incorporating LFC in power systems based on different generating sources has become the most signiﬁcant research area to focus on. To overcome this issue, many researchers presented different approaches to simulate LFC integrated into different power systems. Traditional control techniques, robust control methods, sliding mode control and artiﬁcial controllers have been applied in this ﬁeld. In [3], a PI controller-based ﬁreﬂy optimization algorithm was designed and implemented for LFC in a dual area interconnected power system incorporating a PV system. A PI controller tuned by the Harris Hawks optimizer-based LFC in two different power systems with RERs was successfully implemented to regulate the frequency in the case of sudden load disturbance [4]. A movable damped wave algorithm tuned Fractional Order (FO) PID was equipped as an LFC system in two and four area interconnected power systems with PV and WT plants, and the FOPID-based proposed algorithm outperformed the same controller tuned by other optimization techniques [5]. Another application of a FOPID in LFC for power systems with RERs was suggested in [6]; in this study, a modiﬁed hunger games search optimizer was proposed to ﬁnd the optimal values of the FOPID’s gains. An adaptive PI-based LFC system installed in two different dual area power systems was proposed in [7]; the suggested controller evidenced its superiority over the traditional PI. LFC-based sliding mode control (SMC) was proposed in [8] for a hybrid power system incorporating WT and energy storage systems. 1. Introduction A new SMC based on the disturbance observer was suggested in [9] for LFC in a hybrid power system considering the presence of WT, system parametric uncertainty and non-linear restraints. A linear matrix inequality- based particle swarm (PSO) optimization algorithm was proposed in [10] to stabilize the frequency in a power system with different energy resources. A model predictive control approach was also proposed for frequency stabilization in a hybrid power system consisting of different plants including WT [11]. Notably, in recent years, LFC-based fuzzy logic control has received considerable attention. This is due to the wide range of merits offered by this controller. Furthermore, soft computing techniques have been exploited to achieve the best possible dynamic performance of FLC. Fuzzy PID with a ﬁltered derivative action optimized by the Bees algorithm has been successfully implemented as an LFC system in a simpliﬁed form of the Great Britain power system [12]. Different fuzzy structures were proposed in [13] to damp the frequency/tie-line power variations in a two-area power system. In this study, the proposed fuzzy PI plus Fuzzy PD illustrated a slight supremacy over the other investigated conﬁgurations. A fuzzy cascade FOPI-FOPD-based PSO for LFC purposes in a multi- area electrical system was studied in [14]. Another application of FLC-based frequency regulation was investigated in [15]; in this study the proposed controller was installed in a two-area multi-source power system with a PV unit, WT and redox ﬂow battery, and successfully damped the frequency deviation under various operating conditions. In [16], an adaptive fuzzy FOPID was implemented for LFC in the renewable penetrated electrical power network. Notwithstanding the fact that the above-mentioned techniques have different merits and successfully solved the problem of frequency deviation to a great extent, it is note- Eng. Proc. 2022, 19, 1 3 of 7 merits worthy to mention the limitation of installing these techniques in power systems. For example, the traditional controllers are known not to provide the required response when the controlled system undergoes possible operating conditions of non-linearities or high sensitivity. However, the low cost is the main advantage of classical controllers. In terms of other controllers such as adaptive control and SMC, although they offer an effective performance as LFC systems, the complexity in their design and high computational time and burden are known as the most common limitations. 2. Power System under Study 2. Power System under Study In this study, a two-area interconnected power system is considered to demonstrate the effectiveness of the proposed fuzzy controller for LFC purposes. In this study, a two-area interconnected power system is considered to demonstrate the effectiveness of the proposed fuzzy controller for LFC purposes. As shown in Figure 1, this system consists of a PV plant equipped in area one and a thermal unit installed in area two. This system has extensively been used in the literature [3,7]. The values of the parameters of the investigated system are as follows As shown in Figure 1, this system consists of a PV plant equipped in area one and a thermal unit installed in area two. This system has extensively been used in the literature [3,7]. The values of the parameters of the investigated system are as follows C1 = −18, C2= 900, C3= 100, C4= 50 Tg= 0.08, Tt= 0.18, Kr= 3.3, Tr= 10, Kp= 120, Tp= 20 and T = 0.545. , C2 900, C3 100, C4 50 Tt = 0.18, Kr = 3.3, Tr = 10, Kp = 120, Tp = 20 and T = 0.545. 1 , 2 , 3 , 4 Tg= 0.08, Tt= 0.18, Kr= 3.3, Tr= 10, Kp= 120, Tp= 20 and T = 0.545. Tg= 0.08, Tt = 0.18, Kr = 3.3, Tr = 10, Kp = 120, Tp = 20 and T = 0.545. Figure 1. The testbed dual area power system. Figure 1. The testbed dual area power system. Figure 1. The testbed dual area power system. Figure 1. The testbed dual area power system. 1. Introduction Furthermore, some useful studies have proposed and applied different structures of fuzzy control for frequency regulation; however, most of these techniques lack the consideration of reliability in the design. worthy to mention the limitation of installing these techniques in power systems. For ex- ample, the traditional controllers are known not to provide the required response when the controlled system undergoes possible operating conditions of non-linearities or high sensitivity. However, the low cost is the main advantage of classical controllers. In terms of other controllers such as adaptive control and SMC, although they offer an effective performance as LFC systems, the complexity in their design and high computational time and burden are known as the most common limitations. Furthermore, some useful studies have proposed and applied different structures of fuzzy control for frequency regulation; however, most of these techniques lack the consideration of reliability in the design. Therefore the authors of this paper propose a no el FLC configuration that guaran Therefore, the authors of this paper propose a novel FLC conﬁguration that guaran- tees a high level of reliability for LFC in a dual area power system incorporating a PV unit. The suggested technique is an I controller plus Fuzzy Cascade FOPI-FOPD (I + F C FOPI-FOPD). Moreover, to obtain the best performance from the suggested controller, a well-known/powerful optimization tool called the teaching learning-based optimization algorithm is used in this work to optimally set the parameters of the proposed I + F C FOPI- FOPD. Furthermore, the strong performance of this controller is evidenced by comparing the results with those based on the PI optimized by the ﬁreﬂy algorithm studied in [3]. Therefore, the authors of this paper propose a novel FLC configuration that guaran- tees a high level of reliability for LFC in a dual area power system incorporating a PV unit. The suggested technique is an I controller plus Fuzzy Cascade FOPI-FOPD (I + F C FOPI- FOPD). Moreover, to obtain the best performance from the suggested controller, a well- known/powerful optimization tool called the teaching learning-based optimization algo- rithm is used in this work to optimally set the parameters of the proposed I + F C FOPI- FOPD. Furthermore, the strong performance of this controller is evidenced by comparing the results with those based on the PI optimized by the firefly algorithm studied in [3]. 3.1. I Controller Plus Cascade Fuzzy FOPI-FOPD 3.1. I Controller Plus Cascade Fuzzy FOPI-FOPD q 1 I C ll Pl C d F FOPI FOPD 3.1. I Controller Plus Cascade Fuzzy FOPI-FOPD 3.1. I Controller Plus Cascade Fuzzy FOPI-FOPD q I ll l d O I O Figure 2 demonstrates the structural diagram of the proposed controller. This con- troller comprises two parts; a fuzzy cascade FOPI-FOPID enhanced with a classical I controller. As is obvious, the fuzzy part has two inputs: “the Area Control Error (ACE) and change of ACE” in addition to one output. The output signal of the fuzzy controller is connected with the input of the FOPI. However, the input signal of the latter controller “FOPD” is the output signal of the FOPI in addition to the ACE signal. Finally, the output signal of FOPD is added to the output of the classical I, which in total represent the control signal of the proposed controller. The rule bases of the fuzzy controller are given in Table 1, while the membership function of the inputs and the output are illustrated in Figure 3. Figure 2 demonstrates the structural diagram of the proposed controller. This con- troller comprises two parts; a fuzzy cascade FOPI-FOPID enhanced with a classical I con- troller. As is obvious, the fuzzy part has two inputs: “the Area Control Error (ACE) and change of ACE” in addition to one output. The output signal of the fuzzy controller is connected with the input of the FOPI. However, the input signal of the latter controller “FOPD” is the output signal of the FOPI in addition to the ACE signal. Finally, the output signal of FOPD is added to the output of the classical I, which in total represent the control signal of the proposed controller. The rule bases of the fuzzy controller are given in Table 1, while the membership function of the inputs and the output are illustrated in Figure 3. 3.1. I Controller Plus Cascade Fuzzy FOPI-FOPD Figure 2 demonstrates the structural diagram of the proposed controller. This controller comprises two parts; a fuzzy cascade FOPI-FOPID enhanced with a classical I controller. As is obvious, the fuzzy part has two inputs: “the Area Control Error (ACE) and change of ACE” in addition to one output. The output signal of the fuzzy controller is connected with the input of the FOPI. However, the input signal of the latter controller “FOPD” is the output signal of the FOPI in addition to the ACE signal. 3.1. I Controller Plus Cascade Fuzzy FOPI-FOPD 3.1. I Controller Plus Cascade Fuzzy FOPI-FOPD q 1 I C ll Pl C d F FOPI FOPD The diagram of the proposed I plus fuzzy cascade FOPI-FOPD. Figure 2. The diagram of the proposed I plus fuzzy cascade FOPI-FOPD. Table 1. Fuzzy role bases. ACE 1 ACE 1 ሶ NB NS Z PS PB NB NB NB NB NS Z NS NB NB NS Z PS Z NB NS Z PS PB PS NS Z PS PB PB PB Z PS PB PB PB Figure 3. Membership functions of inputs/output. 3.2. The Optimisation Tool-TLBO and the Objective Function In order to enhance the performance of the proposed LFC system, the values of its parameters are found by employing the TLBO by minimizing the Integral Time Absolut Table 1. Fuzzy role bases. ACE 1 ACE 1 NB NS Z PS PB NB NB NB NB NS Z NS NB NB NS Z PS Z NB NS Z PS PB PS NS Z PS PB PB PB Z PS PB PB PB Table 1. Fuzzy role bases. ACE 1 ACE 1 ̇ NB NS Z PS PB NB NB NB NB NS Z NS NB NB NS Z PS Z NB NS Z PS PB PS NS Z PS PB PB PB Z PS PB PB PB Figure 3. Membership functions of inputs/output. 3.2. The Optimisation Tool-TLBO and the Objective Function In order to enhance the performance of the proposed LFC system, the values of its Figure 3. Membership functions of inputs/output. 3.2. The Optimisation Tool-TLBO and the Objective Function In order to enhance the performance of the proposed LFC system, the values of its ACE 1 ACE 1 ሶ NB NS Z PS PB NB NB NB NB NS Z NS NB NB NS Z PS Z NB NS Z PS PB PS NS Z PS PB PB PB Z PS PB PB PB ACE 1 ACE 1 NB NS Z PS PB NB NB NB NB NS Z NS NB NB NS Z PS Z NB NS Z PS PB PS NS Z PS PB PB PB Z PS PB PB PB Table 1. Fuzzy role bases. ACE 1 ACE 1 ̇ NB NS Z PS PB NB NB NB NB NS Z NS NB NB NS Z PS Z NB NS Z PS PB PS NS Z PS PB PB PB Z PS PB PB PB Figure 3. Membership functions of inputs/output. 3.2. The Optimisation Tool-TLBO and the Objectiv Figure 3. Membership functions of inputs/output. 3. The Proposed LFC System As above-mentioned, this study develops and implements a new fuzzy conﬁguration in order to achieve the required level of reliability. Eng. Proc. 2022, 19, 1 4 of 7 ration 3.1. I Controller Plus Cascade Fuzzy FOPI-FOPD 3.1. I Controller Plus Cascade Fuzzy FOPI-FOPD q 1 I C ll Pl C d F FOPI FOPD Finally, the output signal of FOPD is added to the output of the classical I, which in total represent the control signal of the proposed controller. The rule bases of the fuzzy controller are given in Table Figure 2. The diagram of the proposed I plus fuzzy cascade FOPI-FOPD. T bl 1 F l b Figure 2. The diagram of the proposed I plus fuzzy cascade FOPI-FOPD. T bl 1 F l b 1, while the membership function of the inputs and the output are illustrated in Figur Figure 2. The diagram of the proposed I plus fuzzy cascade FOPI-FOPD. Figure 2. The diagram of the proposed I plus fuzzy cascade FOPI-FOPD. Table 1. Fuzzy role bases. ACE 1 ACE 1 ሶ NB NS Z PS PB NB NB NB NB NS Z NS NB NB NS Z PS Z NB NS Z PS PB PS NS Z PS PB PB PB Z PS PB PB PB Figure 3. Membership functions of inputs/output. 3.2. The Optimisation Tool-TLBO and the Objective Function In order to enhance the performance of the proposed LFC system, the values of its parameters are found by employing the TLBO by minimizing the Integral Time Absolut Figure 2. The diagram of the proposed I plus fuzzy cascade FOPI-FOPD. Table 1. Fuzzy role bases. ACE 1 ACE 1 NB NS Z PS PB NB NB NB NB NS Z NS NB NB NS Z PS Z NB NS Z PS PB PS NS Z PS PB PB PB Z PS PB PB PB Figure 2. The diagram of the proposed I plus fuzzy cascade FOPI-FOPD. Table 1. Fuzzy role bases. ACE 1 ACE 1 ̇ NB NS Z PS PB NB NB NB NB NS Z NS NB NB NS Z PS Z NB NS Z PS PB PS NS Z PS PB PB PB Z PS PB PB PB Figure 3. Membership functions of inputs/output. 3.2. The Optimisation Tool-TLBO and the Objective Function In order to enhance the performance of the proposed LFC system, the values of its parameters are found by employing the TLBO by minimizing the Integral Time Absolut Figure 3. Membership functions of inputs/output. 3.2. The Optimisation Tool-TLBO and the Objective Function In order to enhance the performance of the proposed LFC system, the values of its parameters are found by employing the TLBO by minimizing the Integral Time Absolut Figure 2. 4. Results and Discussion 4. Results and Discussion 4. Results and Discussion The proposed controller is equipped in the system, with a controller in each area; then a load disturbance of 0.1 pu is applied in area two. The optimal values of the controller obtained by TLBO are given in Table 2. The frequency deviation in areas one and two and the tie-line power ﬂuctuation are given in Figures 4–6, respectively, in addition to Table 3. Additionally, these ﬁgures provide a comparison between the results obtained via the proposed fuzzy controller and the results from the PI tuned by FA. Figures 4–6 and Table 3 reveal the effectiveness and superiority of the proposed fuzzy controller. This controller successfully met the requirements of LFC systems and provided excellent responses in all aspects. The proposed controller is equipped in the system, with a controller in each area; then a load disturbance of 0.1 pu is applied in area two. The optimal values of the control- ler obtained by TLBO are given in Table 2. The frequency deviation in areas one and two and the tie-line power fluctuation are given in Figures 4–6, respectively, in addition to Table 3. Additionally, these figures provide a comparison between the results obtained via the proposed fuzzy controller and the results from the PI tuned by FA. Figures 4–6 and Table 3 reveal the effectiveness and superiority of the proposed fuzzy controller. This controller successfully met the requirements of LFC systems and provided excellent re- sponses in all aspects. 4. Results and Discussion The proposed controller is equipped in the system, with a controller in each area; then a load disturbance of 0.1 pu is applied in area two. The optimal values of the control- ler obtained by TLBO are given in Table 2. The frequency deviation in areas one and two and the tie-line power fluctuation are given in Figures 4–6, respectively, in addition to Table 3. Additionally, these figures provide a comparison between the results obtained via the proposed fuzzy controller and the results from the PI tuned by FA. Figures 4–6 and Table 3 reveal the effectiveness and superiority of the proposed fuzzy controller. This controller successfully met the requirements of LFC systems and provided excellent re- sponses in all aspects Figure 4. Frequency drop in area one. Figure 5. Frequency drop in area two. 3.1. I Controller Plus Cascade Fuzzy FOPI-FOPD 3.1. I Controller Plus Cascade Fuzzy FOPI-FOPD q 1 I C ll Pl C d F FOPI FOPD dt t 0 (1) (1) (1) 3.1. I Controller Plus Cascade Fuzzy FOPI-FOPD 3.1. I Controller Plus Cascade Fuzzy FOPI-FOPD q 1 I C ll Pl C d F FOPI FOPD Figure 3. Membership functions of inputs/output. p j In order to enhance the performance of the proposed 3.2. The Optimisation Tool-TLBO and the Objective Function 3.2. The Optimisation Tool-TLBO and the Objective Function p j In order to enhance the performance of the propose 3.2. The Optimisation Tool-TLBO and the Objective Function 3.2. The Optimisation Tool-TLBO and the Objective Function p p p y parameters are found by employing the TLBO by minimizing the Integral Time Absolut p j In order to enhance the performance of the proposed LFC system, the values of its parameters are found by employing the TLBO by minimizing the Integral Time Absolut In order to enhance the performance of the proposed LFC system, the values of its parameters are found by employing the TLBO by minimizing the Integral Time Absolut p p p y parameters are found by employing the TLBO by minimizing the Integral Time Absolut p j In order to enhance the performance of the proposed LFC system, the values of its parameters are found by employing the TLBO by minimizing the Integral Time Absolut In order to enhance the performance of the proposed LFC system, the values of its parameters are found by employing the TLBO by minimizing the Integral Time Absolut Eng. Proc. 2022, 19, 1 5 of 7 Error (ITAE) of the frequency deviation in both areas and the tie-line power ﬂuctuation (see Equation (1)). The mechanism and the variants of the suggested TLBO are given in [17]. (see Equation (1)). The mechanism and the variants of the suggested TLBO are given in [17]. Error (ITAE) of the frequency deviation in both areas and the tie line power fluctuation (see Equation (1)). The mechanism and the variants of the suggested TLBO are given in [17]. Error (ITAE) of the frequency deviation in both areas and the tie-line power ﬂuctuation (see Equation (1)). The mechanism and the variants of the suggested TLBO are given in [17]. (see Equation (1)). The mechanism and the variants of the suggested TLBO are given in [17]. ( ) q y p (see Equation (1)). The mechanism and the variants of the suggested TLBO are given in [17]. Objective Function = ITAE = Z t 0 (\|∆F1\| + \|∆F2\| + \|∆Ptie\|).t. dt Objective Function = ITAE = නሺ\|∆F1\| + \|∆F2\| + \|∆Ptie\|ሻ.t. dt 0 Objective Function = ITAE = නሺ\|∆F1\| + \|∆F2\| + \|∆Ptie\|ሻ.t. 4. Results and Discussion 4. Results and Discussion 4. Results and Discussion Tie-line power deviation. Table 2. The optimum values of the I plus fuzzy C FOPI-FOPD. Table 2. The optimum values of the I plus fuzzy C FOPI-FOPD. Table 2. The optimum values of the I plus fuzzy C FOPI-FOPD. Table 2. The optimum values of the I plus fuzzy C FOPI-FOPD. Table 2. The optimum values of the I plus fuzzy C FOPI FOPD. Controller Parameters Area one controller K1 K2 K3 KP11 KI1 λ1 KP12 KD1 µ1 0.284 0.7304 −1.9193 0.5200 0.4816 0.6971 −1.6179 0.7885 0.1499 Area two controller K4 K5 K6 KP21 KI2 λ2 KP22 KD2 µ2 −1.28 −0.1798 −2 −2 0.0409 0.1631 −1.6188 0.0142 0.2625 Table 3. The dynamic response of the system with the proposed controller. Controller Parameters Area one controller K1 K2 K3 KP11 KI1 λ1 KP12 KD1 µ1 0.284 0.7304 −1.9193 0.5200 0.4816 0.6971 −1.6179 0.7885 0.1499 Area two controller K4 K5 K6 KP21 KI2 λ2 KP22 KD2 µ2 −1.28 −0.1798 −2 −2 0.0409 0.1631 −1.6188 0.0142 0.2625 Table 3. The dynamic response of the system with the proposed controller. Table 3. The dynamic response of the system with the proposed controller. Table 3. The dynamic response of the system with the proposed controller. Controller Frequency in Area One Frequency in Area Two Tie-line Power Deviation ITAE Ushin pu Oshin pu Ts in s Ushin pu Oshin pu Ts in s Ushin pu Oshin pu Ts in s I + F C FOPI- FOPD −0.0835 0 5.697 −0.0751 0 5.9810 −0.0015 0.0052 3.3810 0.267 PI—FA −0.281 0.106 12.283 −0.210 0.0908 13.1132 −0.0382 0.0427 9.9657 4.192 Controller Frequency in Area One Frequency in Area Two Tie-line Power Deviation ITAE Ushin pu Oshin pu Ts in s Ushin pu Oshin pu Ts in s Ushin pu Oshin pu Ts in s I + F C FOPI-FOPD −0.0835 0 5.697 −0.0751 0 5.9810 −0.0015 0.0052 3.3810 0.267 PI—FA −0.281 0.106 12.283 −0.210 0.0908 13.1132 −0.0382 0.0427 9.9657 4.192 4. Results and Discussion 4. Results and Discussion 4. Results and Discussion This controller significantly damped the fre- quency deviation in both areas and also outperformed the previously proposed PI con- troller-based FA. Author Contributions: Both authors have contributed the same in producing this work. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. 0 5 10 15 20 25 30 35 Time (seconds) -0.04 -0.02 0 0.02 0.04 0.06 Tie line power deviation (pu) I + Fuzzy C FOPI-FOPD - TLBO PI - FA Figure 6. Tie-line power deviation. Table 2. The optimum values of the I plus fuzzy C FOPI-FOPD. Controller Parameters Area one controller K1 K2 K3 KP11 KI1 λ1 KP12 KD1 µ1 0.284 0.7304 −1.9193 0.5200 0.4816 0.6971 −1.6179 0.7885 0.1499 Area two controller K4 K5 K6 KP21 KI2 λ2 KP22 KD2 µ2 −1.28 −0.1798 −2 −2 0.0409 0.1631 −1.6188 0.0142 0.2625 Table 3. The dynamic response of the system with the proposed controller. Controller Frequency in Area One Frequency in Area Two Tie-line Power Deviation ITAE Ushin pu Oshin pu Ts in s Ushin pu Oshin pu Ts in s Ushin pu Oshin pu Ts in s I + F C FOPI-FOPD −0.0835 0 5.697 −0.0751 0 5.9810 −0.0015 0.0052 3.3810 0.267 PI—FA −0.281 0.106 12.283 −0.210 0.0908 13.1132 −0.0382 0.0427 9.9657 4.192 5. Conclusions Concisely, this paper developed and implemented a virgin fuzzy control structure named I plus fuzzy cascade FOPI-FOPD employed as an LFC system in a dual area power system incorporating a PV unit in area one. This controller signiﬁcantly damped the frequency deviation in both areas and also outperformed the previously proposed PI controller-based FA. Author Contributions: Both authors have contributed the same in producing this work. All authors have read and agreed to the published version of the manuscript. F di Thi h i d t l f di Figure 6. Tie-line power deviation. 0 5 10 15 20 25 30 35 Time (seconds) -0.04 -0.02 0 0.02 0.04 0.06 Tie line power deviation (pu) I + Fuzzy C FOPI-FOPD - TLBO PI - FA Figure 6. Tie-line power deviation. Table 2 The optimum values of the I plus fuzzy C FOPI FOPD 0 5 10 15 20 25 30 35 Time (seconds) -0.04 -0.02 0 0.02 0.04 0.06 Tie line power deviation (pu) I + Fuzzy C FOPI-FOPD - TLBO PI - FA Figure 6. Tie-line power deviation. Figure 6. 4. Results and Discussion 4. Results and Discussion 4. Results and Discussion 0 5 10 15 20 25 30 35 Time (seconds) -0.3 -0.2 -0.1 0 0.1 0.2 Frequency devaition (pu) I + Fuzzy C FOPI-FOPD - TLBO PI - FA Figure 4. Frequency drop in area one. Figure 4. Frequency drop in area one. Figure 5. Frequency drop in area two. 0 5 10 15 20 25 30 35 Time (seconds) -0.3 -0.2 -0.1 0 0.1 0.2 Frequency devaition (pu) I + Fuzzy C FOPI-FOPD - TLBO PI - FA Figure 5. Frequency drop in area two. 0 5 10 15 20 25 30 35 Time (seconds) -0.3 -0.2 -0.1 0 0.1 0.2 Frequency devaition (pu) I + Fuzzy C FOPI-FOPD - TLBO PI - FA 0 5 10 15 20 25 30 35 Time (seconds) -0.3 -0.2 -0.1 0 0.1 0.2 Frequency devaition (pu) I + Fuzzy C FOPI-FOPD - TLBO PI - FA Figure 4. Frequency drop in area one. Figure 4. Frequency drop in area one. Figure 4. Frequency drop in area one. Figure 4. Frequency drop in area one. Figure 4. Frequency drop in area one. Figure 4. Frequency drop in area one. Figure 5. Frequency drop in area two. Figure 5. Frequency drop in area two. Figure 5. Frequency drop in area two. Eng. Proc. 2022, 19, 1 Eng. Proc. 2022, 19, 1 6 of 7 6 of 7 Figure 6. Tie-line power deviation. Table 2. The optimum values of the I plus fuzzy C FOPI-FOPD. Controller Parameters Area one controller K1 K2 K3 KP11 KI1 λ1 KP12 KD1 µ1 0.284 0.7304 −1.9193 0.5200 0.4816 0.6971 −1.6179 0.7885 0.1499 Area two controller K4 K5 K6 KP21 KI2 λ2 KP22 KD2 µ2 −1.28 −0.1798 −2 −2 0.0409 0.1631 −1.6188 0.0142 0.2625 Table 3. The dynamic response of the system with the proposed controller. Controller Frequency in Area One Frequency in Area Two Tie-line Power Deviation ITAE Ushin pu Oshin pu Ts in s Ushin pu Oshin pu Ts in s Ushin pu Oshin pu Ts in s I + F C FOPI- FOPD −0.0835 0 5.697 −0.0751 0 5.9810 −0.0015 0.0052 3.3810 0.267 PI—FA −0.281 0.106 12.283 −0.210 0.0908 13.1132 −0.0382 0.0427 9.9657 4.192 5. Conclusions Concisely, this paper developed and implemented a virgin fuzzy control structure named I plus fuzzy cascade FOPI-FOPD employed as an LFC system in a dual area power system incorporating a PV unit in area one. 5. Conclusions Concisely t 5. Conclusions Concisely, this paper developed and implemented a virgin fuzzy control structure named I plus fuzzy cascade FOPI-FOPD employed as an LFC system in a dual area power system incorporating a PV unit in area one. This controller significantly damped the fre- quency deviation in both areas and also outperformed the previously proposed PI con- troller-based FA. Concisely, this paper developed and implemented a virgin fuzzy control structure named I plus fuzzy cascade FOPI-FOPD employed as an LFC system in a dual area power system incorporating a PV unit in area one. This controller signiﬁcantly damped the frequency deviation in both areas and also outperformed the previously proposed PI controller-based FA. Author Contributions: Both authors have contributed the same in producing this work. All authors have read and agreed to the published version of the manuscript. F di Thi h i d l f di Author Contributions: Both authors have contributed the same in producing this work. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. I titutio al Re ie Boa d State e t Not a li able Funding: This research received no external funding. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. I f d l bl Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. C fli t f I t t Th th d l Informed Consent Statement: Not applicable. Conflicts of Interest: The authors declare no conflict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 7 of 7 Eng. Proc. 2022, 19, 1 References 1. Tungadio, D.H.; Sun, Y. Load frequency controllers considering renewable energy integration in power system. Energy Rep. 2019, 5, 436–453. [CrossRef] , [ ] 2. Shouran, M.; Anayi, F.; Packianather, M. A State-of-the-Art Review on LFC Strategies in Conventional and Modern Power Systems. In Proceedings of the 2021 International Conference on Advance Computing and Innovative Technologies in Engineering (ICACITE), Greater Noida, India, 4–5 March 2021; pp. 268–277. [CrossRef] ( ) pp 3. Abd-Elazim, S.M.; Ali, E.S. Load frequency controller design of a two-area system composing of PV grid and thermal generator via ﬁreﬂy algorithm. Neural Comput. Appl. 2018, 30, 607–616. [CrossRef] y g p pp 4. Yousri, D.; Babu, T.S.; Fathy, A. Recent methodology based Harris Hawks optimizer for designing load frequency control incorporated in multi-interconnected renewable energy plants. Sustain. Energy Grids Netw. 2020, 22, 100352. [CrossRef] 5. Fathy, A.; Alharbi, A.G. Recent Approach Based Movable Damped Wave Algorithm for Designing Fr Frequency Control Installed in Multi-Interconnected Plants With Renewable Energy. IEEE Access 2021, 9 6. Fathy, A.; Yousri, D.; Rezk, H.; Thanikanti, S.B.; Hasanien, H.M. A Robust Fractional-Order PID Controller Based Load Frequency Control Using Modiﬁed Hunger Games Search Optimizer. Energies 2022, 15, 361. [CrossRef] g g p g 7. Mokhtar, M.; Marei, M.I.; Sameh, M.A.; Attia, M.A. An Adaptive Load Frequency Control for Power Systems with Renewable Energy Sources. Energies 2022, 15, 573. [CrossRef] 8. Xu, Y.; Ma, Y.; Han, Y.; Hu, T.; Lun, X.; Mi, Y. Load Frequency Control for Renewable Energy Power System Based on Sliding Mode control with Hybrid Energy Storage. In Proceedings of the 2020 Chinese Control And Decision Conference (CCDC), Hefei, China, 22–24 August 2020; pp. 1340–1345. [CrossRef] g pp 9. Mi, Y.; Fu, Y.; Li, D.; Wang, C.; Loh, P.C.; Wang, P. The sliding mode load frequency control for hybrid power system based on disturbance observer. Int. J. Electr. Power Energy Syst. 2016, 74, 446–452. [CrossRef] 10. Pandey, S.K.; Mohanty, S.R.; Kishor, N.; Catalão, J.P.S. Frequency regulation in hybrid power systems using particle swarm optimization and linear matrix inequalities based robust controller design. Int. J. Electr. Power Energy Syst. 2014, 63, 887–900. [CrossRef] 11. Loka, R.; Parimi, A.M.; Srinivas, S. Model Predictive Control Design for Fast Frequency Regulation in Hybrid Power System. In Proceedings of the 2022 2nd International Conference on Power Electronics & IoT Applications in Renewable Energy and its Control (PARC), Mathura, India, 21–22 January 2022; pp. 1–5. References [CrossRef] g Control (PARC), Mathura, India, 21–22 January 2022; pp. 1–5. [CrossRef] y pp F.; Packianather, M.; Habil, M. Load Frequency Control Based on the Bees Algorithm for the Great Britain gns 2021, 5, 50. [CrossRef] 12. Shouran, M.; Anayi, F.; Packianather, M.; Habil, M. Load Frequency Control Based on the Bees Alg Power System. Designs 2021, 5, 50. [CrossRef] 13. Shouran, M.; Anayi, F.; Packianather, M.; Habil, M. Different Fuzzy Control Conﬁgurations Tuned by the Bees Algorithm for LFC of Two-Area Power System. Energies 2022, 15, 657. [CrossRef] y 14. Shouran, M.; Alsseid, A. Particle Swarm Optimization Algorithm-Tuned Fuzzy Cascade Fractional Order PI-Fractional Order PD for Frequency Regulation of Dual-Area Power System. Processes 2022, 10, 477. [CrossRef] q y g y 15. Shafei, M.A.R.; Ibrahim, D.K.; Bahaa, M. Application of PSO tuned fuzzy logic controller for LFC of two-area power system with redox ﬂow battery and PV solar park. Ain Shams Eng. J. 2022, 13, 101710. [CrossRef] 16. Annamraju, A.; Nandiraju, S. Robust frequency control in a renewable penetrated power system: An adaptive fractional order-fuzzy approach. Prot. Control Mod. Power Syst. 2019, 4, 16. [CrossRef] y pp y 17. Zou, F.; Chen, D.; Xu, Q. A survey of teaching—Learning-based optimization. Neurocomputing 2019, 33
https://openalex.org/W2766858801	https://www.epj-conferences.org/10.1051/epjconf/201715807003/pdf	English	null	U(1) gauged Q-balls and their properties	EPJ web of conferences	2,017	cc-by	4,824	U(1) gauged Q-balls and their properties Emin Nugaev1,⋆, Alexander Panin1,⋆⋆, and Mikhail Smolyakov2,1,⋆⋆⋆ 1Institute for Nuclear Research of the Russian Academy of Sciences, 60th October Anniversary prospect 7a, Moscow 117312, Russia 2Skobeltsyn Institute of Nuclear Physics, Lomonosov Moscow State University, Moscow 119991, Russia Emin Nugaev1,⋆, Alexander Panin1,⋆⋆, and Mikhail Smolyakov2,1,⋆⋆⋆ 1Institute for Nuclear Research of the Russian Academy of Sciences, 60th October Anniversary prospect 7a, Moscow 117312, Russia 2Skobeltsyn Institute of Nuclear Physics, Lomonosov Moscow State University, Moscow 119991, Russia Emin Nugaev1,⋆, Alexander Panin1,⋆⋆, and Mikhail Smolyakov2,1,⋆⋆⋆ 1Institute for Nuclear Research of the Russian Academy of Sciences, 60th October Anniversary prospect 7a, Moscow 117312, Russia 2Skobeltsyn Institute of Nuclear Physics, Lomonosov Moscow State University, Moscow 119991, Russia Abstract. In the present manuscript, we brieﬂy discuss the diﬀerences between ordinary Q-balls and U(1) gauged Q-balls. We focus on the diﬀerences in the existence of Q-ball solutions for appropriate values of the model parameters, as well as on the diﬀerences in classical stability of such Q-balls. EPJ Web of Conferences 158, 07003 (2017) QFTHEP 2017 EPJ Web of Conferences 158, 07003 (2017) QFTHEP 2017 DOI: 10.1051/epjconf/201715807003 ⋆e-mail: emin@ms2.inr.ac.ru 1 Ordinary Q-balls For Q-balls, this piecewise scalar ﬁeld potential was introduced in a more general form in [1]. The corresponding solution for the scalar ﬁeld takes the 1 2 ΦΦ v 0 1 VΦΦ M2 v2 Figure 1. The scalar ﬁeld potential (7). form [1] f = vRe− √ M2−ω2(r−R) r , for f < v (r > R), (8) f = vR sin(ωR) sin(ωr) r , for f > v (r < R). (9) with R = 1 ω arctan − ω √ M2 −ω2 + π , 0 < ω < M. (10) The solution for ω M = 0.8 is presented in Figure 2. The Q(ω) and E(Q) dependencies for these Q-balls are presented in Figure 3 (see [3] for a more detailed discussion of Q-balls in the model with the scalar ﬁeld potential (7)). 2 U(1) gauged Q-balls 1 2 ΦΦ v 0 1 VΦΦ M2 v2 Figure 1. The scalar ﬁeld potential (7). Figure 1. The scalar ﬁeld potential (7). form [1] f = vRe− √ M2−ω2(r−R) r , for f < v (r > R), (8) f = vR sin(ωR) sin(ωr) r , for f > v (r < R). (9) with −ω2(r−R) r , for f < v (r > R), (8) sin(ωr) r , for f > v (r < R). (9) (8) (9) with R = 1 ω arctan − ω √ M2 −ω2 + π , 0 < ω < M. (10) (10) The solution for ω M = 0.8 is presented in Figure 2. The Q(ω) and E(Q) dependencies for these Q-balls are presented in Figure 3 (see [3] for a more detailed discussion of Q-balls in the model with the scalar ﬁeld potential (7)). The solution for ω M = 0.8 is presented in Figure 2. The Q(ω) and E(Q) dependencies for these Q-balls are presented in Figure 3 (see [3] for a more detailed discussion of Q-balls in the model with the scalar ﬁeld potential (7)). 1 Ordinary Q-balls Q-balls are localized solutions in the theory of a complex scalar ﬁeld, described by the action S = d4x ∂µφ∗∂µφ −V(φ∗φ) , (1) (1) such that such that φ(t, x) = eiωt f(r), f(r)\|r→∞→0, ∂r f(r)\|r=0 = 0, r = √ x2. (2) φ(t, x) = eiωt f(r), f(r)\|r→∞→0, ∂r f(r)\|r=0 = 0, r = √ x2. (2) (2) Without loss of generality one can take f(r) to be real and such that f(r) > 0. Initially, such solutions were proposed in [1] and became popular after the well-known paper [2]. The Q-ball charge is deﬁned as Q = −i d3x φ∗˙φ −˙φ∗φ = 8πω ∞ 0 f 2 r2dr, (3) (3) whereas the Q-ball energy is deﬁned as whereas the Q-ball energy is deﬁned as whereas the Q-ball energy is deﬁned as E = d3x ˙φ∗˙φ + ∂iφ∗∂iφ + V(φ∗φ) . (4) (4) It is easy to show that the relation It is easy to show that the relation dE dQ = ω (5) It is easy to show that the relation dE dQ = ω (5) dE dQ = ω (5) (5) ⋆e-mail: emin@ms2.inr.ac.ru ⋆⋆ ⋆⋆e-mail: panin@ms2.inr.ac.ru ⋆⋆⋆e-mail: smolyakov@theory.sinp.msu.ru © 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/). EPJ Web of Conferences 158, 07003 (2017) EPJ Web of Conferences 158, 07003 (2017) QFTHEP 2017 DOI: 10.1051/epjconf/201715807003 holds for Q-balls. Since there exists the symmetry holds for Q-balls. Since there exists the symmetry holds for Q-balls. Since there exists the symmetry ince there exists the symmetry ω →−ω ⇒ Q →−Q, E →E, (6) (6) without loss of generality one can examine only the case ω ≥0. A i l l f th Q b ll id d l ith th l ﬁld t ti l without loss of generality one can examine only the case ω ≥0. without loss of generality one can examine only the case ω ≥0. A i l l f th Q b ll id d l without loss of generality one can examine only the case ω ≥0. As a simple example of the Q-ball, we can consider a model with the scalar ﬁeld potential V(φ∗φ) = M2φ∗φ θ 1 −φ∗φ v2 + M2v2θ φ∗φ v2 −1 , (7) (7) where θ is the Heaviside step function (see Figure 1). 2 U(1) gauged Q-balls Now we turn to U(1) gauged Q-balls, which were initially proposed in [4], see also [5]. To describe such Q-balls, let us consider the action S = d4x (∂µφ∗−ieAµφ∗)(∂µφ + ieAµφ) −V(φ∗φ) −1 4FµνFµν . (11) (11) 2 2 EPJ Web of Conferences 158, 07003 (2017) DOI: 10.1051/epjconf/201715807003 QFTHEP 2017 EPJ Web of Conferences 158, 07003 (2017) QFTHEP 2017 DOI: 10.1051/epjconf/201715807003 Ω M0.8 2 4 6 8 10 Mr 0.5 1.0 1.5 2.0 2.5 f v Figure 2. The scalar ﬁeld proﬁle for ω M = 0.8. The vertical line indicates the point r = R where f (r) = v. Figure 2. The scalar ﬁeld proﬁle for ω M = 0.8. The vertical line indicates the point r = R where f (r) = v. 400 600 800 1000 QM2 v2 400 600 800 1000 EM v2 0.2 0.4 0.6 0.8 1.0 Ω M 100 104 106 108 1010 QM2 v2 0.2 0.4 0.6 0.8 1.0 Ω M 100 104 106 108 1010 QM2 v2 400 600 800 1000 QM2 v2 400 600 800 1000 EM v2 Figure 3. The Q(ω) and E(Q) dependencies for the Q-ball deﬁned by (8)–(10) (see [3] for details). Figure 3. The Q(ω) and E(Q) dependencies for the Q-ball deﬁned by (8)–(10) (see [3] for details). The U(1) gauged Q-ball is a localized solution to the equations of motion for the ﬁelds, following from action (11), of the form The U(1) gauged Q-ball is a localized solution to the equations of motion for the ﬁelds, following from action (11), of the form φ(t, x) = eiωt f(r), f(r)\|r→∞→0, ∂r f(r)\|r=0 = 0, (12) A0(t, x) = A0(r), A0(r)\|r→∞→0, ∂rA0(r)\|r=0 = 0, (13) Ai(t, x) ≡ 0. (14) (12) (13) (14) (14) Again, without loss of generality we take f(r) > 0. The charge of U(1) gauged Q-ball is deﬁned as Again, without loss of generality we take f(r) > 0. The charge of U(1) gauged Q-ball is deﬁned as Q = 8π (ω + eA0)f 2r2dr, (15) (15) whereas its energy is deﬁned as whereas its energy is deﬁned as E = 4π ∞ 0 (ω + eA0)2 f 2 + ∂r f∂r f + V(f) + 1 2∂rA0∂rA0 r2dr. 2 U(1) gauged Q-balls (16) (16) It is possible to show [6] that the relation dE It is possible to show [6] that the relation dE dQ = ω (17) It is possible to show [6] that the relation It is possible to show [6] that the relation dE dQ = ω (17) dE dQ = ω dE dQ = ω (17) 3 EPJ Web of Conferences 158, 07003 (2017) QFTHEP 2017 DOI: 10.1051/epjconf/201715807003 holds for U(1) gauged Q-balls too. (1) d Q b ll h f ll i i holds for U(1) gauged Q-balls too. ( ) g g Q U(1) gauged Q-balls possess the following properties: • ω →−ω ⇒ A0 →−A0. • The sign of ω + eA0 coincides with the sign of ω [5]. • The sign of ω + eA0 coincides with the sign of ω [5]. • The sign of ω + eA0 coincides with the sign of ω [5]. • The sign of ω + eA0 coincides with the sign of ω [5]. • If A0 ≡0, then ω = 0 [4]. hen ω = 0 [4]. Thus, there exists the symmetry ω →−ω ⇒ Q →−Q, E →E, (18) (18) and without loss of generality one can examine only the case ω ≥0. and without loss of generality one can examine only the case ω ≥0. and without loss of generality one can examine only the case ω ≥0 After this brief introduction, we are ready to turn to examining the diﬀerences between ordinary Q-balls and U(1) gauged Q-balls. 3 Behavior of Q-balls at r →∞ ﬁeld potential V(φ∗φ) such that Let us consider the scalar ﬁeld potential V(φ∗φ) such that Let us consider the scalar ﬁeld potential V(φ∗φ) such that Let us consider the scalar ﬁeld potential V(φ∗φ) such that V(φ∗φ) →M2φ∗φ for φ∗φ →0. (19) (19) For f(r) →0, the equation of motion for ordinary Q-balls reduces to (ω2 −M2)f + 1 r d2 dr2 (r f) ≈0, (20) (20) leading to f(r) ∼e− √ M2−ω2r r . It is clear that there are no Q-balls with ω = M, because Q →∞(as well as E →∞) for ω →M. leading to f(r) ∼e− √ M2−ω2r r . It is clear that there are no Q-balls with ω = M, because Q →∞(as well as E →∞) for ω →M. In the case of U(1) gauged Q-balls the situation is completely diﬀerent. Indeed, suppose that there exists a U(1) gauged Q-ball with the charge Q. Then, for f(r) →0 the equation of motion for the scalar ﬁeld reduces to 2 2 (ω2 −M2)f −2 ωe2Q 4πr f + 1 r d2 dr2 (r f) ≈0. (21) (21) Now we have two cases: • For ω < M f(r) ∼e− √ M2−ω2 r r 1+ ωe2Q 4π √ M2−ω2 for r →∞. (22) (22) • For ω = M f(r) = C K1 2Me2Q π r √r , (23) (23) √ where C is a constant and K1(b, z) is the modiﬁed Bessel function of the second kind, leading to where C is a constant and K1(b, z) is the modiﬁed Bessel function of the second kind, leading to f(r) ∼e− 2Me2Q π r r 3 4 for r →∞. (24) (24) We see that in the gauged case there may exist a solution possessing the ﬁnite charge and energy even for ω = M. This conclusion is supported by the results of numerical simulations (see [7] for details) for the scalar ﬁeld potential (7) (Figures 4 and 5) and for the scalar ﬁeld potential V(f) = M2 f 2 −λf 4 (Figures 6 and 7). We see that in the gauged case there may exist a solution possessing the ﬁnite charge and energy even for ω = M. 3 Behavior of Q-balls at r →∞ This conclusion is supported by the results of numerical simulations (see [7] for details) for the scalar ﬁeld potential (7) (Figures 4 and 5) and for the scalar ﬁeld potential V(f) = M2 f 2 −λf 4 (Figures 6 and 7). 4 DOI: 10.1051/epjconf/201715807003 EPJ Web of Conferences 158, 07003 (2017) QFTHEP 2017 EPJ Web of Conferences 158, 07003 (2017) QFTHEP 2017 102 104 106 108 0 0.2 0.4 0.6 0.8 1 Q~ ω/M √α2  = 0.005 102 104 106 108 0 0.2 0.4 0.6 0.8 1 Q~ ω/M √α2  = 0.02 102 104 106 108 0 0.2 0.4 0.6 0.8 1 Q~ ω/M √α2  = 0.07 102 104 106 108 0 0.2 0.4 0.6 0.8 1 Q~ ω/M √α2  = 0.1 Figure 4. ˜Q = M2 v2 Q for diﬀerent values of the parameter α2 = e2v2 M2 (thick lines). The thin lines stand for the nongauged case. The ﬁgure is taken from [7]. 102 104 106 108 0 0.2 0.4 0.6 0.8 1 Q~ ω/M √α2  = 0.02 102 104 106 108 0 0.2 0.4 0.6 0.8 1 Q~ ω/M √α2  = 0.1 Figure 4. ˜Q = M2 v2 Q for diﬀerent values of the parameter α2 = e2v2 M2 (thick lines). The thin lines stand for the nongauged case. The ﬁgure is taken from [7]. 0 1 2 3 4 5 6 7 8 0 10 20 30 40 50 F R ω/M = 1 ω/M ≈ 0.96 ω/M ≈ 0.46 ω/M = 1 Figure 5. Proﬁles of the scalar ﬁeld for diﬀerent values of ω M . Here √α2 = 0.02, R = Mr, F(R) = 1 v f (r). The ﬁgure is taken from [7]. 0 1 2 3 4 5 6 7 8 0 10 20 30 40 50 F R ω/M = 1 ω/M ≈ 0.96 ω/M ≈ 0.46 ω/M = 1 R Figure 5. Proﬁles of the scalar ﬁeld for diﬀerent values of ω M . Here √α2 = 0.02, R = Mr, F(R) = 1 v f (r). The ﬁgure is taken from [7]. where ∆= ∂i∂i, has only one negative eigenvalue. The proof of the classical stability criterion based on the use of the energy functional of the system can be found in [8], the proof based on the use of the linearized equations of motion along the lines of the Vakhitov-Kolokolov method proposed in [9] can be found in [10]. It should be noted that we are not aware of any explicit example of ordinary Q-ball, for which the operator L has more than one negative eigenvalues and which is classically unstable for dQ dω < 0. dω In order to examine the gauged case, we consider the perturbations above the U(1) gauged Q-ball solution in the form [10] φ(t, x) = eiωt f(r) + eiωteγt u(x) + iv(x) , (27) A0(t, x) = A0(r) + eγta0(x), (28) Ai(t, x) = eγtai(x), (29) φ(t, x) = eiωt f(r) + eiωteγt u(x) + iv(x) A0(t, x) = A0(r) + eγta0(x), Ai(t, x) = eγtai(x), (27) (28) (29) (28) (29) Ai(t, x) = eγtai(x), (29) where γ is real, which passes through the linearized equations of motion. It is possible to show (see the detailed derivation in [10]) that U(1) gauged Q-balls are classically stable if the following conditions hold: where γ is real, which passes through the linearized equations of motion. It is possible to show (see the detailed derivation in [10]) that U(1) gauged Q-balls are classically stable if the following conditions hold: 1. dQ dω < 0 (30) dQ dω < 0 (30) dQ dω < 0 (30) (30) 2. The corresponding operator ˆL has only one negative eigenvalue. Here the operator ˆL takes the form rresponding operator ˆL has only one negative eigenvalue. 2. The corresponding operator ˆL has only one negative eigenvalue. Here the operator ˆL takes the form 2. The corresponding operator ˆL has only one negative eigenvalue. Here the operator ˆL takes the form ˆL =  −∆+ W(r) −2e(ω + eA0)f 0 −2e(ω + eA0)f ∆ 2 −e2 f 2 0 0 0 ∆ 2 −e2 f 2 −γ2 2 I3×3  , (31) (31) where I3×3 is the 3 × 3 unit matrix and where I3×3 is the 3 × 3 unit matrix and W(r) = dV d(φ∗φ) φ∗φ= f 2(r) +2 d2V d(φ∗φ)2 φ∗φ= f 2(r) f 2(r) −(ω + eA0)2. 3 Behavior of Q-balls at r →∞ 5 5 EPJ Web of Conferences 158, 07003 (2017) QFTHEP 2017 DOI: 10.1051/epjconf/201715807003 0 50 100 150 200 250 300 0 0.2 0.4 0.6 0.8 1 Q~ ω/M √α1  = 0.05 0 50 100 150 200 250 300 0 0.2 0.4 0.6 0.8 1 Q~ ω/M √α1  = 0.1 0 50 100 150 200 250 300 0 0.2 0.4 0.6 0.8 1 Q~ ω/M √α1  = 0.2 0 50 100 150 200 250 300 0 0.2 0.4 0.6 0.8 1 Q~ ω/M √α1  = 0.5 Figure 6. ˜Q = λQ for diﬀerent values of the parameter α1 = e2 λ (thick lines). The thin lines stand for the nongauged case. The ﬁgure is taken from [7]. Figure 6. ˜Q = λQ for diﬀerent values of the parameter α1 = e2 λ (thick lines). The thin lines stand for the nongauged case. The ﬁgure is taken from [7]. 0 0.5 1 1.5 2 2.5 3 3.5 0 2 4 6 8 10 12 14 F R ω/M = 0 ω/M = 0.8 ω/M = 0.95 ω/M = 1 Figure 7. Proﬁles of the scalar ﬁeld for diﬀerent values of ω M . Here √α1 = 0.05, R = Mr, F(R) = √ λ M f (r). The ﬁgure is taken from [7]. 0 0.5 1 1.5 2 2.5 3 3.5 0 2 4 6 8 10 12 14 F R ω/M = 0 ω/M = 0.8 ω/M = 0.95 ω/M = 1 Figure 7. Proﬁles of the scalar ﬁeld for diﬀerent values of ω M . Here √α1 = 0.05, R = Mr, F(R) = √ λ M f (r). The ﬁgure is taken from [7]. 6 EPJ Web of Conferences 158, 07003 (2017) QFTHEP 2017 DOI: 10.1051/epjconf/201715807003 4 Classical stability of U(1) gauged Q-balls: theoretical considerations Now we turn to examining another diﬀerence between ordinary and U(1) gauged Q-balls — the clas sical stability, i.e., stability of the Q-ball solution with respect to small perturbations of the ﬁelds. It is known that ordinary Q-balls are stable with respect to small perturbations if the following conditions hold: 1. dQ dω < 0 (25) 2. The operator ˆL = −∆+ dV d(φ∗φ) φ∗φ= f 2(r) +2 d2V d(φ∗φ)2 φ∗φ= f 2(r) f 2(r) −ω2, (26) where ∆= ∂i∂i, has only one negative eigenvalue. dQ dω < 0 (25) (25) 2. The operator 2. The operator ˆL = −∆+ dV d(φ∗φ) φ∗φ= f 2(r) +2 d2V d(φ∗φ)2 φ∗φ= f 2(r) f 2(r) −ω2, (26) (26) where ∆= ∂i∂i, has only one negative eigenvalue. where ∆= ∂i∂i, has only one negative eigenvalue. where ∆= ∂i∂i, has only one negative eigenvalue. On the other hand, it is possible, due to the repulsive nature of the electromagnetic ﬁeld, that nonspherically symmetric perturbations destroy any U(1) gauged Q-ball. It is necessary to note that the question remains open as to whether it is possible to have only one negative eigenvalue of the operator ˆL even in the case of spherically symmetric perturbations, in which the operator ˆL takes a simpler form (see the detailed discussion in [10]). where ∆= ∂i∂i, has only one negative eigenvalue. (32) (32) One can see that there always exist negative eigenvalues of the operator ˆL, corresponding to the perturbations ai (because of the elements ∆ 2 −e2 f 2 −γ2 2 I3×3). Thus, the corresponding negative part 7 EPJ Web of Conferences 158, 07003 (2017) QFTHEP 2017 DOI: 10.1051/epjconf/201715807003 of the spectrum is deﬁned only by the spectrum of the operator ∆ 2 −e2 f 2 −γ2 2 . Moreover, due to the fact that f(r) rapidly tends to zero at r →∞, one expects that the operator ∆ 2 −e2 f 2 −γ2 2 has of the spectrum is deﬁned only by the spectrum of the operator ∆ 2 −e2 f 2 −γ2 2 . Moreover, due to the fact that f(r) rapidly tends to zero at r →∞, one expects that the operator ∆ 2 −e2 f 2 −γ2 2 has a negative continuous spectrum starting at −γ2 2 . Thus, fulﬁllment of the condition dQ dω < 0 does not provide any information about possible stability regions of the U(1) gauged Q-ball, at least within the framework of the generalized Vakhitov-Kolokolov approach used in [10]. At the moment it is not clear what is the origin of the problem described above. On the one hand, it is possible that the existence of the negative spectrum is just a technical artifact of the approach used in [10]. On the other hand, it is possible, due to the repulsive nature of the electromagnetic ﬁeld, that nonspherically symmetric perturbations destroy any U(1) gauged Q-ball. It is necessary to note that the question remains open as to whether it is possible to have only one negative eigenvalue of the operator ˆL even in the case of spherically symmetric perturbations, in which the operator ˆL takes a simpler form (see the detailed discussion in [10]). a negative continuous spectrum starting at −γ2 2 . Thus, fulﬁllment of the condition dQ dω < 0 does not provide any information about possible stability regions of the U(1) gauged Q-ball, at least within the framework of the generalized Vakhitov-Kolokolov approach used in [10]. At the moment it is not clear what is the origin of the problem described above. On the one hand, it is possible that the existence of the negative spectrum is just a technical artifact of the approach used in [10]. The second scalar ﬁeld potential has the form ar ﬁeld potential has the form V(φ∗φ) = −µ2φ∗φ ln(β2φ∗φ). (33) (33) In the nongauged case this scalar ﬁeld potential was proposed in [11]. An interesting feature of this model is that the linearized equations of motion for perturbations can be solved exactly in the nongauged case [12]. The dQ dω < 0 stability criterion is valid for ordinary Q-balls in this model — Q-balls with dQ dω < 0 are classically stable (and Q-balls with dQ dω > 0 are classically unstable). dω th dQ dω < 0 are classically stable (and Q-balls with dQ dω > 0 are classically unstable). dω dω The results of the numerical simulation in the gauged case can be found in dω dω The results of the numerical simulation in the gauged case can be found in Figure 9. One can easily Μ2Β2Q 0 1 2 3 4 0 20 40 60 80 100 120 ΩΜ Μ2Β2Q 0 1 2 3 4 0 20 40 60 80 100 120 ΩΜ 25 50 75 100 125 0 1 2 3 4 µ2β2Q ω/µ 80 90 100 3.4 3.5 3.6 3.7 stable unstable Figure 9. Ordinary (left plot) and U(1) gauged Q-balls for e/βµ = 1.1 (right plot) for the logarithmic scalar ﬁeld potential. The solid lines stand for the classically stable Q-balls. The short-dashed lines stand for the classically unstable Q-balls. The right plot is taken from [10]. Μ2Β2Q 0 1 2 3 4 0 20 40 60 80 100 120 ΩΜ 25 50 75 100 125 0 1 2 3 4 µ2β2Q ω/µ 80 90 100 3.4 3.5 3.6 3.7 stable unstable Figure 9. Ordinary (left plot) and U(1) gauged Q-balls for e/βµ = 1.1 (right plot) for the logarithmic scalar ﬁeld potential. The solid lines stand for the classically stable Q-balls. The short-dashed lines stand for the classically unstable Q-balls. The right plot is taken from [10]. see from Figure 9 that there are classically unstable U(1) gauged Q-balls with dQ dω < 0 (for example, the Q-ball marked by the dot in Figure 9). This diﬀers considerably from the case of ordinary Q-balls. 5 Classical stability of U(1) gauged Q-balls: numerical simulations In order to examine the classical stability regions of U(1) gauged Q-balls, the numerical simulations in the spherically symmetric case were performed (see the details in [10]). Here we present the results for two diﬀerent scalar ﬁeld potentials. The ﬁrst one is the potential (7). We see from Figure 8 102 104 106 0.4 ω0 M 0.6 0.8 ωc M 1 QM 2/v2 ω/M ev/M = 0.02 nongauged stable unstable Figure 8. The stability and instability regions of Q-balls in the model with the scalar ﬁeld potential (7). The long- dashed line stands for the nongauged Q-balls. The solid line stands for the classically stable gauged Q-balls. The short-dashed line stands for the classically unstable gauged Q-balls. The ﬁgure is taken from [10]. Figure 8. The stability and instability regions of Q-balls in the model with the scalar ﬁeld potential (7). The long- dashed line stands for the nongauged Q-balls. The solid line stands for the classically stable gauged Q-balls. The short-dashed line stands for the classically unstable gauged Q-balls. The ﬁgure is taken from [10]. that the inequality dQ dω > 0 holds for classically unstable solutions, which formally is in agreement with the classical stability criterion. Meanwhile, the inequality dQ dω > 0 also holds for gauged Q- balls on the upper branch of the Q(ω) dependence, which appear to be stable at least with respect to spherically symmetric perturbations. Although formally this fact does not contradict the classical stability criterion (in the case of ordinary Q-balls the classical stability criterion does not guarantee that there exist unstable modes in the case dQ dω > 0, it just states that there are no classical instabilities if dQ dω < 0), this situation diﬀers from the case of ordinary Q-balls, for which usually there exist spherically symmetric unstable modes for Q-balls with dQ dω > 0. 8 8 DOI: 10.1051/epjconf/201715807003 EPJ Web of Conferences 158, 07003 (2017) QFTHEP 2017 The second scalar ﬁeld potential has the form The second scalar ﬁeld potential has the form 6 Conclusion In the present manuscript, we brieﬂy discussed the diﬀerences between ordinary and U(1) gauged Q-balls. It is shown that U(1) gauged Q-balls may exist for the values of the parameters (ω = M), for which ordinary Q-balls do not exist. It is also shown that the classical stability criterion for ordinary Q-balls cannot be applied to U(1) gauged Q-balls. An explicit example of classically unstable U(1) gauged Q-ball with dQ dω < 0 is presented. These results demonstrate that addition of the gauge ﬁeld changes drastically the properties of Q-balls. The work was supported by the Grant 16-12-10494 of the Russian Science Foundation. The second scalar ﬁeld potential has the form An example of time evolution of the perturbed U(1) gauged Q-ball is shown in Figure 10, where the 0.5 1 0 2.5 5 7.5 10 12.5 \|φ\|/µ µr µt = 0 5 10 15 0.5 1 0 2.5 5 7.5 10 12.5 \|φ\|/µ µr µt = 0 5 10 15 Figure 10. The scalar ﬁeld proﬁle of the classically unstable gauged Q-ball at diﬀerent moments of time. The initial solution (at µt = 0) is marked by the dot in the upper right corner of Figure 9. The ﬁgure is taken from [10]. Figure 10. The scalar ﬁeld proﬁle of the classically unstable gauged Q-ball at diﬀerent moments of time. The initial solution (at µt = 0) is marked by the dot in the upper right corner of Figure 9. The ﬁgure is taken from [10]. scalar ﬁeld proﬁle is presented at diﬀerent moments of time. 9 DOI: 10.1051/epjconf/201715807003 EPJ Web of Conferences 158, 07003 (2017) QFTHEP 2017 References [1] G. Rosen, J. Math. Phys. 9, 996 (1968) [1] G. Rosen, J. Math. Phys. 9, 996 (1968) [2] S. R. Coleman, Nucl. Phys. B 262, 263 (1985) [Erratum-ibid. B 269, 744 (1986)] [3] I E G l E Y N d M N S l k Ph R D 87 085043 (2013) [2] S. R. Coleman, Nucl. Phys. B 262, 263 (1985) [Erratum-ibid. B 269, 744 (1986)] [3] I. E. Gulamov, E. Y. Nugaev and M. N. Smolyakov, Phys. Rev. D 87, 085043 (2013 [3] I. E. Gulamov, E. Y. Nugaev and M. N. Smolyakov, Phys. Rev. D 87, 085043 (2013) [4] G. Rosen, J. Math. Phys. 9, 999 (1968) [3] I. E. Gulamov, E. Y. Nugaev and M. N. Smolyakov, Phys. Rev. D 87, 085043 (2013 [4] G. Rosen, J. Math. Phys. 9, 999 (1968) 5] K.-M. Lee, J. A. Stein-Schabes, R. Watkins and L. M. Widrow, Phys. Rev. D 39, 1665 [6] I. E. Gulamov, E. Y. Nugaev and M. N. Smolyakov, Phys. Rev. D 89, 085006 (2014) [7] I. E. Gulamov, E. Y. Nugaev, A. G. Panin and M. N. Smolyakov, Phys. Rev. D 92, 0 8] T. D. Lee and Y. Pang, Phys. Rept. 221, 251 (1992) [8] T. D. Lee and Y. Pang, Phys. Rept. 221, 251 (1992) [9] N. G. Vakhitov and A. A. Kolokolov, Radiophys. Quantum Electron. 16, 783 (1973 [10] A. G. Panin and M. N. Smolyakov, Phys. Rev. D 95, 065006 (2017) [11] G. Rosen, Phys. Rev. 183, 1186 (1969) [12] G.C. Marques and I.Ventura, Phys. Rev. D 14, 1056 (1976) 10
https://openalex.org/W2067360604	https://researchrepository.wvu.edu/cgi/viewcontent.cgi?article=3430&context=faculty_publications	English	null	Nobiletin suppresses cell viability through AKT Pathways in PC-3 and DU-145 prostate cancer cells	BMC pharmacology & toxicology	2,014	cc-by	7,633	Follow this and additional works at: https://researchrepository.wvu.edu/faculty_publications Follow this and additional works at: https://researchrepository.wvu.edu/faculty_publications Nobiletin suppresses cell viability through AKT Pathways in PC-3 Nobiletin suppresses cell viability through AKT Pathways in PC-3 and DU-145 prostate cancer cells and DU-145 prostate cancer cells See next page for additional authors This article is available at The Research Repository @ WVU: https://researchrepository.wvu.edu/faculty_publications/ 2482 Authors Authors Jianchu Chen, Ashley Creed, Allen Y. Chen, Haizhi Huang, Zhaoliang Li, Gary O. Rankin, Xingqian Ye, Guihua Xu, and Yi C. Chen Digital Commons Citation Digital Commons Citation Chen, Jianchu; Creed, Ashley; Chen, Allen Y.; Huang, Haizhi; Li, Zhaoliang; Rankin, Gary O.; Ye, Xingqian; Xu, Guihua; and Chen, Yi C., "Nobiletin suppresses cell viability through AKT Pathways in PC-3 and DU-145 prostate cancer cells" (2014). Faculty & Staff Scholarship. 2482. https://researchrepository.wvu.edu/faculty_publications/2482 This Article is brought to you for free and open access by The Research Repository @ WVU. It has been accepted for inclusion in Faculty & Staff Scholarship by an authorized administrator of The Research Repository @ WVU. For more information, please contact researchrepository@mail.wvu.edu. This article is available at The Research Repository @ WVU: https://researchrepository.wvu.edu/faculty_publications/ 2482 RESEARCH ARTICLE Open Access © 2014 Chen et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Nobiletin suppresses cell viability through AKT Pathways in PC-3 and DU-145 prostate cancer cells Jianchu Chen1,2, Ashley Creed2, Allen Y Chen3, Haizhi Huang1,2, Zhaoliang Li2, Gary O Rankin4, Xingqian Ye1, Guihua Xu1 and Yi Charlie Chen2* * Correspondence: chenyc@ab.edu 2College of Science, Technology and Mathematics, Alderson Broaddus University, Philippi, WV 26416, USA Full list of author information is available at the end of the article Open Access Keywords: Nobiletin, Prostate cancer, VEGF, NF-κB, HIF-1α, cMyc Keywords: Nobiletin, Prostate cancer, VEGF, NF-κB, HIF-1α, cMyc Abstract Background: Nobiletin is a non-toxic dietary flavonoid that possesses anti-cancer properties. Nobiletin has been reported to reduce the risk of prostate cancer, but the mechanism is not well understood. In this study, we investigated the effects of nobiletin in prostate cancer cell lines PC-3 and DU-145. Methods: Nobiletin was isolated from a polymethoxy flavonoid mixture using HPLC, cell viability was analyzed with MTS-based assays. Protein expression was examined by ELISA and western blotting. Gene expression was examined by luciferase assay. And the pathways were examined by manipulating genetic components with plasmid transfection. Results: Data showed that nobiletin decreased cell viability in both prostate cell lines, with a greater reduction in viability in PC-3 cells. HIF-1α expression and AKT phosphorylation were decreased in both cell lines. The VEGF expression was inhibited in PC-3 but not DU-145 cells. cMyc expression was decreased in DU-145 cells. Nobiletin down-regulated NF-κB (p50) expression in nuclei of DU145 cells but not whole cells. It also suppressed NF-κB expression in both whole cells and nuclei of PC-3 cells. Increasing HIF-1α levels reversed nobiletin’s inhibitory effects on VEGF expression, and up-regulating AKT levels reversed its inhibitory effects on HIF-1α expression. We speculate that AKT influences cell viability probably by its effect on NF-κB in both prostate cells. The effect of nobiletin on VEGF expression in PC-3 cell lines was through the AKT/HIF-1α pathway. Conclusion: Taken together, our results show that nobiletin suppresses cell viability through AKT pathways, with a more profound effect against the more metastatic PC-3 line. Due to this enhanced action against a more malignant cell type, nobiletin may be used to improve prostate cancer survival rates. Authors Authors Chen et al. BMC Pharmacology and Toxicology 2014, 15:59 http://www.biomedcentral.com/2050-6511/15/59 Cell proliferation assay levels of sex hormones, preventing oxidation or inflam- mation, diminishing angiogenesis or cell proliferation, or stimulating apoptosis [10]. There are more than 400 flavonoids found in our food supply; however, in this research we focused our attention on nobiletin [11]. Effects of nobiletin on prostate cancer cells (PC-3 and DU-145) viability were colorimetrically determined with a “Cell Titer 96 Aqueous One Solution Cell Proliferation Assay” kit from Promega (Madison, WI). Cells (5 × 103/ well) were seeded into 96-well plates and incubated for 16 h before being treated with 0 to 160 μg/ml nobiletin in triplicates for 24 h with DMSO as solvent control. After removing the medium, cells were washed with phosphate buffered saline (PBS), and then 100μL Aqueous One Reagent dilute solution (80 μL PBS +20 μL Aqueous One Reagent) was added to each well. Cells were incubated at 37°C for 1.5 h and measured for optical density (OD) values at 490 nm. Cell viability was expressed as a percent- age of control from three independent experiments. Nobiletin is an O-methylated flavonoid found in citrus peels with an empirical formula of C21H22O8 and molecu- lar weight of 402.39 [12]. An inverse relationship has been identified between nobiletin and cancer risk, which is likely due to nobiletin’s anticancer, antiviral, and anti- inflammatory activities [13,14]. More specifically, recent findings have identified nobiletin as a cell differentiation modulator. Cell differentiation is a crucial step in angio- genesis and therefore could affect tumor growth and metastasis which both depend on angiogenesis [15]. Research has also shown that a diet high in flavonoids reduced oxidative damage to deoxyribonucleic acid (DNA), blocking a significant step in the onset of some types of cancers [16]. These findings support the proposition that nobiletin is functionally unique and could be a possible chemopreventive agent in inflammation-associated tumori- genesis [17]. ELISA for VEGF d l Secreted vascular endothelial growth factor (VEGF) protein levels were analyzed by sandwich enzyme-linked immuno- sorbent assay (ELISA) with a Quantikine Human VEGF Immunoassay Kit from R&D Systems (Minneapolis, MN) targeting VEGF in cell culture supernates. Cells (104/well) were seeded into 96-well plates and incubated for 16 h before being treated with 0 to 160 μg/ml nobiletin in tripli- cates for 24 h with DMSO as solvent control. Culture supernates were collected for VEGF assay. VEGF levels were determined following the manufacturer's instructions. A total of 3 independent experiments, each in triplicates, were assayed, and the mean VEGF protein level from each duplicate was used for statistical analysis. Currently, metastatic prostate cancer is incurable and ultimately claims the life of patients [18,19]. An import- ant factor in the relative seriousness of prostate cancer is the invasiveness of the constituent tumor cells causing metastasis [19]. Nobiletin has been reported to reduce the risk of prostate cancer, but the mechanism is not well understood. Therefore we studied the effects of nobiletin in prostate cancer cell lines PC-3 and DU-145. The pathways that affect the viability and VEGF expression of these cell lines have also been investigated in this paper. DU-145 and PC-3 are prostate cancer cell lines with moderate and high metastatic potential, respectively [20]. In the present study, we isolated nobiletin from a polymethoxy flavonoid mixture. Then we investigated the effect of nobiletin on cell viability in prostate cancer cell lines PC-3 and DU-145 and also performed western blot- ting and ELISA to identify changes in protein expression. Moreover, we examined the VEGF changes through trans- fection of AKT and HIF-1α plasmids in luciferase assays. Western blot Prostate cancer cells (106) were seeded in 60-mm dishes and incubated for 16 h before treatment with nobiletin for 24 h. After washing with PBS, cells were harvested with 100 μL Mammalian Protein Extraction Reagent including 1 μL Halt Protease, 1 μL Phosphatase Inhibitor and 2 μL EDTA (Thermo Scientific, Rockford, IL). Cells were then frozen at -80°C for 30 min, melted, centrifuged at 12,000 g at 4°C for 10 min, and collected in aqueous phase for measurement. Nuclear protein was extracted by NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, Rockford, IL). Total protein levels were assayed with a BCA Protein Assay Kit (Pierce, Rockford, IL), and lysates were separated by 10% SDS-PAGE and blotted into nitrocellulose membrane. For immune detection, antibodies against HIF-1α, NF-κB (p50), PTEN, cMyc, GAPDH, p-AKT, total AKT (Santa Cruz Biotechnology, Santa Cruz, CA) and PCNA (Cell Signaling Technology, Boston, MA) were applied and signals visualized with x-ray film (Pierce Biotechnology, Rockford, IL). Protein bands were quantitated with NIH ImageJ software, nor- malized to corresponding GAPDH, PCNA or total AKT bands, and expressed as percentages of control. A total Background risk factors, such as advancing age, African American ethnicity, and a positive family history, are associated with the likelihood of developing prostate cancer [4]. However, research has also shown that prostate cancer is not solely due to genetic factors, but is also related to lifestyle, diet, and environmental factors [4-6]. It is now believed that 90-95% of all cancers are caused by life- style [7]. This observation has encouraged researchers to identify dietary components, such as flavonoids like nobiletin, which may have anticancer properties. Prostate cancer is the second most common cancer in the United States, as well as, the second leading cause of mortality among males in the western world [1]. Approximately 25% of all newly diagnosed cancers in American men are prostate cancer [2]. Studies relating lifestyle to the risk of prostate cancer have become more prevalent in recent years due the escalating num- ber of prostate cancer cases over the past decade [1]. Nevertheless, the etiology of prostate cancer is still uncertain because no specific carcinogen is known to cause this disease [3]. Research has found that certain It has been suggested that dietary intake of natural products rich in citrus flavonoids can play an important role in chemoprevention [8,9]. Flavonoids are phyto- chemicals found in fruits, vegetables, teas, and wines. Flavonoids display anti-carcinogenic characteristics in vitro and might be able to decrease cancer risk by changing * Correspondence: chenyc@ab.edu 2College of Science, Technology and Mathematics, Alderson Broaddus University, Philippi, WV 26416, USA Full list of author information is available at the end of the article Page 2 of 10 Chen et al. BMC Pharmacology and Toxicology 2014, 15:59 http://www.biomedcentral.com/2050-6511/15/59 Page 2 of 10 Isolation and identification of nobiletin Nobiletin was prepared from a polymethoxy flavonoid mixture, which was provided by Zhejiang Quzhou Tian- sheng Plant Extraction Co. Ltd. in China, containing about 60% nobiletin and tangeretin. The polymethoxy flavonoid mixture was dissolved in methanol-dimethyl sulfoxide (1:1) to a concentration of 50 mg/mL. Then it was chromate graphed with high-performance liquid chromatography (HPLC), eluted with methanol-H2O (70:30) in 8 mL/min at room temperature, separated into two fractions (Fractions I and II), collected individually, and evaporated. Fraction I and fraction II were obtained by HPLC (Figure 1(a)). Fraction I was identified as nobiletin by HPLC-MS (Figure 1(b)), UV-vis chromatography (Figure 1(c)) and comparing peak time with that of nobiletin sample from Sigma (Figure 1(d)) and previous reports. Its purity was above 98%. Cell culture and treatment PC-3 cells were cultured in F-12K medium (ATCC, Manassas, VA) supplemented with 10% US-qualified fetal bovine serum (FBS) (Invitrogen, Grand Island, NY). DU-145 cells were cultured in Eagle’s minimum essential medium (ATCC, Manassas, VA) supplemented with 10% US-qualified fetal bovine serum. All cells were cultured in a cell culture incubator with 5% CO2 at 37°C. Nobiletin was dissolved in dimethyl sulfoxide (DMSO) to make stock solutions of 100 mM and equal amount of DMSO was included in controls for every experiment. Page 3 of 10 Page 3 of 10 Chen et al. BMC Pharmacology and Toxicology 2014, 15:59 http://www.biomedcentral.com/2050-6511/15/59 Chen et al. BMC Pharmacology and Toxicology 2014, 15:59 http://www.biomedcentral.com/2050-6511/15/59 gradually inhibited to 46% by a 160 μM nobiletin treat- ment (p < 0.01). An overall inhibitory effect on cell via- bility was observed for both cell lines, although DU-145 (IC-50 = 137 μM nobiletin) cells appear more resistant than PC-3 cells (IC-50 = 117 μM nobiletin) to the inhi- biting effect of nobiletin. It is in agreement with the cell viability determined by WST-1 assay [21]. of three independent experiments were carried out for statistical analysis. of three independent experiments were carried out for statistical analysis. Nobiletin inhibits VEGF expression in prostate cancer cell line PC-3 The levels of VEGF protein in PC-3 cell culture super- nates were down-regulated to 70% at a concentration of 10 μM nobiletin (p < 0.01) and to 18% at a concentration of 160 μM nobiletin (p < 0.01) (Figure 3). However, the levels of VEGF protein in DU-145 cells ranged from 90-110% with no consistency with respect to nobiletin concentration. Our study revealed that VEGF expression was significantly (p < 0.01) reduced in PC-3 cancer cells by nobiletin treatment. Statistical analysis Results were expressed as mean ± standard error of mean (SEM). Statistical assessment was carried out with the program system of SPSS (Version 16.0 for Windows). The results were analyzed using one-way analysis of variance (ANOVA) and post hoc test (2-sided Dunnett's t) to test both overall differences and specific differences between each treatment and control. A p value of less than 0.05 was considered significant. Nobiletin inhibits HIF-1α (Hypoxia inducible factor) protein expression in prostate cancer cell lines HIF-1α protein levels in PC-3 cells showed intense and consistent down-regulation by nobiletin treatment (Figure 4). A 20 μM nobiletin treatment led to inhibition of HIF-1α protein to 70% (p < 0.01). Higher concentra- tions of nobiletin resulted in greater inhibition, with the levels of HIF-1α protein down to 48% by a 40 μM nobiletin treatment (p < 0.05) and 10% by a 80 μM nobiletin treatment (p < 0.01). HIF-1α protein levels in DU-145 cells showed consistent but gradual down- regulation by nobiletin treatment. DU-145 cells seem to be much more resistant to nobiletin treatment with down-regulation ranging from 94% at a concentration of 20 μM nobiletin to 79% at a concentration of 80 μM nobiletin (p < 0.05). HIF-1α expression in both cells lines (PC-3 and DU-145) was inhibited by nobiletin treatment, with a greater inhibition in PC-3 cells. Transient transfection and luciferase assay PC-3 prostate cancer cells were seeded in 96-well plate at 104 cells/well and incubated overnight. The cells were then transfected with 0.05 μg VEGF (Hif-1α) luciferase reporter, 0-0.25 μg HIF-1α (AKT) or SR-α plasmids by 0.6 μL jetPRIME reagent (VWR, West Chester, PA) for 4 hours, followed by 16 hour treatment with 0 or 40 μM nobiletin. The cells were harvested and analyzed for luciferase and total protein levels, and the levels of VEGF (HIF-1α) reporter were normalized by corresponding total protein levels. Data represent Means ± SE from three independent experiments. Nobiletin inhibits phosphorylation of AKT in prostate cancer cell lines P-AKT levels were down-regulated from 65% by a 20 μM nobiletin treatment to 56% by a 80 μM nobiletin treatment (p < 0.01) in PC-3 cells (Figure 5). In DU-145 cells, p-AKT levels were down-regulated to 67% by a 20 μM nobiletin treatment (p < 0.05), to 51% by a 80 μM nobiletin treatment (p < 0.01). The percentage of down-regulation in each cell line was similar at each treatment concentration and indi- cates that nobiletin could decrease AKT phosphorylation for both PC-3 and DU-145. However, the observed down- regulation seemed to subside at 40 μM nobiletin treatment, with not much difference resulting at 80 μM nobiletin treatment for both cell lines. Therefore, higher concentra- tions may not be any more beneficial. Nobiletin inhibits cell viability in prostate cancer cell lines Cell viability steadily decreased as nobiletin concentra- tion increased in both cell lines (Figure 2). Beginning at a concentration of 10 μM nobiletin, PC-3 cell viability consistently decreased from 95% to 40% at a concentra- tion of 160 μM nobiletin (p < 0.01). Similarly, DU-145 cell viability was also inhibited with each successive doubling of concentration. At a concentration of 10 μM nobiletin cell viability was 92% (p < 0.05), which was Chen et al. BMC Pharmacology and Toxicology 2014, 15:59 http://www.biomedcentral.com/2050-6511/15/59 Page 4 of 10 a b c AU 0.00 1.00 2.00 3.00 4.00 5.00 6.00 Minutes 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 nm 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 229.7 249.8 269.9 336.5 AU d e 1 Isolation and identification of nobiletin. (a) Preparation HPLC graph of nobiletin and tangeretin. (b) HPLC-MS graph of nobiletin. V-vis chromatography of nobiletin. (d) HPLC graph of nobiletin. a AU 0.00 1.00 2.00 3.00 4.00 5.00 6.00 Minutes 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 b b c 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 nm 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 229.7 249.8 269.9 336.5 AU d Figure 1 Isolation and identification of nobiletin. (a) Preparation HPLC graph of nobiletin and tangeretin. (b) HPLC-MS graph of nobiletin. (c) UV-vis chromatography of nobiletin. (d) HPLC graph of nobiletin. d Figure 1 Isolation and identification of nobiletin. (a) Preparation HPLC graph of nobiletin and tangeretin. Nobiletin inhibits phosphorylation of AKT in prostate cancer cell lines (b) HPLC-MS graph of nobiletin. (c) UV-vis chromatography of nobiletin. (d) HPLC graph of nobiletin. Chen et al. BMC Pharmacology and Toxicology 2014, 15:59 http://www.biomedcentral.com/2050-6511/15/59 Page 5 of 10 DU-145 PC-3 a b Figure 2 Effect of nobiletin on viability of PC-3 and DU-145 cells. (a) DU-145 cells, (b) PC-3 cells. Cells (5.0 × 103 /well) were seeded in 96-well plates, incubated for 16 h, and treated with nobiletin for 24 h. Cell viability was colorimetrically determined by a MTS-based method and expressed as percentages of control. Data represents mean ± SE from 3 independent experiments. P < 0.05 as compared to control. P < 0.01 as compared to control. DU-145 PC-3 a b Figure 2 Effect of nobiletin on viability of PC-3 and DU-145 cells. (a) DU-145 cells, (b) PC-3 cells. Cells (5.0 × 103 /well) were seeded in 96-well plates, incubated for 16 h, and treated with nobiletin for 24 h. Cell viability was colorimetrically determined by a MTS-based method and expressed as percentages of control. Data represents mean ± SE from 3 independent experiments. P < 0.05 as compared to control. *P < 0.01 as compared to control. b a Figure 2 Effect of nobiletin on viability of PC-3 and DU-145 cells. (a) DU-145 cells, (b) PC-3 cells. Cells (5.0 × 103 /well) were seeded in 96-well plates, incubated for 16 h, and treated with nobiletin for 24 h. Cell viability was colorimetrically determined by a MTS-based method and expressed as percentages of control. Data represents mean ± SE from 3 independent experiments. P < 0.05 as compared to control. *P < 0.01 as compared to control. Effect of nobiletin on PTEN expression Nobiletin inhibits cMyc expression in prostate cancer cell line DU-145 Nobiletin inhibits cMyc expression in prostate cancer cell line DU-145 Our study found that nobiletin had no significant effect on PTEN expression in DU-145 cells (Figure 8). PTEN was not expressed in PC-3 cells. In PC-3 cells, cMyc levels were slightly down regulated to 93% by a 40 μM nobiletin treatment and to 90% by a 80 μM nobiletin treatment (Figure 6). The results were very gradual, revealing that the PC-3 cells have a high level of resistance and there was no statistical significance among treatments. The cMyc levels in DU-145 cells were down-regulated to 79-84% by nobiletin treatment. Nobiletin inhibits VEGF expression through regulating AKT and HIF-1α gene in prostate cancer cell line PC-3 AKT and HIF-1α gene in prostate cancer cell line PC-3 We used PC-3 cancer cells with low levels of AKT and HIF-1α expression to test whether nobiletin affects VEGF expression. Our study found that transfected plasmid AKT and HIF-1α concentration-dependently reversed nobiletin’s inhibitory effects (Figure 9). Our findings sug- gest that nobiletin regulates VEGF expression through down-regulating AKT and HIF-1α in prostate cancer cells. Nobiletin inhibits NF-κB (p50) expression in nucleus of prostate cancer cell NF-κb (p50) expression was down-regulated in both whole cells and nuclei of PC-3 cells when treated with nobiletin. At the concentration of 80 μM nobiletin, its expression was inhibited by 28% (p < 0.01) and 37% (p < 0.05) respectively in whole cells and nuclei. In nuclei of DU145 cells, p50 expression was inhibited to 42% (p < 0.01) by a 40 μM nobi- letin treatment and 10% (p < 0.01) by a 80 μM nobiletin treatment (Figure 7). However, its expression in whole cells was up-regulated to 103-112% with higher concentrations of nobiletin resulting in greater promotion. Nobiletin inhibits phosphorylation of AKT in prostate cancer cell lines There- fore, nobiletin reduced cMyc expression in DU-145 cells, but showed no evidence of inhibition in PC-3 cells. Discussion Approximately 25% of all newly diagnosed cancers in American men are prostate cancer [2]. The risk of devel- oping prostate cancer is associated with advancing age, African American ethnicity, and a positive family history [4]. However, research has also shown that diet and other lifestyle factors may influence prostate cancer risk [4]. Studies relating lifestyle to the risk of prostate cancer Figure 3 Effect of nobiletin on VEGF expression in PC-3 and DU-145 cells. (a) DU-145. (b) PC-3. Cells (1.0 × 104/well) were seeded into 96-well plates , incubated for 16 h, and treated with nobiletin for 24 h. Vascular endothelial growth factor (VEGF) in cellculture supernate were analyzed with a Quantikine Human VEGF Immunoassay Kit from R&D Systems (Minneapolis, MN). Data represents mean ± SE from 3 independent experiments. P < 0.05 as compared to control. *P < 0.01 as compared to control. Figure 3 Effect of nobiletin on VEGF expression in PC-3 and DU-145 cells. (a) DU-145. (b) PC-3. Cells (1.0 × 104/well) were seeded into 96-well plates , incubated for 16 h, and treated with nobiletin for 24 h. Vascular endothelial growth factor (VEGF) in cellculture supernate were analyzed with a Quantikine Human VEGF Immunoassay Kit from R&D Systems (Minneapolis, MN). Data represents mean ± SE from 3 independent experiments. P < 0.05 as compared to control. *P < 0.01 as compared to control. Chen et al. BMC Pharmacology and Toxicology 2014, 15:59 http://www.biomedcentral.com/2050-6511/15/59 Page 6 of 10 Nobiletin(µM) 0 20 40 80 Nobiletin(µM) 0 20 40 80 PC-3 HIF-1 DU-145 HIF-1 PC-3 GAPDH DU-145 GAPDH Figure 4 Nobiletin's effects on HIF-1α expression in PC-3 and DU-145 cells. Nobiletin's effect on HIF-1α expression. PC-3 and DU-145 cells (1.0 × 106) were seeded in 60-mm dishes, incubated overnight, and treated with nobiletin for 24 hours. Cells were harvested and analyzed by SDS-PAGE and Western Blotting. HIF-1α protein levels were normalized by GAPDH protein levels. Data represents means ± SE from 3 independent experiments. P < 0.05 compared to control. *P < 0.01 compared to control. Nobiletin(µM) 0 20 40 80 DU-145 HIF-1 DU-145 GAPDH Figure 4 Nobiletin's effects on HIF-1α expression in PC-3 and DU-145 cells. Nobiletin's effect on HIF-1α expression. PC-3 and DU-145 cells (1.0 × 106) were seeded in 60-mm dishes, incubated overnight, and treated with nobiletin for 24 hours. Cells were harvested and analyzed by SDS-PAGE and Western Blotting. Discussion BMC Pharmacology and Toxicology 2014, 15:59 http://www.biomedcentral.com/2050-6511/15/59 Page 7 of 10 Page 7 of 10 Nobiletin(µM) 0 20 40 80 Nobiletin(µM) 0 20 40 80 PC-3 cMyc DU-145 cMyc PC-3 GAPDH DU-145 GAPDH Figure 6 Nobiletin’s effect on cMyc. PC-3 and DU-145 cells (1.0 × 106) were seeded in 60-mm dishes, incubated overnight, and treated with nobiletin for 24 hours. Cells were harvested and analyzed by SDS-PAGE and Western Blotting. cMyc levels were normalized by GAPDH protein levels and expressed as percentages of control. Data represents means ± SE from 3 independent experiments. P < 0.05 compared to control. *P < 0.01 compared to control. Nobiletin(µM) 0 20 40 80 DU-145 cMyc DU-145 GAPDH Figure 6 Nobiletin’s effect on cMyc. PC-3 and DU-145 cells (1.0 × 106) were seeded in 60-mm dishes, incubated overnight, and treated with nobiletin for 24 hours. Cells were harvested and analyzed by SDS-PAGE and Western Blotting. cMyc levels were normalized by GAPDH protein levels and expressed as percentages of control. Data represents means ± SE from 3 independent experiments. P < 0.05 compared to control. *P < 0.01 compared to control. Figure 6 Nobiletin’s effect on cMyc. PC-3 and DU-145 cells (1.0 × 106) were seeded in 60-mm dishes, incubated overnight, and treated with nobiletin for 24 hours. Cells were harvested and analyzed by SDS-PAGE and Western Blotting. cMyc levels were normalized by GAPDH protein levels and expressed as percentages of control. Data represents means ± SE from 3 independent experiments. P < 0.05 compared to control. *P < 0.01 compared to control. We tested the effectiveness of the preventive and/or treatment measures that nobiletin exhibits on PC-3 and DU-145 prostate cancer cells and showed that PC-3 and DU-145 cell viability was suppressed concentration- dependently by nobiletin treatment. Several pathways including VEGF, HIF-1α, AKT phosphorylation, cMyc, and NF-κB influence cell viability inhibition. In both cell lines, nobiletin inhibited phosphorylation of AKT, which is known to be the major signal for cell survival and proliferation [22]. Nobiletin treatment also reduced NF-κB (p50) expression in nuclei of both prostate cancer cells. NF-κB activation plays many roles when it enter into Figure 7 Nobiletin's effect on NF-κB (p50) expression. (a) Effect of nobiletin on total NF-kB expression, (b) Effect of nobiletin on nuclear NF-kB expression. PC-3 and DU-145 cells (1.0 × 106) were seeded in 60 mm dishes, incubated overnight, and treated with nobiletin for 24 hours. Discussion HIF-1α protein levels were normalized by GAPDH protein levels. Data represents means ± SE from 3 independent experiments. P < 0.05 compared to control. *P < 0.01 compared to control. Nobiletin, a citrus polymethoxy flavonoid, possesses an- ticancer, antiviral, and anti-inflammatory activities [14]. More specifically, recent findings have identified nobiletin as a cell differentiation modulator. Cell differentiation is a crucial step in angiogenesis and therefore could affect tumor growth and metastasis which both depend on angiogenesis [15]. These findings support the proposition that nobiletin is functionally unique and could be a pos- sible chemopreventive agent in inflammation-associated tumorigenesis [17]. have become more prevalent in recent years due to the escalating number of prostate cancer cases. It has been suggested that dietary intake of natural products rich in flavonoids from citrus fruits may play a role in the prevention of cancer [8]. Tangeretin, nobiletin, hesperetin, hesperidin, naringenin, and naringin are just a few examples of citrus flavonoids that have the potential to be used as chemotherapeutic agents. Research has shown that these flavonoids possess inhibition activity on certain cancer cells' growth through various mechanisms [8]. Nobiletin(µM) 0 20 40 80 Nobiletin(µM) 0 20 40 80 PC-3 p-AKT DU-145 p-AKT PC-3 t-AKT DU-145 t-AKT Figure 5 Nobiletin's effect on AKT phosphorylation in PC-3 and DU-145 cells. PC-3 and DU-145 cells (1.0 × 106) were seeded in 60-mm dishes, incubated overnight, and treated with nobiletin for 24 hours. Cells were harvested and analyzed by SDS-PAGE and Western Blotting. P-AKT protein levels were normalized by total AKT protein levels and expressed as percentages of control. Data represents means ± SE from 3 independent experiments. P < 0.05 compared to control. *P < 0.01 compared to control. Figure 5 Nobiletin's effect on AKT phosphorylation in PC-3 and DU-145 cells. PC-3 and DU-145 cells (1.0 × 106) were seeded in 60-mm dishes, incubated overnight, and treated with nobiletin for 24 hours. Cells were harvested and analyzed by SDS-PAGE and Western Blotting. P-AKT protein levels were normalized by total AKT protein levels and expressed as percentages of control. Data represents means ± SE from 3 independent experiments. P < 0.05 compared to control. *P < 0.01 compared to control. Chen et al. Discussion Cells were harvested and analyzed by SDS-PAGE and Western Blotting. NF-кB protein levels whole cells were normalized by GAPDH protein levels and its protein levels of nucleus were normalized by PCNA protein levels. They were expressed as percentages of control. Data represents means ± SE from 3 independent experiments. P < 0.05 compared to control. *P < 0.01 compared to control. Figure 7 Nobiletin's effect on NF-κB (p50) expression. (a) Effect of nobiletin on total NF-kB expression, (b) Effect of nobiletin on nuclear NF-kB expression. PC-3 and DU-145 cells (1.0 × 106) were seeded in 60 mm dishes, incubated overnight, and treated with nobiletin for 24 hours. Cells were harvested and analyzed by SDS-PAGE and Western Blotting. NF-кB protein levels whole cells were normalized by GAPDH protein levels and its protein levels of nucleus were normalized by PCNA protein levels. They were expressed as percentages of control. Data represents means ± SE from 3 independent experiments. P < 0.05 compared to control. *P < 0.01 compared to control. Chen et al. BMC Pharmacology and Toxicology 2014, 15:59 http://www.biomedcentral.com/2050-6511/15/59 Page 8 of 10 Nobiletin(µM) 0 20 40 80 Nobiletin(µM) 0 20 40 80 PC-3 PTEN DU-145 PTEN PC-3 GAPDH DU-145 GAPDH Figure 8 Nobiletin’s effect on PTEN expression. PC-3 and DU-145 cells (1.0 × 106) were seeded in 60-mm dishes, incubated overnight, and treated with nobiletin for 24 hours. Cells were harvested and analyzed by SDS-PAGE and Western Blotting. PTEN protein levels were normalized by GAPDH protein levels and expressed as percentages of control. Data represents means ± SE from 3 independent experiments. P < 0.05 compared to control. *P < 0.01 compared to control. Figure 8 Nobiletin’s effect on PTEN expression. PC-3 and DU-145 cells (1.0 × 106) were seeded in 60-mm dishes, incubated overnight, and treated with nobiletin for 24 hours. Cells were harvested and analyzed by SDS-PAGE and Western Blotting. PTEN protein levels were normalized by GAPDH protein levels and expressed as percentages of control. Data represents means ± SE from 3 independent experiments. P < 0.05 compared to control. *P < 0.01 compared to control. and AKT phosphorylation, indicating that AKT may influ- ence cell viability by its effect on NF-κB in both prostate cells. It was also found that HIF-1α promoter is responsive to selective NF-κB subunits, indicating that NF-κB is a direct modulator of HIF-1α expression [29]. Discussion VEGF is a signal protein produced by cells related to vasculogenesis and angiogenesis, and it is also the downstream gene of HIF-1α. Our study revealed that VEGF expression was significantly (p < 0.01) reduced in PC-3 cancer cells by nobiletin treatment. HIF-1α expression in both cells lines (PC-3 and DU-145) was also inhibited by nobiletin the nucleus, including initiating cellular transformation, mediating cellular proliferation, mediating cellular inva- sion and angiogenesis, mediating metastasis, and linking inflammation and cancer [23,24]. Suppression of NF-κB in tumor samples also inhibits proliferation, causes cell cycle arrest, and leads to apoptosis, indicating the crucial role of NF-κB in cell proliferation and survival [25]. Some researchers have found that the expression, activation and translocation of NF-κB were regulated by AKT pathways [26-28]. Our results showed that nobiletin treatment could decrease NF-κB expression in nuclei of both cells Figure 9 Nobiletin inhibits VEGF expression by regulating AKT and HIF-1α gene in PC-3 cells. (a) Nobiletin inhibits VEGF expression by regulating HIF-1 α gene, (b) Nobiletin inhibits VEGF expression by regulating AKT gene. PC-3 prostate cancer cells were seeded in 96-well plate at 10,000 cells/well and incubated overnight. The cells were then transfected with 0.05 ug VEGF (Hif-1α) luciferase reporter, 0-0.25 μg HIF-1α (AKT) or SR-α plasmids by 0.6 μL jetPRIME reagent for 4 hours, followed by 16 hour treatment with 0 or 40 μM nobiletin. The cells were harvested and analyzed for luciferase and total protein levels, and the levels of VEGF (Hif-1α) reporter were normalized by corresponding total protein levels. Data represent Means ± SE from 3 independent experiments. #p < 0.05 as compared to control. ##p <0.01 as compared to control. p <0.05 as compared to nobiletin-treated control. *p <0.01 as compared to nobiletin-treated control. Figure 9 Nobiletin inhibits VEGF expression by regulating AKT and HIF-1α gene in PC-3 cells. (a) Nobiletin inhibits VEGF expression by regulating HIF-1 α gene, (b) Nobiletin inhibits VEGF expression by regulating AKT gene. PC-3 prostate cancer cells were seeded in 96-well plate at 10,000 cells/well and incubated overnight. The cells were then transfected with 0.05 ug VEGF (Hif-1α) luciferase reporter, 0-0.25 μg HIF-1α (AKT) or SR-α plasmids by 0.6 μL jetPRIME reagent for 4 hours, followed by 16 hour treatment with 0 or 40 μM nobiletin. Discussion The cells were harvested and analyzed for luciferase and total protein levels, and the levels of VEGF (Hif-1α) reporter were normalized by corresponding total protein levels. Data represent Means ± SE from 3 independent experiments. #p < 0.05 as compared to control. ##p <0.01 as compared to control. p <0.05 as compared to nobiletin-treated control. *p <0.01 as compared to nobiletin-treated control. Figure 9 Nobiletin inhibits VEGF expression by regulating AKT and HIF-1α gene in PC-3 cells. (a) Nobiletin inhibits VEGF expression by regulating HIF-1 α gene, (b) Nobiletin inhibits VEGF expression by regulating AKT gene. PC-3 prostate cancer cells were seeded in 96-well plate at 10,000 cells/well and incubated overnight. The cells were then transfected with 0.05 ug VEGF (Hif-1α) luciferase reporter, 0-0.25 μg HIF-1α (AKT) or SR-α plasmids by 0.6 μL jetPRIME reagent for 4 hours, followed by 16 hour treatment with 0 or 40 μM nobiletin. The cells were harvested and analyzed for luciferase and total protein levels, and the levels of VEGF (Hif-1α) reporter were normalized by corresponding total protein levels. Data represent Means ± SE from 3 independent experiments. #p < 0.05 as compared to control. ##p <0.01 as compared to control. p <0.05 as compared to nobiletin-treated control. **p <0.01 as compared to nobiletin-treated control. Chen et al. BMC Pharmacology and Toxicology 2014, 15:59 http://www.biomedcentral.com/2050-6511/15/59 Page 9 of 10 treatment, with a greater inhibition in PC-3 cells. Fur- thermore, it was found that nobiletin inhibited VEGF expression through regulating AKT/HIF-1α pathways in prostate cancer cell line PC-3. Increasing HIF-1α levels actually reversed nobiletin’s inhibitory effects on VEGF expression. Similarly up-regulating AKT levels reversed its inhibitory effects on HIF-1α expression. These results correspond to a previous study that HIF-1α/VEGF expression can be regulated by AKT pathways [30]. Nobi- letin reduced cMyc expression in DU-145 cells, but showed no evidence of inhibition in PC-3 cells. PTEN has been shown to play a pivotal role in apoptosis, cell cycle arrest, and possibly cell migration. PTEN is the most frequently mutated gene in prostate cancer, loss of hetero- zygosity at 10q23 can be detected in approximately 50% of human prostate cancers, whereas homozygous deletions of PTEN can be detected in approximately 10% of these cases [31]. However, nobiletin appears to lower cell viability through a mechanism independent of PTEN, as it does not seem to affect PTEN concentrations. distribution, metabolism, and excretion of nobiletin into consideration. References Meiyanto E, Hermawan A, Anindyajati: Natural products for cancer-targeted therapy: citrus flavonoids as potent chemopreventive agents. Asian Pac J Cancer Prev 2012, 13:427–436. Acknowledgments This research was supported by a West Virginia Experimental Program to Stimulate Competitive Research grant and an NIH grant (5P20RR016477 and 8P20GM104434) from the National Center for Research Resources awarded to the West Virginia IDeA Network of Biomedical Research Excellence. Authors’ contributions d h JC carried out the majority of experimental work. YC drafted the manuscript. All authors participated in experimental design and read and approved the final manuscript. References 1. Mohile SG, Shelke AR: Treating prostate cancer in elderly men: how does aging affect the outcome? Curr Treat Options Oncol 2011, 12:263–275. 1. Mohile SG, Shelke AR: Treating prostate cancer in elderly men: how does aging affect the outcome? Curr Treat Options Oncol 2011, 12:263–275. aging affect the outcome? Curr Treat Options Oncol 2011, 12:263–275. 2. Crawford E: Understanding the epidemiology, natural history, and key pathways involved in prostate cancer. Urology 2009, 73:4–10. 3. Bosland MC: The role of steroid hormones in prostate carcinogenesis. J Natl Cancer Inst Monogr 2000 27:39 66 2. Crawford E: Understanding the epidemiology, natural history, and key pathways involved in prostate cancer. Urology 2009, 73:4–10. 3. Bosland MC: The role of steroid hormones in prostate carcinogenesis. J Natl Cancer Inst Monogr 2000, 27:39–66. 4. Crawford E: Epidemiology of prostate cancer. Urology 2003, 62:3–12. 4. Crawford E: Epidemiology of prostate cancer. Urology 2003, 62:3–12. 5. Gronberg H: Prostate cancer epidemiology. Lancet 2003, 361:859–864. 4. Crawford E: Epidemiology of prostate cancer. Urology 2003, 62:3–12. 5 G b H P t t id i l L t 2003 361 859 864 4. Crawford E: Epidemiology of prostate cancer. Urology 2003, 62:3–12. 5. Gronberg H: Prostate cancer epidemiology. Lancet 2003, 361:859–864. 5. Gronberg H: Prostate cancer epidemiology. Lancet 2003, 361:859–864. 6. Whittemore AS, Kolonel LN, Wu AH, John EM, Gallagher RP, Howe GR: Prostate cancer in relation to diet, physical activity, and body size in blacks, whites, and Asians in the United States and Canada. J Natl Cancer Inst 1995, 87:652–661. 6. Whittemore AS, Kolonel LN, Wu AH, John EM, Gallagher RP, Howe GR: Prostate cancer in relation to diet, physical activity, and body size in blacks, whites, and Asians in the United States and Canada. J Natl Cance Inst 1995, 87:652–661. 7. Gupta S, Kim J, Prasad S, Aggarwal B: Regulation of survival, proliferation, invasion, angiogenesis, and metastasis of tumor cells through modulation of inflammatory pathways by nutraceuticals. Cancer and Metastasis Reviews 2010, 29:405–434. 7. Gupta S, Kim J, Prasad S, Aggarwal B: Regulation of survival, proliferation, invasion, angiogenesis, and metastasis of tumor cells through modulation of inflammatory pathways by nutraceuticals. Cancer and Metastasis Reviews 2010, 29:405–434. 8. Meiyanto E, Hermawan A, Anindyajati: Natural products for cancer-targeted therapy: citrus flavonoids as potent chemopreventive agents. Asian Pac J Cancer Prev 2012, 13:427–436. 8. Discussion Further studies in animal models and human trials are warranted to determine if physicians can promote this natural compound toward chemoprevention of prostate cancer cells. Competing interest The authors state that they have no competing interest. Author details 1 1College of Biosystems Engineering and Food Science, Fuli Institute of Food Science, Zhejiang Key Laboratory for Agro-Food Processing, Zhejiang University, Hangzhou 310058, China. 2College of Science, Technology and Mathematics, Alderson Broaddus University, Philippi, WV 26416, USA. 3Department of Pharmaceutical Science, West Virginia University, Morgantown, WV 26506, USA. 4Department of Pharmacology, Physiology and Toxicology, Joan C. Edwards School of Medicine, Marshall University, Huntington, WV 25755, USA. Our research indicated that nobiletin is a good candidate for the chemoprevention of prostate cancer in humans and could be an effective measure in inhibiting prostate cancer cell viability. Nobiletin has the apparent ability to suppress cell viability through multiple pathways, thus inhibiting tumor growth. Most encouraging is its capacity to suppress the more metastatic PC-3 cell line. Since the lethality of a tumor links directly to its ability to spread, nobiletin promises to increase the prostate cancer survival rate. However, more data needs to be obtained on nobiletin’s toxicity and tolerable dosages before it can become part of prostate cancer prevention and/or treatment. Also, as an in vitro model, cell culture cannot take absorption, distribution, metabolism, and excretion of nobiletin into consideration. Further studies in animal models and hu- man trials are warranted to determine if physicians can promote this natural compound toward chemoprevention of prostate cancer cells. Received: 6 June 2014 Accepted: 14 October 2014 Published: 24 October 2014 Chen et al. BMC Pharmacology and Toxicology 2014, 15:59 http://www.biomedcentral.com/2050-6511/15/59 Chen et al. BMC Pharmacology and Toxicology 2014, 15:59 http://www.biomedcentral.com/2050-6511/15/59 angiogenesis-related endothelial cell functions and angiogenesis in vivo. Cancer Sci 2010, 101:2462–2469. 16. Nichenametla S, Taruscio TG, Barney DL, Exon JH: A review of the effects and mechanisms of polyphenolics in cancer. Crit Rev Food Sci Nutr 2006, 46:161–183. 17. Murakami A, Nakamura Y, Torikai K, Tanaka T, Koshiba T, Koshimizu K, Kuwahara S, Takahashi Y, Ogawa K, Yano M, Tokuda H, Nishino H, Mimaki Y, Sashida Y, Kitanaka S, Ohigashi H: Inhibitory effect of citrus nobiletin on phorbol ester-induced skin inflammation, oxidative stress, and tumor promotion in mice. Cancer Res 2000, 60:5059–5066. 18. Kim SJ, Uehara H, Yazici S, Langley RR, He J, Tsan R, Fan D, Killion JJ, Fidler IJ: Simultaneous blockade of platelet-derived growth factor-receptor and epidermal growth factor-receptor signaling and systemic administration of paclitaxel as therapy for human prostate cancer metastasis in bone of nude mice. Cancer Res 2004, 64:4201–4208. 19. Kim SJ, Johnson M, Koterba K, Herynk MH, Uehara H, Gallick GE: Reduced c-Met expression by an adenovirus expressing a c-Met ribozyme inhibits tumorigenic growth and lymph node metastases of PC3-LN4 prostate tumor cells in an orthotopic nude mouse model. Clin Cancer Res 2003, 9:5161–5170. 20. Pulukuri SM, Gondi CS, Lakka SS, Jutla A, Estes N, Gujrati M, Rao JS: RNA interference-directed knockdown of urokinase plasminogen activator and urokinase plasminogen activator receptor inhibits prostate cancer cell invasion, survival, and tumorigenicity in vivo. Journal of Biological Chemisry 2005, 280:36529–36540. 21. Tang M, Ogawa K, Asamoto M, Hokaiwado N, Seeni A, Suzuki S, Takahashi S, Tanaka T, Ichikawa K, Shirai T: Protective effects of citrus nobiletin and auraptene in transgenic rats developing adenocarcinoma of the prostate (TRAP) and human prostate carcinoma cells. Cancer Sci 2007, 98:471–477. 22. Fang J, Zhou Q, Shi XL, Jiang BH: Luteolin inhibits insulin-like growth factor 1receptor signaling in prostate cancer cells. Carcinogenesis 2007, 28:713–723. 23. Aggarwal BB: Nuclear-factor-κB: The enemy within. Cancer Cell 2004, 6:203–206. 24. Gilmore TD: Introduction to NF-κB: players, pathways, perspectives. Oncogene 2006, 25:6680–6684. 25. Bharti AC, Aggarwal BB: Nuclear factor-κB and cancer: Its role in prevention and therapy. Biochem Pharmacol 2002, 64:883–888. 26. Kar S, Palit S, Ball WB, Das PK: Carnosic acid modulates Akt/IKK/NF-κB signaling by PP2A and induces intrinsic and extrinsic pathway mediated apoptosis in human prostate carcinoma PC-3 cells. Apoptosis 2012, 17:735–747. 27. Conclusion Our research indicated that nobiletin is a good candidate for the chemoprevention of prostate cancer in humans and could be an effective measure in suppressing pros- tate cancer cell viability. For these two prostate cancer cell lines, nobiletin has the apparent ability to suppress cell viability concentration-dependently through multiple pathways (VEGF, HIF-1α, AKT phosphorylation, cMyc, and NF-κB). Because nobiletin seems to work better against the more dangerous PC-3 cell line, nobiletin holds real potential in improving prostate cancer outcomes. However, more data needs to be obtained on nobiletin’s toxicity and tolerable dosages before it can become part of prostate cancer prevention and/or treatment. Also, as an in vitro model, cell culture cannot take absorption, 9. Yang C, Wang X, Lu G, Picinich S: Cancer prevention by tea: animal studies, molecular mechanisms and human relevance. Nat Rev Cancer 2009, 9:429–439. 9. Yang C, Wang X, Lu G, Picinich S: Cancer prevention by tea: animal studies, molecular mechanisms and human relevance. Nat Rev Cancer 2009, 9:429–439. 10. Gates M, Vitonis A, Tworoger S, Rosner B, Titus-Ernstoff L, Hankinson S, Cramer H: Flavonoid intake and ovarian cancer risk in a population based case-control study. Int J Cancer 2009, 124:1918–1925. 11. McCann S, Freudenheim J, Marshall J, Saxon G: Risk of human ovarian cancer is related to dietary intake of selected nutrients, phytochemicals and food groups. Journal of Nutrition 2003, 133:1937–1942. 12. Bernini R, Crisante F, Ginnasi MC: A convenient and safe O-methylation offlavonoids with dimethyl carbonate (DMC). Molecules 2011, 16:1418–1425. 13. Knekt P, Järvinen R, Seppänen R, Heliövaara M, Teppo L, Pukkala E, Aromaa A: Dietary flavonoids and the risk of lung cancer and other malignant neoplasms. Am J Epidemiol 1997, 146:223–230. 14. Li S, Yu H, Ho CT: Nobiletin: efficient and large quantity isolation from orange peel extract. Biomed Chromatogr 2006, 20:133–138. 15. Kunimasa K, Ikekita M, Sato M, Ohta T, Yamori Y, Ikeda M, Kuranuki S, Oikawa T: Nobiletin, a citrus polymethoxy flavonoid, suppresses multiple 15. Kunimasa K, Ikekita M, Sato M, Ohta T, Yamori Y, Ikeda M, Kuranuki S, Oikawa T: Nobiletin, a citrus polymethoxy flavonoid, suppresses multiple Page 10 of 10 Page 10 of 10 Chen et al. BMC Pharmacology and Toxicology 2014, 15:59 http://www.biomedcentral.com/2050-6511/15/59 Kim MO, Moon DO, Heo MS, Lee JD, Jung JH, Kim SK, Choi YH, Kim GY: Pectenotoxin-2 abolishes constitutively activated NF-κB, leading to suppression of NF-κB related gene products and potentiation of apoptosis. Cancer Letter 2008, 271:25–33. 28. Ozes ON, Mayo LD, Gustin JA, Pfeffer SR, Pfeffer LM, Donner DB: NF-κB activation by tumour necrosis factor requires the Akt serine-threonine kinase. Nature 1999, 401:82–85. 29. Uden PV, Kenneth NS, Rocha S: Regulation of hypoxia-inducible factor-1α by NF-κB. Biochemical Journal 2008, 412:477–484. 30. Shi YH, Wang YX, Bingle L, Gong LH, Heng WJ, Li Y, Fang WG: In vitro study of HIF-1 activation and VEGF release by bFGF in the T47D breast cancer cell line under normoxic conditions: involvement of PI-3K/Akt and MEK1/ERK pathways. Journal of Pathology 2005, 205:530–536. 31. Chu EC, Tarnawski AS: PTEN regulatory functions in tumor suppression and cell biology. Med Sci Monit 2004, 10:RA235–241. 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BMC Pharmacology and Toxicology 2014 15:59. • Convenient online submission
https://openalex.org/W4207002079	https://link.springer.com/content/pdf/10.1007/s44192-022-00004-3.pdf	English	null	Men are the main COVID-19 transmitters: behavior or biology?	Discover Mental Health	2,022	cc-by	5,654	Monize V. R. Silva1 · Mateus V. de Castro1 · Maria Rita Passos‑Bueno1 · Paulo A. Otto2 · Michel S. Naslavsky2 · Mayana Zatz1 Received: 25 October 2021 / Accepted: 12 January 2022 © The Author(s) 2022 OPEN Abstract Background COVID-19 has affected millions of people worldwide. Clinical manifestations range from severe cases with lethal outcome to mild or asymptomatic cases. Although the proportion of infected individuals does not differ between sexes, men are more susceptible to severe COVID-19, with a higher risk of death than women. Also, men are pointed out as more lax regarding protective measures, mask wearing and vaccination. Thus, we questioned whether sex-bias may be explained by biological pathways and/or behavioral aspects or both. Methods Between July 2020 and July 2021, we performed an epidemiological survey including 1744 unvaccinated adult Brazilian couples, with there was at least one infected symptomatic member, who were living together during the COVID- 19 infection without protective measures. Presence or absence of infection was confirmed by RT-PCR and/or serology results. Couples were divided into two groups: (1) both partners were infected (concordant couples) and (2) one partner was infected and the spouse remained asymptomatic despite the close contact with the COVID-19 symptomatic partner (discordant couples). Statistical analysis of the collected data was performed aiming to verify a differential transmission potential between genders in couples keeping contact without protective measures. p g p p g p Results The combination of our collected data showed that the man is the first (or the only) affected member in most cases when compared to women and that this difference may be explained by biological and behavioral factors.if Results The combination of our collected data showed that the man is the first (or the only) affected member in most cases when compared to women and that this difference may be explained by biological and behavioral factors. Conclusions The present study confirmed the existence of gender differences not only for susceptibility to infection and resistance to COVID-19 but also in its transmission rate. Conclusions The present study confirmed the existence of gender differences not only for susceptibility to infection and resistance to COVID-19 but also in its transmission rate. Monize V. R. Silva and Mateus V. de Castro contributed equally for the manuscript * Mayana Zatz, mayazatz@usp.br \| 1Human Genome and Stem Cell Research Center (HUG‑CELL), Biosciences Institute, University of Sao Paulo, Sao Paulo, SP, Brazil. 2Department of Genetics and Evolutionary Biology, Biosciences Institute, University of Sao Paulo, Sao Paulo, SP, Brazil. Monize V. R. Silva and Mateus V. de Castro contributed equally for the manuscript Discover Mental Health (2022) 2:1 \| https://doi.org/10.1007/s44192-022-00004-3 1 Introduction Independently of age, men are more likely to have complications by COVID-19 than women and if hospitalization is required, males are more at risk of death than females [19]. Interestingly, comparable gender differences also occur for other viral infections [20]. Furthermore, some behavioral aspects such as COVID-19 prevention and control measures vary between genders. A survey conducted in March–April 2020 indicated that men are more reluctant than women to wear protective masks and respect social distancing [21]. A recent survey conducted with almost 2000 American adults showed that the importance of masks for protection differ considerably between genders and males are more likely to consider masks as an infringement on their independ- ence and freedom [22]. Another survey with 2500 Americans showed that males are less inclined to wearing masks than females, although both genders consider masks shameful [23]. Other protective practices as handwashing and sanitizer use had been previously reported as more common among females [24] and this tendency is the same for COVID-19 pandemic [25].f Interestingly, preliminary surveys to evaluate sex-differences for COVID-19 vaccination intention showed that females intended less to be vaccinated, probably due to the fear as to side effects or even the desire to “not the first to be vac- cinated” [26–29]. Nonetheless, updated data compiled by The COVID-19 Sex-Disaggregated Data Tracker initiative by Global Health 5050 [30] has shown that more females are being vaccinated than males in many countries, including Brazil and U.S., as also shown by local media [31–33]. Considering the mental health impact due the pandemic, it was also observed that females experienced more depres- sion symptoms after lockdown while males experienced more anxiety symptoms [34], but after a few weeks the gender- differences disappeared and both groups experienced resilience feelings [35]. The mental impact of the pandemic in LMICs varied from immediate distress and discrimination experienced mainly by healthcare workers to long-term side effects such as higher prevalence of depression and other mood disorders as a result of social isolation, job insecurity, unemployment and economic distress [36]. It has been suggested that some individuals (named “superspreaders”) are able to transmit the virus to a great number of persons [37]. However, it is not known if this could be explained by behavior (men speaking louder without mask) or biologically (differences in lung capacity between sexes and ages). Highlights • There are sex differences in COVID-19 susceptibility and transmission between couples highly exposed to SARS-Cov-2 without protective measures; M ffi i t i t itt th • There are sex differences in COVID-19 susceptibility and transmission between couples highly exposed to SARS-Cov-2 without protective measures; • Men are more efficient virus transmitters than women; • Men are more efficient virus transmitters than women; • Sex-bias in COVID-19 transmission can be explained by differences in viral load in saliva, immune response and also behavioral protective differences between genders. • Sex-bias in COVID-19 transmission can be explained by differences in viral load in saliva, immune response and also behavioral protective differences between genders. Keywords COVID-19 · Household transmission · Behavior · Adult couples · SARS-CoV-2 Monize V. R. Silva and Mateus V. de Castro contributed equally for the manuscript Monize V. R. Silva and Mateus V. de Castro contributed equally for the manuscript * Mayana Zatz, mayazatz@usp.br \| 1Human Genome and Stem Cell Research Center (HUG‑CELL), Biosciences Institute, University of Sao Paulo, Sao Paulo, SP, Brazil. 2Department of Genetics and Evolutionary Biology, Biosciences Institute, University of Sao Paulo, Sao Paulo, SP, Brazil. * Mayana Zatz, mayazatz@usp.br \| 1Human Genome and Stem Cell Research Center (HUG‑CELL), Biosciences Institute, University of Sao Paulo, Sao Paulo, SP, Brazil. 2Department of Genetics and Evolutionary Biology, Biosciences Institute, University of Sao Paulo, Sao Paulo, SP, Brazil. tal Health (2022) 2:1 \| https://doi.org/10.1007/s44192-022-00004-3 1 3 1 3 3 \| https://doi.org/10.1007/s44192-022-00004-3 Brief Communication Discover Mental Health Discover Mental Health Brief Communication (2022) 2:1 1 Introduction 1 Introduction Since the first reported case of COVID-19 in December 2019, some 200 million individuals have been infected by the novel coronavirus. Global epidemiological data revealed that age increases the risk of dying from COVID-19 because of the significant number of older adults with comorbidities [1–4]. Moreover, although there are no gender differences in the proportion of people infected by SARS-CoV-2, men are more susceptible to develop severe COVID-19 [5, 6]. Since the first reported case of COVID-19 in December 2019, some 200 million individuals have been infected by the novel coronavirus. Global epidemiological data revealed that age increases the risk of dying from COVID-19 because of the significant number of older adults with comorbidities [1–4]. Moreover, although there are no gender differences in the proportion of people infected by SARS-CoV-2, men are more susceptible to develop severe COVID-19 [5, 6].i COVID-19 pandemic caused a significant number of deaths worldwide, but the prevalence of comorbidities [7, 8], lower education levels, socioeconomic inequalities as the healthcare system inexperience to deal with the pandemic [9, 10] contributed to the increase of COVID-19 cases and deaths in low- and middle-income countries (LMICs) [11–13]. Most of these countries still show inadequate vaccination programs, lack of an accurate and rapid diagnosis as well as poor viral surveillance [10, 14–16], contributing for the emergence of COVID-19 waves and new SARS-CoV-2 variants [17, 18]. COVID-19 pandemic caused a significant number of deaths worldwide, but the prevalence of comorbidities [7, 8], lower education levels, socioeconomic inequalities as the healthcare system inexperience to deal with the pandemic [9, 10] contributed to the increase of COVID-19 cases and deaths in low- and middle-income countries (LMICs) [11–13]. Most of these countries still show inadequate vaccination programs, lack of an accurate and rapid diagnosis as well as poor viral surveillance [10, 14–16], contributing for the emergence of COVID-19 waves and new SARS-CoV-2 variants [17, 18]. Independently of age, men are more likely to have complications by COVID-19 than women and if hospitalization is required, males are more at risk of death than females [19]. Interestingly, comparable gender differences also occur for other viral infections [20]. Furthermore, some behavioral aspects such as COVID-19 prevention and control measures vary between genders. A survey conducted in March–April 2020 indicated that men are more reluctant than women to wear protective masks and respect social distancing [21]. 2 Methods We present an epidemiological survey (from July 2020 to July 2021) including 1744 adult Brazilian couples who were liv- ing together during the COVID-19 infection without protective measures. Their ages ranged from 20 to 70 years (overall mean age of 45 years, 44 for women and 46 for men). The positive diagnosis in symptomatic individuals was confirmed by RT-PCR and/or serology for the infected partners while negative results in both tests were confirmed in non-infected partners. These individuals presented complete information related to the infection event, sex, age, and diagnostic tests results. They were divided in two groups: (a) concordant couples where one spouse infected with confirmed COVID- 19 transmitted the infection to the partner and (b) discordant couples where one SARS-CoV-2 infected spouse was symptomatic while the partner was not, as confirmed by negative RT-PCR and serology results after viral exposure. The concordant and discordant couples were then subdivided according to which partner was infected first: men or women and women to men. Our whole data collection rationale from partners is presented in Fig. 1. Discover Mental Health (2022) 2:1 \| https://doi.org/10.1007/s44192-022-00004-3 Brief Communication \| https://doi.org/10.1007/s44192-022-00004-3 Brief Communication Discover Mental Health (2022) 2:1 \| https://doi.org/10.1007/s44192-022-00004-3 Brief Communication Discover Mental Health 3 Results As seen in Table 1, concordant couples analysis showed that men are significantly more infectious than women, with an estimated chance of 146/230 = 0.635 (95% confidence interval: 0.569–0.697) of being the first one affected. The cor- responding female values were 84/230 = 0.365 (95% confidence interval 0.303–0.431). These differences in male/female values are statistically significant (χ2 = 16.713, P ≪ 0.0005). The analysis of the group of discordant couples showed again that men are preferentially affected, with a prob- ability estimated as 797/1367 = 0.583 (95% confidence interval: 0.557–0.609). The corresponding female values were 570/1367 = 0.417 (95% confidence interval: 0.391–0.443). The combination of data from concordant and dis- cordant couples showed that the man is preferentially affected in 146 + 797 = 943 occurrences and the woman in 84 + 576 = 660 instances (χ2 = 49.962, P ≪ 0.0005). χ Interestingly, the analysis of COVD-19 reports of the present cohort showed that some 62% among those who men- tioned keywords as “fear”, “excessive care”, “pandemic worries” and “anxiety” were sent by females. This observation is consistent with the results of a Brazilian survey with 4693 adults which showed that among 55% who declared that they were worried about the pandemic 62% were females and 45% males [39]. Such observation reporting that men were less worried about the pandemic than women could provide an explanation for their less protective behavior. 1 Introduction Biologically, viral transmission capability could be influenced by less aerosol emission by females and children [37]. These sex-based differences may be also associated with variances in biological pathways such as immune responses against SARS-CoV-2, the expression of X-chromo- some–encoded genes [20], or both. These observations led us to question whether the virus could be transmitted more frequently by men than by women, independently of protection measures, age and socio-economic status. In order to circumvent such differences, we have analyzed the virus transmission in couples who kept neither conjugal distancing during the infection period nor the use of protective measures. Moreover, the mean age did not differ significantly between spouses and both partners had a comparable socio-economic status. In a previous genetic study with 81 discordant couples for COVID-19, where one person was infected and symptomatic while the partner remained asymptomatic and serum-negative (despite remaining in close contact and sharing the same bed throughout the disease), we observed that there were significantly more women in the asymptomatic group [38]. Subsequently, there were reports of couples where the wife was infected by SARS-CoV-2 and clinically affected while the husband remained asymptomatic despite the close contact throughout the infection period. Some months later, the previously asymptomatic husbands beame infected and symptomatic after contact with male patients. Vol:1 :.(123456789 3 4 Discussion All the results obtained in the present study strongly suggest that males are not only more susceptible to COVID-19 severity, as shown in worldwide epidemiological surveys, but they are also more likely to transmit the virus to their partners when compared to females in the household transmission context. The epidemiological findings in the present survey are consistent with the results of other published studies involving couples where one of the partners was infected by their spouses [40, 41]. Female individuals aged between 17 and 65 years were also frequently found to be secondary cases [41]. Aiming to analyze a more homogeneous cohort and since age is an important predictor of severity and risk of death by COVID-19, we focused our survey on couples of comparable ages and economic status and therefore similar access to health care. It is also important to note that the survey was performed before the vaccination was started. One of the possible current biological hypotheses for such gender variable transmission rate is a differential viral load in saliva, which has been explored as an important clinical measure of disease severity due to its positive association with many COVID-19 inflammatory markers [42]. These factors, together with the higher adoption of hygiene and protective measures among females, may justify the lower transmission rates in this group. One of the possible current biological hypotheses for such gender variable transmission rate is a differential viral load in saliva, which has been explored as an important clinical measure of disease severity due to its positive association with many COVID-19 inflammatory markers [42]. These factors, together with the higher adoption of hygiene and protective measures among females, may justify the lower transmission rates in this group. Interestingly, in a recent study of our group [43] it was observed that, although there were no observed gender dif- ferences in viral load in nasopharyngeal samples, adult males showed a significantly higher viral load in saliva samples (verified by RT-LAMP viral testing) than adult women. (0123456 1 3 23456789) 3 Brief Communication Discover Mental Health (2022) 2:1 \| https://doi.org/10.1007/s44192-022-00004-3 Brief Communication Discover Mental Health \| https://doi.org/10.1007/s44192-022-00004-3 mmunication Discover Mental Health (2022) 2:1 \| https://doi.org/10.1007/s4 Group Men Women Total Chi-square (χ2) values Discordant 797 570 1367 37.6950 Concordant 146 84 230 16.7130 Fig. 4 Discussion 1 Survey data collection rational diagram Table 1 Numerical description of couple groups composition and chi-square (χ2) values Group Men Women Total Chi-square (χ2) values Discordant 797 570 1367 37.6950 Concordant 146 84 230 16.7130 Table 1 Numerical description of couple groups composition and chi-square (χ2) values Group Men Women Total Chi-square (χ2) values Discordant 797 570 1367 37.6950 Concordant 146 84 230 16.7130 Table 1 Numerical description of couple groups composition and chi-square (χ2) values Group Men Women Total Chi-square (χ2) values Discordant 797 570 1367 37.6950 Concordant 146 84 230 16.7130 Vol:1 :.(123456789 3 \| https://doi.org/10.1007/s44192-022-00004-3 Brief Communication Discover Mental Health \| https://doi.org/10.1007/s44192-022-00004-3 th (2022) 2:1 (2022) 2:1 Brief Communication These observations, together with the evidence of higher aerosol emission by men which makes them more likely to be “superspreaders” than women, support the hypothesis that male individuals are more efficient virus transmitters than females, which is related to biological and behavioral aspects. These observations, together with the evidence of higher aerosol emission by men which makes them more likely to be “superspreaders” than women, support the hypothesis that male individuals are more efficient virus transmitters than females, which is related to biological and behavioral aspects. This study has some limitations regarding the relatively modest number of couples included in the present cohort when compared with other epidemiological surveys of in-house transmission [40, 41]. Additionally, the couples who responded the questionnaire are on average younger than the mean age of the population since, in Brazil, younger peo- ple have more access and familiarity with internet than older adults [44]. Nevertheless, our study brings new knowledge to the field of public health regarding SARS-CoV-2 transmission dynamics.if i In short, the present study confirmed the existence of gender differences not only for susceptibility to infection and resistance to COVID-19 but also in the transmission rate. Acknowledgements The authors are extremely grateful to all volunteers for their participation and collaboration. This work was supported by the Sao Paulo Research Foundation (FAPESP/Brazil) [Grant Numbers 2013/08028-1, 2014/50931-3 and 2020/09702-1], the National Council for Scientific and Technological Development (CNPq) [Grant Number 465355/2014-5], JBS S.A [Grant Number 69004]. Special thanks to Brazilian senator Mara Gabrilli for financial support. Authors’ contributions MVRS and MVC: methodology, formal analysis, investigation, data curation, writing—original draft, writing—review and editing and visualization. MRPB: conceptualization and writing—review and editing. PAO: methodology, formal analysis, investigation, data curation, writing—original draft and writing—review and editing. Declarations Ethics approval and consent to participate This study was approved by the Committee for Ethics in Research of the Institute of Biosciences at the University of São Paulo (CAAE 34786620.2.0000.5464) in accordance with de Declaration of Helsinki principles, ICH06 Good Clinical Prac- tices and Brazilian Health Regulatory Agency (ANVISA) resolution number 466 from 2012 that regulates research with humans. An informed consent term was obtained from all participants. Competing interests The authors declare no conflicts of interest. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons 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/. 4 Discussion MSN: conceptualization and writing—review and editing. MZ: super- vision, project administration, conceptualization, funding acquisition and writing—review and editing. All authors read and approved the final manuscript. Data availability Data sharing is not applicable to this article as no datasets were generated during the current study. References 1. Chai S, Li Y, Li X, Tan J, Abdelrahim MEA, Xu X. Effect of age of COVID-19 inpatient on the severity of the disease: a meta-analysis. Int J Clin Pract. 2021;75(10): e14640.f 1. Chai S, Li Y, Li X, Tan J, Abdelrahim MEA, Xu X. Effect of age of COVID-19 inpatient on the severity of the disease: a meta-analysis. Int J Clin Pract. 2021;75(10): e14640.f 2. Thakur B, Dubey P, Benitez J, Torres JP, Reddy S, Shokar N, et al. A systematic review and meta-analysis of geographic differences in comorbidities and associated severity and mortality among individuals with COVID-19. Sci Rep. 2021;11(1):8562. 2. Thakur B, Dubey P, Benitez J, Torres JP, Reddy S, Shokar N, et al. A systematic review and meta-analysis of geographic differences in comorbidities and associated severity and mortality among individuals with COVID-19. Sci Rep. 2021;11(1):8562. 2. Thakur B, Dubey P, Benitez J, Torres JP, Reddy S, Shokar N, et al. A systematic review and meta-analysis of geographic differences in comorbidities and associated severity and mortality among individuals with COVID-19. Sci Rep. 2021;11(1):8562. 3. Zhou F, Yu T, Du R, Fan G, Liu Y, Liu Z, et al. Clinical course and risk factors for mo a retrospective cohort study. Lancet. 2020;395(10229):1054–62. 4. Mallapaty S. The coronavirus is most deadly if you are older and male new data reveal the risks. Nature. 2020;585(7823):16 7. 5. Peckham H, de Gruijter NM, Raine C, Radziszewska A, Ciurtin C, Wedderburn LR, et al. Male sex identified by global COVID-19 meta-analysis as a risk factor for death and ITU admission. Nat Commun. 2020;11(1):6317.f 5. Peckham H, de Gruijter NM, Raine C, Radziszewska A, Ciurtin C, Wedderburn LR, et al. Male sex identified by global COVID-19 meta-analysis as a risk factor for death and ITU admission. Nat Commun. 2020;11(1):6317.f 6. Dehingia N, Raj A. Sex differences in COVID-19 case fatality: do we know enough? Lancet Glob Health. 2021;9 f 7. Lam AA, Lepe A, Wild SH, Jackson C. Diabetes comorbidities in low- and middle-income countries: an umbrella review. J Glob Health. 2021;11:04040. 7. Lam AA, Lepe A, Wild SH, Jackson C. Diabetes comorbidities in low- and middle-income countries: an umbrella review. J Glob Health. 2021;11:04040. 8. Geldsetzer P, Manne-Goehler J, Marcus M-E, Ebert C, Zhumadilov Z, Wesseh CS, et al. g 9. Taylor L. Covid-19: Brazil’s hospitals close to collapse as cases reach record high. BMJ. 2021;23(372): n800. References Vahidy FS, Pan AP, Ahnstedt H, Munshi Y, Choi HA, Tiruneh Y, et al. Sex differences in susceptibility, severity disease 2019: cross-sectional analysis from a diverse US metropolitan area. PLoS ONE. 2021;16(1): e02455 y p 20. Klein SL, Flanagan KL. Sex differences in immune responses. Nat Rev Immunol. 2016;16(10):626–38.f y Klein SL, Flanagan KL. Sex differences in immune responses. Nat Rev Immunol. 2016;16(10):626–38.f gf p 21. Galasso V, Pons V, Profeta P, Becher M, Brouard S, Foucault M. Gender differences in COVID-19 attitudes and behavior: panel evidence from eight countries. Proc Natl Acad Sci. 2020;117(44):27285–91. gf 21. Galasso V, Pons V, Profeta P, Becher M, Brouard S, Foucault M. Gender differences in COVID-19 attitudes and behavior: panel evidence from eight countries. Proc Natl Acad Sci. 2020;117(44):27285–91. g 22. Howard MC. Gender, face mask perceptions, and face mask wearing: are men being dangerous during the COVID-19 pandemic? Personal Individ Differ. 2021;15(170): 110417.f g 22. Howard MC. Gender, face mask perceptions, and face mask wearing: are men being dangerous during the COVID-19 pandemic? Personal Individ Differ. 2021;15(170): 110417.f f 23. Capraro V, Barcelo H. The effect of messaging and gender on intentions to wear a face covering to slow down COVID-19 transmission. PsyArXiv; 2020. https://psyarxiv.com/tg7vz/. Accessed 24 Oct 2021. f 23. Capraro V, Barcelo H. The effect of messaging and gender on intentions to wear a face covering to slow down COVID-19 transmission. PsyArXiv; 2020. https://psyarxiv.com/tg7vz/. Accessed 24 Oct 2021. y y Suen LKP, So ZYY, Yeung SKW, Lo KYK, Lam SC. Epidemiological investigation on hand hygiene knowledge and beha study on gender disparity. BMC Public Health. 2019;11(19):401. y y 25. Kollamparambil U, Oyenubi A. Behavioural response to the Covid-19 pandemic in South Africa. PLoS ONE. 2021;16(4): e0250269. 25. Kollamparambil U, Oyenubi A. Behavioural response to the Covid-19 pandemic in South Africa. PLoS ONE. 2021;16(4): e0250269. 26 B k BN COVID 19 i ti d i t t d lt d 18 39 U it d St t M h M 2021 MMWR M b 27. Hesse, M. Women might be more skeptical of the covid vaccine. Why? The Washington Post. https://ww style/women-covid-vaccine-skeptical/2020/12/15/63551cac-3a61-11eb-9276-ae0ca72729be_story.h y p _ y 28. Loomba S, de Figueiredo A, Piatek SJ, de Graaf K, Larson HJ. Measuring the impact of COVID-19 vaccine misinformation on vaccination intent in the UK and USA. Nat Hum Behav. 2021;5(3):337–48. 28. Loomba S, de Figueiredo A, Piatek SJ, de Graaf K, Larson HJ. References Measuring the impact of COVID-19 vaccine misinformation on vaccination intent in the UK and USA. Nat Hum Behav. 2021;5(3):337–48. 29. Solís Arce JS, Warren SS, Meriggi NF, Scacco A, McMurry N, Voors M, et al. COVID-19 vaccine acceptance and hesitancy in low- and middle- income countries. Nat Med. 2021;27(8):1385–94. 29. Solís Arce JS, Warren SS, Meriggi NF, Scacco A, McMurry N, Voors M, et al. COVID-19 vaccine acceptance and hesitancy in low- and middle- income countries. Nat Med. 2021;27(8):1385–94. 30. Global Health 50/50. The COVID-19 sex-disaggregated data tracker \| global health 50/50. https://globalhealth5050.org/the-sex-gender- and-covid-19-project/the-data-tracker/. Accessed 24 Oct 2021. 30. Global Health 50/50. The COVID-19 sex-disaggregated data tracker \| global health 50/50. https://globalhealth5050.org/the-sex-gender- and-covid-19-project/the-data-tracker/. Accessed 24 Oct 2021. p j M. January 14, 2021 coronavirus news. https://edition.cnn.com/world/live-news/coronavirus-pandemic-vaccine-u e7c384a6d1b631c1ed48cef287256be. Accessed 24 Oct 2021. 31. Nedelman M. January 14, 2021 coronavirus news. https://edition.cnn.com/world/live-news/coronavirus-p 14-21/h_4e7c384a6d1b631c1ed48cef287256be. Accessed 24 Oct 2021. 32. Viana T. Mulheres se vacinam mais contra a Covid-19 do que homens no Ceará; 56% dos óbitos são masculinos - Metro - Diário do Nordeste. https://diariodonordeste.verdesmares.com.br/metro/mulheres-se-vacinam-mais-contra-a-covid-19-do-que-homens-no-ceara-56-dos- obitos-sao-masculinos-1.3100266. Accessed 24 Oct 2021. 32. Viana T. Mulheres se vacinam mais contra a Covid-19 do que homens no Ceará; 56% dos óbitos são masculinos - Metro - Diário do Nordeste. https://diariodonordeste.verdesmares.com.br/metro/mulheres-se-vacinam-mais-contra-a-covid-19-do-que-homens-no-ceara-56-dos- obitos-sao-masculinos-1.3100266. Accessed 24 Oct 2021. 33. Ungar L. The gender vaccine gap: more women than men are getting Covid shots \| Kaiser health news. https://khn.org/news/article/ gender-vaccine-gap-more-women-than-men-vaccinated-against-covid/. Accessed 24 Oct 2021.f 33. Ungar L. The gender vaccine gap: more women than men are getting Covid shots \| Kaiser health news. https://khn.org/news/article/ gender-vaccine-gap-more-women-than-men-vaccinated-against-covid/. Accessed 24 Oct 2021.f g g p g 34. Vloo A, Alessie RJM, Mierau JO, Boezen MH, Mierau JO, Franke L, et al. Gender differences in the mental health impact of the COVID-19 lockdown: longitudinal evidence from the Netherlands. SSM Popul Health. 2021;15: 100878.fi g g p g 34. Vloo A, Alessie RJM, Mierau JO, Boezen MH, Mierau JO, Franke L, et al. Gender differences in the mental health impact of the COVID-19 lockdown: longitudinal evidence from the Netherlands. SSM Popul Health. 2021;15: 100878.fi 35. Fenollar-Cortés J, Jiménez Ó, Ruiz-García A, Resurrección DM. Gender differences in psychological impact of the confinement during the COVID-19 outbreak in Spain: a longitudinal study. Front Psychol. 2021;12:2279. 35. Fenollar-Cortés J, Jiménez Ó, Ruiz-García A, Resurrección DM. Gender differences in psychological impact of the confinement during the COVID-19 outbreak in Spain: a longitudinal study. Front Psychol. 2021;12:2279. 36. References Kola L, Kohrt BA, Hanlon C, Naslund JA, Sikander S, Balaji M, et al. COVID-19 mental health impact and resp middle-income countries: reimagining global mental health. Lancet Psychiatry. 2021;8(6):535–50. middle-income countries: reimagining global mental health. Lancet Psychiatry. 2021;8(6):535–50. 37 Lewis D Superspreading drives the COVID pandemic and could help to tame it Nature 2021;590(7847):544 6 g g g y y ; ( ) 37. Lewis D. Superspreading drives the COVID pandemic—and could help to tame it. Nature. 2021;590(7847):544–6. 37. Lewis D. Superspreading drives the COVID pandemic—and could help to tame it. Nature. 2021;590(7847):544 p p g p p 38. Castelli EC, de Castro MV, Naslavsky MS, Scliar MO, Silva NSB, Andrade HS, et al. MHC variants associated with symptomatic versus asymp- tomatic SARS-CoV-2 infection in highly exposed individuals. Front Immunol. 2021;12:3898. 39. Sponchiato D. Pesquisa mostra o grau de medo e tensão dos brasileiros com o coronavírus \| Veja Saúde. h mente-saudavel/medo-e-tensao-brasileiros-coronavirus/. Accessed 24 Oct 2021. 40. Bi Q, Lessler J, Eckerle I, Lauer SA, Kaiser L, Vuilleumier N, et al. Insights into household transmission of SARS-CoV-2 from a population- based serological survey. Nat Commun. 2021;12(1):3643. 40. Bi Q, Lessler J, Eckerle I, Lauer SA, Kaiser L, Vuilleumier N, et al. Insights into household transmission of SARS-CoV-2 from a population- based serological survey. Nat Commun. 2021;12(1):3643. g y 41. Hall J, Harris R, Zaidi A, Woodhall S, Dabrera G, Dunbar J. HOSTED—England’s household transmission evaluation dataset: preliminary findings from a novel passive surveillance system of COVID-19. Int J Epidemiol. 2021;50(3):743–52. g y 41. Hall J, Harris R, Zaidi A, Woodhall S, Dabrera G, Dunbar J. HOSTED—England’s household transmission evaluation dataset: preliminary findings from a novel passive surveillance system of COVID-19. Int J Epidemiol. 2021;50(3):743–52. i y 42. Silva J, Lucas C, Sundaram M, Israelow B, Wong P, Klein J, et al. Saliva viral load is a dynamic unifying correlate of COVID-19 severity and mortality. MedRxiv Prepr Serv Health Sci. 2021. https://doi.org/10.1101/2021.01.04.21249236.li 42. Silva J, Lucas C, Sundaram M, Israelow B, Wong P, Klein J, et al. Saliva viral load is a dynamic unifying correlate of COVID-19 severity and mortality. MedRxiv Prepr Serv Health Sci. 2021. https://doi.org/10.1101/2021.01.04.21249236. y p p g 43. Kobayashi GS, Brito LA, Moreira DDP, Suzuki AM, Hsia GSP, Pimentel LF, et al. A novel saliva RT-LAMP workflow COVID-19 cases and restraining viral spread. Diagnostics. 2021;11(8):1400. 44. Brazil: share of internet users by age group 2020 \| Statista. References Emergence of highly infectious SARS-CoV-2 vari- ants in Bangladesh: the need for systematic genetic surveillance as a public health strategy. Trop Med Health. 2021;49(1):69.f g y g p gy p ; ( ) 15. Rocha R, Atun R, Massuda A, Rache B, Spinola P, Nunes L, et al. Effect of socioeconomic inequalities and vulnerabilities on health-system preparedness and response to COVID-19 in Brazil: a comprehensive analysis. Lancet Glob Health. 2021;9(6):e782–92. 5. Rocha R, Atun R, Massuda A, Rache B, Spinola P, Nunes L, et al. Effect of socioeconomic inequalities and vulne preparedness and response to COVID-19 in Brazil: a comprehensive analysis. Lancet Glob Health. 2021;9(6):e 15. Rocha R, Atun R, Massuda A, Rache B, Spinola P, Nunes L, et al. Effect of socioeconomic inequalities and vulnerabilities on health-system preparedness and response to COVID-19 in Brazil: a comprehensive analysis Lancet Glob Health 2021;9(6):e782–92 p p p p y 16 Guedes MBOG, de Assis SJC, Sanchis GJB, Araujo DN, Oliveira AGRDC, Lopes JM. COVID-19 in Brazilian cities: impact of social determinants, coverage and quality of primary health care. PLoS ONE. 2021;16(9): e0257347. p p p p y 16 Guedes MBOG, de Assis SJC, Sanchis GJB, Araujo DN, Oliveira AGRDC, Lopes JM. COVID-19 in Brazilian cities: impact of social determinants, coverage and quality of primary health care. PLoS ONE. 2021;16(9): e0257347. g q y p y 17. Rackimuthu S, Hasan MM, Bardhan M, Essar MY. COVID-19 vaccination strategies and polic is a pressing priority. Int J Health Plan Manag. 2021. https://doi.org/10.1002/hpm.3321. 18. Ghosh S, Moledina N, Hasan MM, Jain S, Ghosh A. Colossal challenges to healthcare workers combating the second wave of coronavirus disease 2019 (COVID-19) in India. Infect Control Hosp Epidemiol. 2021;2:1–2.f 18. Ghosh S, Moledina N, Hasan MM, Jain S, Ghosh A. Colossal challenges to healthcare workers combating the second wave of coronavirus disease 2019 (COVID-19) in India. Infect Control Hosp Epidemiol. 2021;2:1–2.f ( ) p p ; 19. Vahidy FS, Pan AP, Ahnstedt H, Munshi Y, Choi HA, Tiruneh Y, et al. Sex differences in susceptibility, severity, and outcomes of coronavirus disease 2019: cross-sectional analysis from a diverse US metropolitan area. PLoS ONE. 2021;16(1): e0245556.f 19. Vahidy FS, Pan AP, Ahnstedt H, Munshi Y, Choi HA, Tiruneh Y, et al. Sex differences in susceptibility, severity, and outcomes of coronavirus disease 2019: cross-sectional analysis from a diverse US metropolitan area. PLoS ONE. 2021;16(1): e0245556.f 19. References The state of hypertension care in 44 low-income and middle-income countries: a cross-sectional study of nationally representative individual-level data from 1.1 million adults. Lancet Lond Engl. 2019;394(10199):652–62. 8. Geldsetzer P, Manne-Goehler J, Marcus M-E, Ebert C, Zhumadilov Z, Wesseh CS, et al. The state of hypertension care in 44 low-income and middle-income countries: a cross-sectional study of nationally representative individual-level data from 1.1 million adults. Lancet Lond Engl. 2019;394(10199):652–62. 10. Aborode AT, Corriero AC, Fajemisin EA, Hasan MM, Kazmi SK, Olajiga O. Dengue and coronavirus disease (COVID-19) syndemic: double threat to an overburdened healthcare system in Africa. Int J Health Plan Manag. 2021. https://doi.org/10.1002/hpm.3334. 10. Aborode AT, Corriero AC, Fajemisin EA, Hasan MM, Kazmi SK, Olajiga O. Dengue and coronavirus disease (COVID-19) syndemic: double threat to an overburdened healthcare system in Africa. Int J Health Plan Manag. 2021. https://doi.org/10.1002/hpm.3334. 23456789) 1 3 (01231 Brief Communication \| https://doi.org/10.1007/s44192-022-00004-3 Discover Mental Health (2022) 2:1 11. Bong C-L, Brasher C, Chikumba E, McDougall R, Mellin-Olsen J, Enright A. The COVID-19 pandemic: effects on low- and middle-income countries. Anesth Analg. 2020;131(1):86–92. 11. Bong C-L, Brasher C, Chikumba E, McDougall R, Mellin-Olsen J, Enright A. The COVID-19 pandemic: effec countries. Anesth Analg. 2020;131(1):86–92. 11. Bong C-L, Brasher C, Chikumba E, McDougall R, Mellin-Olsen J, Enright A. The COVID-19 pandemic: effects on low- and middle-income countries. Anesth Analg. 2020;131(1):86–92. g ; ( ) 12 Islam Z, Kokash DM, Babar MS, Uday U, Hasan MM, Rackimuthu S, et al. Food security, conflict, and COVID-19: perspective from Afghani- stan. Am J Trop Med Hyg. 2021. https://doi.org/10.4269/ajtmh.21-1058. g 12 Islam Z, Kokash DM, Babar MS, Uday U, Hasan MM, Rackimuthu S, et al. Food security, conflict, and COVID-19: perspective from Afghani- stan. Am J Trop Med Hyg. 2021. https://doi.org/10.4269/ajtmh.21-1058. p yg p g j 3. Li SL, Pereira RHM, Prete CA Jr, Zarebski AE, Emanuel L, Alves PJH, et al. Higher risk of death from COVID-19 in l populations of São Paulo, Brazil. BMJ Glob Health. 2021;6(4): e004959. p p 14. Hasan MM, Rocha ICN, Ramos KG, Cedeño TDD, dos Santos Costa AC, Tsagkaris C, et al. Emergence of highly infectious SARS-CoV-2 vari- ants in Bangladesh: the need for systematic genetic surveillance as a public health strategy. Trop Med Health. 2021;49(1):69. p p 14. Hasan MM, Rocha ICN, Ramos KG, Cedeño TDD, dos Santos Costa AC, Tsagkaris C, et al. References https://www.statista.com/statistics/254724/age-distribution-of-internet-users- in-brazil/. Accessed 27 Dec 2021. 44. Brazil: share of internet users by age group 2020 \| Statista. https://www.statista.com/statistics/254724/age-distribution-of-internet-users- in-brazil/. Accessed 27 Dec 2021. 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https://openalex.org/W2599986806	https://europepmc.org/articles/pmc5364512?pdf=render	English	null	Circulating tumor cell status monitors the treatment responses in breast cancer patients: a meta-analysis	Scientific reports	2,017	cc-by	8,315	Circulating tumor cell status monitors the treatment responses in breast cancer patients: a meta- analysis 16 17 N received: 10 August 2016 accepted: 25 January 2017 Published: 24 March 2017 Wen-Ting Yan1, Xiang Cui1, Qing Chen2, Ya-Fei Li3, You-Hong Cui4, Yan Wang4 & Jun Jiang1 Whether circulating tumor cells (CTCs) can be used as an indicator of treatment response in breast cancer (BC) needs to be clarified. We addressed this issue by a meta-analysis. PubMed, EMBase and Cochrane library databases were searched in June 2016. Effect measures were estimated as pooled risk ratio (RR), odds ratio (OR) or mean difference by fixed- or random-effect models, according to heterogeneity of included studies. In total, 50 studies with 6712 patients were recruited. Overall analysis showed that there was a significant reduction of CTC-positive rate (RR = 0.68, 95% CI: 0.61–0.76, P < 0.00001) after treatment. Subgroup analyses revealed that neoadjuvant treatment, adjuvant treatment, metastatic treatment or combination therapy could reduce the CTC-positive rate, but surgery could not; moreover, the reduction was only found in HER2+ or HER2- patients but not in the triple-negative ones. Reduction of CTC-positive rate was associated with lower probability of disease progression (OR = 0.54, 95% CI: 0.33–0.89, P = 0.01) and longer overall survival period (mean difference = 11.61 months, 95% CI: 8.63–14.59, P < 0.00001) as well as longer progression-free survival period (mean difference = 5.07 months, 95% CI: 2.70–7.44, P < 0.0001). These results demonstrate that CTC status can serve as an indicator to monitor the effectiveness of treatments and guide subsequent therapies in BC. Metastasis is the major cause of cancer-related death in patients with breast cancer (BC)1. Despite the improve- ments in treatment, metastatic relapse may occur in about 30% of BC patients with lymph node-negative axilla and about 50% of BC patients with positive axilla within 5 years2. During the process of metastasis, cancer cells shed from primary tumors and migrate to distal sites through the blood or lymphatic system. Those migrating cells found in peripheral blood are known as circulating tumor cells (CTCs). CTCs are the metastatic precursors, which may have potential roles not only in predicting the risk of metastatic relapse and monitoring the treatment efficacy, but also in acting as a therapeutic target for preventing metastasis of cancers, including BC3–9. fi Up to now, CTCs have been well studied and are currently being used in the clinical setting10. However, there are controversial results about the effectiveness of different treatments to reduce CTCs. For example, Martin M. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports \| 7:43464 \| DOI: 10.1038/srep43464 Results St d h Results Study characteristics. We initially retrieved 1004 articles through database searching. 846 articles were excluded based on the title and abstract. Further, 108 articles were excluded after reviewing full-texts, in which the results of CTC status before and after treatment were not completely contained, or sample size was less than 20, and or duplicated data with other studies was reported. Finally, 50 studies were eligible for meta-analysis (Fig. 1)4,5,9,11–57.h g The eligible studies were conducted between 2009 and 2016 in America (USA), Asia (China, Japan and Korea) and Europe (Belgium, Czech, Denmark, France, Germany, Greece, Italy, Norway, Russia, Slovakia, Spain, and Turkey). The sample sizes of studies ranged from 21 to 2026 patients, accumulating 8019 patients before treat- ments and 6712 patients after treatments in total. Some studies reported the results in patients with particular molecular subtype BC, such as the HER2-positve or the triple-negative ones. The treatments performed in the studies included surgery, neoadjuvant treatment, adjuvant treatment, metastatic treatment, etc., either alone or in combination. The main characteristics of these studies are listed in Table S1. The quality of the studies was also estimated (Table S2). Overall CTC-positive rate is significantly decreased after therapies. The CTC status was detected with different platforms or methods and presented with different indicators in the 50 studies with 6712 patients. The CTC-positive rate was reported in all of the studies, in which the different cut-off values of CTC count for CTC-capturing methods were used, such as ≥5 CTCs/7.5 mL, ≥1 CTCs/7.5 mL and so on, and the different expression thresholds of epithelial genes (EpCAM, CK18, CK19) were used for RT-PCR method. Some studies also presented the CTC status as CTC count. We first performed an overall analysis of the 50 studies with 6712 patients with CTC-positive rate by the random-effects model, and found that the CTC-positive rate was signifi- cantly decreased after treatment compared to the baseline (RR = 0.68, 95% CI: 0.61 to 0.76, P < 0.00001; I2 = 73%, P < 0.00001) (Fig. 2). We then analyzed the change of CTC counts in the 7 studies (2324 cases) that simultaneously reported CTC-positive rate and CTC count before and after treatments. No significant change of CTC counts was observed (mean difference = −1.17 CTCs/7.5 mL, 95% CI: −3.17 to 0.84, P = 0.25; I2 = 65%, P = 0.009) (Fig. Circulating tumor cell status monitors the treatment responses in breast cancer patients: a meta- analysis 16 17 N et al. analyzed the change of CTCs in 117 BC patients and observed a substantial decline in CTC-positive rate after chemotherapy11, but Rack B. et al. conducted a larger perspective study with 2026 BC patients and found that the detection rate of CTCs after chemotherapy (22.1%) was even a bit higher than the baseline condition (21.5%)12. In addition, it needs also to be clarified whether molecular subtypes of BC affect CTC status under same treatments13. Hence, we conducted a meta-analysis of the published researches with measurement of CTCs before and after treatment in BC patients, and estimated the CTC-reducing effect of the current anti-tumor therapies. The CTC-reducing effects by different treatments were investigated separately in subgroup analyses, so were the effects in patients with different molecular subtypes. Then we also analyzed the relationship between reduction of CTCs and disease progression probability as well as survival period. This study followed the PRISMA criterions. 1Breast Disease Center, Southwest Hospital, Third Military Medical University, Chongqing 400038, China. 2Institute of Toxicology, College of Preventive Medicine, Third Military Medical University, Chongqing 400038, China. 3Department of Epidemiology, College of Preventive Medicine, Third Military Medical University, Chongqing 400038, China. 4Institute of Pathology and Southwest Cancer Center, Southwest Hospital, Third Military Medical University Chongqing 400038, China. Correspondence and requests for materials should be addressed to J.J. (email: jcbd@ medmail.com.cn) Scientific Reports \| 7:43464 \| DOI: 10.1038/srep43464 1 www.nature.com/scientificreports/ Figure 1. Flowchart for article search. Figure 1. Flowchart for article search. Figure 1. Flowchart for article search. Scientific Reports \| 7:43464 \| DOI: 10.1038/srep43464 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 2. Forest plot for the comparison of CTC-positive rate before and after treatment: overall analysis. The black diamond and its extremities indicate the pooled risk ratio center and 95% confidential interval. Figure 2. Forest plot for the comparison of CTC-positive rate before and after treatment: overall analysis. The black diamond and its extremities indicate the pooled risk ratio center and 95% confidential interval. Figure 2. Forest plot for the comparison of CTC-positive rate before and after treatment: overall analysis. The black diamond and its extremities indicate the pooled risk ratio center and 95% confidential interval. Figure 2. Forest plot for the comparison of CTC-positive rate before and after treatment: overall analys The black diamond and its extremities indicate the pooled risk ratio center and 95% confidential interval. Figure 3. Forest plot for the comparison of CTC count before and after treatment: overall analysis. The black diamond indicates the difference of CTC counts (cells/7.5 mL peripheral blood; the post-therapeutic counts minus the pre-therapeutic counts). Its center indicates the mean and the extremities indicate the 95% confidential interval. Figure 3. Forest plot for the comparison of CTC count before and after treatment: overall analysis. The black diamond indicates the difference of CTC counts (cells/7.5 mL peripheral blood; the post-therapeutic counts minus the pre-therapeutic counts). Its center indicates the mean and the extremities indicate the 95% confidential interval. Figure 3. Forest plot for the comparison of CTC count before and after treatment: overall analysis. The black diamond indicates the difference of CTC counts (cells/7.5 mL peripheral blood; the post-therapeutic counts minus the pre-therapeutic counts). Its center indicates the mean and the extremities indicate the 95% confidential interval. CTC-positive rates are decreased after neoadjuvant treatment, adjuvant treatment, me static treatment and combination therapy, but not after surgery. Clinically, many therape h d l d i f BC Th h i h d i l d i h l d 50 di l CTC-positive rates are decreased after neoadjuvant treatment, adjuvant treatment, meta- static treatment and combination therapy, but not after surgery. Clinically, many therapeutic methods are employed in treatment of BC. The therapeutic methods involved in the pooled 50 studies could be roughly classified into neoadjuvant setting, adjuvant setting, metastatic setting, surgery and combination therapy. To clarify the efficacy of various therapies on decreasing CTC-positive rate, we performed a subgroup analy- sis. Results St d h 3), while a significant reduction was observed with CTC-positive rate in the 7 studies investigated after treatment (RR = 0.65, 95% CI: 0.48 to 0.87, P = 0.004; I2 = 85%, P < 0.00001) (Figure S1). This inconsistent results between CTC-positive rate and CTC count might attribute to the heterogeneity among the studies, because the CTC count, a continuous variable, could be more susceptible to the variance caused by individual study than the CTC-positive rate, a dichotomized variable. So we made a sensitivity analysis by removing each study, and found that one of them (Bidard FC 2012) substantially affected the result. Once it was excluded, the significant decrease of CTC level after treatment was observed compared to pre-treatment (mean difference = −0.94, 95% CI: −1.49 to −0.38, P = 0.0010 by fixed-effects model). These results confirmed the promising application of CTCs in mon- itoring the effectiveness of treatments for BC. gf In addition, different methods as well as different cut-off values for a method were used in the studies. Hence, we also investigated the CTC-positive rate before and after treatment in the subgroups of different CTC-measuring methods. A significant reduction of CTC-positive rate after treatment was observed no matter how the CTC positivity was defined as ≥5CTCs/7.5 mL, ≥1CTCs/7.5 mL, or other threshold including a thresh- old for RT-PCR technique (Fig. 4). Scientific Reports \| 7:43464 \| DOI: 10.1038/srep43464 2 www.nature.com/scientificreports/ Compared to pre-treatment, CTC-positive rate were decreased after treatment in the neoadjuvant setting CTC-positive rates are decreased after neoadjuvant treatment, adjuvant treatment, meta- static treatment and combination therapy, but not after surgery. Clinically, many therapeutic methods are employed in treatment of BC. The therapeutic methods involved in the pooled 50 studies could be roughly classified into neoadjuvant setting, adjuvant setting, metastatic setting, surgery and combination therapy. To clarify the efficacy of various therapies on decreasing CTC-positive rate, we performed a subgroup analy- sis. Compared to pre-treatment, CTC-positive rate were decreased after treatment in the neoadjuvant setting Scientific Reports \| 7:43464 \| DOI: 10.1038/srep43464 3 CTC-positive rates are decreased after therapies in the HER2 -positive or -negative patients, ut not in the triple-negative patients Currently the clinical management of breast cancer mainly The disease progression of patients after treatments was available in 11 studies with 1363 patients. A significantly lower probability of disease progression was observed in the CTC-reduced patients (OR = 0.54, 95% CI: 0.33 to 0.89, P = 0.01; I2 = 45%, P = 0.05) (Fig. 7). The 11 studies could be divided into 3 subgroups, namely metastatic setting (6 studies with 244 patients), adjuvant setting (3 studies with 1095 patients), neoadjuvant set- ting (2 studies with 24 patients). Significantly lower probability of disease progression in CTC-reduced patients was observed in the metastatic setting subgroup (OR = 0.37, 95% CI: 0.20 to 0.66, P = 0.0008; I2 = 13%, P = 0.33) (Fig. 8). But there was no significant difference of probability of disease progression in patients with or without CTC reduction in the adjuvant setting subgroup (OR = 0.73, 95% CI: 0.44 to 1.22, P = 0.23; I2 = 54%, P = 0.11) and in the neoadjuvant setting subgroup (OR = 0.30, 95% CI: <0.01 to 77.67, P = 0.67; I2 = 80%, P = 0.03) (Fig. 8). There was a significant heterogeneity in the adjuvant-setting subgroup. When a study (Rack B 2014) was excluded, the heterogeneity was relieved (P = 0.68, I2 = 0%) and the correlation of CTC status and disease progression became significant (OR = 0.57, 95%CI: 0.37 to 0.86, P = 0.008 by fixed-effect model). In the neoadju- vant setting subgroup, the sample size was extremely small, implying that did not have adequate statistic power. The results showed that the reduction of CTCs was significantly associated with decreased probability of disease progression, increased overall survival and progression-free survival period. Sensitivity Analysis and Publication Bias. Among the 50 studies included for the pooled RR estimation, no single one contributed substantial influence. When we analyzed the change of CTC counts post-treatments with 7 studies reported CTC status both in CTC-positive rate and CTC counts, the sensitivity analysis was tested by removal of each study. One of them (Bidard FC 2012) was found to substantially affect the heterogeneity and the significance of overall effect: when it was excluded, the heterogeneity was relieved (P = 0.69, I2 = 0%) and the change of CTC counts after treatment became significant (mean difference = −0.94, 95%CI: −1.49 to −0.38, P = 0.0010 by fixed-effect model). In the adjuvant-setting subgroup analysis of disease progression (Fig. CTC-positive rates are decreased after therapies in the HER2 -positive or -negative patients, ut not in the triple-negative patients Currently the clinical management of breast cancer mainly CTC-positive rates are decreased after therapies in the HER2 -positive or -negative patients, but not in the triple-negative patients. Currently, the clinical management of breast cancer mainly relies on the molecular subtypes based on the expression of estrogen receptor, progesterone receptor and HER2 in primary tumors. It is well known that different molecular subtypes of BC are associated with distinct malig- nant nature and drug response. Therefore, we further assessed the effects of therapies on reduction of CTCs in different subgroups, including HER2-positive, HER2-negative and triple-negative BC. Compared to pre-therapy, CTC-positive rates were significantly decreased after treatment in HER2-positive patients (RR = 0.68, 95% CI: 0.57 to 0.82, P < 0.0001; I2 = 0%, P = 0.59) and HER2-negative patients (RR = 0.52, 95% CI: 0.31 to 0.86, P = 0.01; I2 = 66%, P = 0.01), but not in the triple-negative ones (RR = 0.38, 95% CI: 0.06 to 2.33, P = 0.29; I2 = 72%, P = 0.06) (Fig. 6). These results indicate that different molecular subtypes of BC affect the efficacy of therapeutics on reducing CTCs. The poor reduction of CTC-positive rate in triple-negative BC is consistent with clinical outcome, implying that current therapies should be further optimized and the new therapeutic methods should be developed for this specific molecular subtype. Correlation between status of CTCs after treatment and prognosis of the patients. Because CTCs are shed from the primary tumor and serve as the metastatic precursors, the changes of CTC status after therapies may associate with the risk of metastasis as well as the outcome of patients. We compared the prognosis of the CTC-reduced patients with that of the CTC-unchanged or -elevated patients after treatment. The overall survival (OS) of patients after treatment was available in 2 studies (71 cases), in both of which the patients received metastatic setting. The CTC-reduced patients had a longer overall survival period compared to the CTC-unchanged or -elevated patients (mean difference = 11.61 months, 95% CI: 8.63 to 14.59, P < 0.00001; I2 = 69%, P = 0.07) (Fig. 7). The progression-free survival (PFS) of patients after treatment were available in 3 studies (125 patients), in which all the patients received metastatic setting. The CTC-reduced patients had a longer PFS than the CTC-unchanged or -elevated patients (mean difference = 5.07 months, 95% CI: 2.70 to 7.44, P < 0.0001; I2 = 96%, P < 0.00001) (Fig. 7). CTC-positive rates are decreased after therapies in the HER2 -positive or -negative patients, ut not in the triple-negative patients Currently the clinical management of breast cancer mainly 8), one of the three recruited studies (Rack B 2014) was found to substantially affect the heterogeneity and the significance of overall effect: when it was excluded, the heterogeneity was relieved (P = 0.68, I2 = 0%) and the correlation of CTC status and disease progression became significant (OR = 0.57, 95%CI: 0.37 to 0.86, P = 0.008 by fixed-effect model). No substantial publication bias was found according to the Funnel plot (Fig. 9). Scientific Reports \| 7:43464 \| DOI: 10.1038/srep43464 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 4. Forest plot for the comparison of CTC-positive rate before and after treatment: subgroup analysis of different CTC determination methods (threshold). The results of ≥5 CTCs/7.5 mL as positi (A) ≥1 CTCs/7.5 mL as positive (B) other threshold as positive (C) and RT-PCR method (D) are shown, respectively. The center of black diamond and its extremities indicate the pooled risk ratio and 95% conf interval. t Figure 4. Forest plot for the comparison of CTC-positive rate before and after treatment: subgroup analysis of different CTC determination methods (threshold). The results of ≥5 CTCs/7.5 mL as positive (A) ≥1 CTCs/7.5 mL as positive (B) other threshold as positive (C) and RT-PCR method (D) are shown, respectively. The center of black diamond and its extremities indicate the pooled risk ratio and 95% confidential interval. Scientific Reports \| 7:43464 \| DOI: 10.1038/srep43464 4 www.nature.com/scientificreports/ (RR = 0.65, 95% CI: 0.48 to 0.88, P = 0.006; I2 = 40%, P = 0.08), and adjuvant setting (RR = 0.89, 95% CI: 0.76 to 1.02, P = 0.10; I2 = 62%, P = 0.007), the metastatic setting (RR = 0.59, 95% CI: 0.50 to 0.70, P < 0.00001; I2 = 66%, P < 0.0001) and the combination therapy (RR = 0.78, 95% CI: 0.62 to 0.97, P = 0.03; I2 = 29%, P = 0.20), but not in the surgery (RR = 1.27, 95% CI: 0.71 to 2.27, P = 0.42; I2 = 45%, P = 0.16) (Fig. 5). These results indicate that surgery as a local treatment can not eliminate CTCs timely, because CTCs can survive in peripheral blood for a certain amount of time58, suggesting that patients with CTC positive should be further treated with other thera- pies after surgery, in order to decrease the risk of metastasis and recurrence. Discussion The black diamond and its extremities indicate the pooled risk ratio center and 95% confidential interval. The actual application of CTCs in clinical setting relies on the progression of detection technologies. In the past decades, a number of technically diverse platforms have been developed for CTC assay60. However, for any technology to be used in the clinic, demonstration of analytic validity, clinical validity, and clinical utility is required60. Up to now, the only system approved by the Food and Drug Administration (FDA) as an aid in mon- itoring patients with metastatic breast, colorectal, or prostate cancer is CellSearch® (Veridex, Raritan, NJ, USA). Also, CellSearch® is the only semi-automated system and has contributed considerably to the development of standards for CTC enumeration. Nevertheless, there are disadvantages to be perfect for it. Its enrichment/capture technology is based on epithelial marker EpCAM, which is usually with low sensitivity and efficiency, due to the lost expression of EpCAM in CTCs by EMT process61,62. In our meta-analysis, other CTC detection methods are employed in some studies, including RT-PCR, which determined the CTC status by detecting the mRNA expression of epithelial markers, such as EpCAM or CKs. There is a probability that the CTC detection meth- ods based on different labels or rationales would find different counts of CTCs in the same individual patients. Given the CTC rarity, especially in non-metastatic breast cancer, continuous training and central image review is required in order to gain best inter-reader agreement. A recent study evaluated the inter-reader agreement in 22 readers from 15 academic laboratories and 8 readers from two Veridex laboratories with non-metastatic (M0) and metastatic (M1) breast cancer samples. For CTC definition (No CTC vs. CTC), the median agreement between academic readers and VC was 92% (range 69 to 97%) with a median κ of 0.83 (range: 0.37 to 0.93). The inter-reader agreement for CTC definition was high. Reduced agreement was observed in M0 patients with low CTC counts63. In addition, the inconsistence of the cut-off value to determine CTC-positive amongst studies would be a limitation for actual application of CTCs in clinical setting (Fig. 4). Standard or uniform protocol for CTC measurement would be required before this indicator could be clinically adopted.h There were several limitations in the present research. First, most of studies included in this meta-analysis were consisted of patients receiving certain therapy alone without an appropriate negative control. Discussion It has been well known that even localized tumors without clinically apparent metastasis give rise to CTCs. Because generation of CTCs is an indispensable step of the metastatic process of tumors, the promising appli- cation as a noninvasive blood biomarker in prognosis and response to therapy are very attractive. Although the actual utility of CTCs remains largely academic59, many studies have reported the detection of CTCs to facilitate early diagnosis of relapse or metastasis and improve the treatment decisions. In present meta-analysis, we ana- lyzed the changes of CTC status after therapies compared to that of before therapies in pooled 50 studies with 6712 BC patients, and demonstrated that CTC status was a useful indicator to monitor the treatment response, and predict the outcome of patients. Scientific Reports \| 7:43464 \| DOI: 10.1038/srep43464 5 www.nature.com/scientificreports/ Figure 5. Forest plot for the comparison of CTC-positive rate before and after treatment: subgroup analysis of different therapy strategies. The results of neoadjuvant setting (A), adjuvant setting (B), meta setting (C), surgery (D) and combination therapy (E) are shown, respectively. The center of black diamon its extremities indicate the pooled risk ratio and 95% confidential interval. Figure 5. Forest plot for the comparison of CTC-positive rate before and after treatment: subgroup analysis of different therapy strategies. The results of neoadjuvant setting (A), adjuvant setting (B), metastatic setting (C), surgery (D) and combination therapy (E) are shown, respectively. The center of black diamond and its extremities indicate the pooled risk ratio and 95% confidential interval. 6 Scientific Reports \| 7:43464 \| DOI: 10.1038/srep43464 www.nature.com/scientificreports/ Figure 6. Forest plot for the comparison of CTC-positive rate before and after treatment: subgroup analysis of different molecular subtypes. The results of HER2-positive subtype (A) HER2-negative subtype (B) and triple-negative subtype (C) are shown, respectively. The black diamond and its extremities indicate the pooled risk ratio center and 95% confidential interval. Figure 6. Forest plot for the comparison of CTC-positive rate before and after treatment: subgroup analysis of different molecular subtypes. The results of HER2-positive subtype (A) HER2-negative subtype (B) and triple-negative subtype (C) are shown, respectively. The black diamond and its extremities indicate the pooled risk ratio center and 95% confidential interval. Figure 6. Forest plot for the comparison of CTC-positive rate before and after treatment: subgroup analysis of different molecular subtypes. The results of HER2-positive subtype (A) HER2-negative subtype (B) and triple-negative subtype (C) are shown, respectively. Scientific Reports \| 7:43464 \| DOI: 10.1038/srep43464 Discussion Second, some studies with substantial sample heterogeneities, which were caused by the complexity of patient characteristics (race, age), therapeutic details (drug, dose, treatment periodicity, etc.) or other factors, were excluded in our Scientific Reports \| 7:43464 \| DOI: 10.1038/srep43464 7 www.nature.com/scientificreports/ Figure 7. Forest plot for the comparison of prognosis between the CTC-reduced patients and those without CTC-unchanged or -elevated. The diamond indicated the odds ratio of disease progression (A) the difference of progression-free survival (B) or overall survival period (C). The centers of the diamonds indicated the pooled odds ratio (A) or the mean difference (B,C) and the extremities indicated the 95% confidential interval. Figure 7. Forest plot for the comparison of prognosis between the CTC-reduced patients and those without CTC-unchanged or -elevated. The diamond indicated the odds ratio of disease progression (A) the difference of progression-free survival (B) or overall survival period (C). The centers of the diamonds indicated the pooled odds ratio (A) or the mean difference (B,C) and the extremities indicated the 95% confidential interval. Figure 7. Forest plot for the comparison of prognosis between the CTC-reduced patients and those without CTC-unchanged or -elevated. The diamond indicated the odds ratio of disease progression (A) the difference of progression-free survival (B) or overall survival period (C). The centers of the diamonds indicated the pooled odds ratio (A) or the mean difference (B,C) and the extremities indicated the 95% confidential interval. subgroup analyses. Third, the sample sizes of some subgroups were relatively small, which might affect the detec- tion of potential difference. Fourth, CTCs were thought to be a set of cells with different characteristics, so it would be more meaningful to investigate the correlation of change of the CTC subpopulations after treatment with patient prognosis. However, so far few data are available to perform an analysis.fi p p g p y In summary, the present meta-analysis demonstrated that the status of CTCs is a useful indicator of the effi- cacy of therapies for BC, which may help clinicians make a decision for further personalized therapy of patients. However, it is on the way for application of CTCs in clinical setting because there are still challenges presented in analytic validity, clinical validity, and clinical utility of CTCs. Methods h Forest plot for the comparison of prognosis between the CTC-reduced patients and those without CTC-unchanged or –elevated: subgroup analysis of disease progression. The diamond indicated the odds ratio of disease progression in the metastatic setting (A), the adjuvant setting (B) or the neoadjuvant setting (C). The centers of the diamonds indicated the pooled odds ratio and the extremities indicated the 95% confidential interval. Figure 9. Funnel plot for the studies included for comparison of CTC-positive rate before and after treatment. Figure 9. Funnel plot for the studies included for comparison of CTC-positive rate before and after treatment. molecular subtypes of tumor), assessment of CTCs (i.e., methods of CTC detection, blood volume, the cut-off value of CTCs, the count of CTCs and/or the positive rate of CTCs) and the type of the treatments. If CTCs at more than one phase of follow ups were reported in a study, the latest phase with follow-up rate ≥75% was cho- sen; if no phase met the criteria, the earliest phase was chosen. The prognosis (progression-free survival, overall survival) was also extracted for assessing prognostic value of reducing CTCs. molecular subtypes of tumor), assessment of CTCs (i.e., methods of CTC detection, blood volume, the cut-off value of CTCs, the count of CTCs and/or the positive rate of CTCs) and the type of the treatments. If CTCs at more than one phase of follow ups were reported in a study, the latest phase with follow-up rate ≥75% was cho- sen; if no phase met the criteria, the earliest phase was chosen. The prognosis (progression-free survival, overall survival) was also extracted for assessing prognostic value of reducing CTCs. Quality assessment of primary studies. The quality of each included study was evaluated by a scale based on the Newcastle–Ottawa Quality Assessment Scale. Quality assessment of primary studies. The quality of each included study was evaluated by a scale ased on the Newcastle–Ottawa Quality Assessment Scale. Statistical analysis. The pooled RR (relative risk) and mean difference were calculated to analyze the dif- ference of CTCs between pre-therapy and post-therapy by fixed model or random-effects model according to the heterogeneity of the studies, which was estimated by the Cochran’s Q test and the I2 index (P value < 0.10 or I2 over 50% was define as substantial heterogeneity). RR less than 1 or mean difference less than 0 indicated declined CTCs in the peripheral blood. Methods h Search strategy. A comprehensive literary search for potential studies was searched in June 2016 without time or language restrictions. The electronic databases include PubMed, EMBase and Cochrane library. The key- words and MeSH terms were variably combined: “circulating tumor cell (s)”, “breast cancer”, “therapy”. The search strategy was intended to exclude reviews, comments, letters and editorials, which have irrelevant study data, by screening the titles and abstracts of publications. Eligibility criteria. Inclusion criteria: (i) enrolled patients with BC were pathologically diagnosed; (ii) CTCs were detected by any method, including cell capture and quantitative PCR; (iii) the patients’ CTC status both pre- and post-therapy was reported. p py p Exclusion criteria: (i) cell-line experiments or animal models; (ii) a small size <20 patients; (iii) when infor- mation of the same patients was reported in different studies, only the latest and most informative one was included; (iv) CTC status was reported pre- or post-therapy alone; (v) CTC status pre- and post-therapy was reported in different cohort of patients; (vi) Studies which reported CTCs only in continuous style were excluded, as their results could not be integrated with the majority of studies which reported the results in dichotomous pattern (positive vs. negative). Data extraction. Studies were reviewed and extracted independently by two reviewers (WT Yan and Q Chen). The primary data of the included studies were following: the general information (i.e., the first author, the year of publication, the nationality of studies), sample size, the patients’ characteristics (i.e., ages, tumor stage, Scientific Reports \| 7:43464 \| DOI: 10.1038/srep43464 8 www.nature.com/scientificreports/ Figure 8. Forest plot for the comparison of prognosis between the CTC-reduced patients and those without CTC-unchanged or –elevated: subgroup analysis of disease progression. The diamond indicated the odds ratio of disease progression in the metastatic setting (A), the adjuvant setting (B) or the neoadjuvant setting (C). The centers of the diamonds indicated the pooled odds ratio and the extremities indicated the 95% confidential interval. Figure 8. Forest plot for the comparison of prognosis between the CTC-reduced patients and those without CTC-unchanged or –elevated: subgroup analysis of disease progression. The diamond indicated the odds ratio of disease progression in the metastatic setting (A), the adjuvant setting (B) or the neoadjuvant setting (C). The centers of the diamonds indicated the pooled odds ratio and the extremities indicated the 95% confidential interval. Figure 8. Methods h Pooled OR (odds ratio) and mean difference were calculated to analyze Scientific Reports \| 7:43464 \| DOI: 10.1038/srep43464 9 www.nature.com/scientificreports/ the difference of progression between the patients with different dynamic conditions of CTCs (decreasing vs. increasing or persistent elevated). Subgroup analyses were performed for CTC determination methods, treat- ments and molecular subtypes of primary tumor as long as two or more studies were available to be included. Forest plot was used to illustrate the pooled RR mean difference. All analyses were run by Review Manager Version 5.3 (Cochrane Collaboration, Copenhagen, Denmark). the difference of progression between the patients with different dynamic conditions of CTCs (decreasing vs. increasing or persistent elevated). Subgroup analyses were performed for CTC determination methods, treat- ments and molecular subtypes of primary tumor as long as two or more studies were available to be included. Forest plot was used to illustrate the pooled RR mean difference. All analyses were run by Review Manager Version 5.3 (Cochrane Collaboration, Copenhagen, Denmark). Sensitivity analysis and estimation of publication bias. To evaluate the influence of individual study on the pooled RR or mean difference, sensitivity analysis was performed by removing each eligible study sepa- rately. Funnel plot developed by Begg was used to detect potential publication bias which might affect the validity of the results. References Circulating Tumor Cells (Ctcs) and Metronomic Oral Vinorelbine (Ov) in Advanced Breast Cancer (Abc) Patient (Pts): Exploratory Prospective Analysis. Annals of Oncology 23, 26–26, doi: 10.1093/annonc/mds040 (2012). 9. Aurilio, G. et al. Circulating Tumor Cells (Ctcs) and Metronomic Oral Vinorelbine (Ov) in Advanced Breast Cancer (Abc) Patients (Pts): Exploratory Prospective Analysis. Annals of Oncology 23, 26–26, doi: 10.1093/annonc/mds040 (2012). d l l l d b l l fi f l ll l l d 9. Aurilio, G. et al. Circulating Tumor Cells (Ctcs) and Metronomic Oral Vinorelbine (Ov) in Advanced Breast Cancer (Abc) Patients (Pts): Exploratory Prospective Analysis. Annals of Oncology 23, 26–26, doi: 10.1093/annonc/mds040 (2012).i 9. Aurilio, G. et al. Circulating Tumor Cells (Ctcs) and Metronomic Oral Vinorelbine (Ov) in Advanced Breast Cancer (Abc) Patient (Pts): Exploratory Prospective Analysis. Annals of Oncology 23, 26–26, doi: 10.1093/annonc/mds040 (2012).i y y f gy 0. Masuda, T. et al. Clinical and biological significance of circulating tumor cells in cancer. Mol Oncol 10, 408–417, doi: 10.1016/j molonc.2016.01.010 (2016).i 11. Martin, M. et al. Circulating tumor cells following first chemotherapy cycle: An early and strong predictor of outcome in patients with metastatic breast cancer. Oncologist 18, 917–923 (2013). g 2. Rack, B. et al. Circulating tumor cells predict survival in early average-to-high risk breast cancer patients. J Natl Cancer Inst 106, doi 10.1093/jnci/dju066 (2014).t j j 3. Van Dalum, G. et al. Circulating tumor cells before and during follow-up after breast cancer surgery. International Journal o Oncology 46, 407–413 (2015). gy ( ) 4. Cabinakova, M. et al. Predictive factors for the presence of tumor cells in bone marrow and peripheral blood in breast cance patients. Neoplasma 62, 259–268, doi: 10.4149/neo_2015_031 (2015). p p 5. Magbanua, M. J. et al. Circulating tumor cell analysis in metastatic triple-negative breast cancers. Clin Cancer Res 21, 1098–1105 doi: 10.1158/1078-0432.CCR-14-1948 (2015). 6. Maltoni, R. et al. Circulating tumor cells in early breast cancer: A connection with vascular invasion. Cancer Lett 367, 43–48, doi 10.1016/j.canlet.2015.06.020 (2015). j ( ) 17. Kasimir-Bauer, S. et al. Does primary neoadjuvant systemic therapy eradicate minimal residual disease? Analysis of disseminated and circulating tumor cells before and after therapy. Breast Cancer Res 18, 20, doi: 10.1186/s13058-016-0679-3 (2016).il 18. Pierga, J. Y. et al. Pathological response and circulating tumor cell count identifies treated HER2+ inflammatory breast cancer patients with excellent prognosis: BEVERLY-2 survival data. Clin Cancer Res 21, 1298–1304, doi: 10.1158/1078-0432.CCR-14-1705 (2015).i 19. Wang, H. References Y. et al. Detection of circulating tumor cell-specific markers in breast cancer patients using the quantitative RT-PCR assay. International Journal of Clinical Oncology (2015).f f gy 0. Zhang, J. L. et al. Effects of Herceptin on circulating tumor cells in HER2 positive early breast cancer. Genet Mol Res 14, 2099–2103 doi: 10.4238/2015.March.20.20 (2015). 1. Barnadas, A. et al. Bone turnover markers as predictive indicators of outcome in patients with breast cancer and bone metastase treated with bisphosphonates: Results from a 2-year multicentre observational study (ZOMAR study). Bone 68, 32–40 (2014). 2. Bian, L. et al. Prediction value for dynamic changes of circulating tumor cell in therapeutic response and prognosis of Chinese metastatic breast cancer patients. Zhonghua Yi Xue Za Zhi 94, 265–268 (2014). p g 3. Horn, P., Jakobsen, E. H., Madsen, J. S. & Brandslund, I. New approach for interpreting changes in circulating tumour cells (CTC for evaluation of treatment effect in metastatic breast cancer. Translational Oncology 7, 694–701 (2014). f gy 24. Lavrov, A. V. et al. Analysis of Circulating Tumor Cells in Patients with Triple Negative Breast Cancer during Preoperative Chemotherapy. Bulletin of Experimental Biology and Medicine (2014). f 24. Lavrov, A. V. et al. Analysis of Circulating Tumor Cells in Patients w Chemotherapy. Bulletin of Experimental Biology and Medicine (2014). Chemotherapy. Bulletin of Experimental Biology and Medicine (2014 py f p gy 25. Mikulova, V. et al. Detection of circulating tumor cells during follow-up of patients with early breast cancer: Clinical utilit monitoring of therapy efficacy. Scand J Clin Lab Invest 74, 132–142, doi: 10.3109/00365513.2013.864784 (2014).i g pyfi y 26. Peeters, D. J. et al. Detection and prognostic significance of circulating tumour cells in patients with metastatic breast ca according to immunohistochemical subtypes. Br J Cancer 110, 375–383, doi: 10.1038/bjc.2013.743 (2014). g y j 27. Usiakova, Z. et al. Circulating tumor cells in patients with breast cancer: monitoring chemotherapy success. In Vivo 28, 605–614 (2014). 8. Azim, H. A. Jr. et al. Circulating tumor cells and response to neoadjuvant paclitaxel and HER2-targeted therapy: a sub-study from the NeoALTTO phase III trial. Breast 22, 1060–1065, doi: 10.1016/j.breast.2013.08.014 (2013). 28. Azim, H. A. Jr. et al. Circulating tumor cells and response to neoadjuvant paclitaxel and HER2-targ the NeoALTTO phase III trial. Breast 22, 1060–1065, doi: 10.1016/j.breast.2013.08.014 (2013). 9. Bidard, F. C. et al. References 1. Ursaru, M., Jari, I., Naum, A., Scripcariu, V. & Negru, D. Causes of Death in Patients with Stage 0-Ii Breast Cancer. Revista medico chirurgicala a Societatii de Medici si Naturalisti din Iasi 119, 374–378 (2015). g 2. Maltoni, R., Gallerani, G., Fici, P., Rocca, A. & Fabbri, F. CTCs in early breast cancer: A path worth taking. Cancer Lett 376, 205–210, doi: 10.1016/j.canlet.2016.03.051 (2016). g 2. Maltoni, R., Gallerani, G., Fici, P., Rocca, A. & Fabbri, F. CTCs in early breast cancer: A path worth taking. Cancer Lett 376, 205–210 doi: 10.1016/j.canlet.2016.03.051 (2016). j ( ) 3. Beije, N., Jager, A. & Sleijfer, S. Circulating tumor cell enumeration by the CellSearch system: the clinician’s guide to breast cance treatment? Cancer Treat Rev 41, 144–150, doi: 10.1016/j.ctrv.2014.12.008 (2015). j 4. Wallwiener, M. et al. Serial enumeration of circulating tumor cells predicts treatment response and prognosis in metastatic b cancer: a prospective study in 393 patients. BMC Cancer 14, 512, doi: 10.1186/1471-2407-14-512 (2014).t p p y p 5. Pierga, J. Y. et al. Circulating tumor cell detection predicts early metastatic relapse after neoadjuvant chemotherapy in large operable and locally advanced breast cancer in a phase II randomized trial. Clin Cancer Res 14, 7004–7010, doi: 10.1158/1078-0432.CCR-08- 0030 (2008). 5. Pierga, J. Y. et al. Circulating tumor cell detection predicts early metastatic relapse after neoadjuvant chemotherapy in large operable and locally advanced breast cancer in a phase II randomized trial. Clin Cancer Res 14, 7004–7010, doi: 10.1158/1078-0432.CCR-08- 0030 (2008). ( ) 6. Janni, W. et al. Pooled Analysis of the Prognostic Relevance of Circulating Tumor Cells in Primary Breast Cancer. Clin Cancer Res, doi: 10.1158/1078-0432.ccr-15-1603 (2016).i ( ) 6. Janni, W. et al. Pooled Analysis of the Prognostic Relevance of Circulating Tumor Cells in Primary Breast Cancer. Clin Cancer Res doi: 10.1158/1078-0432.ccr-15-1603 (2016).i 7. Pierga, J. Y. et al. Impact of targeted therapy on circulating tumor cells in metastatic breast cancer treated by first-line chemotherapy IC 2006-04 study. Cancer Research 71 (2011). y R ( ) 8. Smerage, J. B. et al. Circulating tumor cells and response to chemotherapy in metastatic breast cancer: SWOG S0500. J Clin Oncol 32, 3483–3489, doi: 10.1200/JCO.2014.56.2561 (2014). y 8. Smerage, J. B. et al. Circulating tumor cells and response to chemotherapy in metastatic breast cancer: SWOG S0500. J Clin Onco 32, 3483–3489, doi: 10.1200/JCO.2014.56.2561 (2014). ( ) 9. Aurilio, G. et al. www.nature.com/scientificreports/ Assessment of circulating tumor cells and serum markers for progression-free survival prediction in metastatic breast cancer: a prospective observational study. Breast Cancer Res 14, R29, doi: 10.1186/bcr3114 (2012). 43. Hayashi, N. et al. Prognostic value of HER2-positive circulating tumor cells in patients with metastatic breast cancer. Int J Clin Oncol 17, 96–104, doi: 10.1007/s10147-011-0260-0 (2012). 44. Mego, M. et al. Prognostic value of EMT-circulating tumor cells in metastatic breast can-cer patients undergoing high- chemotherapy with autologous hematopoietic stem cell transplantation. Journal of Cancer 3, 369–380 (2012). 45. Nadal, R. et al. Biomarkers characterization of circulating tumour cells in breast cancer patients. Breast Cancer Res 14, R71, doi: 10.1186/bcr3180 (2012).h 46. Serrano, M. J. et al. Dynamics of circulating tumor cells in early breast cancer under neoadjuvant therapy. Exp Ther Med 4, 43–48, doi: 10.3892/etm.2012.540 (2012).f 47. Daskalakis, M. et al. Assessment of the effect of surgery on the kinetics of circulating tumour cells in patients with operable b cancer based on cytokeratin-19 mRNA detection. Eur J Surg Oncol 37, 404–410, doi: 10.1016/j.ejso.2011.02.003 (2011). 47. Daskalakis, M. et al. Assessment of the effect of surgery on the kinetics of circulating tumour cells in patients with opera cancer based on cytokeratin-19 mRNA detection. Eur J Surg Oncol 37, 404–410, doi: 10.1016/j.ejso.2011.02.003 (2011). 48. Hartkopf, A. D., Wagner, P., Wallwiener, D., Fehm, T. & Rothmund, R. Changing levels of circulating tumor chemotherapy response in patients with metastatic breast cancer. Anticancer Res 31, 979–984 (2011). 48. Hartkopf, A. D., Wagner, P., Wallwiener, D., Fehm, T. & Rothmund, R. Changing levels of circulating tumor cells in monitoring chemotherapy response in patients with metastatic breast cancer. Anticancer Res 31, 979–984 (2011). 49. Reinholz, M. M. et al. Cytokeratin-19 and mammaglobin gene expression in circulating tumor cells from metastatic breast cancer patients enrolled in North Central Cancer Treatment Group trials, N0234/336/436/437. Clin Cancer Res 17, 7183–7193, doi: 10.1158/1078-0432.CCR-11-0981 (2011). 50. Tokudome, N. et al. Detection of circulating tumor cells in peripheral blood of heavily treated metastatic breast cancer patients. Breast Cancer 18, 195–202, doi: 10.1007/s12282-011-0259-4 (2011). 1. Bidard, F. C. et al. Clinical value of circulating endothelial cells and circulating tumor cells in metastatic breast cancer patients treated first line with bevacizumab and chemotherapy. Ann Oncol 21, 1765–1771, doi: 10.1093/annonc/mdq052 (2010). i py q 52. Cristofanilli, M. et al. Circulating tumor cells and FDG-PET/CT: Biological and functional methods for therapeutic monitoring in metastatic breast cancer. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 2. Karaba, M. et al. Circulating tumor cells (CTCs) and epithelial mesenchymal transition (EMT) in primary breast cancer. Journal o Clinical Oncology 31 (2013). Clinical Oncology 31 (2013). 33. Mathiesen, R. R. et al. Detection and monitoring of circulating endothelial cells, circulating tumor cells and disseminated tumor cells during neoadjuvant breast cancer treatment including bevacizumab Cancer Research 73 (2013) gy 33. Mathiesen, R. R. et al. Detection and monitoring of circulating endothelial cells, circulating tumor cells and disseminated tu cells during neoadjuvant breast cancer treatment including bevacizumab. Cancer Research 73 (2013). 4. Nakayama, Y., Takahashi, S. & Ito, Y. A phase II clinical trial of neoadjuvant therapy with zoledronic acid for operable breast cancer Journal of Clinical Oncology 31 (2013).t f gy ( ) 5. Neugebauer, J. K. et al. Persistence of HER2 overexpression on circulating tumor cells in patients after systemic treatment for HER2 positive breast cancer: Follow-up results of the German Success B trial. Journal of Clinical Oncology 31 (2013). p p f gy 36. Pierga, J. Y. et al. Circulating tumor cells and brain metastasis outcome in patients with HER2-positive breast cancer: The LANDSCAPE trial. Annals of Oncology 24, 2999–3004 (2013).f g g LANDSCAPE trial. Annals of Oncology 24, 2999–3004 (2013). f gy 7. Roop, R. P. et al. A randomized phase II trial investigating the effect of platelet function inhibition on circulating tumor cells in patients with metastatic breast cancer. Clin Breast Cancer 13, 409–415, doi: 10.1016/j.clbc.2013.08.006 (2013). p j 8. Smerage, J. B. et al. Monitoring apoptosis and Bcl-2 on circulating tumor cells in patients with metastatic breast cancer. Molecula Oncology 7, 680–692 (2013). gy 39. Tryfonidis, K. et al. A multicenter phase I-II study of docetaxel plus epirubicin plus bevacizumab as first-line treatment in women with HER2-negative metastatic breast cancer. Breast 22, 1171–1177, doi: 10.1016/j.breast.2013.08.017 (2013). k l f l ll b d l fi l f g j 40. Turker, I. et al. Detection of circulating tumor cells in breast cancer patients: Prognostic predictive role. Asian Pacific Journal of Cancer Prevention 14, 1601–1607 (2013).ff ( ) 1. Xenidis, N. et al. Differential effect of adjuvant taxane-based and taxane-free chemotherapy regimens on the CK-19 mRNA-positive circulating tumour cells in patients with early breast cancer. Br J Cancer 108, 549–556, doi: 10.1038/bjc.2012.597 (2013). g p y j 42. Bidard, F. C. et al. www.nature.com/scientificreports/ Negative enrichment by immunomagnetic nanobeads for unbiased characterization of circulating tumor cells h l bl d f J l f l l d d / ( ) y 61. Liu, Z. et al. Negative enrichment by immunomagnetic nanobeads for unbiased characterization of circulating tumor cells from peripheral blood of cancer patients. Journal of translational medicine 9, 70, doi: 10.1186/1479-5876-9-70 (2011). y 1. Liu, Z. et al. Negative enrichment by immunomagnetic nanobeads for unbiased characterization of circulating tumor cells from peripheral blood of cancer patients Journal of translational medicine 9 70 doi: 10 1186/1479-5876-9-70 (2011) 61. Liu, Z. et al. Negative enrichment by immunomagnetic nanobeads for unbiased characterization of circulating tumor cells from peripheral blood of cancer patients. Journal of translational medicine 9, 70, doi: 10.1186/1479-5876-9-70 (2011). p p p f 2. Cen, P. T., Ni, X. L., Yang, J. X., Graham, D. Y. & Li, M. Circulating tumor cells in the diagnosis and management of pancreatic cancer. Bba-Rev Cancer 1826, 350–356, doi: 10.1016/j.bbcan.2012.05.007 (2012). j 3. Ignatiadis, M. et al. International study on inter-reader variability for circulating tumor cells in breast cancer. Breast Cancer Res 16 R43, doi: 10.1186/bcr3647 (2014). 63. Ignatiadis, M. et al. International study on inter-reader variability for circulating tumor cells in breast cancer. Breast Cancer Res 16, R43, doi: 10.1186/bcr3647 (2014). Author Contributions J.J. and C.Y.H. conceived the study, participated in drafting the final manuscript. Y.W.T., C.Q. and C.X. analyzed the data and completed the final draft of the manuscript. L.Y.F. and W.Y. prepared all the figures. All authors reviewed the manuscript. References Time-dependent prognostic impact of circulating tumor cells detection in non-metastatic breast cancer: 70-month analysis of the REMAGUS02 study. International Journal of Breast Cancer 2013 (2013). y y J f ( ) 30. Boutrus, R. R. et al. Circulating tumor cells as predictors of response and failure in breast cancer patients treated with preoperative chemotherapy. International Journal of Biological Markers 28, 17–23 (2013). py f g 31. Jiang, Z. F. et al. Circulating tumor cells predict progression-free and overall survival in Chinese patients with metastatic breast cancer, HER2-positive or triple-negative (CBCSG004): a multicenter, double-blind, prospective trial. Ann Oncol, doi: 10.1093/ annonc/mdt246 (2013). Scientific Reports \| 7:43464 \| DOI: 10.1038/srep43464 10 Acknowledgementsh Acknowledgementsh www.nature.com/scientificreports/ Cancer Research 69 (2009). 3. Serrano, M. J., Sanchez-Rovira, P., Delgado-Rodriguez, M. & Gaforio, J. J. Detection of circulating tumor cells in the context o treatment: prognostic value in breast cancer. Cancer Biol Ther 8, 671–675 (2009).t gh 4. Xenidis, N. et al. Cytokeratin-19 mRNA-positive circulating tumor cells after adjuvant chemotherapy in patients with early breas cancer. J Clin Oncol 27, 2177–2184, doi: 10.1200/JCO.2008.18.0497 (2009). 55. Nole, F. et al. Variation of circulating tumor cell levels during treatment of metastatic breast cancer: prognostic and therapeutic implications. Ann Oncol 19, 891–897, doi: 10.1093/annonc/mdm558 (2008). p 56. Muller, V. et al. Circulating tumor cells in breast cancer: correlation to bone marrow micrometastases, heterogeneous respon systemic therapy and low proliferative activity. Clin Cancer Res 11, 3678–3685, doi: 10.1158/1078-0432.ccr-04-2469 (2005). systemic therapy and low proliferative activity. Clin Cancer Res 11, 3678–3685, doi: 10.1158/1078-0432.ccr-04-2469 (2005). 57. Cristofanilli, M. et al. Circulating tumor cells, disease progression, and survival in metastatic breast cancer. The New England journal of medicine 351, 781–791, doi: 10.1056/NEJMoa040766 (2004). y py p y 57. Cristofanilli, M. et al. Circulating tumor cells, disease progression, and survival in metastatic breast cancer. The New England jo of medicine 351, 781–791, doi: 10.1056/NEJMoa040766 (2004). f J ( ) 58. Broersen, L. H., van Pelt, G. W., Tollenaar, R. A. & Mesker, W. E. Clinical application of circulating tumor cells in breast cancer. Cell Oncol (Dordr) 37, 9–15, doi: 10.1007/s13402-013-0160-6 (2014).i f 58. Broersen, L. H., van Pelt, G. W., Tollenaar, R. A. & Mesker, W. E. Clinical application of circulating tumor cells in breast cancer Oncol (Dordr) 37, 9–15, doi: 10.1007/s13402-013-0160-6 (2014).i dams, D. L. et al. Mitosis in circulating tumor cells stratifies highly aggressive breast carcinomas. Breast Cancer Res 18, 44, doi: 0.1186/s13058-016-0706-4 (2016).f 59. Adams, D. L. et al. Mitosis in circulating tumor cells stratifies highly aggressive breast carcinomas. Breast Cancer Res 18, 44 10.1186/s13058-016-0706-4 (2016).f ( ) 60. Ignatiadis, M., Lee, M. & Jeffrey, S. S. Circulating Tumor Cells and Circulating Tumor DNA: Challenges and Opportunities on the Path to Clinical Utility. Clinical Cancer Research 21, 4786–4800, doi: 10.1158/1078-0432.CCR-14-1190 (2015). 60. Ignatiadis, M., Lee, M. & Jeffrey, S. S. Circulating Tumor Cells and Circulating Tumor DNA: Challenges and Opportunities on the Path to Clinical Utility. Clinical Cancer Research 21, 4786–4800, doi: 10.1158/1078-0432.CCR-14-1190 (2015). y 61. Liu, Z. et al. Scientific Reports \| 7:43464 \| DOI: 10.1038/srep43464 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/ Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Additional Information Supplementary information accompanies this paper at http://www.nature.com/srepi Competing financial interests: The authors declare no competing financial interests. Scientific Reports \| 7:43464 \| DOI: 10.1038/srep43464 11 www.nature.com/scientificreports/ How to cite this article: Yan, W.-T. et al. Circulating tumor cell status monitors the treatment responses in breast cancer patients: a meta-analysis. Sci. Rep. 7, 43464; doi: 10.1038/srep43464 (2017). How to cite this article: Yan, W.-T. et al. Circulating tumor cell status monitors the treatment responses in breast cancer patients: a meta-analysis. Sci. Rep. 7, 43464; doi: 10.1038/srep43464 (2017). 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/ © The Author(s) 2017 Scientific Reports \| 7:43464 \| DOI: 10.1038/srep43464 12
https://openalex.org/W2170947739	https://eprints.soton.ac.uk/66751/1/1471-2377-9-45.pdf	English	null	Protocol for the saMS trial (supportive adjustment for multiple sclerosis): a randomized controlled trial comparing cognitive behavioral therapy to supportive listening for adjustment to multiple sclerosis	BMC neurology	2,009	cc-by	13,363	This article is available from: http://www.biomedcentral.com/1471-2377/9/45 This article is available from: http://www.biomedcentral.com/1471-2377/9/45 © 2009 Moss-Morris 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. BioMed Central BioMed Central BioMed Central BMC Neurology Open Access Trial registration: Current Controlled Trials ISRCTN91377356 y p Protocol for the saMS trial (supportive adjustment for multiple sclerosis): a randomized controlled trial comparing cognitive behavioral therapy to supportive listening for adjustment to multiple sclerosis p Rona Moss-Morris1, Laura Dennison1, Lucy Yardley1, Sabin Suzanne Roche3, Paul McCrone4 and Trudie Chalder5 Address: 1School of Psychology, University of Southampton, Highfield Campus, Southampton, SO17 1BJ, UK , 2Department of Biostatistics, Institute of Psychiatry, King's College London, De Crespigny Park, London, SE5 8AF, UK , 3Chronic Fatigue Unit, Maudsley Hospital, Denmark Hill, London, SE5 9RS, UK , 4Health Service and Population Research Department, Institute of Psychiatry, King's College London, De Crespigny Park, London, SE5 8AF, UK and 5Department of Psychological Medicine, Institute of Psychiatry, King's College London, Weston Education Centre, Cutcombe Road, London, SE5 9RJ, UK Email: Rona Moss-Morris - remm@soton.ac.uk; Laura Dennison - L.k.dennison@soton.ac.uk; Lucy Yardley - Ly3@soton.ac.uk; Sabine Landau - S.Landau@iop.kcl.ac.uk; Suzanne Roche - Suzanne.roche@slam.nhs.uk; Paul McCrone - p.mccrone@iop.kcl.ac.uk; Trudie Chalder - sphatrc@iop.kcl.ac.uk * Corresponding author Received: 15 July 2009 Accepted: 23 August 2009 Published: 23 August 2009 BMC Neurology 2009, 9:45 doi:10.1186/1471-2377-9-45 Published: 23 August 2009 Published: 23 August 2009 Published: 23 August 2009 BMC Neurology 2009, 9:45 doi:10.1186/1471-2377-9-45 BMC Neurology 2009, 9:45 doi:10.1186/1471-2377-9-45 g Psychological adjustment to Multiple Sclerosis Psychological adjustment to Multiple Sclerosis y g j p Multiple Sclerosis (MS) is an incurable, chronic and unpredictable disease of the central nervous system. The disease is characterized by the destruction of the myelin sheath surrounding the nerves resulting in the formation of plaques. These plaques disrupt the transmission of nerve impulses leading to the symptoms of the illness which include, but are not limited to, spasticity, loss of balance and co-ordination, blurred or double vision, blindness, numbness, speech distortions, bladder and bowel problems, fatigue, pain and cognitive dysfunction [1]. Plaques can occur in a variety of sites resulting in sub- stantial variation in the type and nature of the symptoms across individuals. The course of the illness is also highly variable and unpredictable [1]. The majority have either a relapsing-remitting or a relapsing-progressive course. Patients experience periods of partial or total remission where the illness is inactive, interspersed with symptom relapses. MS can also have a chronic-progressive course, in which there is a progressive worsening of symptoms and disability. Patients may be initially diagnosed with one type of MS, but over time progress to another. In the intervention literature to date targets of psycholog- ical interventions for people with MS, particularly Cogni- tive Behavioral Therapy (CBT), have been mostly centred on the alleviation of depression [27-29]. Two systematic reviews of psychological interventions for MS suggest that there is some evidence that cognitive behavioural approaches can be beneficial in the treatment of depres- sion, and in helping people to adjust to, and cope with having MS[15,30] However, both reviews also criticise the methodological quality of the intervention studies to date. Many have small sample sizes and many inade- quately report details of their intervention and trial meth- odology. This makes it hard to draw definitive conclusions about the efficacy of CBT in this area. Both reviews conclude that further, well-designed trials of CBT- based approaches are warranted. One in particular, con- cludes that existing research on adjustment and coping looks encouraging and that approaches that target the time period soon after diagnosis and see adjustment as an ongoing process would be useful [15]. This review also draws particular attention to the importance of following the CONSORT statement [31] for reporting such trials and using manuals to make replication of interventions possi- ble. g Psychological adjustment to Multiple Sclerosis MS is thought to affect more than 2.5 million people worldwide and around 400,000 people in the United States and 85,000 people in the United Kingdom cur- rently live with the disease [2,3]. The illness is more com- mon in females than males. The cause of the illness is largely unknown and there is currently no cure. Treatment focuses on the management of the patient's symptoms and reducing the number and severity of relapses. Individuals who have MS are faced with uncertainty about the future, unpleasant and unpredictable symptoms, treatment regimes and drug side effects. MS can have pro- found consequences including disruptions to life goals, employment, income, relationships, social and leisure activities and activities of daily living. Therefore it is not surprising that it poses multiple challenges for psycholog- ical adjustment. A large body of empirical literature attests to poor adjustment outcomes in MS including elevated rates of depressive symptomology or distress [4-7], increased anxiety [5,8], poor subjective well-being and quality of life [5,9], and social role and relationship diffi- culties [4,10]. On the other hand, research and clinical experience suggests that a substantial proportion of peo- ple with MS manage to adapt well to living with the illness [11,12]. Abstract Background: Multiple Sclerosis (MS) is an incurable, chronic, potentially progressive and unpredictable disease of the central nervous system. The disease produces a range of unpleasant and debilitating symptoms, which can have a profound impact including disrupting activities of daily living, employment, income, relationships, social and leisure activities, and life goals. Adjusting to the illness is therefore particularly challenging. This trial tests the effectiveness of a Cognitive Behavioural intervention compared to Supportive Listening to assist adjustment in the early stages of MS. Methods/Design: This is a two arm randomized multi-centre parallel group controlled trial. 122 consenting participants who meet eligibility criteria will be randomly allocated to receive either Cognitive Behavioral Therapy or Supportive Listening. Eight one hour sessions of therapy (delivered over a period of 10 weeks) will be delivered by general nurses trained in both treatments. Self-report questionnaire data will be collected at baseline (0 weeks), mid-therapy (week 5 of therapy), post-therapy (15 weeks) and at six months (26 weeks) and twelve months (52 weeks) follow-up. Primary outcomes are distress and MS-related social and role impairment at twelve month follow-up. Analysis will also consider predictors and mechanisms of change during therapy. In-depth interviews to examine participants' experiences of the interventions will be conducted with a purposively sampled sub-set of the trial participants. An economic analysis will also take place. Discussion: This trial is distinctive in its aims in that it aids adjustment to MS in a broad sense. It is not a treatment specifically for depression. Use of nurses as therapists makes the interventions potentially viable in terms of being rolled out in the NHS. The trial benefits from incorporating patient input in the development and evaluation stages. The trial will provide important information about the efficacy, cost-effectiveness and acceptability of the interventions as well as mechanisms of psychosocial adjustment. Trial registration: Current Controlled Trials ISRCTN91377356 Trial registration: Current Controlled Trials ISRCTN91377356 Page 1 of 17 (page number not for citation purposes) Page 1 of 17 (page number not for citation purposes) http://www.biomedcentral.com/1471-2377/9/45 BMC Neurology 2009, 9:45 Page 2 of 17 (page number not for citation purposes) Background ual differences in adjustment [14,15]. A systematic review conducted as pilot work for this trial demonstrated that a range of psychological factors were associated with adjust- ment outcomes in MS [16]. For instance, people reporting high levels of perceived social support showed better adjustment [17,18], as did individuals with high levels of optimism and hope [19,20] and those who engaged in health behaviours such as exercise [21]. Use of problem- focussed coping strategies such as planning was also con- sistently associated with more positive outcomes. On the other hand, emotion-focussed coping strategies including wishful thinking and escape/avoidance were consistently linked with worse adjustment [22,23]. High perceived lev- els of stress were also linked to worse adjustment [23]. Similarly, a tendency to interpret events, the illness and MS symptoms in an overly negative fashion has been linked to worse adjustment [24-26]. Since many of these factors are potentially modifiable through psychological intervention there is reason to conclude that a programme of therapy that addresses a number of these factors may lead to improvements in adjustment. Methods/Design Design The trial is a two arm randomized multi-centre parallel group controlled trial. Consenting participants who meet eligibility criteria are randomly allocated to receive either CBT for adjustment to MS or Supportive Listening (SL). The SL arm of the trial has been chosen over standard medical care as a comparison condition as it is an accept- able and plausible approach that will control for non-spe- cific treatment factors such as support, therapist time and attention, and patient expectations of improvement. The full trial design is summarized in Figure 1. Because most MS patients receive little structured psycho- logical support, CBT will be compared to Supportive Lis- tening therapy rather than treatment as usual. This will allow us to ascertain whether there are any particular ben- efits to CBT or whether just having the opportunity to talk to a supportive, empathetic therapist can make a differ- ence to adjustment outcomes. In line with suggestions for research in this area, general nurses will be trained and supervised to deliver the interventions [15]. Background to the project Given the evidence that CBT is helpful in improving men- tal health and psychological aspects of chronic health conditions, we wanted to investigate the efficacy of CBT in helping people with MS adjust to the psychological and social challenges of living with the disease. Although there are some differences between regional MS services in the United Kingdom in availability of information, advice Illness factors such as the extent of neurological disability, symptom severity, remission status and length of illness can influence levels of adjustment or a sense of well-being and quality of life [13,14]. However, psychological factors can be as important in predicting and explaining individ- Page 2 of 17 (page number not for citation purposes) Page 2 of 17 (page number not for citation purposes) http://www.biomedcentral.com/1471-2377/9/45 BMC Neurology 2009, 9:45 BMC Neurology 2009, 9:45 and support in the early stages of MS, formal psychologi- cal interventions are not routinely available. 6) To evaluate both interventions from the perspective of the person with MS using in-depth interviews and qualita- tive analysis methods to elicit their experiences of the ther- apy, any changes it brought, and any helpful and unhelpful aspects. The aim of this project is to assess whether CBT can enhance the adjustment of people in the relatively early years of dealing with MS. Unlike most previous studies which have evaluated the use of CBT in this group [15,30], the focus of this study is to improve broadly defined adjustment outcomes rather than restricting the scope of the intervention to reducing psychiatric morbidity in MS populations where individuals meet criteria for psychiat- ric disorder. By focusing the intervention on the earlier years of MS it is hoped that patients will learn strategies that will assist in managing the illness in the future. Primary Aim 1) To determine whether patients with early stage MS who undertake an 8 session CBT programme for adjustment to MS will demonstrate better adjustment (defined as psy- chological well-being and social and role adjustment) than those undergoing 8 sessions of Supportive Listening (SL). Sample size calculation Since there were no available MS CBT trials using either of our primary outcomes we computed a power analysis cal- culation based on a recent CBT trial for depression in peo- ple with MS, which used the Beck Depression Inventory (BDI) as the primary outcome [28]. The BDI, like the GHQ (one of our primary outcomes) is a measure of affec- tive distress. Using a conservative estimate for the within- group standard deviation the effect size from this study was .60. To achieve 80% power at alpha = 0.025 signifi- cance level (to adjust for two primary outcomes) we would need 55 patients in each group to detect an effect of this magnitude by an independent samples t-test. To allow for attrition of around 10% at follow-up we need to initially include 61 participants in each group. In our CBT for MS fatigue trial we only lost 3/72 patients to follow-up (4%). Setting The trial has two centers (Southampton and South Lon- don) and each center has one therapist who delivers both CBT and SL. Secondary Aims 2) To examine whether the CBT and SL groups differ on a range of secondary outcomes including quality of life, acceptance of illness and dysfunctional cognitions. 3) To examine whether changes in predefined psycholog- ical mechanisms act as mediators through which change/ adjustment in our primary outcomes occurs. These pro- posed mediating factors are dysfunctional or unhelpful cognitions and behaviours, and acceptance of illness. 4) To examine whether patients' responses to treatment is moderated by therapeutic alliance factors, patients' treat- ment preference, engagement in homework tasks, and perceived social support. Ethical approval pp This study has been reviewed and approved by the Thames Valley Multi-centre Research Ethics Committee (ref: 07/MRE12/6) Source of participants Participants will be recruited from two National Health Service (NHS) MS centers in the Southampton area (Southampton University Hospital Trust; SUHT) and South London (King's College Hospital Trust; KCHT). http://www.biomedcentral.com/1471-2377/9/45 BMC Neurology 2009, 9:45 - have a definite diagnosis of MS, of any type, con- firmed by a neurologist. exclusion criteria, to identify suitable patients. The nurse/ neurologist will hand over a Participant Information Sheet with a contact details reply slip, consent form, and freepost envelope to all that are potentially eligible and interested. If the participant is keen to be involved the nurse/neurologist will ask permission to write down the participant's contact details and freepost it back to the trial co-ordinator so that she can contact them about the project. Alternatively the patient can choose to initiate contact with the research team by posting the contact details form back to the trial co-ordinator, or getting in touch by telephone or email. - be diagnosed with MS within the last 10 years. - have some degree of ambulation (with assistance if needed), equivalent to an Expanded Disability Status Scale (EDSS) score [32] of 6.5 or less. - be stabilised on disease modifying or anti-depressant medication (if taking). For disease modifying drugs (e.g. interferon) patients must have been on medica- tion for a minimum of 3 months. For anti-depressants, patients must be on a stable dose for a minimum of 2 months. Once contact is made with the trial co-ordinator the patient's questions will be answered and their under- standing of what is involved in the trial verified. Consent forms will be returned to the trial co-ordinator by freepost for those who wish to take part. On the form, participants are given the choice to consent to the therapy trial and questionnaire assessments only, or to an additional in- depth telephone interview after their therapy programme has been completed. Randomization Randomization is at the patient level and will take place after the baseline questionnaire has been received. The randomization will be stratified by centre to ensure equal proportions in each treatment arm. Randomization is block stratified with varying block sizes. Randomization will be handled by an independent service at the King's College Mental Health and Neuroscience Clinical Trials Unit (CTU). On receipt of the baseline questionnaire, the trial co-ordinator will electronically submit details of each Recruitment and consent process Exclusion criteria Patients will be excluded from the study if they: - have gross cognitive impairment that would make participation in 8 one hour sessions of talking therapy problematic or distressing. This will be assessed using the Telephone Interview for Cognitive Status-Modi- fied; TICS-M [33] administered by the trial co-ordina- tor during screening. Patients with a score of less than 20 will be excluded. Eligibility screening and enrolment Eligibility screening and enrolment After obtaining written consent, participants will be screened for eligibility. Screening will be completed by the trial co-ordinator over the telephone using a screening checklist of the inclusion/exclusion criteria. - have serious psychological disorders for which treat- ment would be inappropriate (including psychotic disorders or active substance abuse problems). If potential participants do not meet the medication or current/recent psychological therapy criteria at the time of the eligibility screening they will be placed on hold and there will be the necessary delay (see inclusion/exclusion criteria) before they are enrolled into the trial. After screening, participants will be notified on the telephone by the trial co-ordinator whether they are eligible or inel- igible for the trial. - have other co-morbid serious chronic illnesses (e.g. a malignancy). - are currently participating in other psychological therapies or have participated in other therapies within the last 2 months. - are considered by their treating physician to have needs that are more appropriately addressed by a refer- ral to another psychological service (e.g. psychiatrist, clinical psychologist, MS special mental health nurse). Baseline questionnaire assessments Eligible participants will be sent the baseline question- naire and freepost return envelope together with a copy of their consent form. The schedule of assessments in the baseline questionnaire and subsequent follow-up ques- tionnaires is outlined in Table 1. Source of participants Inclusion criteria 5) To examine the cost-effectiveness of both interventions by taking into account benefits to patients, effects on health service usage and other costs to society, and cost of providing the interventions. Participants do not need to be currently experiencing dis- tress, depression or any particular coping difficulties to be included in the study. However, they must: Page 3 of 17 (page number not for citation purposes) Page 3 of 17 (page number not for citation purposes) http://www.biomedcentral.com/1471-2377/9/45 BMC Neurology 2009, 9:45 T flowchart of trial design RT flowchart of trial design. Screened for eligibility Baseline assessment (0 weeks) Excluded 1. Not meeting inclusion criteria 2. Refused to participate 3. Other reasons Not randomized 1. Not meeting eligibility criteria 2. Refused to participate 3. Other reasons Randomized (n=122) Allocation Completed therapy Dropped out of therapy Cognitive Behavior Therapy Delivered over 10 weeks Dropped out of therapy Supportive Listening Delivered over 10 weeks Assessed 5 weeks Assessed 15 weeks Assessed 5 weeks Assessed 5 weeks Assessed 5 weeks Assessed 15 weeks Assessed 15 weeks Assessed 15 weeks Assessed 26 weeks Assessed 52 weeks Assessed 26 weeks Assessed 52 weeks Assessed 26 weeks Assessed 52 weeks Mid-therapy assessment 6 month follow-up Post-therapy assessment Assessed 26 weeks Allocated to either London centre (i.e. Nurse-therapist 1) or Southampton centre (i.e. Nurse- therapist 2) Allocated to either London centre (i.e. Nurse-therapist 1) or Southampton centre (i.e. Nurse- therapist 2) Completed therapy Allocation: therapists Allocation: patients Assessed 52 weeks Enrolment 12 month follow-up Analyzed Analyzed Analyzed Analyzed Data analysis Screened for eligibility Excluded 1. Not meeting inclusion criteria 2. Refused to participate 3. Other reasons Baseline assessment (0 weeks) Not randomized 1. Not meeting eligibility criteria 2. Refused to participate 3. Other reasons Randomized (n=122) Supportive Listening Delivered over 10 weeks Cognitive Behavior Therapy Delivered over 10 weeks Analyzed Analyzed CONSORT flowchart of trial design Figure 1 CONSORT flowchart of trial design. http://www.biomedcentral.com/1471-2377/9/45 Page 5 of 17 (page number not for citation purposes) Therapist training Both therapies will be delivered in 8 sessions over a 10 week period. The first six sessions will be scheduled weekly and the last two fortnightly. The first session will be 80–90 minutes long and the remainder will be between 50 minutes to one hour. The first and fourth ses- sions will be held face-to-face and the remaining six will be telephone sessions. Telephone sessions make CBT more accessible to patients, particularly those with mobil- ity problems, and have been shown to be an effective form of therapy for people with MS [27]. The therapists in this trial are two general nurses without prior experience in delivering psychological interventions, or specialist MS experience. They were given 2 weeks of intensive classroom training in understanding MS, CBT and SL. The training included a combination of didactic teaching, homework readings, and discussion. Role play and vid- eoed therapy sessions were also used throughout the CBT and SL training sessions. A summary of the classroom training package is presented in Table 2. Both interventions will be carried out in accordance with written, structured manuals. Participants will be issued either a CBT or SL therapy manual in their first session with the nurse-therapist. The classroom training was followed by 12 weeks of closely supervised practice with 5 pilot patients. Each nurse saw 3 pilot patients for 8 weeks of CBT and 2 pilot patients for 8 weeks of Supportive Listening. These patients had a diagnosis of MS but did not necessarily meet other trial criteria. RMM supervised the SL and TC and SR supervised the CBT. Booking in participants During the pilot/training stage the nurse-therapists audio- taped every therapy session using digital recorders. The clinical supervisors rated two randomly selected record- ings (sessions 5, 6, 7 or 8) using the Therapy Competence and Fidelity Rating scale described above. The therapists were required to score at least 4 (i.e. a 'acceptable-good' level of competence demonstrated) on all the relevant items in order to be deemed competent in the therapy and ready to commence therapy with trial participants. On receiving the randomisation outcome from the CTU the nurse-therapist will contact the new participant, notify them of their group allocation and set a date for their first session. Nurse-therapists will endeavour to book patients within one week of randomisation. Participants will be consulted about special considerations needed for their visit (e.g. disabled parking) and reimbursed for their travel expenses. http://www.biomedcentral.com/1471-2377/9/45 http://www.biomedcentral.com/1471-2377/9/45 http://www.biomedcentral.com/1471-2377/9/45 BMC Neurology 2009, 9:45 http://www.biomedcentral.com/1471-2377/9/45 participant to an electronic randomization system set up and maintained by the CTU. The system then notifies the relevant nurse-therapist (i.e. London or Southampton) of the randomisation outcome by email immediately. This email will be printed and placed on the participant's ther- apy file. in the pilot phase of the training, both supervisors and nurses met to discuss pilot patients and any difficulties encountered. Difficult situations were role played and new skills or ways of managing the situations were prac- ticed. Recruitment and consent process Recruitment and consent process Patients seen or in contact with these services during the trial period will be informed about the trial if they appear to meet eligibility criteria. During routine consultations, either one of the MS nurses or one of the neurologists will use an eligibility checklist, detailing the key inclusion and Page 5 of 17 (page number not for citation purposes) Page 5 of 17 (page number not for citation purposes) rial assessment procedures Baseline (week 0) Mid-therapy (week 5) Post-therapy (week 15) 6 month follow-up (week 26) 12 month follow (week 52) X - - - - me since symptoms started, type of for depression, supplements or ganizations) X - - - - cation, interventions, recent and - - X X X X - - - - s - - X - - - - X - - - - - X X 1]) X X X X X [43]) X X X X X X X X X X X - X X X Scale (ACHC [45]) X X X X X X X X X X Q [48]) X X X X X mptoms Questionnaire (CBRSQ; in preparation) X X X X X ) X - - X X X - - X X (EDSS [50]) X - - - X Page 7 of 17 (page number not for citation purposes) CBT Development of the manual and therapy package Beck's cognitive model of emotion which incorporates a developmental perspective [35] in conjunction with a sys- tematic review of 72 studies which looked at psychologi- cal factors associated with adjustment in MS [16] was used to guide the development of the CBT model and therapy manual for this trial. Details of our CBT model of adjust- ment to MS are presented in the review [16]. Because acceptance of MS appeared to be a significant factor in adjusting to the illness, we incorporated some basic prin- ciples for facilitating acceptance into our manual from Steven Hayes' Acceptance and Commitment Therapy [36]. Supervision of pilot patients occurred once a week as soon after a session as possible. The supervisor listened to one of the recent sessions and rated the session using the Ther- apy Competence and Fidelity Rating scale modified spe- cifically for this trial [34] This scale includes 14 items, 7 of which assess SL and 7 CBT. Example items are 'Does the therapist reflect or paraphrase appropriately' (SL) and 'Does the therapist help the client to identify specific types of cognitive distortions or errors (e.g. all-or-nothing thinking, over-generalization) (CBT item). The nurse- therapist also listened to the session and completed self- ratings of competence using the same rating scale. Super- visor and nurse ratings were discussed during supervision and strengths highlighted. Areas for improvements and strategies for these were also identified. Checks were made for treatment fidelity and to assess that CBT techniques were not used in the SL sessions. Approximately midway We also conducted two qualitative studies where 30 peo- ple with early stage MS and 15 of their partners engaged in in-depth interviews about their experiences of having MS, problems they encountered, and the things they found helpful and unhelpful in adjusting to living with MS [[37]; Dennison, Yardley, Devereux and Moss-Morris, in preparation]. CBT • Emotional processing and how to facilitate it. • Psychiatric emergencies and risk assessment • Identifying MS specific cognitions • How to use thought records to link, thoughts, behaviors and emotions • Challenging unhelpful thoughts • How to deal with patients "realistic" negative thoughts • Behavioral Activation and behavioral experiments • Problem solving • Dealing with resistance • Dealing with hopelessness • Behavioral Activation and behavioral experiments • Dealing with resistance • Dealing with hopelessness • Introduction to therapist and patient manuals • Issues around housekeeping • Introduction to concepts of unconditional regard, warmth and empathy • Describing the active listening techniques • Facilitator demonstration of techniques • Nurse role play using MS case studies • Nurse role play using a personally relevant difficult or painful situation • Practice and feedback using the variety of techniques • Introduction to therapist and patient manuals • Issues around housekeeping balance between looking after your own well-being and the needs of your partner with MS. lysed using thematic analysis. Core themes were covered in the manual and verbatim quotes from the interviews were used for illustrating points. lysed using thematic analysis. Core themes were covered in the manual and verbatim quotes from the interviews were used for illustrating points. Supportive Listening (13 hours) Page 8 of 17 (page number not for citation purposes) CBT Transcripts of these interviews were ana- Page 7 of 17 (page number not for citation purposes) Page 7 of 17 (page number not for citation purposes) http://www.biomedcentral.com/1471-2377/9/45 BMC Neurology 2009, 9:45 Table 2: Details of the two week classroom training program for the nurse-therapists Pragmatics of the trial and house keeping (6 hours) • Line management and orientation to the units • Record keeping for the trial, general paperwork, keeping patient notes and writing GP letters Understanding MS: (6.5 hours) • Introduction to MS including the pathogenesis of the disease, types of MS, symptoms and medical treatment • Understanding adjustment to MS and introduction to the CBT model and manual • The role of partners and family members Cognitive Behavioral Therapy (32.5 hours) • Introduction to Beck's systems model of CBT • Introduction to CBT, psychological therapies, and psychiatric diagnosis • Process of treatment and how change takes place over time • Assessment, conceptualization and formulation in CBT • Supervision and how to use it • Exploring the nurse-therapists' personal beliefs and assumptions • Motivational Interviewing Skills • Setting homework • Overview of anxiety and depression, including prevalence and diagnostic criteria. • Emotional processing and how to facilitate it. • Psychiatric emergencies and risk assessment • Identifying MS specific cognitions • How to use thought records to link, thoughts, behaviors and emotions • Challenging unhelpful thoughts • How to deal with patients "realistic" negative thoughts • Behavioral Activation and behavioral experiments • Problem solving • Dealing with resistance • Dealing with hopelessness Supportive Listening (13 hours) • Introduction to therapist and patient manuals • Issues around housekeeping • Introduction to concepts of unconditional regard, warmth and empathy • Describing the active listening techniques • Facilitator demonstration of techniques • Nurse role play using MS case studies • Nurse role play using a personally relevant difficult or painful situation • Practice and feedback using the variety of techniques Table 2: Details of the two week classroom training program for the nurse-therapists • Introduction to Beck's systems model of CBT • Introduction to CBT, psychological therapies, and psychiatric diagnosis • Process of treatment and how change takes place over time • Assessment, conceptualization and formulation in CBT • Supervision and how to use it • Exploring the nurse-therapists' personal beliefs and assumptions • Motivational Interviewing Skills • Setting homework • Overview of anxiety and depression, including prevalence and diagnostic criteria. The CBT therapy package py p g The aim of this CBT package is to enable patients to adapt appropriately to their illness. The focus is to achieve opti- mal day-to-day functioning within the constraints of the disease, to minimize distress and manage symptoms in the short and long term. The treatment is structured but also individualized to the needs of the patient as it is clear from the literature and our qualitative interviews that the process of adjustment can vary across individuals. Once the 100 page draft manual was developed, it was reviewed by people with MS and their families, neurolo- gists, MS nurses and CBT therapists. Feedback from these sources was then used to make appropriate amendments to the manual. We also developed a short 10 page booklet for the partic- ipant to give to a partner or significant other entitled "Coping when somebody close to you has MS". This booklet covers information which closely reflects the themes from the qualitative study on spouses of people with MS [37]. These include dealing with feelings of help- lessness and difficult emotions, how to support someone with MS, dealing with other people's reactions to MS, maintaining a social life in the face of MS, and finding a In the early sessions, participants will work with their nurse-therapist in developing a formulation of their par- ticular areas of strengths and difficulties. The manual con- sists of nine chapters which can be used as appropriate depending on the formulation (see Table 3 for details of the manualized sessions). The nurse-therapist will work Page 8 of 17 (page number not for citation purposes) Page 8 of 17 (page number not for citation purposes) BMC Neurology 2009, 9:45 http://www.biomedcentral.com/1471-2377/9/45 discuss an appropriate solution. If the session can be rear- ranged to stay in line with the standard scheduling of ses- sions it should be rescheduled. together with the participant in deciding which areas to focus on and in setting tasks or homework to do in between the sessions. The nurses will record which of the nine chapters are covered in each session for each patient. Both nurses and patients will work from the same detailed manual. Inevitably there will be times when MS symptoms or relapse, other sickness, holidays, or unexpected events interfere with the standard scheduling of sessions such that therapy exceeds or alters the 10 week window (described earlier). The CBT therapy package However, generally therapy should be completed within a maximum of 12 weeks. In exceptional circumstances (e.g. the participant experiences a severe relapse of MS or has a family/personal crisis) timing of sessions can be discussed with principal investigators and rearranged. However, a participant will never have more than the 8 sessions. Therapy record keeping Therapy record keeping Deviations from the standard therapy schedule will be logged so they can be reported and analysed. Details of the date and length of each session (and for CBT partici- pants, the chapters covered and a rating on a scale of 1–10 of homework engagement) will be recorded by the nurse- therapist. The SL therapist manual is based on manuals used in pre- vious trials comparing CBT to counselling [38] and prag- matic rehabilitation to Supportive Listening therapy [39]. The manual outlines how SL is different to CBT and which interventions are prohibited such as offering explanations for symptoms, eliciting symptom information or changes, suggesting explicit coping strategies, interpreting informa- tion rather than responding reflectively, leading or direct- ing the client and giving homework assignments or tasks. All therapy sessions will be digitally recorded with permis- sion from the participant and saved in an encrypted anon- ymous format. These recordings will be used for ongoing supervision and to assess treatment fidelity and therapist effects at the end of the trial. The SL therapist manual also includes a description of the listening skills to be used in SL which are based on the the- ories and counselling techniques of Carl Rogers [40]. These core skills include asking open questions, active lis- tening skills such as minimal encouragers, paraphrasing, empathising, reflecting and summarising. The purpose is to provide the participant the opportunity to talk and express themselves in a non-judgemental, safe environ- ment. The person should experience empathy from the therapist and feel listened to. Informing the participants' health professionals regarding involvement in the trial After the first session, the nurse-therapist will write to the participant's GP and MS service to inform them of the patient's participation in the trial. For CBT patients this will also include a summary of the assessment made and the areas that are likely to be concentrated on in the remaining sessions (discussed and agreed with the partic- ipant and a copy sent to the participant for their own records). More advanced Rogerian counselling skills such as prob- lem clarification, accurate understanding and challenging are not included in the therapist training or manual. The SL was designed to control for the non-specific effects of therapy such as warmth and positive regard rather than being formal counselling therapy per se. Ongoing supervision of nurse-therapists Two experienced CBT therapists (SR and TC) will super- vise the CBT and an experienced health psychologist (RM) will supervise the SL. For the first two months of the trial each nurse-therapist will have a separate CBT and SL supervision session once per week. Supervision will be fortnightly for the rest of the trial. Sessions will be between 30 – 60 minutes long and may be either by tele- phone, or face-to-face. At least once every two months the nurse-therapists will have a face-to-face session. Tape recordings of a session will be shared with the supervisor before the sessions and discussed during supervision. Each nurse-therapist will keep a log of dates and timings of their supervision sessions. Once per month the super- The participant or client manual for SL is a short 7 page document. It provides brief information about MS and why Supportive Listening may be beneficial for people with MS. It also outlines what patients can expect from the therapy sessions and a timetable for scheduling the meet- ings. Missed or postponed sessions Supportive Listening Supportive Listening (SL) will be presented to patients as a treatment based on the idea that they will be able to help themselves if given the opportunity to talk freely, exten- sively and confidentially about their experiences, thoughts and feeling about MS and its effect on their lives. If participants prefer not to focus on their MS, they will be encouraged to choose other topics to talk about which, they feel, are currently relevant to them. Page 9 of 17 (page number not for citation purposes) Missed or postponed sessions p p In the case of cancellation or non-attendance at therapy the nurse-therapist will contact the participant by tele- phone to ascertain the problem of attendance and will Page 9 of 17 (page number not for citation purposes) Page 9 of 17 (page number not for citation purposes) http://www.biomedcentral.com/1471-2377/9/45 http://www.biomedcentral.com/1471-2377/9/45 BMC Neurology 2009, 9:45 visor will check that session documentation has been properly completed. Therapeutic fidelity ratings Fidelity rating for the interventions will occur throughout the trial in the supervision sessions. In addition, at the end f h i l l i f di f h i will be assessed by an independent experienced clinician using a modified version of the therapy rating scale [34] to ensure treatment integrity. Sessions to be rated will be selected at the end of the therapy part of the trial without input from nurses and supervisors. Approximately one third (randomly chosen) will be co-rated by a second rater i d bli h li bili f i Table 3: Summary of the content of the CBT manual for adjustment to MS Chapter 1: Introduction to adjusting to MS What is MS and what does adjusting to MS mean? Factors which have been shown to affect adjustment in MS. A CBT model of adjusting to MS which includes interactions between thoughts, behavior, biology and emotions. Assessment of current strengths and difficulties. Chapter 2: Adapting to living with MS What do we mean by acceptance? Strategies for becoming accepting. Dealing with negative emotions such as sadness, grief, loss, frustration, anger, anxiety, depression, shame and embarrassment. Chapter 3. Setting goals and problem solving Exploring values and setting treatment goals where change may be needed across different areas of life. Once problems are identified, developing a stepped approach to problem solving drawing on the patient's strengths and support network. Chapter 4. Managing symptoms Helping make the link between symptoms, thoughts and behaviors. May involve a discussion on accepting limitations. The pitfalls of becoming overly symptom focused and avoidant and strategies for managing these. Understanding MS symptoms, and which symptoms are likely to be a sign of relapse, medication side effects, or stress/distress. Diaries of patterns of rest and activity to see how these may influence symptom experience. Chapter 5. How to tackle negative and unhelpful thoughts Demonstrations of how perceptions of events can influence coping with illness. There are two primary outcomes. There are two primary outcomes. 1. Cognitive and behavioural responses to symptoms. The Cognitive and Behavioural Response to Symp- toms Questionnaire (CBRSQ) [Moss-Morris, Chalder, Skerrett & Baldwin, in preparation] is a newly devised 34 item questionnaire which was designed to measure patients' cognitive and behavioural responses to symptoms. The subscales of this questionnaire have been shown to predict a significant amount of the var- iance in MS related disability and fatigue over and above EDSS scores, remission status, and mood [47] 1. Cognitive and behavioural responses to symptoms. The Cognitive and Behavioural Response to Symp- toms Questionnaire (CBRSQ) [Moss-Morris, Chalder, Skerrett & Baldwin, in preparation] is a newly devised 34 item questionnaire which was designed to measure patients' cognitive and behavioural responses to symptoms. The subscales of this questionnaire have been shown to predict a significant amount of the var- iance in MS related disability and fatigue over and above EDSS scores, remission status, and mood [47] 1. The General Health Questionnaire (GHQ) [41] is designed to measure general levels of distress in peo- ple in the community and medical settings. The meas- ure is uncontaminated by the experience of MS related somatic symptoms and a recent study showed the GHQ was the most treatment responsive measure of psychological distress in three discrete MS samples [42]. 2. The Work and Social Adjustment Scale (WSAS) [43] is a self-report scale of functional impairment attribut- able to an identified illness, in this case MS. The scale measures how much MS interferes with a person's work, home management, social leisure activities, pri- vate leisure activities, and the ability to form and maintain close relationships. 2. Illness perceptions will be assessed using the Brief Illness Perceptions Questionnaire (BIPQ) [48]. This 8 item questionnaire assesses cognitive and emotional illness representations (e.g. its timeline, conse- quences, controllability). Across a range of illnesses, negative illness perceptions are related to worse adjustment outcomes, whilst changes to illness per- ceptions may improve adjustment. Mediators of the treatment effect In order to gain a clearer idea of the possible mechanisms and to help refine our CBT model of MS adjustment we have included a number of measures as possible media- tors of the treatment effect. These are cognitive behav- ioural factors which have been demonstrated to be associated with adjustment outcomes, and/or are addressed within either or both interventions. We hypoth- esize that the mediators of improvements in psychologi- cal well-being (GHQ) will be different to the mediators of improvements in functional impairment (WSAS). If participants are unable to complete questionnaires themselves (e.g. due to relapse) the trial co-ordinator will go through the questionnaire on the telephone and this will be logged as such on the trial database. Spouses/part- ners/friends will not be allowed to complete the question- naire on the participant's behalf or assist them with writing their answers. Completed, returned question- naires will be checked by the trial co-ordinator and partic- ipants contacted if there is a large amount of missing data. Outcome assessments Outcome assessments of mobility, self-care, usual activities, pain/discomfort and anxiety/depression. of mobility, self-care, usual activities, pain/discomfort and anxiety/depression. Questionnaires (see Table 1) will be completed at base- line (prior to randomisation), mid-therapy (session 5 of therapy sessions), post-therapy follow-up (15 weeks after randomisation), 6 month follow-up (26 weeks after ran- domisation) and 12 month follow-up (52 weeks after ran- domisation). All assessments are questionnaire-based. Data collection will be managed by the trial co-ordinator who will be blind to treatment allocation. Participants will be able to choose whether to complete online or paper-based questionnaires (returned by freepost). Dates that questionnaires are sent out and received will be recorded. A reminder phone call or reminder emails will be used if the questionnaire is not received within two weeks of send-out. After four weeks another copy of the questionnaire pack will be reissued. 2. Acceptance of illness will be measured using the Acceptance of Chronic Health Conditions Scale (ACHC) [45]. This scale assesses acceptance of and adjustment to change in one's life due to a chronic health condition (in this case MS). 2. Acceptance of illness will be measured using the Acceptance of Chronic Health Conditions Scale (ACHC) [45]. This scale assesses acceptance of and adjustment to change in one's life due to a chronic health condition (in this case MS). 3. Dysfunctional cognitions will be measured by the Psychological Vulnerability Scale (PVS) [46]. This short scale taps maladaptive cognitive responses which are proposed to promote unhelpful adjustment to stressors (e.g. perfectionism, need for approval). Missed or postponed sessions Identifying traps or 'errors' in thinking and finding alternatives can help with adjustment and levels of distress. Examples of unhelpful thoughts are covered such as fears about the illness and future, and high personal expectations. Using daily thought records of unhelpful thoughts, challenging these thoughts and coming up with alternative thoughts. Chapter 6. Improving the quality of your sleep Basic sleep hygiene including establishing a good sleep/wake routine which encourages natural sleep and addressing factors which interrupt sleep. Goal setting to improve sleep. Chapter 7. Managing stress Exploring skills to call on in times of stress such as distraction, problem solving, relaxation, prioritising, saying no and planning. Goal setting to improve stress management. Chapter 8. Managing social relationships Becoming more assertive. Managing relationships with care providers. Getting the right type of support for one's needs and sharing emotions. Chapter 9. Preparing for the future Identifying physical and emotional warning signs of relapse and normalising these. Developing a future management plan using personal strengths, newly learnt skills and support from others in difficult times. Table 3: Summary of the content of the CBT manual for adjustment to MS visor will check that session documentation has been properly completed. visor will check that session documentation has been properly completed. will be assessed by an independent experienced clinician using a modified version of the therapy rating scale [34] to ensure treatment integrity. Sessions to be rated will be selected at the end of the therapy part of the trial without input from nurses and supervisors. Approximately one third (randomly chosen) will be co-rated by a second rater in order to establish reliability of ratings. Therapeutic fidelity ratings Fidelity rating for the interventions will occur throughout the trial in the supervision sessions. In addition, at the end of the trial a selection of recordings of therapy sessions Page 10 of 17 (page number not for citation purposes) http://www.biomedcentral.com/1471-2377/9/45 BMC Neurology 2009, 9:45 1. Quality of life will be measured using the EuroQol 5 (EQ 5D) [44] which is a 5 item composite measure 1. Quality of life will be measured using the EuroQol 5 (EQ-5D) [44] which is a 5-item composite measure Blinding g The trial co-ordinator will administer all quantitative assessment procedures and will be kept blind to the treat- ment allocation of participants until final follow-up has been completed. The trial co-ordinator will not deal with paperwork that links a participant to their therapy condi- tion, or be present when any discussions revealing this information take place. However, if she becomes aware or suspicious of the treatment condition of any participant she will log this lapse of blinding on the trial database for later consideration in a sensitivity analysis. Either way, as all assessments are by self-report, rather than rated by a member of the research team, influences of observer biases are not expected. Social support is measured by the Significant Other Scale (SOS) [49]. We are using a shortened version of this scale (8 items) where the participant is asked to state one key support person and then rate desired and received support in different domains (e.g. practical help and socialising). Due to the nature of the interventions it is not possible to blind the principal investigators and nurse-therapists as they will be delivering and supervising the therapy ses- sions. It is also not possible to blind participants to treat- ment allocation. However, the trial is set up as comparison of two treatments rather than as a treatment versus control condition. The participant information presents both treatments as a way of assisting adjustment to MS with neither treatment being given preference. Dur- ing statistical analysis the treatment groups will be assigned a code rather than their name so that the statisti- cians are unaware what the treatment group code repre- sents. In addition to these measures we have included a number of questions to measure illness severity, progression and relapse (see Table 1) including the self-reported Expanded Disability Status Scale (EDSS)[50]. This will allow us to assess whether there has been any noticeable disease pro- gression. The self-reported EDSS is a relatively new instru- ment which allows MS patients to self-report their current disease status, rather than this being assessed by a neurol- ogist during a clinical examination. The questionnaire includes items which relate to mobility, strength, co-ordi- nation, sensation, bladder, vision, speech, swallowing, and cognition. Using the responses from these items, functional system scores are computed and an EDSS score is assigned to the participant ranging from 0 (no neuro- logical impairment) to 10 (death from MS). 2. Dysfunctional cognitions (PVS)[46] as described above under secondary outcomes. This will record health and social service contacts in the six months prior to baseline assessment, and the periods prior to 6- and 12-month follow-up interviews. Time lost from work because of MS-related problems will also be measured. 3. Acceptance of illness (ACHC) [45]as described above. 3. Acceptance of illness (ACHC) [45]as described above. Moderators of Treatment Improvement Data inputting Data will be input into a flat SPSS spreadsheet by the trial co-ordinator. All primary data (GHQ and WSAS) at all time points will be double entered by an administrator. This will then be checked for errors and discrepancies, which will then be corrected by consulting the raw ques- tionnaire data (paper based or online). A percentage of all other data inputting will also be double entered to ensure accuracy. Any follow-up action (e.g. double entry of more data) will be taken as necessary. Patients will also be asked to rate their treatment prefer- ence at the beginning of the trial and their feelings about treatment efficacy at the end of the trial. Blinding The trial co- ordinator will review and score each questionnaire, which is then co-scored by a neurologist specialising in MS in order to ensure reliability of scoring. Moderators of Treatment Improvement The EuroQol (EQ-5D) [44], a 5 item composite measure which considers mobility, self-care, usual activities, pain/ discomfort and anxiety/depression will be used to assess quality of life. The EQ-5D will be used to generate quality adjusted Life Years (QALYs) [52]. We will also look at two potential moderators of patients' response to treatment, the therapeutic alliance and per- ceived social support. The therapeutic alliance is measured using the Alliance subscale of the Therapy Competence and Fidelity Rating scale [34]. Independent raters blind to treatment outcome will use this scale to rate therapy tapes from each of the two therapists. The scale includes items including emo- tional expression by the patient and level of empathy expressed by the therapist. Items are rated on a 7-point Likert Rating Scale, with anchors at four points along the scale [e.g. 'not at all' (1), 'somewhat' (3), 'considerably' (5) and 'extensively']. Mediators for GHQ These include quality of life and factors which have been shown to be related to adjustment outcome in MS and are key components of our model of adjustment to MS. 1. Unhelpful beliefs about emotions are measured using the recently developed Beliefs About Emotions Scale (BES) [Rimes & Chalder; in preparation]. This 6 item questionnaire measures the extent to which the person holds unhelpful beliefs about experiencing, expressing, and controlling emotions. 1. Quality of life will be measured using the EuroQol 5 (EQ-5D) [44] which is a 5-item composite measure Page 11 of 17 (page number not for citation purposes) Page 11 of 17 (page number not for citation purposes) BMC Neurology 2009, 9:45 http://www.biomedcentral.com/1471-2377/9/45 http://www.biomedcentral.com/1471-2377/9/45 2. Dysfunctional cognitions (PVS)[46] as described above under secondary outcomes. 2. Dysfunctional cognitions (PVS)[46] as described above under secondary outcomes. Page 12 of 17 (page number not for citation purposes) Withdrawals from therapy If a participant expresses the wish to withdraw from the trial, the nurse-therapist will contact the participant to ascertain the reason for drop-out if the participant is will- ing to share this. They will be offered the option of talking to a principal investigator instead if they wish. If the par- ticipant considers that they are deteriorating a principal investigator will contact them and discuss an appropriate solution. Independent monitoring and quality control A Trial Steering Committee (TSC) including a Data Moni- toring Group has been set up to monitor the conduct of the trial. They will provide overall supervision for the trial and safeguard its integrity. Authority for continuation of the trial lies with the TSC. The committee includes an independent chairperson, the lead and principal investi- gators, the trial co-ordinator, a statistician, a representa- tive from the MS Society and a patient representative. The committee will meet at least annually during the trial period, and more frequently and/or via telephone confer- ence or email communication if pressing issues arise. The nurse-therapist (or principal investigator if they make contact) should ascertain whether consent is withdrawn from: a) further treatment only a) further treatment only b) further treatment and follow-up (questionnaires plus the in-depth telephone interview) Compliance The trial will be conducted in compliance with the Decla- ration of Helsinki, MRC Good Clinical Practice (GCP) guidance, the Data Protection Act (1998), the National Research Ethics Service (NRES) approvals, NHS Trust reg- ulations, and other regulatory requirements as appropri- ate. The final trial publication will include the items recommended under the extended CONSORT statement for randomized trials of nonpharmacologic treatment [53]. c) retaining data already collected for use in final anal- ysis. The reason for withdrawal (e.g. adverse events, relapse, ill- ness progression, inability to adhere, inability to attend) will be communicated to the principal investigator for that centre as well as the trial co-ordinator who will record and report this information as appropriate. Adverse events/reactions Adverse events (AE), adverse reactions (AR), serious adverse events (SAE) and serious adverse reactions (SAR) will be defined according to the usual clinical trial defini- tions. Where these occur they will be recorded on the trial spreadsheet and reported to the appropriate authorities and followed up in the standard manner. The interviewer will contact each participant to schedule a telephone interview and conduct the interview according to an interview schedule. Questions address expectations of therapy, experiences of therapy sessions, features of therapy liked and disliked, and the process of change (or not) as a result of therapy. Each interview will be audio- taped or digitally recorded. Interviews will be transcribed by an administrator, omitting any information that may compromise confidentiality. Completed recordings and transcripts will be stored in a locked cabinet (if printed) or in a password protected file (if digital) for analysis after the trial has concluded. Departures from the trial protocol, changes to the manu- alized treatments, and breaking of the randomization code will only occur with the advice of the TSC if it becomes apparent that a particular treatment arm is caus- ing a consistent pattern of deterioration, or if there is another obvious and significant clinical necessity. http://www.biomedcentral.com/1471-2377/9/45 http://www.biomedcentral.com/1471-2377/9/45 BMC Neurology 2009, 9:45 toral researcher who has not had any involvement in other aspects of the trial. assess the severity of the situation. If necessary, a referral will be made to ensure the patient gets the support they need. Around 15 CBT participants and 15 SL participants will be interviewed regarding their experiences of therapy. If a large enough pool of consenting participants is available, purposive sampling will be employed on the basis of questionnaire-based reports of therapy satisfaction, per- ceived improvement, and demographic and illness char- acteristics. Qualitative interviews Service use (additional to the CBT intervention or Sup- portive Listening which will both be centrally recorded) by patients in both arms of the trial will be measured using the Client Service Receipt Inventory (CSRI) [51]. On receipt of completed post-therapy questionnaires, details of those participants who have consented to take part in the in-depth interview will be passed to a post-doc- Page 12 of 17 (page number not for citation purposes) Page 12 of 17 (page number not for citation purposes) http://www.biomedcentral.com/1471-2377/9/45 Statistical analysis plan life years (QALYs), generated from the EQ-5D to assess cost-effectiveness. If the intervention has lower costs and better outcomes then it will be 'dominant'. In the event of the intervention having higher service costs and better outcomes, cost-effectiveness will be assessed using incre- mental cost-effectiveness ratios. To address uncertainty in cost and outcome differences we will use cost-effective- ness planes (CEPs) which will show the probability of the intervention being (i) cost-saving and more effective, (ii) cost-saving and less effective, (iii) cost-increasing and more effective and (iv) cost-increasing and less effective. Cost-effectiveness acceptability curves (CEACs) will be generated to show the probability that the intervention is cost-effective for different values placed on a unit improvement in the GHQ/WSAS or one extra QALY gained. CEPs will involve producing a large number of cost-outcome combinations using bootstrap methods and plotting these on a plane where one axis represents incre- mental costs and the other incremental outcome. CEACs will be generated by computing 'net benefits' for each par- ticipant (defined as the monetised value of outcome minus service costs). The value of a unit improvement in outcome is unknown and therefore a range of values will be used resulting in a number of different net benefits for each participant. Regression analyses will be used to esti- mate the difference in net benefits between the two arms for each value placed on a unit improvement in outcome. Bootstrapped regression coefficients of these differences will be saved and the proportion that are above zero will indicate the probability that one arm is more cost-effec- tive than the other. y p Statistical analysis will be carried out in SPSS and/or Stata general purpose statistical analysis packages. Mediation modeling may require use of specialist structural equation modeling software (e.g. AMOS or M-Plus). All treatment group comparisons will be carried out on an intention-to-treat basis, that is subjects will be analyzed in the group to which they were randomized irrespectively of the treatment received. The primary and secondary longi- tudinal outcomes will be analyzed using linear mixed modeling. In these models the outcome variable at the post-treatment time points will be the dependent variable and baseline values of the outcome variable, centre, time (post-treatment, 6 M follow-up or 12 M follow-up), group (CBT or Supportive Listening) and a time × group interac- tion terms will feature as explanatory variables. Qualitative analysis plan Qualitative analysis plan A thematic analysis of the participants' therapy experience interviews will be conducted based upon procedures described by Boyatzis and Joffe and Yardley [59,60]. This will involve an inductive approach whereby transcripts are coded in order to develop conceptual categories which describe salient themes in the data. Elements of grounded theory practice [61,62] will also be incorporated; constant comparison, "in vivo" coding, attention to discrepant cases and memoing. Once all transcripts are coded and a comprehensive coding manual has been developed and refined we will inspect the data for patterns and relation- ships in the themes within and between the two therapy groups. An approach similar to Baron and Kenny's mediation modeling [55]will be used to explore whether change of psychological outcomes mediates the path from therapy to change in MS adjustment. Specifically we will use instrumental variable methods advocated by Dunn and colleagues to try and adjust for unobserved confounders of the path from mediations to outcomes [56,57]. Participant safety If it is felt that a participant should be withdrawn from the trial this will be discussed with the nurse-therapist, princi- pal investigator and clinical supervisor. One of the princi- pal investigators will assess the participant clinically within a week and arrange appropriate ongoing care. The nurse-therapist will write to the participant's GP and MS service to confirm withdrawal from treatment. Both CBT and SL are expected to be of low risk to partici- pants. They are non-invasive talking therapies and similar interventions have been previously used in chronic illness populations. If the nurses are concerned about a patient's safety, particularly if they believe the person may be vul- nerable to self-harm, they immediately contact one of the therapy supervisors who will telephone the patients to Page 13 of 17 (page number not for citation purposes) Statistical analysis plan To account for correlation between measures taken on the same individual at various time points subject-varying random intercepts and slopes of time will also be included in the model. Further baseline variables might be used as explanatory variables, to provide more power- ful group comparisons if they are found to be predictive of the outcome variable. As the model fitting will by maxi- mum likelihood such analyses are valid if missing data arises at random (MAR). The effect of informative miss- ingness processes will be explored by means of a formal sensitivity analysis [54]. more effective and (iv) cost-increasing and less effective. Cost-effectiveness acceptability curves (CEACs) will be generated to show the probability that the intervention is cost-effective for different values placed on a unit improvement in the GHQ/WSAS or one extra QALY gained. CEPs will involve producing a large number of cost-outcome combinations using bootstrap methods and plotting these on a plane where one axis represents incre- mental costs and the other incremental outcome. CEACs will be generated by computing 'net benefits' for each par- ticipant (defined as the monetised value of outcome minus service costs). The value of a unit improvement in outcome is unknown and therefore a range of values will be used resulting in a number of different net benefits for each participant. Regression analyses will be used to esti- mate the difference in net benefits between the two arms for each value placed on a unit improvement in outcome. Bootstrapped regression coefficients of these differences will be saved and the proportion that are above zero will indicate the probability that one arm is more cost-effec- tive than the other. We will further investigate whether results were unduly influenced by patients who have shown marked disease progression during the trial or who have altered medica- tion during the trial period. We will also assess the sensi- tivity of the results to excluding patients who did not receive a sufficient number of treatment sessions or those who had low expectations of the therapy they received. http://www.biomedcentral.com/1471-2377/9/45 http://www.biomedcentral.com/1471-2377/9/45 BMC Neurology 2009, 9:45 Statistical analysis plan Competing interests The authors declare that they have no competing interests. Study status The trial opened to recruitment in January 2008. Enrol- ment and the therapy interventions will continue until early 2009. Throughout 2009 follow-up assessments of participants will be conducted. Results will be analyzed and reported in 2010. • Cross-sectional study of factors involved in adjustment from baseline data We expect to present the findings at various scientific forums including MS specific and neurology conferences, health and clinical psychology conferences, and behav- ioural medicine meetings. We will also report back to our funder the MS Society and patient led meetings such MS Life. A lay summary of the results will be sent to trial par- ticipants. The trial is set up to be methodologically robust and to conform to best practice for the conduct and reporting of RCTs. We have taken care to address key sources of bias; the manualized therapies are subject to ongoing supervi- sion to ensure therapy fidelity and this will also be checked post-therapy by independent raters. The assess- ments of patient outcomes are self-reported and collected by the trial co-ordinator who is blind to treatment alloca- tion and uninvolved in therapy delivery. The CBT is being exposed to a stringent test of it's efficacy since it is being compared to a potentially therapeutic intervention, SL. Although the SL is designed to control for non-specific therapeutic effects such as of the considerable time and attention from an interested and caring health profes- sional, it is based on principles of counseling which allows patients to explore and work through issues is a non-directive format. Discussion To our knowledge this is the first RCT to look specifically at assisting with broadly defined psychological adjust- ment in the early stages of the MS. The relatively inclusive eligibility criteria for this trial mean that we are offering therapy to a group of patients who would not otherwise routinely have any formal psychosocial interventions, but who nonetheless are faced with considerable adjustment challenges. The saMS trial benefits from patient user input from the early stages of therapy design and manual development, right through to the in depth evaluation of the therapy sessions. The use of qualitative methods alongside the RCT means that the voice of those who would be using the intervention has been considered during both the design and evaluation of the therapy. This qualitative data will be particularly important in guiding any changes to the ther- apy format. This trial will answer important questions about the effi- cacy of an 8 session manualized CBT treatment in reduc- ing distress and MS-related social and role impairment as well as a number of secondary outcomes. It has a relatively long follow-up period (12 months) which allows for the assessment of both short and longer-term effects of the interventions. The trial will also provide important data regarding the cost-effectiveness of both interventions and their acceptability to patients. The trial is also set up in such a way to study mediators of change during therapy and therefore shed light on the process of adjustment and the important factors involved. • Predictors of treatment outcome professionals with high-level training in clinical psychol- ogy (who are in short supply and expensive to services) to deliver adjustment interventions to those with MS. Indeed, they could potentially be offered by health profes- sionals who are already working with MS patients in the NHS (e.g. specialist MS nurses and occupational thera- pists). Furthermore, the use of telephone contact for the majority of the therapy sessions means that the interven- tion could be delivered in a flexible way that could reach more patients. Mobility difficulties and other disabling and unpredictable symptoms might be significant barriers for people with MS accessing face-to-face services. • Mechanisms of change during therapy Economic evaluation plan Service use measured with the CSRI will be combined with relevant unit costs (e.g. [58]). The cost of the inter- ventions will be calculated using information on therapist pay as well as training and supervision costs and over- heads. Cost comparisons will be made between the two groups using bootstrapping methods to account for non normality in the data distribution. Reporting and Dissemination Reporting and Dissemination A number of publications are expected from this trial including: • Main outcome paper • Economic analysis-costs/benefits Service cost data will be combined with the primary out- come measures (GHQ and WSAS) and quality-adjusted • Qualitative analysis of patient feedback and experiences • Qualitative analysis of patient feedback and experiences Page 14 of 17 (page number not for citation purposes) http://www.biomedcentral.com/1471-2377/9/45 http://www.biomedcentral.com/1471-2377/9/45 BMC Neurology 2009, 9:45 Page 15 of 17 (page number not for citation purposes) References Mohr D, Hart S, Julian L, Catledge C, Honos-Webb L, Vella L, Tasch E: Telephone-administered psychotherapy for depression. Archives of General Psychiatry 2005, 62:1007-1014. gy 9. Benito-Leon J, Morales MJ, Rivera-Navarro J, Mitchell A: A review about the impact of multiple sclerosis on health-related quality of life. Disability and Rehabilitation 2003, 25:1291-1304. y y 30. Malcomson KS, Dunwoody L, Lowe-Strong AS: Psychosocial inter- ventions in people with multiple sclerosis: a review. Journal of Neurology 2007, 254:1-13. 10. Mohr DC, Dick PP, Russo D, Pinn J, Boudewyn AC, Likosky W, Goodkin DE: The Psychosocial Impact of Multiple Scleoris: Exploring the Patient's Perspective. Health Psychology 1999, 18:376-382. Neurology 2007, 254:1 13. 31. Altman DG, Schulz KF, Moher D, Egger M, Davidoff F, Elbourne D, Gotzsche PC, Lang T, for the CONSORT Group: The revised CONSORT statement for reporting randomized trials: Explanation and elaboration. Ann Intern Med 2001, 134:663-694. 32. Kurtzke J: Rating neurological impairment in multiple sclero- sis: an expanded disability status scale (EDSS). Neurology 1983, 33:1444-1452. gy 31. Altman DG, Schulz KF, Moher D, Egger M, Davidoff F, Elbourne D, Gotzsche PC, Lang T, for the CONSORT Group: The revised CONSORT statement for reporting randomized trials: Explanation and elaboration. Ann Intern Med 2001, 134:663-694. 11. Antonak RF, Livneh H: Psychosocial adaptation to disability and its investigation among persons with multiple sclerosis. Social Science and Medicine 1995, 40:1099-1108. p 32. Kurtzke J: Rating neurological impairment in multiple sclero- sis: an expanded disability status scale (EDSS). Neurology 1983, 33:1444-1452. 12. Brooks NA, Matson RR: Social-psychological adjustment to multiple sclerosis. A longitudinal study. Social Science & Medi- cine 1982, 16:2129-2135. 33. Brandt J, Welsh K, Breitner J, Folstein M, Helms M, Christian J: Hereditary influences on cognitive functioning in older men; a study of 4000 pairs. Archives of Neurology 1993, 50:599-603. 13. Chwastiak L, Ehde DM, Gibbons LE, Sullivan M, Bowen JD, Kraft GH: Depressive symptoms and severity of illness in multiple scle- rosis: Epidemiologic study of a large community sample. Am J Psychiatry 2002, 159:1862-1868. p gy 34. Godfrey E, Chalder T, Ridsdale L, Seed P, Ogden J: Investigating the 'active ingredients' of cognitive behaviour therapy and coun- selling for patients with chronic fatigue in primary care: developing a new process measure to assess treatment fidel- ity and predict outcome. British Journal of Clinical Psychology 2007, 46:253-272. J y y 14. References p g y gy 24. Bruce JM, Polen D, Arnett PA: Pain and affective memory biases interact to predict depressive symptoms in multiple sclero- sis. Multiple Sclerosis 2007, 13:58-66. 1. Robinson I: Multiple Sclerosis London: Routledge; 1988. 1. Robinson I: Multiple Sclerosis London: Routledge; 1988. p g 2. National MS Society: Who gets MS? 2009 [http:// www.nationalmssociety.org/about-multiple-sclerosis/who-gets-ms/ index.aspx]. p g 2. National MS Society: Who gets MS? 2009 [http:// www.nationalmssociety.org/about-multiple-sclerosis/who-gets-ms/ index.aspx]. p 25. Kneebone II, Dunmore EC, Evans E: Symptoms of depression in older adults with multiple sclerosis (MS): comparison with a matched sample of younger adults. Aging & Mental Health 2003, 7:182-185. p ] 3. UK MS Society: What is MS? 2009 [http://www.mssociety.org.uk/ about_ms/what_is_ms/index.html]. 26. Shnek ZM, Foley FW, LaRocca NG, Gordon WA, DeLuca J, Schwartzman HG, Halper J, Lennox S, Irvine J: Helplessness, self- efficacy, cognitive distortions, and depression in multiple sclerosis and spinal cord injury. Annals of Behavioral Medicine 1997, 19:287-294. 4. Hakim EA, Bakheit AMO, Bryant TN, Roberts MWH, McIntosh- Michaelis SA, Spackman AJ, Martin JP, McLellan DL: The social impact of multiple sclerosis-a study of 305 patients and their relatives. Disability and Rehabilitation 2000, 22:288-293. y 5. Janssens A, van Dorn P, de Boer J, Meche F van der, Passchier J, Hintzen R: Impact of recently diagnosed multiple sclerosis on quality of life, anxiety, depression and distress of patients and partners. Acta Neurologica Scandinavica 2003, 108:389-395. 27. Mohr D, Likosky W, Bertagnolli A, Goodkin D, Wende J Van Der, Dwyer P, Dick L: Telephone-administered cognitive-behavio- ral therapy for the treatment of depressive symptoms in multiple sclerosis. Journal of Consulting and Clinical Psychology 2000, 68:356-361. p g 6. Patten SC, Metz LM: Depression in Multiple Sclerosis. Psychother- apy and Psychosomatics 1997, 66:286-292. 28. Mohr D, Boudewyn A, Goodkin D, Bostrom A, Epstein L: Compar- ative outcomes for individual cognitive-behavior therapy, supportive-expressive group psychotherapy, and Sertraline for the treatment of depression in multiple sclerosis. Journal of Consulting and Clinical Psychology 2001, 69:942-949. py y 7. Siegert RJ, Abernethy DA: Depression in multiple sclerosis: a review. Journal of Neurology, Neurosurgery and Psychiatry 2005, 76:469-475. 8. Zorzon M, de Masi R, Nasuelli D, Ukmar M, Pozzi Mucelli R, Cazzato G, Bratina A, Zivadinov R: Depression and anxiety in Multiple Sclerosis: a clinical and MRI study in 95 subjects. Journal of Neu- rology 2001, 248:1432-1459. 29. http://www.biomedcentral.com/1471-2377/9/45 http://www.biomedcentral.com/1471-2377/9/45 BMC Neurology 2009, 9:45 uted to the training and competency assessment of the therapists, conducts ongoing supervision of the CBT ther- apy and contributed to these sections of the manuscript .SL developed the statistical analysis plan. PM developed the economic analysis plan. All authors read and approved the final version of the manuscript. 16. Dennison L, Moss-Morris R, Chalder T: A review of psychological correlates of adjustment in patients with multiple sclerosis. Clinical Psychology Review 2009, 29:141-153. y gy 17. McCabe MP, McKern S, McDonald E: Coping and psychological adjustment among people with multiple sclerosis. Journal of Psychosomatic Research 2004, 56:355-361. 18. 18. Schwartz C, Frohner R: Contribution of demographic, medical, and social support variables in predicting the mental health dimension of quality of life among people with multiple scle- rosis. Health Soc Work 2005, 30:203-212. Acknowledgements 19. de Ridder D, Schreurs K, Bensing J: The relative benefits of being optimistic: Optimism as a coping resource in multiple scle- rosis and Parkinson's disease. British Journal of Health Psychology 2000, 5:141-155. g This project was funded by a grant from the UK MS Society. Thank you to the nurse-therapists Sarah Morton and Sally Baynes. 20. 20. Patten SB, Metz LM: Hopelessness ratings in relapsing-remit- ting and secondary progressive multiple sclerosis. International Journal of Psychiatry in Medicine 2002, 32:155-165. Thank you also to our colleagues at the NHS MS Services at Southampton University Hospital Trust and King's College Hospital Trust: Alan Turner, Chris Halfpenny, Ian Galea, Sheila Chartres, Jane Ware, Jane Cameron, Hazel Daniel, Stephanie Heath, Eli Silber, Joan Regan, Pauline Shaw, Fiona Barnes and Sally Jones. 21. Stuifbergen AK, Blozis SA, Harrison TC, Becker HA: Exercise, func- tional limitations, and quality of life: A longitudinal study of persons with multiple sclerosis. Archives of Physical Medicine & Rehabilitation 2006, 87:935-943. 22. Aikens JE, Fischer JS, Namey M, Rudick RA: A replicated prospec- tive investigation of life stress, coping, and depressive symp- toms in multiple sclerosis. Journal of Behavioral Medicine 1997, 20:433-445. Thank you to the independent members of our Trial Steering Committee: Alison Wearden, Charlie Bloom, Rebecca Walwyn and Ed Holloway. Thank you to the independent members of our Trial Steering Committee: Alison Wearden, Charlie Bloom, Rebecca Walwyn and Ed Holloway. 23. Pakenham KI: Adjustment to multiple sclerosis: application of a stress and coping model. Health Psychology 1999, 18:383-392. 36. Hayes SC, Follette VM, Lineham M: Mindfulness and acceptance: Expanding the cognitive behavioral tradition New York: The Guilford Press; 2004. Authors' contributions RM and TC were involved in the conception and design of the study and applied for funding. RM wrote the initial grant, is the lead investigator and the Principal Investiga- tor for the Southampton site, supervises the SL therapy and led the write up of the protocol. TC is the Principal Investigator for the London site. LY developed the idea for and supervised the qualitative studies and assisted in the initial grant application. LD helped develop and refine the study protocol, co-ordinates the trial, and has had signifi- cant input into the write-up of the protocol. SR contrib- If the CBT (or indeed the SL) is an efficacious and cost- effective intervention for assisting with adjustment to MS, the fact that it has been manualized and the details of training, supervision and therapy delivery are intricately described means that it should be straightforward to rep- licate on a larger scale. The use of general nurses as thera- pists in this trial is a significant advantage. If therapy can be successfully learned and delivered by general nurses with no prior experience of psychological interventions, it suggests that it would not be necessary to employ health Page 15 of 17 (page number not for citation purposes) 35. Beck AT: Cognitive therapy and the emotional disorders New York: Pen- guin Books; 1976. Pre-publication history p The pre-publication history for this paper can be accessed here: The pre-publication history for this paper can be accessed here: 39. Wearden AJ, Riste L, Dowrick C, Chew-Graham C, Bentall RP, Mor- riss RK, Peters S, Dunn G, Richardson G, Lovell K, et al.: Fatigue Intervention by Nurses Evaluation – The FINE Trial. A ran- domised controlled trial of nurse led self-help treatment for patients in primary care with chronic fatigue syndrome: study protocol. [ISRCTN74156610]. BMC Medicine 2006, 4:9. http://www.biomedcentral.com/1471-2377/9/45/prepub y p [ ] , 40. Rogers C: Client-centered therapy: its current practice, implications and theory London: Constable; 1951. y 41. Goldberg D: General Health Questionnaire (GHQ-12). Wind- sor, NFER-NELSON; 1992. 42. Hobart J, Riazi A, Lamping D, Fitzpatrick R, Thompson A: How responsive is the Multiple Sclerosis Impact Scale (MSIS-29)? A comparison with some other self report scales. Journal of Neurology Neurosurgery and Psychiatry 2006, 76:1539. 43. Mundt J, Marks I, Shear K, Greist J: The Work and Social Adjust- ment Scale: a simple measure of impairment in functioning. British Journal of Psychiatry 2002, 180:461-464. J f y y 44. Curtis L, Netten A: Unit costs of health and social care Canterbury: PSSRU; 2006. 45. Stuifbergen A: Conceptualization and development of the Acceptance of Chronic Health Conditions Scale. Issues in Men- tal Health Nursing 2008, 29:101-114. g 46. Sinclair VG, Wallston KA: The development and validation of the Psychological Vulnerability Scale. Cognitive Therapy and Research 1999, 23:119-129. 47. Jopson NM, Moss-Morris R: The role of illness severity and ill- ness representations in adjusting to multiple sclerosis. Journal of Psychosomatic Research 2003, 54:503-511. 48. Broadbent E, Petrie KJ, Main J, Weinman J: The Brief Illness Per- ception Questionnaire. Journal of Psychosomatic Research 2006, 60:631-637. 49. Power M, Champion LA, Aris SJ: The development of a measure of social support: The Significant Others (SOS) Scale. Br J Clin Psychol 1988, 27:349-358. y 50. Bowen J, Gibbons L, Gianas A, Kraft G: Self-administered Expanded Disability Status Scale with functional system scores correlates well with physician-administered test. Mul- tiple Sclerosis 2001, 7:201-206. p 51. Beecham J, Knapp M: Costing psychiatric interventions. In Meas- uring mental health needs Edited by: Thornicroft G. London: Gaskell; 2001. 52. Williams A: The role of the EuroQol instrument in QALY cal- culations. University of York, Centre for Health Economics; 1995. 53. http://www.biomedcentral.com/1471-2377/9/45 http://www.biomedcentral.com/1471-2377/9/45 http://www.biomedcentral.com/1471-2377/9/45 BMC Neurology 2009, 9:45 61. Charmaz K: Constructing Grounded Theory: A Practical Guide Through Qualitative Analysis London: Sage; 2006. 37. Bogosian A, Moss-Morris R, Yardley L, Dennison L: Experiences of partners of people in the early stages of multiple sclerosis. Multiple Sclerosis 2009, 15:876-884. y g 62. Glaser BG, Strauss SA: Discovery of Grounded Theory. Strategies for Qual- itative Resarch New York: Aldine; 1967. p 38. Risdale L, Godfrey E, Chalder T, Seed P, King M, Wallace P, Wessely p 38. Risdale L, Godfrey E, Chalder T, Seed P, King M, Wallace P, Wessely S: Chronic fatigue in general practice: is counselling as good as cognitive behaviour therapy? A UK randomised trial. Brit- ish Journal of General Practice 2001, 51:19-24. References McIvor G, Rikland M, Reznikoff M: Depression in multiple sclero- sis as a function of length and severity of illness, age, remis- sions, and perceived social support. Journal of Clinical Psychology 1984, 40:1028-1033. 35. Beck AT: Cognitive therapy and the emotional disorders New York: Pen- guin Books; 1976. 35. Beck AT: Cognitive therapy and the emotional disorders New York: Pen- guin Books; 1976. 15. Thomas PW, Thomas S, Hiller C, Galvin K, Baker R: Psychological interventions for multiple sclerosis. Cochrane Database of Sys- tematic Reviews 2006, 1:CD004431. 36. Hayes SC, Follette VM, Lineham M: Mindfulness and acceptance: Expanding the cognitive behavioral tradition New York: The Guilford Press; 2004. Page 16 of 17 (page number not for citation purposes) 61. Charmaz K: Constructing Grounded Theory: A Practical Guide Through Qualitative Analysis London: Sage; 2006. y g 62. Glaser BG, Strauss SA: Discovery of Grounded Theory. Strategies for Qual- itative Resarch New York: Aldine; 1967. Pre-publication history Boutron I, Moher D, Altman D, Schulz K, Ravaud P: Extending the CONSORT statement to randomized trials of nonpharma- cologic treatment: Explanation and Elaboration. Ann Intern Med 2008, 148:295-310. 54. Carpenter J, Kenward MG, White IR: Sensitivity analysis after multiple imputation under missing at random: a weighting approach. Statistical Methods in Medical Research 2007, 16:259-275. ' pp 55. Baron R, Kenny D': The moderator-mediator variable distinc- tion in social psychological research: conceptual, strategic and statistical considerations. Journal of Personality and Social Psy- chology 1986, 51:1173-1182. Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Page 17 of 17 (page number not for citation purposes) Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Publish with BioMed Central and every scientist can read your work free of charge gy 56. Dunn G, Bentall R: Modelling treatment effect heterogeneity in randomised controlled trials of complex interventions (psy- chological treatments). Statistics in Medicine 2007, 26:4709-4745. g ) 57. Emsley R, White I, Dunn G: Mediation and moderation of treat- ment effects in randomised controlled trials of complex interventions. Statistical Methods in Medical Reserach 2009 in press. 58. Williams P: The psychopathology of self-assessed health: a cognitive approach to health anxiety and hypochondriasis. Cognitive Therapy and Research 2004, 28:629-644. g py 59. Boyatzis RE: Transforming Qualitative Information Thousand Oaks, CA: Sage Publications; 1998. g 60. Joffe H, Yardley L: Content and Thematic Analysis. In Research Methods for Clinical and Health Psychology Edited by: Marks D, Yardley L. London: Sage; 2000.
https://openalex.org/W2888171838	https://europepmc.org/articles/pmc6102935?pdf=render	English	null	Age dependency of risk factors for cognitive decline	BMC geriatrics	2,018	cc-by	8,489	Open Access © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: Risk factors for cognitive decline might depend on chronological age. The aim of the study was to explore the age dependency of risk factors for cognitive decline in cognitively healthy subjects aged 55–85 years at baseline. Methods: We included 2527 cognitively healthy subjects from the Longitudinal Aging Study Amsterdam (LASA). Median follow-up was 9.1 (IQR: 3.2–19.0) years. The association of genetic and cardiovascular risk factors, depressive symptoms, inflammation markers and lifestyle risk factors with decline in MMSE and memory function was tested using spline regression analyses. Results: Subjects were on average 70.1 (SD 8.8) years old at baseline. Based on a spline regression model, we divided our sample in three age groups: ≤70 years (young-old), > 70–80 years (old) and > 80 years (oldest-old). The association of LDL cholesterol, homocysteine, hypertension, history of stroke, depressive symptoms, interleukin-6, a1-antichymotrypsin, alcohol use and smoking with cognitive decline significantly differed between the age groups. In general, the presence of these risk factors was associated with less cognitive decline in the oldest-old group compared to the young-old and old group. Conclusions: The negative effect of various risk factors on cognitive decline decreases with higher age. A combination of epidemiological factors, such as the selection towards healthier subjects during follow-up, but also risk factor specific features, for example ensuring the cerebral blood flow in case of hypertension, explain this diminished association at higher age. It is important to take these age differences into account when applying preventive strategies to avert cognitive decline. Keywords: Cognitive decline, Risk factors, Aging, Oldest-old lifestyle factors [2–8]. However, whether the effect of risk factors on cognitive decline in cognitively healthy subjects is dependent on age is not clear, as the majority of the pre- vious studies did not discriminate between younger and older subjects and the number of subjects aged 80 years and older in these studies was generally low [8, 9]. Still, there is increasing evidence that the association of risk factors with cognitive decline becomes less strong at higher age and may even have a protective effect [10]. For example, the risk of the APOE ε4 allele on Alzheimer’s dementia (AD) decreases after the age of 70 years [11]. In addition, the association of cardiovascular risk factors with cognitive decline might decrease with increasing age [10, 12–14]. N. Legdeur1* , M. W. Heymans2, H. C. Comijs3, M. Huisman2,4, A. B. Maier5,6 and P. J. Visser1,7 N. Legdeur1* , M. W. Heymans2, H. C. Comijs3, M. Huisman2,4, A. B. Maier5,6 and P. J. Visser1,7 Background Dementia is a growing health problem with an expected number of 115 million cases worldwide in 2050 [1]. The prevalence of dementia increases steeply with age from a prevalence of 2.6% in subjects aged 65–69 years and a prevalence of 43.1% in subjects aged 90 years and older [1]. Insight in the risk factors for cognitive decline is essential in the search for preventive strategies for cogni- tive impairment and dementia. Former studies identified a range of potential risk factors including the APOE (apolipoprotein E) ε4 allele, cardiovascular risk factors, depressive symptoms, inflammation markers and * Correspondence: n.legdeur@vumc.nl 1Alzheimer Center Amsterdam, Department of Neurology, Amsterdam Neuroscience, Vrije Universiteit Amsterdam, Amsterdam UMC, PO Box 7057, 1007, MB, Amsterdam, the Netherlands Full list of author information is available at the end of the article The aim of the present study was to explore whether the association of risk factors with cognitive decline in * Correspondence: n.legdeur@vumc.nl 1Alzheimer Center Amsterdam, Department of Neurology, Amsterdam Neuroscience, Vrije Universiteit Amsterdam, Amsterdam UMC, PO Box 7057, 1007, MB, Amsterdam, the Netherlands Full list of author information is available at the end of the article Legdeur et al. BMC Geriatrics (2018) 18:187 https://doi.org/10.1186/s12877-018-0876-2 Legdeur et al. BMC Geriatrics (2018) 18:187 https://doi.org/10.1186/s12877-018-0876-2 Legdeur et al. BMC Geriatrics (2018) 18:187 Legdeur et al. BMC Geriatrics (2018) 18:187 Page 2 of 10 Page 2 of 10 Cholesterol levels (total cholesterol, High-Density Lipoprotein (HDL) cholesterol and Low-Density Lipo- protein (LDL) cholesterol) and homocysteine (in com- bination with vitamin B12) were determined in morning blood samples collected in 1995–1996 (second LASA cycle). Subjects were allowed to eat toast and drink tea, but no dairy products. The EDTA plasma samples were stored at -80 °C and analyzed by the Department of Clinical Chemistry of the VUmc in 2001/2002 (homo- cysteine) and 2005 (cholesterol). For determination of total cholesterol and HDL cholesterol enzymatic colori- metric tests were used. LDL cholesterol was calculated as total cholesterol minus HDL-cholesterol minus VLDL-cholesterol; VLDL-cholesterol was calculated as total triglyceride concentration expressed in mmol/L multiplied by 0.456 [18]. This method is less reliable when the triglyceride level is ≥5.0 mmol/L. Therefore, this analysis was only done for triglyceride levels of < 5.0 mmol/L. Total homocysteine was determined with the Abbott IMx analyser which uses fluorescence polarization immunoassay (FPIA) technology. Serum levels of vitamin B12 were determined at the Endocrine Laboratory of the VUmc with a competitive immuno- assay luminescence on the automated ACS 180 System (Bayer Diagnostics, Mijdrecht, The Netherlands). cognitively healthy subjects across the age range of 55 to 85 years was dependent on age. We hypothesized that predictive accuracy would change with age for APOE ε4 allele and cardiovascular risk factors based on previous studies and we performed exploratory ana- lyses to test whether age effects were also present for other established risk factors including depressive symptoms, inflammation markers, alcohol use, smoking and physical activity. Study sample y p Data were derived from the on-going Longitudinal Aging Study Amsterdam (LASA) [15]. This is a longitudinal, population-based study in the Netherlands focusing on trajectories of physical, psychological, social and cognitive functioning in subjects aged 55 years and older. In 1992– 1993 a random sample of men and women aged 55– 85 years, stratified for age and sex, from three geographic areas of the Netherlands (Amsterdam, Zwolle and Oss) was included. Follow-up measurements were conducted about every 3 years. Data collection included a main and medical interview conducted in the homes of the subjects. The main interview was done by trained and supervised interviewers and the medical interview was performed by trained nurses. All subjects gave informed consent and the study was approved by the Ethical Review Board of the VU University Medical Center (VUmc), Amsterdam, the Netherlands and conducted according to the principles of the Helsinki declaration. ( y g j ) For determination of the inflammation markers (interleukin-6 (IL-6), C-reactive protein (CRP) and a1-antichymotrypsin (ACT)) serum collected in 1992– 1993 (only in Amsterdam and Zwolle) was stored at -80 °C until determination in 2002–2004. Sensitive regular immunoassays (ELISA) were used at Sanquin Research (Amsterdam) to determine IL-6, CRP and ACT. CRP was expressed in ug/ml, IL-6 in pg/ml and ACT in % of normal plasma. The normal human plasma pool (% NHP) used as a standard for ACT contained ~ 300 mg ACT per L. For part of the sub- jects, CRP levels were determined directly after blood sampling. At the start of the study in 1992–1993, 3107 subjects were enrolled. To select cognitively healthy subjects at baseline, subjects with an age and education corrected MMSE lower than 27 points were excluded (this cut-off is based on the lowest 10th percentile of the MMSE in the Maastricht Aging Study (MAAS) [16], leaving 2527 subjects at baseline. In 1995–1996, 2545 subjects were re-examined. See for further details about the following cycles of this LASA cohort and for the sample size per risk factor Additional file 1: Table S1 and S2. Both the cholesterol as the inflammation markers, were added to the analyses as continuous variables. Comorbidity The ApoE phenotyping was done either in 1992–1993 or 1995–1996 at the Immunochemisch Laboratorium of the VUmc. The blood samples were frozen at -80 °C until determination in 1997–1998. The method used is described by Havekes et al. (1987) and consisted of iso- electric focusing of delipidated serum samples, followed by immunoblotting [17]. In the analyses, we used the presence of an ApoE ε4 isoform (phenotypes ε2/4, ε3/4, ε4/4) as a dichotomous variable [17]. The ApoE ε4 iso- form was used as proxy for the presence of an APOE ε4 allele. Hypertension was defined as a blood pressure > 140/ 90 mmHg measured at the upper arm or the use of anti- hypertensive medication collected in the first follow-up measurement in 1995–1996 (at baseline blood pressure was measured only at the finger). Post-hoc we also ana- lyzed the association of a measured high blood pressure and the use of antihypertensive medication with cogni- tive decline separately. The presence of a history of myocardial infarction (MI), DM or stroke was assessed by self-report. The as- sessment of comorbidity by self-report was found to be Legdeur et al. BMC Geriatrics (2018) 18:187 Page 3 of 10 Page 3 of 10 comparable with the medical information reported by the general practitioner [19]. determined which of these two types of splines showed a better fit with our data. We examined the positions where the splines join smoothly together, referred to as knots in spline regression analyses. We identified the op- timal position of the knots by testing both a model with one and two knots and moving those 5 years up and down. The ages that corresponded to the position of the knots were used to separate our sample into different age groups, to facilitate interpretation of results. Lastly, for all the different risk factors and outcome measures we compared our final model with a linear regression model without splines to test whether the model with splines showed a better fit (based on the LR test). Depressive symptoms were assessed using the Center for Epidemiologic Studies Depression scale (CES-D) [20]. The CES-D is a self-report scale containing 20 items describing depressive symptoms. The maximum score is 60 with higher scores indicating more depressive symptoms. In the analyses, the CES-D was used as a continuous variable. Spline regression analyses Former studies, have shown that the association between age and cognition is nonlinear [24]. Linear regression techniques are therefore not sufficient enough to esti- mate this association and spline regression analyses are indicated to fit the nonlinear longitudinal associations between age and the cognitive outcome measures more precisely (Additional file 2: Spline regression analyses) [25]. To achieve the best fit of the data with a spline re- gression model, either linear or cubic splines can be used. Based on the likelihood-ratio (LR) test we Differences between age groups at baseline Statistically significant differences in baseline character- istics between the age groups were determined by using ANOVA for continuous variables, chi-square for cat- egorical variables and Kruskal-Wallis test for skewed variables. Mixed model analysis was used to determine the difference in cognitive test score change per year in the age groups. For the assessment of physical activity, the LASA Physical Activity Questionnaire (LAPAQ) was used ad- dressing walking outdoors, bicycling, light household, heavy household, and two sports activities [21]. The sub- jects are asked how often and how long they carried out these activities in the past 2 weeks. In the analyses, total physical activity in minutes per day was used as a con- tinuous variable. Association of risk factors with cognitive outcome measures We performed three different analyses to determine the association of the risk factors (measured at baseline or, for hypertension and some biomaterial measurements, in the second cycle) with the cognitive outcome mea- sures. In all these analyses, splines (determined as de- scribed in 2.3.1) were added to the model to estimate the association between age and the cognitive outcome measure. First, we determined the association of the risk factors with the three cognitive outcome measures in the total sample by using a linear mixed model (includ- ing a random intercept and fixed slopes). Secondly, we added the interaction of the risk factors with the splines to the analyses to assess the age dependency of the risk factors. Because the splines represent different age groups, a significant interaction means that the associ- ation of that risk factor with the cognitive outcome measure is different between age groups. If this inter- action was statistically significant for a categorized risk factor, we visualized the association in a figure. Lastly, we determined the association coefficient per age group of the risk factors with the cognitive outcome measures. This last step helps us to interpret the results we found with the interaction analyses (we also performed these analyses for the risk factors that did not show a signifi- cant interaction). All the analyses were adjusted for sex and education (in years). Lifestyle The number of alcohol consumptions was categorized into three categories: 0 alcoholic drinks per day (‘none’ group), 1–2 alcoholic drinks for men and 1 alcoholic drink for women per day (‘minimal’ group) or > 2 alcohol drinks for men and > 1 alcohol drink for women per day (‘moderate’ group) [8]. Smoking was dichotomized in ‘yes (or stopped within one year)’ or ‘no’. Cognitive outcome measures Two different neuropsychological tests were used as out- come measures: The Mini-Mental State Examination (MMSE) and 15 Words Test (15WT). The MMSE is the most used screening instrument for global cognitive dys- function [22]. The score ranges from 0 to 30 points, with higher scores indicating better cognitive functioning. The 15WT is the Dutch version of the Auditory Verbal Learning Test [23]. Fifteen words have to be learned over five trials. In LASA the 15WT is restricted to three trials due to a limitation in time. In this study we used the maximum immediate recall score and delayed recall score, both ranging from 0 to 15 words. The delayed re- call was assessed after 20 minutes of distraction. Differences between age groups at baseline Most characteristics differed between age groups (Table 1). Years of education, follow-up time, scores on the cognitive tests, total and LDL cholesterol levels, alcohol use, smoking and physical activity all decreased with age. The level of homocysteine and inflammation markers and the presence of cardiovascular comorbidities and depressive symptoms increased with age. Age dependency of risk factors The association of LDL cholesterol, homocysteine, hypertension, history of stroke, depressive symptoms, IL-6, ACT, alcohol use and smoking with cognitive de- cline differed between the age groups (Table 3 and Fig. 2). The presence of APOE ε4, total and HDL choles- terol level, a history of DM or MI, CRP level and phys- ical activity did not show an age effect. In general, the regression coefficient changed from a negative associ- ation in the young-old and old subjects to a positive as- sociation in the oldest-old subjects. This means that on top of the decline in MMSE and 15WT as visualized in Fig. 1, the presence of these risk factors was associated with more decline in MMSE or 15WT in the young-old and old subjects and less decline in MMSE or 15WT in the oldest-old subjects. If we determined the association of the age-dependent risk factors with cognitive decline per age group, we found that hypertension, high IL-6 levels, and alcohol use were significantly associated with less cognitive decline in the oldest-old subjects (Table 3 and Additional file 1: Tables S5–S6). Smoking was sig- nificantly associated with more memory decline in the young-old subjects and high LDL cholesterol with more MMSE decline in the young-old subjects (Table 3 and Additional file 1: Table S6). Statistical software The spline regression analyses were performed with the stat- istical software R version 3.2.5 (http://www.r-project.org). The statistical significance of the association of the risk factor with cognitive decline per age group was determined with Stata version 15. The differences in baseline charac- teristics between the three age groups were analyzed with SPSS Statistics version 22. The level of significance was set to p = 0.05. Selection during follow-up To determine whether there was a selection towards healthier subjects during follow-up, we determined the Page 4 of 10 Legdeur et al. BMC Geriatrics (2018) 18:187 smoking and physical activity were not associated with cognitive decline. baseline values of the risk factors and cognitive outcome measures of the subjects that were present in the sample during each LASA cycle. Decreasing baseline values dur- ing follow-up would be indicative of selection towards healthier subjects. baseline values of the risk factors and cognitive outcome measures of the subjects that were present in the sample during each LASA cycle. Decreasing baseline values dur- ing follow-up would be indicative of selection towards healthier subjects. Determination of the best-fitted spline regression model Determination of the best-fitted spline regression model For the longitudinal associations between the cognitive outcome measures and age in the total group, a linear spline regression model showed a better fit then a cubic spline regression model. A model with two knots placed at the ages 70 and 80 years showed the best fit for the three outcome measures, dividing the sample in three age groups: ≤70 years (young-old subjects), > 70–80 years (old subjects) and > 80 years (oldest-old subjects) (Fig. 1). The spline regression model showed a better fit then a linear regression model without splines for all the different associations. Post-hoc analyses with hypertension defined by the measured high blood pressure only or the use of anti- hypertensive medication only, yielded similar results (Additional file 1: Table S3). Selection during follow-up Subjects who were retained in the later LASA cycles had a lower age, higher level of education, higher scores on the cognitive outcome measures, higher cholesterol levels, less comorbidities, lower levels of inflammation markers, higher alcohol use, lower levels of smoking and had a higher level of physical activity at baseline com- pared to subjects who dropped out during follow-up (Additional file 1: Table S4). Results We included 2527 subjects (51.2% women) who were on average 70.1 (SD: 8.8, range: 54.8–85.6) years at baseline and had 9.1 (SD: 3.4) years of education (Table 1). Me- dian follow-up was 9.1 (IQR: 3.2–19.0) years. Association of risk factors with cognitive decline in the total sample This study showed that the association of LDL choles- terol, homocysteine, hypertension, history of stroke, de- pressive symptoms, IL-6, ACT, alcohol use and smoking with cognitive decline was age-dependent. In general, these risk factors were associated with more cognitive decline in the young-old and old subjects and less cogni- tive decline in the oldest-old subjects. APOE ε4 geno- type and DM were negatively associated with cognitive decline regardless of age. In the total sample, the presence of high homocysteine levels, history of stroke and depressive symptoms were associated with more decline in MMSE and the 15WT (Table 2). Alcohol use was associated with less decline in MMSE and the 15WT. The presence of APOE ε4 was associated with more decline in MMSE and a history of DM with more decline in the 15WT. Cholesterol levels, hypertension, history of MI, inflammation markers, Legdeur et al. Association of risk factors with cognitive decline in the total sample BMC Geriatrics (2018) 18:187 Page 5 of 10 Table 1 Baseline characteristics of subjects in the total sample Characteristic Total sample ≤70 years > 70–80 years > 80 years P-valueb Sample sizea 2527 1292 794 441 Age, y 70.1 (8.8) 62.6 (4.2) 75.5 (2.9) 82.6 (1.5) Female, % 51.2 52.3 50.0 50.3 0.48 Education, y 9.1 (3.4) 9.5 (3.3) 8.7 (3.2) 8.6 (3.7) < 0.01 Follow-up, y (median, IQR) 9.1 (3.2–19.0) 13.3 (8.9–19.2) 6.2 (3.0–13.0) 4.9 (3.3–8.9) < 0.01 MMSE, points (median, IQR) 28 (27–29) 29 (26–30) 28 (24–30) 27 (24–30) < 0.01 Change in MMSE per year (SE) −0.11 (0.00) −0.06 (0.00) −0.18 (0.01) −0.25 (0.02) < 0.01 15WT immediate recall, words 8.1 (2.5) 8.9 (2.3) 7.6 (2.4) 6.3 (2.1) < 0.01 Change in 15WT immediate recall per year (SE) −0.11 (0.00) −0.07 (0.00) −0.11 (0.01) −0.07 (0.02) < 0.01 15WT delayed recall, words 5.3 (2.7) 6.2 (2.6) 4.7 (2.5) 3.5 (2.2) < 0.01 Change in 15WT delayed recall per year (SE) −0.11 (0.00) −0.05 (0.01) −0.10 (0.01) −0.06 (0.02) < 0.01 APOE ε4, %c 26.3 27.8 25.2 23.6 0.29 Total cholesterol, mmol/L 5.7 (1.0) 5.9 (1.0) 5.6 (1.0) 5.3 (1.1) < 0.01 LDL cholesterol, mmol/L 3.7 (1.0) 3.8 (0.9) 3.6 (0.9) 3.4 (1.1) < 0.01 HDL cholesterol, mmol/L 1.3 (0.4) 1.3 (0.4) 1.4 (0.4) 1.3 (0.4) 0.48 Homocysteine, mmol/L 14.5 (6.1) 13.5 (5.2) 14.7 (5.0) 17.2 (9.2) < 0.01 Vitamin B12, pMol/L (median, IR) 266 (212–333) 268 (219–335) 264 (213–331) 249 (196–333) 0.12 Hypertension, % 76.7 72.9 82.2 73.7 < 0.01 Myocardial infarction, % 8.8 6.4 11.1 11.7 < 0.01 Diabetes mellitus, % 7.0 4.3 8.6 12.3 < 0.01 Stroke, % 4.4 1.6 6.7 8.7 < 0.01 CES-D total score (median, IQR) 5 (2–11) 5 (2–9) 6 (3–11) 7 (3–12) < 0.01 IL-6, pg/ml (median, IQR) 1.4 (0.6–2.5) 1.3 (0.6–2.4) 1.6 (0.7–2.7) 1.8 (1.0–3.1) < 0.01 CRP, ug/ml (median, IQR) 2.2 (1.0–4.7) 2.0 (0.9–3.9) 2.5 (1.3–5.6) 2.8 (1.4–5.7) < 0.01 ACT, % of NHP 173.6 (57.6) 169.6 (53.0) 179.3 (66.1) 177.2 (54.1) 0.01 Alcohol consumption, % None 20.1 15.6 25.3 24.7 < 0.01 Minimald 20.6 17.8 23.6 23.6 Moderatee 59.4 66.6 51.1 51.7 Smokers, % 24.6 30.4 23.1 19.8 < 0.01 Total physical activity, min per day 169.2 (114.2) 188.4 (121.3) 160.0 (103.7) 126.8 (95.3) < 0.01 15WT 15 Words Test, ACT a1-antichymotrypsin, APOE apolipoprotein E, CES-D Center for Epidemiologic Studies Depression scale, CRP C-reactive protein, HDL High- Density Lipoprotein, IL-6 interleukin-6, IQR interquartile range, LASA Longitudinal Aging Study Amsterdam, LDL Low-Density Lipoprotein, MMSE Mini-Mental State Examination, NHP normal human plasma, SE standard error aSample size varies per characteristic (Additional file 1: Table S2). Association of risk factors with cognitive decline in the total sample bDifferences between the three age groups tested with ANOVA for continuous variables, chi- square for categorical variables, Kruskal-Wallis test for non-parametric variables and mixed model analysis for the change in cognitive test scores per year. cPercentage of subjects with an apolipoprotein E ε4 isoform as proxy for an APOE ε4 allele. dWomen:1 drink/day, men: 1–2 drinks/day. eWomen: > 1 drink/day, men: > 2 drinks/day. Values are means (SD) unless stated otherwise Total physical activity, min per day Before we discuss these findings in more detail, it should be noted that the baseline age of the subjects with follow-up was lower than that of the subjects who were lost to follow up. The subjects with follow-up also showed a better overall health with less comorbidity. The selection towards younger and healthier subjects at follow-up may explain why the negative impact of the risk factors was strongest in younger subjects. However, it does not explain why these risk factors became protective at higher age. We reduced the potential selec- tion bias by combining baseline data and follow-up data across the age span in the spline regression model. In this way follow-up data of the selected younger, healthier subjects (Additional file 1: Table S4) were combined with baseline data of the older, less healthy subjects (Table 1). In addition, most subjects dropped out of the study because of mortality [15]. A comparison of one-year Legdeur et al. BMC Geriatrics (2018) 18:187 Page 6 of 10 Fig. 1 Association between age and Mini-Mental State Examination (MMSE, figure a) and 15 Words Test (15WT, figure b and c). The grey dots represent all the longitudinal data points. The lines represent the splines with the two knots (black dots) at the ages 70 and 80 years Fig. 1 Association between age and Mini-Mental State Examination (MMSE, figure a) and 15 Words Test (15WT, figure b and c). The grey dots represent all the longitudinal data points. The lines represent the splines with the two knots (black dots) at the ages 70 and 80 years explained by differences in selection of subjects (normal cognition or MCI) and outcome measure (progression to dementia or cognitive decline). mortality rates of LASA subjects with those in the gen- eral Dutch population showed that mortality in the LASA subjects was slightly higher than in the general population, but that this difference exceeded 1% only in women aged 80–85 years (unpublished data). Cardiovascular factors Most cardiovascular factors were associated with less de- cline at higher age than at younger age, which is in line with previous studies [12, 28]. High cholesterol in late-life can be an indicator of a better nutritional status and a better overall health and therefore associated with less cognitive decline [28, 29]. Additionally, cholesterol synthesis is thought to decrease with aging, but only in AD patients and not in subjects with a normal cognition [30]. The association of low cholesterol with more Association of risk factors with cognitive decline in the total sample There- fore, it may not necessarily affect the generalizability of our findings. , y p p , y , aNo alcohol use is reference group. bWomen:1 drink/day, men: 1–2 drinks/day. cWomen: > 1 drink/day, men: > 2 drinks/day 15WT 15 Words Test, ACT a1-antichymotrypsin, APOE apolipoprotein E, CRP C-reactive protein, DM Diabetes mellitus, HDL High-De interleukin-6, LDL Low-Density Lipoprotein, MI Myocardial infarction, MMSE Mini-Mental State Examination B’s are determined by linear mixed models in combination with splines and adjusted for sex and education B’s are determined by linear mixed models in combination with splines and adjusted for sex and education 15WT 15 Words Test ACT a1-antichymotrypsin APOE apolipoprotein E CRP C-reactive protein DM Diabetes mellitus APOE genotype Our finding that the APOE ε4 genotype increases the risk for cognitive decline regardless of age is at odds with some earlier studies that reported a decrease of risk for dementia with age [11, 26] but is consistent with others [27]. Differences between studies may be Table 2 The association of risk factors with cognitive decline in the total sample Table 2 The association of risk factors with cognitive decline in the total sample Risk factor MMSE 15WT immediate recall 15WT delayed recall B SE P-value B SE P-value B SE P-value APOE ε4 −0.25 0.24 < 0.01 −0.07 1.27 0.49 −0.09 1.44 0.42 Total cholesterol 0.05 0.17 0.34 0.09 1.22 0.11 0.08 1.39 0.21 LDL cholesterol 0.05 0.17 0.41 0.08 1.24 0.20 0.08 1.41 0.25 HDL cholesterol 0.16 0.16 0.20 0.23 1.21 0.08 0.07 1.42 0.67 Homocysteine −0.05 0.12 < 0.01 −0.05 1.16 < 0.01 −0.06 1.33 < 0.01 Hypertension 0.13 0.28 0.26 −0.02 1.26 0.88 −0.03 1.43 0.85 DM −0.16 0.29 0.25 −0.42 1.28 0.01 −0.42 1.45 0.04 MI 0.05 0.29 0.69 0.08 1.28 0.58 0.22 1.48 0.21 Stroke −0.40 0.28 0.02 −0.55 1.27 0.01 −0.58 1.44 0.02 Depressive symptoms −0.01 0.33 < 0.01 −0.02 1.33 < 0.01 −0.02 1.50 < 0.01 CRP 0.00 0.27 0.57 −0.01 1.27 0.15 −0.01 1.45 0.47 IL-6 0.01 0.26 0.46 0.01 1.27 0.72 0.02 1.44 0.42 ACT 0.00 0.27 0.68 0.00 1.27 0.74 0.00 1.45 0.87 Alcohola: minimalb 0.27 0.10 < 0.01 0.35 0.12 < 0.01 0.35 0.14 0.01 Alcohola: moderatec 0.24 0.08 < 0.01 0.41 0.10 < 0.01 0.42 0.12 < 0.01 Smoking −0.09 0.25 0.26 −0.16 1.27 0.09 −0.03 1.46 0.77 Physical activity 0.00 0.30 0.78 0.00 1.28 0.59 0.00 1.45 0.24 B’s are determined by linear mixed models in combination with splines and adjusted for sex and education 15WT 15 Words Test, ACT a1-antichymotrypsin, APOE apolipoprotein E, CRP C-reactive protein, DM Diabetes mellitus, HDL High-Density Lipoprotein, IL-6 interleukin-6 LDL Low-Density Lipoprotein MI Myocardial infarction MMSE Mini-Mental State Examination Legdeur et al. APOE genotype BMC Geriatrics (2018) 18:187 Page 7 of 10 Table 3 The association of risk factors with cognitive decline per age group Risk factor MMSE 15WT immediate recall 15WT delayed recall ≤70 > 70–80 > 80 ≤70 > 70–80 > 80 ≤70 > 70–80 > 80 APOE ε4 0.72 −4.24 −10.17 −0.65 −3.85 −5.32 0.18 −4.15 −8.52 Total cholesterol −3.37 −0.10 1.26 −1.08 −1.09 1.53 0.97 −1.53 1.27 LDL cholesterol −5.40* 0.66 1.83* −0.18 −1.92* 1.96* 0.68 −2.62 1.41 HDL cholesterol 7.35 −2.17 −1.07 −1.84 2.64 0.50 6.21 1.45 2.97 Homocysteine 0.39 −0.40 −0.92 −0.39 −0.21 0.13 −0.76* −0.08 0.13* Hypertension 0.04 −2.14* 6.52* −5.65* −0.22 5.06* −4.04 3.54 0.43 DM −0.95 −0.46 −9.32 −0.11 −3.49 −0.29 −1.68 −5.44 4.90 MI 2.42 −1.59 2.23 0.41 1.93 5.64 4.90 0.62 10.14 Stroke 1.25 −9.16* 9.16* 2.60 −0.34 6.70 0.79 −1.58 −4.30 Depressive symptoms −0.07 0.02 −0.06 −0.21 0.00 0.07 −0.11* −0.23 0.21* CRP −0.18 0.18 0.19 −0.21 0.03 0.19 −0.19 −0.11 0.16 IL-6 0.02* −0.10* 1.31* 0.27 −0.11 0.64 0.31 −0.03 0.88 ACT 0.00 0.01 0.06 −0.01 −0.01 0.03 −0.01* −0.04 0.03* Alcohola: minimalb 2.82* −0.26 7.93* 3.19 −3.09 −0.31 5.91 −3.03 0.25 Alcohola: moderatec 0.78 −0.95 3.48 2.85 −3.51 1.42 3.19 −0.47 −0.69 Smoking −0.30 −1.05 −5.15 −5.84* 0.53* −1.13 −4.52 −2.88 −0.61 Physical activity 0.00 0.00 0.02 −0.01 0.01 0.00 0.01 0.01 0.00 Shown are beta’s (multiplied by 100) of the associations of a risk factor with cognitive decline within each age group. They show the extra cognitive decline (next to the overall cognitive decline as visualized in Fig. 1) per age group in the presence of a risk factor. A negative beta indicates that a unit increase in the risk factor is associated with more cognitive decline. Bold beta’s indicate a significant (p < 0.05) association with cognitive decline in that age group (in Additional file 1: Tables S5–S7 we present the standard errors and p-values corresponding to the beta’s in this table per age group) 15WT 15 Words Test, ACT a1-antichymotrypsin, IL-6 interleukin-6, LDL Low-Density Lipoprotein, MMSE Mini-Mental State Examination Association of risk factor with MMSE or 15WT decline is significantly different between these two age groups. In case of three : difference is significant between ≤70 and > 80 years old group and between > 70–80 and > 80 years old group. APOE genotype Beta’s are determined by linear mixed models in combination with splines and adjusted for sex and education aNo alcohol use is reference group. bWomen:1 drink/day, men: 1–2 drinks/day. cWomen: > 1 drink/day, men: > 2 drink/day Table 3 The association of risk factors with cognitive decline per age group djusted for sex and education No alcohol use is reference group. bWomen:1 drink/day, men: 1–2 drinks/day. cWomen: > 1 drink/day, men: > 2 drink/day adjusted for sex and education aNo alcohol use is reference group. bWomen:1 drink/day, men: 1–2 drinks/day. cWomen: > 1 drink/day, men: > 2 drink/day Fig. 2 Association of risk factors with Mini-Mental State Examination (MMSE, figure a, c and d) or 15 Words Test (15WT) immediate recall (figure b and e). Shown are the categorized risk factors (hypertension, stroke, alcohol use (‘minimal’: 1 drink/day for women and 1–2 drinks/day for men, ‘moderate’: > 1 drink/day for women and > 2 drinks/day for men) and smoking) which have a significant age-dependent association with the outcome measure (MMSE or 15WT immediate recall). B’s can be found in Table 3 Fig. 2 Association of risk factors with Mini-Mental State Examination (MMSE, figure a, c and d) or 15 Words Test (15WT) immediate recall (figure b and e). Shown are the categorized risk factors (hypertension, stroke, alcohol use (‘minimal’: 1 drink/day for women and 1–2 drinks/day for men, ‘moderate’: > 1 drink/day for women and > 2 drinks/day for men) and smoking) which have a significant age-dependent association with the outcome measure (MMSE or 15WT immediate recall). B’s can be found in Table 3 Legdeur et al. BMC Geriatrics (2018) 18:187 Page 8 of 10 Page 8 of 10 cognitive decline in the oldest-old subjects might there- fore be an expression of underlying AD pathology. Hypertension may prevent cognitive decline at old age by ensuring the cerebral blood flow [31, 32]. On the other hand, low blood pressure can be a consequence of neurodegenerative disease and therefore an early sign of dementia onset, although this is an aspect which one can also expect in the young-old subjects [33, 34]. probably the most important phenomena to explain this finding [40–42]. cognitive decline in the oldest-old subjects might there- fore be an expression of underlying AD pathology. Hypertension may prevent cognitive decline at old age by ensuring the cerebral blood flow [31, 32]. Strengths and limitations This is the first study that analyzes the influence of age on the association of different types of risk factors with cognitive decline in one prospective cohort study. Earlier studies have indicated age differences but never studied the various risk factors in one cohort. Additionally, the use of a nonlinear analyze technique, is an important added value of this study to earlier literature. A limita- tion of this study is that we did not have data about the presence of cardiovascular risk factors, such as hyperten- sion and high cholesterol, before age 55 years. Therefore, we could not discriminate between a high blood pres- sure and cholesterol emerging at high age or already present at younger ages. While we tested many risk fac- tors with different outcomes at the same time, this also increased the risk of false-positive findings. However, we decided not to correct for multiple testing given the ex- ploratory nature of the study and the increased risk of missing important findings when applying Bonferroni adjustments (type II errors) [46, 47]. For simplification purposes, we describe our results in relation to cognitive decline, although we only used the MMSE and 15WT to assess cognition. It therefore needs to be noted that these results cannot automatically be extrapolated to other forms of cognition. In addition, the 15WT is a test for verbal episodic memory and does not assess other types of memory such as visual and semantic memory. Depressive symptoms Earlier studies have shown that depressive symptoms are an important risk factor for cognitive decline and de- mentia, also among the oldest-old subjects [7, 35]. We replicated this finding for the total sample but also found that the association of depressive symptoms with memory decline was less in the oldest-old group than in the young-old group. This may be explained by the fact that older subjects score higher on the CES-D question- naire for reasons other than depression, such as somatic morbidity [36]. In line with this explanation, earlier cross-domain latent growth models on LASA data dem- onstrated that delayed recall was associated with increas- ing levels of depressed affect, but not with depressive somatic symptoms [37]. APOE genotype On the other hand, low blood pressure can be a consequence of neurodegenerative disease and therefore an early sign of dementia onset, although this is an aspect which one can also expect in the young-old subjects [33, 34]. In contrast to a meta-analysis of observational studies, we did not find an association between physical activity and cognitive decline [43]. However, a meta-analysis of intervention studies on the effect of aerobic exercise on cognitive decline in cognitively normal subjects did not find an effect [44]. We found that in the oldest age group less physical activity was associated with more cognitive decline, but because the over age interaction effect was not statistically significant, this finding should be interpreted cautiously. Cognitive outcome measures We used different cognitive tests as outcome measure (MMSE, 15WT total and delayed recall) and in the total sample, most variables showed similar findings for the different outcome measures and the course of the tests was very similar with age (Table 2 and Fig. 1). However, we also found different results for the different out- comes, which may be explained by the fact that the tests measure different disease processes; memory decline is presumed to be an early marker of Alzheimer’s disease and decline in MMSE can be caused by a broader range of diseases [45]. Inflammation markers Inflammation has been described as an important mech- anism underlying cognitive decline but most of these studies were performed in younger subjects [38]. We did not find an association of inflammation markers with cognitive decline in the total sample but noted differ- ences between age groups showing that higher IL-6 and ACT levels were associated with less cognitive decline in subjects aged 80 years and older compared to younger subjects. Potentially, higher inflammation markers in older subjects are a sign of a better inflammatory re- sponse and therefore related to better overall health and cognitive functioning. Conclusions The associations of LDL cholesterol, homocysteine, hyper- tension, history of stroke, depressive symptoms, IL-6, ACT, alcohol use and smoking with cognitive decline were different per age group. They were all less strongly associ- ated with cognitive decline in the older subjects compared to younger subjects. Selection towards healthier subjects during follow-up need to be considered as possible explanation but also risk factor specific considerations, such as ensuring the cerebral blood flow in case of hyper- tension, need to be taken into account. These age differ- ences are important when applying preventive strategies to avert cognitive decline. Funding 6. Cheng G, Huang C, Deng H, Wang H. Diabetes as a risk factor for dementia and mild cognitive impairment: a meta-analysis of longitudinal studies. Intern Med J. 2012;42:484–91. https://doi.org/10.1111/j.1445-5994.2012.02758.x. 6. Cheng G, Huang C, Deng H, Wang H. Diabetes as a risk factor for dementia and mild cognitive impairment: a meta-analysis of longitudinal studies. Intern Med J. 2012;42:484–91. https://doi.org/10.1111/j.1445-5994.2012.02758.x. g This work has received support from the EU/EFPIA Innovative Medicines Initiative Joint Undertaking EMIF grant agreement no. 115372. The Longitudinal Aging Study Amsterdam (LASA) is primarily funded by the Dutch Ministry of Health, Welfare and Sports and the VU University Amsterdam. 7. Van Den Kommer TN, Comijs HC, Aartsen MJ, Huisman M, Deeg DJH, Beekman ATF. Depression and cognition: how do they interrelate in old age? Am J Geriatr Psychiatry. 2013;21:398–410. https://doi.org/10.1016/j.jagp.2012.12.015. 7. Van Den Kommer TN, Comijs HC, Aartsen MJ, Huisman M, Deeg DJH, Beekman ATF. Depression and cognition: how do they interrelate in old age? Am J Geriatr Psychiatry. 2013;21:398–410. https://doi.org/10.1016/j.jagp.2012.12.015. 8. Anstey KJ, Mack HA, Cherbuin N. Alcohol consumption as a risk factor for dementia and cognitive decline: Meta-analysis of prospective studies. Am J Geriatr Psychiatry. 2009;17:542–55. https://doi.org/10.1097/JGP.0b013e3181 a2fd07. 8. Anstey KJ, Mack HA, Cherbuin N. Alcohol consumption as a risk factor for dementia and cognitive decline: Meta-analysis of prospective studies. Am J Geriatr Psychiatry. 2009;17:542–55. https://doi.org/10.1097/JGP.0b013e3181 a2fd07. Author details 1 1Alzheimer Center Amsterdam, Department of Neurology, Amsterdam Neuroscience, Vrije Universiteit Amsterdam, Amsterdam UMC, PO Box 7057, 1007, MB, Amsterdam, the Netherlands. 2Department of Epidemiology and Biostatistics, Amsterdam Public Health Research Institute, Vrije Universiteit Amsterdam, Amsterdam, the Netherlands. 3GGZ inGeest / Department of Psychiatry, Amsterdam Public Health Research Institute, Vrije Universiteit Amsterdam, Amsterdam, the Netherlands. 4Department of Sociology, Vrije Universiteit Amsterdam, Amsterdam, the Netherlands. 5Department of Medicine and Aged Care, @AgeMelbourne, Royal Melbourne Hospital, University of Melbourne, Melbourne, Australia. 6Department of Human Movement Sciences, @AgeAmsterdam, Faculty of Behavioural and Movement Sciences, Vrije Universiteit Amsterdam, Amsterdam, the Netherlands. 7Department of Psychiatry & Neuropsychology, School for Mental Health and Neuroscience, Maastricht University, Maastricht, the Netherlands. Lifestyle factors Minimal and moderate alcohol use were positively asso- ciated with decline in MMSE and memory functioning compared to no alcohol use, which is in line with earlier studies [3, 8]. We found that the positive association of alcohol use with cognition was strongest in subjects aged 80 years and older. In this age group, no alcohol use is frequently related to poor physical functioning. There- fore, the negative association of no alcohol use with cog- nitive decline is potentially an indirect effect [39]. In accordance with our findings, a meta-analysis in 2015 showed that the negative association of smoking with cognitive decline is decreasing with age [40]. Survival bias and the presence of competing risks are Legdeur et al. BMC Geriatrics (2018) 18:187 Page 9 of 10 Page 9 of 10 Competing interests PJV i l i PJV is currently acting as a Section Editor for BMC Geriatrics. The other authors declare that they have no competing interests. Authors’ contributions With this study we showed that age needs to be taken into consideration when studying risk factors for cogni- tive decline. It is not only needed to add age as a con- founder but especially as an effect modifier to analyses as it changes the relation between a risk factor and cognitive decline. From a clinical perspective, these re- sults suggest that different preventive strategies might be effective in young-old versus oldest-old subjects. Antihypertensive and cholesterol lowering medication might not be appropriate for the oldest-old subjects who develop hypertension and hypercholesterolemia at a high age. MH and HCC are both involved in the design of the Longitudinal Aging Study Amsterdam and are responsible for the data collection. PJV created the concept for this manuscript. MWH and NL performed the statistical analyses. NL wrote the first and final drafts of the manuscript. MWH, HCC, MH, ABM and PJV contributed to and approved the final version. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Additional files Additional file 1: Additional tables about sample sizes, the association of hypertension with cognitive decline separately for use of antihypertensive medication and blood pressure, baseline characteristics per LASA cycle and the associations of the risk factors with the three cognitive tests in the three age groups. (DOCX 72 kb) Received: 4 May 2018 Accepted: 7 August 2018 Received: 4 May 2018 Accepted: 7 August 2018 References Additional file 2: Additional explanation about the spline regression analyses. (DOCX 375 kb) 1. World Health Organization and Alzheimer’s Disease International. Dementia - A public health priority. 2012. 1. World Health Organization and Alzheimer’s Disease International. Dementia - A public health priority. 2012. 2. Kramer AF, Colcombe SJ, McAuley E, Scalf PE, Erickson KI. Fitness, aging and neurocognitive function. Neurobiol Aging. 2005;26:124–7. https://doi.org/10. 1016/j.neurobiolaging.2005.09.009. 2. Kramer AF, Colcombe SJ, McAuley E, Scalf PE, Erickson KI. Fitness, aging and neurocognitive function. Neurobiol Aging. 2005;26:124–7. https://doi.org/10. 1016/j.neurobiolaging.2005.09.009. Consent for publication Not applicable. Consent for publication Not applicable. Abbreviations 15WT: 15 Words Test; ACT: A1-antichymotrypsin; AD: Alzheimer’s dementia; APOE: Apolipoprotein E; CES-D: Center for Epidemiologic Studies Depression scale; CRP: C-reactive protein; DM: Diabetes mellitus; HDL cholesterol: High-Density Lipoprotein cholesterol; IL-6: Interleukin-6; IQR: Interquartile range; LAPAQ: LASA Physical Activity Questionnaire; LASA: Longitudinal Aging Study Amsterdam; LDL cholesterol: Low-Density Lipoprotein cholesterol; LR: Likelihood-ratio; MAAS: Maastricht Aging Study; MI: Myocardial infarction; MMSE: Mini-Mental State Examination; NHP: Normal human plasma; SD: Standard deviation; SE: Standard error; VLDL cholesterol: Very-low-density lipoprotein cholesterol; VUmc: VU University Medical Center 3. Baumgart M, Snyder HM, Carrillo MC, Fazio S, Kim H, Johns H. Summary of the evidence on modifiable risk factors for cognitive decline and dementia: a population-based perspective. Alzheimers Dement. 2015;11:1–9. https://doi.org/10.1016/j.jalz.2015.05.016. 3. Baumgart M, Snyder HM, Carrillo MC, Fazio S, Kim H, Johns H. Summary of the evidence on modifiable risk factors for cognitive decline and dementia: a population-based perspective. Alzheimers Dement. 2015;11:1–9. https://doi.org/10.1016/j.jalz.2015.05.016. 4. Smith RG, Todd S, Passmore PA. Chronic medical disease and cognitive aging: toward a healthy body and brain. Inflammation and Cognitive Decline. Edited by Kristine Yaffe. Can Geriatr J. 2013;16(3):143–44. 4. Smith RG, Todd S, Passmore PA. Chronic medical disease and cognitive aging: toward a healthy body and brain. Inflammation and Cognitive Decline. Edited by Kristine Yaffe. Can Geriatr J. 2013;16(3):143–44. 5. Bretsky P, Guralnik JM, Launer L, Albert M, Seeman TE. The role of APOE- epsilon4 in longitudinal cognitive decline: MacArthur studies of successful aging. Neurology. 2003;60:1077–81. https://doi.org/10.1212/01.WNL.00000 55875.26908.24. 5. Bretsky P, Guralnik JM, Launer L, Albert M, Seeman TE. The role of APOE- epsilon4 in longitudinal cognitive decline: MacArthur studies of successful aging. Neurology. 2003;60:1077–81. https://doi.org/10.1212/01.WNL.00000 55875.26908.24. Ethics approval and consent to participate All subjects gave written informed consent and the study was approved by the Ethical Review Board of the VU University Medical Center (VUmc) and conducted according to the principles of the Helsinki declaration. Availability of data and materials The datasets used and/or analyzed during the current study can be requested through http://www.lasa-vu.nl/data/availability_data/availability_data.htm. Page 10 of 10 Page 10 of 10 Legdeur et al. BMC Geriatrics (2018) 18:187 9. Anstey KJ, Lipnicki DM, Low L-F. Cholesterol as a risk factor for dementia and cognitive decline: a systematic review of prospective studies with Meta-analysis. Am J Geriatr Psychiatry. 2008;16:343–54. https://doi.org/10. 1097/01.JGP.0000310778.20870.ae. 30. van den Kommer TN, Dik MG, Comijs HC, Lutjohann D, Lips P, Jonker C, et al. The role of extracerebral cholesterol homeostasis and ApoE e4 in cognitive decline. Neurobiol Aging. 2012;33:622.e17–28. https://doi.org/10.1016/j. neurobiolaging.2011.02.019. 10. Bullain SS, Corrada MM. Dementia in the oldest old. Continuum (Minneap Minn). 2013;19:457–69. https://doi.org/10.1212/01.CON.0000429172.27815.3f. 31. Corrada MM, Hayden KM, Paganini-Hill A, Bullain SS, DeMoss J, Aguirre C, et al. Age of onset of hypertension and risk of dementia in the oldest-old: the 90+ study. Alzheimers Dement. 2017;13:103–10. https://doi.org/10.1016/j.jalz.2016. 09.007. 11. Farrer LA, Cupples LA, Haines JL, Hyman B, Kukull WA, Mayeux R, et al. Effects of age, sex, and ethnicity on the association between apolipoprotein E genotype and Alzheimer disease. A meta-analysis. APOE and Alzheimer disease Meta analysis consortium. JAMA. 1997;278:1349–56. https://doi.org/ 10.1001/jama.1997.03550160069041. 32. Kennelly SP, Lawlor BA, Kenny RA. Blood pressure and the risk for dementia- a double edged sword. Ageing Res Rev. 2009;8:61–70. https://doi.org/10. 1016/j.arr.2008.11.001. 33. Ruitenberg A, Skoog I, Ott A, Aevarsson O. Blood pressure and risk of dementia: results from the Rotterdam study and the Gothenburg H-70 study. Blood Press. 2001:33–9. https://doi.org/10.1159/000051233. 12. Qiu C, Winblad B, Fratiglioni L. The age-dependent relation of blood pressure to cognitive function and dementia. Lancet Neurol. 2005;4:487–99. https://doi.org/10.1016/S1474-4422(05)70141-1. 34. Skoog I, Lernfelt B, Landahl S, Palmertz B, Andreasson LA, Nilsson L, et al. 15-year longitudinal study of blood pressure and dementia. Lancet (London, England). 1996;347:1141–5. https://doi.org/10.1016/S0140-6736(96)90608-X. 13. Harrison SL, Stephan BCM, Siervo M, Granic A, Davies K, Wesnes KA, et al. Is there an association between metabolic syndrome and cognitive function in very old adults? The Newcastle 85+ study. J Am Geriatr Soc. 2015;63:667– 75. https://doi.org/10.1111/jgs.13358. 35. Spira AP, Rebok GW, Stone KL, Kramer JH, Yaffe K. Depressive symptoms in oldest-old women: risk of mild cognitive impairment and dementia. Am J Geriatr Psychiatry. 2011;19:S56–7. 14. van den Berg E, Biessels GJ, de Craen a J, Gussekloo J, RGJ W. The metabolic syndrome is associated with decelerated cognitive decline in the oldest old. Neurology. 2007;69:979–85. https://doi.org/10.1212/01.wnl.0000271381.30143.75. 36. Availability of data and materials Berkman LF, Berkman CS, Kasl S, Freeman DHJ, Leo L, Ostfeld AM, et al. Depressive symptoms in relation to physical health and functioning in the elderly. Am J Epidemiol. 1986;124:372–88. https://doi.org/10.1093/ oxfordjournals.aje.a114408. 15. Huisman M, Poppelaars J, van der Horst M, Beekman ATF, Brug J, van Tilburg TG, et al. Cohort profile: the longitudinal aging study Amsterdam. Int J Epidemiol. 2011;40:868–76. https://doi.org/10.1093/ije/dyq219. 37. Brailean A, Aartsen MJ, Muniz-Terrera G, Prince M, Prina AM, Comijs HC, et al. Longitudinal associations between late-life depression dimensions and cognitive functioning: a cross-domain latent growth curve analysis. Psychol Med. 2017;47:690–702. https://doi.org/10.1017/S003329171600297X. 16. Reijs BLR, Ramakers IHGB, Elias-Sonnenschein L, Teunissen CE, Koel-Simmelink M, Tsolaki M, et al. Relation of odor identification with Alzheimer’s disease markers in cerebrospinal fluid and cognition. J Alzheimers Dis. 2017;60:1025–34. https://doi.org/10.3233/JAD-170564. 38. Gorelick PB. Role of inflammation in cognitive impairment: results of observational epidemiological studies and clinical trials. Ann N Y Acad Sci. 2010;1207:155–62. https://doi.org/10.1111/j.1749-6632.2010.05726.x. 17. Havekes LM, De Knijff P, Beisiegel U. A rapid micromethod for apolipoprotein E phenotyping directly in serum. J Lipid Res. 1987;28:455–63. 18. Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem. 1972;18:499–502. 39. Anstey KJ, Windsor TD, Rodgers B, Jorm AF, Christensen H. Lower cognitive test scores observed in alcohol abstainers are associated with demographic, personality, and biological factors: the PATH through life project. Addiction. 2005;100:1291–301. https://doi.org/10.1111/j.1360-0443.2005.01159.x. 19. Kriegsman DM, Penninx BW, van Eijk JT, Boeke AJ, Deeg DJ. Self-reports and general practitioner information on the presence of chronic diseases in community dwelling elderly. A study on the accuracy of patients’ self-reports and on determinants of inaccuracy. J Clin Epidemiol. 1996;49:1407–17. https://doi.org/10.1016/S0895-4356(96)00274-0. 40. Zhong G, Wang Y, Zhang Y, Guo JJ, Zhao Y. Smoking is associated with an increased risk of dementia: a meta-analysis of prospective cohort studies with investigation of potential effect modifiers. PLoS One. 2015;10 https://doi.org/10.1371/journal.pone.0118333. p g 20. Radloff LS, Teri L. Use of the Center for Epidemiological Studies-Depression Scale with older adults. Clin Gerontol. 1986;5:119–36. https://doi.org/10. 1007/BF01537606. 41. Chang C-CH, Zhao Y, Lee C-W, Ganguli M. Smoking, death, and Alzheimer’s disease: a case of competing risks. Alzheimer Dis Assoc Disord. 2012;26: 300–6. https://doi.org/10.1097/WAD.0b013e3182420b6e. 21. Stel VS, Smit JH, Pluijm SMF, Visser M, Deeg DJH, Lips P. Comparison of the LASA physical activity questionnaire with a 7-day diary and pedometer. J Clin Epidemiol. 2004;57:252–8. https://doi.org/10.1016/j.jclinepi.2003.07.008. 42. Availability of data and materials Hernán MA, Alonso ALG. Cigarette smoking and dementia: potential selection bias in the elderly. Epidemiol (Cambridge, Mass). 2008;19:242–6. https://doi. org/10.1097/EDE.0b013e31816bbe14. 22. Folstein MF, Folstein SE, McHugh PR. “Mini-mental state”. A practical method for grading the cognitive state of patients for the clinician. J Psychiatr Res. 1975;12:189–98. https://doi.org/10.1016/0022-3956(75)90026-6. 43. Blondell SJ, Hammersley-Mather R, Veerman J. Does physical activity prevent cognitive decline and dementia?: a systematic review and meta- analysis of longitudinal studies. BMC Public Health. 2014;14:1–12. https://doi. org/10.1186/1471-2458-14-510. 23. Rey A. L’examen clinique en psychologie. Paris: Presses Universitaires de France; 1964. 44. Young J, Angevaren M, Rusted J, Tabet N. Aerobic exercise to improve cognitive function in older people without known cognitive impairment (review). Cochrane Database Syst Rev. 2015:1–141. https://doi.org/10.1002/ 14651858.CD005381.pub4. www.cochranelibrary.com 24. Verhaeghen P, Salthouse TA. Meta-analyses of age-cognition relations in adulthood: estimates of linear and nonlinear age effects and structural models. Psychol Bull. 1997;122:231–49. https://doi.org/10.1037//0033-2909. 122.3.231. 45. Jansen WJ, Ossenkoppele R, Tijms BM, Fagan AM, Hansson O, Klunk WE, et al. Association of cerebral amyloid-β aggregation with cognitive functioning in persons without dementia. JAMA Psychiatry. 2018;75:84–95. https://doi.org/10. 1001/jamapsychiatry.2017.3391. 25. Chen H, Zhao B, Cao G, Proges EC, O’Shea A, Woods AJ, et al. Statistical approaches for the study of cognitive and brain aging. Front Aging Neurosci. 2016;8:1–10. https://doi.org/10.3389/fnagi.2016.00176. 26. Corrada MM, Paganini-Hill A, Berlau DJ, Kawas CH. Apolipoprotein e genotype, dementia, and mortality in the oldest old: the 90+ study. Alzheimers Dement. 2013;9:12–8. https://doi.org/10.1016/j.jalz.2011.12.004. 46. Bender R, Lange S. Adjusting for multiple testing - when and how? J Clin Epidemiol. 2001;54:343–9. https://doi.org/10.1016/S0895-4356(00)00314-0. 47. Perneger TV. What’s wrong with Bonferroni adjustments. BMJ. 1998;316 https://doi.org/10.1136/bmj.316.7139.1236. 27. Elias-Sonnenschein LS, Viechtbauer W, Ramakers IHGB, Verhey FRJ, Visser PJ. Predictive value of APOE-ε4 allele for progression from MCI to AD-type dementia: a meta-analysis. J Neurol Neurosurg Psychiatry. 2011;82:1149–56. https://doi.org/10.1136/jnnp.2010.231555. 28. Mielke MM, Zandi PP, Sjögren M, Gustafson D, Ostling S, Steen B, et al. High total cholesterol levels in late life associated with a reduced risk of dementia. Neurology. 2005;64:1689–95. https://doi.org/10.1212/01.WNL. 0000161870.78572.A5. 29. Weverling-Rijnsburger AW, Blauw GJ, Lagaay AM, Knook DL, Meinders AE, Westendorp RG. Total cholesterol and risk of mortality in the oldest old. Lancet (London, England). 1997;350:1119–23. https://doi.org/10.1016/S0140- 6736(97)04430-9. 29. Weverling-Rijnsburger AW, Blauw GJ, Lagaay AM, Knook DL, Meinders AE, Westendorp RG. Total cholesterol and risk of mortality in the oldest old. Lancet (London, England). 1997;350:1119–23. https://doi.org/10.1016/S0140- 6736(97)04430-9.
https://openalex.org/W2964552444	https://europepmc.org/articles/pmc6720615?pdf=render	English	null	Regulation of Wound Healing by the NRF2 Transcription Factor—More Than Cytoprotection	International journal of molecular sciences	2,019	cc-by	9,770	Regulation of Wound Healing by the NRF2 Transcription Factor—More Than Cytoprotection * Correspondence: paul.hiebert@biol.ethz.ch (P.H.); sabine.werner@biol.ethz.ch (S.W.) Received: 17 July 2019; Accepted: 7 August 2019; Published: 8 August 2019 Abstract: The nuclear factor-erythroid 2-related factor 2 (NRF2) transcription factor plays a central role in mediating the cellular stress response. Due to their antioxidant properties, compounds activating NRF2 have received much attention as potential medications for disease prevention, or even for therapy. Accumulating evidence suggests that activation of the NRF2 pathway also has a major impact on wound healing and may be beneﬁcial in the treatment of chronic wounds, which remain a considerable health and economic burden. While NRF2 activation indeed shows promise, important considerations need to be made in light of corresponding evidence that also points towards pro-tumorigenic eﬀects of NRF2. In this review, we discuss the evidence to date, highlighting recent advances using gain- and loss-of-function animal models and how these data ﬁt with observations in humans. Keywords: NRF2; wound healing; keratinocyte; ﬁbroblast; inﬂammation International Journal of Molecular Sciences International Journal of Molecular Sciences Review Regulation of Wound Healing by the NRF2 Transcription Factor—More Than Cytoprotection Paul Hiebert * and Sabine Werner * Institute for Molecular Health Sciences, Department of Biology, Swiss Federal Institute of Technology Zürich, 8093 Zurich, Switzerland * Correspondence: paul.hiebert@biol.ethz.ch (P.H.); sabine.werner@biol.ethz.ch (S.W.) Int. J. Mol. Sci. 2019, 20, 3856; doi:10.3390/ijms20163856 www.mdpi.com/journal/ijms 1. The Wound Healing Process As the organ continuously exposed to the outside world, the skin is charged with many important functions, including maintaining body temperature, hydration, and sensing the environment. Among its most important functions is to provide a barrier to an external environment rich in pathogens and toxins. Furthermore, it is frequently exposed to harmful ultraviolet (UV) radiation. Given the importance of maintaining the integrity of the skin, mammals have evolved a complex and sophisticated wound healing response, whereby damaged tissue is eﬃciently repaired following injury. While the complex nature of this response contributes to its eﬀectiveness, it also leads to challenges when attempting to remedy pathological scenarios where wound healing goes wrong, such as in chronic, non-healing wounds/ulcers and in hypertrophic scars and keloids [1,2]. The normal wound healing response involves a series of overlapping phases beginning with haemostasis and the formation of a ﬁbrin clot. Haemostasis is initiated by platelets immediately after injury and functions critically to reduce blood loss from damaged vessels. It results in the deposition of a provisional matrix made primarily of ﬁbrin that provides a rapid seal of the wound. This temporary ﬁbrin mesh also functions as a scaﬀold for cells to migrate into the wound and as a reservoir of cytokines and growth factors. The inﬂammatory phase follows soon after, with neutrophils being the ﬁrst immune cells to arrive at the wound site due to their abundance in circulation. They function as an innate response to invading pathogens through their production of proteinases and reactive oxygen species (ROS), which, however, can also damage the cells at the wound site. In addition, neutrophils assist in the recruitment of other immune cell types, such as macrophages and lymphocytes, which accumulate in the following days. Inﬂammatory cells also function critically to produce cytokines and growth factors, which serve to attract ﬁbroblasts, stimulate keratinocyte migration and proliferation, and initiate the formation of new blood vessels [3,4]. This provides a ‘kick-start’ for the tissue formation Int. J. Mol. Sci. 2019, 20, 3856; doi:10.3390/ijms20163856 www.mdpi.com/journal/ijms 2 of 13 Int. J. Mol. Sci. 2019, 20, 3856 phase of wound healing, where keratinocytes at the wound edge begin to migrate into the wound along the injured dermis and then across the provisional wound matrix. Keratinocytes behind the migrating tongue accelerate their proliferation rate to produce a pool of new keratinocytes that replenish those that are lost by the wound [5]. 1. The Wound Healing Process Slightly after the onset of re-epithelialization, dermal ﬁbroblasts at the wound edge migrate into the wound bed, where they proliferate and produce large amounts of extracellular matrix (ECM). A large percentage of the wound ﬁbroblasts diﬀerentiate into myoﬁbroblasts, which strongly contribute to wound contraction [6]. Additional sources of wound (myo) ﬁbroblasts are bone marrow-derived cells and adipocyte precursors, which diﬀerentiate into (myo) ﬁbroblasts at the wound site [7–9]. In parallel, endothelial sprouting occurs at the wound edge, and massive angiogenesis leads to the formation of a new vasculature in the newly forming stromal part of the wound, which is called granulation tissue [1,2]. Once keratinocytes have fully covered and closed the wound, ﬁbroblasts and immune cells continue to remodel the underlying tissue, replacing the provisional matrix with one composed primarily of collagen type I. This remodeling phase also features increased apoptosis of immune cells, ﬁbroblasts and endothelial cells of vessels that do not contribute to the vasculature of the mature scar tissue. Fibroblasts that remain in the scar tissue can continue to remodel the healed skin for up to several years. While the resulting scar lacks the full mechanical strength and elasticity of the original skin and also all appendages, it functions eﬀectively as a restored skin barrier that once again oﬀers protection to environmental dangers [1,2,10,11]. The wound healing response, therefore, involves many carefully orchestrated events that need to provide the right signals to the right cells at the right time. Defects in any one of these wound healing phases can have disastrous consequences and lead to chronic, non-healing wounds followed by infection, hospitalization, or even death [12,13]. Alternatively, healing can be excessive, resulting in the formation of hypertrophic scars and keloids, which can cause severe functional and cosmetic impairments [14,15]. The complex gene expression pattern during wound healing is controlled by various key transcription factors, which orchestrate diﬀerent processes at the wound site [16,17]. One such transcription factor, mainly known for its cytoprotective activities, is NRF2, which will be introduced below. 2. The Transcription Factor NRF2 Nuclear factor-erythroid 2-related factor 2 (NRF2; NFE2L2) is a member of the cap‘n‘collar family of transcription factors, which also includes NFE2, NRF1 (NFE2L1), NRF3 (NFE2L3), BACH1, and BACH2 [18]. NRF2 is particularly famous for its central role in regulating the transcription of cytoprotective genes, including genes encoding various ROS-detoxifying enzymes, antioxidant proteins, and drug transporters [18–20]. Over the last three decades, research has demonstrated the remarkable importance of NRF2 in maintaining the cellular redox status in virtually all organs and tissues. Under homeostatic conditions, when only low amounts of the cytoprotective target proteins are needed, NRF2-mediated transcription is restricted to baseline levels by the NRF2 antagonist Kelch-like ECH-associated protein 1 (KEAP1). Binding of NRF2 to KEAP1 sequesters it in the cytoplasm and leads to its rapid proteasomal degradation. The presence of ROS and/or electrophilic compounds weakens the NRF2-KEAP1 interaction, allowing NRF2 to become stabilized. This results in accumulation of newly produced NRF2 in the nucleus. NRF2 acts by dimerizing with small musculoaponeurotic ﬁbrosarcoma (MAF) proteins and binding to speciﬁc regions of DNA called antioxidant response elements (AREs), where it initiates the transcription of its target genes [18,21] (Figure 1). In this way, the NRF2 antioxidant pathway serves to protect cells from dangerous levels of ROS that are generated in response to environmental stressors (e.g., UV irradiation, pathogens, and toxins) and during excessive inﬂammation. However, multiple studies identiﬁed additional NRF2 targets that are not directly involved in the antioxidant response and many of them are expressed in a tissue-speciﬁc and situation-dependent manner [22–25]. This is also relevant for wound healing and will be described in detail below. 3 of 13 Int. J. Mol. Sci. 2019, 20, 3856 Figure 1. The nuclear factor-erythroid 2-related factor 2 (NRF2) signaling pathway. NRF2 strongly binds to its cytoplasmic inhibitor Kelch-like ECH-associated protein 1 (KEAP1) under homeostatic conditions and only low levels of NRF2 are present in the nucleus. In response to reactive oxygen species (ROS) and/or electrophiles, the NRF2-KEAP1 interaction is weakened and newly formed NRF2 accumulates in the nucleus. Here, NRF2 dimerizes with small musculoaponeurotic fibrosarcoma (MAF) proteins and binds to antioxidant response elements (AREs) in the promoters or enhancers of its target genes, of which many encode ROS detoxifying enzymes and other antioxidant proteins, thereby initiating a cytoprotective response. Figure 1. The nuclear factor-erythroid 2-related factor 2 (NRF2) signaling pathway. 2. The Transcription Factor NRF2 NRF2 strongly binds to its cytoplasmic inhibitor Kelch-like ECH-associated protein 1 (KEAP1) under homeostatic conditions and only low levels of NRF2 are present in the nucleus. In response to reactive oxygen species (ROS) and/or electrophiles, the NRF2-KEAP1 interaction is weakened and newly formed NRF2 accumulates in the nucleus. Here, NRF2 dimerizes with small musculoaponeurotic ﬁbrosarcoma (MAF) proteins and binds to antioxidant response elements (AREs) in the promoters or enhancers of its target genes, of which many encode ROS detoxifying enzymes and other antioxidant proteins, thereby initiating a cytoprotective response. The benefits of activating the NRF2 pathway have generated intense interest in recent years, and NRF2 activating compounds continue to be tested for their health benefits and prevention of disease [26–28]. However, the cytoprotective benefits of NRF2 have been met with increased caution as evidence continues to accumulate suggesting a detrimental role in cancer, where NRF2-mediated protection of cancer cells can contribute to accelerated tumor growth and to chemo- and radio- resistance. Examples like these point to both a good and bad side of NRF2 in health and disease The beneﬁts of activating the NRF2 pathway have generated intense interest in recent years, and NRF2 activating compounds continue to be tested for their health beneﬁts and prevention of disease [26–28]. However, the cytoprotective beneﬁts of NRF2 have been met with increased caution as evidence continues to accumulate suggesting a detrimental role in cancer, where NRF2-mediated protection of cancer cells can contribute to accelerated tumor growth and to chemo- and radio-resistance. Examples like these point to both a good and bad side of NRF2 in health and disease [27,28]. 2. The Transcription Factor NRF2 NRF2 strongly binds to its cytoplasmic inhibitor Kelch-like ECH-associated protein 1 (KEAP1) under homeostatic conditions and only low levels of NRF2 are present in the nucleus. In response to reactive oxygen species (ROS) and/or electrophiles, the NRF2-KEAP1 interaction is weakened and newly formed NRF2 accumulates in the nucleus. Here, NRF2 dimerizes with small musculoaponeurotic ﬁbrosarcoma (MAF) proteins and binds to antioxidant response elements (AREs) in the promoters or enhancers of its target genes, of which many encode ROS detoxifying enzymes and other antioxidant proteins, thereby initiating a cytoprotective response. Figure 1. The nuclear factor-erythroid 2-related factor 2 (NRF2) signaling pathway. NRF2 strongly binds to its cytoplasmic inhibitor Kelch-like ECH-associated protein 1 (KEAP1) under homeostatic conditions and only low levels of NRF2 are present in the nucleus. In response to reactive oxygen species (ROS) and/or electrophiles, the NRF2-KEAP1 interaction is weakened and newly formed NRF2 accumulates in the nucleus. Here, NRF2 dimerizes with small musculoaponeurotic fibrosarcoma (MAF) proteins and binds to antioxidant response elements (AREs) in the promoters or enhancers of its target genes, of which many encode ROS detoxifying enzymes and other antioxidant proteins, thereby initiating a cytoprotective response. Figure 1. The nuclear factor-erythroid 2-related factor 2 (NRF2) signaling pathway. NRF2 strongly binds to its cytoplasmic inhibitor Kelch-like ECH-associated protein 1 (KEAP1) under homeostatic conditions and only low levels of NRF2 are present in the nucleus. In response to reactive oxygen species (ROS) and/or electrophiles, the NRF2-KEAP1 interaction is weakened and newly formed NRF2 accumulates in the nucleus. Here, NRF2 dimerizes with small musculoaponeurotic ﬁbrosarcoma (MAF) proteins and binds to antioxidant response elements (AREs) in the promoters or enhancers of its target genes, of which many encode ROS detoxifying enzymes and other antioxidant proteins, thereby initiating a cytoprotective response. Figure 1. The nuclear factor-erythroid 2-related factor 2 (NRF2) signaling pathway. NRF2 strongly binds to its cytoplasmic inhibitor Kelch-like ECH-associated protein 1 (KEAP1) under homeostatic conditions and only low levels of NRF2 are present in the nucleus. In response to reactive oxygen species (ROS) and/or electrophiles, the NRF2-KEAP1 interaction is weakened and newly formed NRF2 accumulates in the nucleus. Here, NRF2 dimerizes with small musculoaponeurotic fibrosarcoma (MAF) proteins and binds to antioxidant response elements (AREs) in the promoters or enhancers of its target genes, of which many encode ROS detoxifying enzymes and other antioxidant proteins, thereby initiating a cytoprotective response. Figure 1. The nuclear factor-erythroid 2-related factor 2 (NRF2) signaling pathway. [27,28]. 3. Expression and Activity of NRF2 in Healing Wounds 3. Expression and Activity of NRF2 in Healing Wounds Wounds produce large amounts of ROS to combat invading pathogens [16], to attract immune cells, and to regulate different cell signaling events [29]. As such, the role of NRF2 during the wound healing process has been of interest to many researchers. Several lines of evidence suggest an important role for NRF2 during tissue repair. This includes the observation that Nrf2 gene expression increases early after full-thickness wounding of mice along with ROS production, followed by a gradual reduction as wound healing progresses [30]. Keratinocytes of the hyperproliferative epithelium of skin wounds were shown to strongly express Nrf2, but expression of this gene was also seen in cells of the granulation tissue [30]. NRF2 has also been shown to become activated after tissue Wounds produce large amounts of ROS to combat invading pathogens [16], to attract immune cells, and to regulate diﬀerent cell signaling events [29]. As such, the role of NRF2 during the wound healing process has been of interest to many researchers. Several lines of evidence suggest an important role for NRF2 during tissue repair. This includes the observation that Nrf2 gene expression increases early after full-thickness wounding of mice along with ROS production, followed by a gradual reduction as wound healing progresses [30]. Keratinocytes of the hyperproliferative epithelium of skin wounds were shown to strongly express Nrf2, but expression of this gene was also seen in cells of the granulation tissue [30]. NRF2 has also been shown to become activated after tissue damage and possibly synergizes with other transcription factors to promote wound repair [31]. g [ ] damage and possibly synergizes with other transcription factors to promote wound repair [31]. A number of studies have tested NRF2-activating compounds for their impact on wound healing and have shown interesting results. These include purified molecules (including nitric oxide (NO) or NO donors), as well as bee venom, olive oil, and various plant extracts that include these compounds A number of studies have tested NRF2-activating compounds for their impact on wound healing and have shown interesting results. These include puriﬁed molecules (including nitric oxide (NO) or NO donors), as well as bee venom, olive oil, and various plant extracts that include these compounds [32–38]. However, it is important to note that these chemicals often have multiple eﬀects and modes of action, Int. J. Mol. Sci. 4. Consequences of NRF2 Loss-of-Function during Wound Healing Global NRF2 knockout (KO) mice have no obvious skin phenotype under normal conditions and were ﬁrst used in wound healing studies nearly two decades ago. Surprisingly, NRF2 KO mice healed wounds at normal rates and displayed no obvious histological diﬀerences compared to control animals [30]. Similar rates of cell proliferation and apoptosis between NRF2 KO and wild-type control mice were also observed in this study. Loss of NRF2 did, however, lead to a delayed induction, but prolonged overexpression of pro-inﬂammatory cytokines at the wound site and persistence of macrophages, even after closure of the wounds. Among the aﬀected cytokines was transforming growth factor β 1 (TGF-β1), whose delay at the beginning of wound healing may also lead to the reduced collagen production observed in wounds of NRF2 KO mice [30]. Despite the observed diﬀerences in cytokine production, the lack of a diﬀerence in wound healing kinetics in NRF2 KO vs. control mice may be due, at least in part, to compensatory actions of other transcription factors (e.g., NRF1 or NRF3) [30]. To address the issue of compensation, mice were generated that express a dominant-negative NRF2 mutant (dnNRF2) in keratinocytes [39]. The dnNRF2 mutant lacks the KEAP1 binding domain and the transactivation domains, leading to the nuclear accumulation of dnNRF2 that binds to AREs without facilitating transcription. In doing so, other cap‘n‘collar transcription factors are prevented from binding to these AREs, thereby avoiding potential compensatory eﬀects caused by a loss of NRF2. Similar to global NRF2 KO mice, however, keratinocyte-speciﬁc dnNRF2 mice showed no diﬀerences either in normal skin morphology or during wound healing [39]. Furthermore, mice lacking either NRF2 in keratinocytes or NRF3 in all cells healed normally [40,41]. This was surprising given previous ﬁndings showing NRF2 and NRF3 predominantly and highly expressed in the hyperproliferative wound epithelium [30]. However, keratinocytes possess multiple, and partially redundant antioxidant defense pathways [40,42], and loss of NRF2 activity may be compensated by other cytoprotective factors and pathways, allowing the epithelium to properly regenerate. It is likely that certain actions of NRF2 diﬀer substantially depending on cell type, as cells located in diﬀerent organs and tissues have diverse functions, speciﬁc NRF2 targets [22,23], and are exposed to varying degrees of environmental stressors. Skin ﬁbroblasts for example, are located within the dermis and are shielded from many of the environmental dangers faced by keratinocytes. [27,28]. 3. Expression and Activity of NRF2 in Healing Wounds 2019, 20, 3856 4 of 13 making the precise role of NRF2 in these studies unclear. Genetic approaches in mice using NRF2 gain- and loss-of-function models oﬀer an alternative strategy to investigating NRF2 function during tissue repair. Recent studies employing this strategy have added to the evidence for NRF2 as a key player in the wound healing process, with actions extending beyond its antioxidant eﬀects. However, as is typical for NRF2, this evidence occasionally oﬀers a mixed view of both the beneﬁts and possible detriments of activating the NRF2 pathway. In this review, we will discuss the accumulating evidence suggesting an important role for NRF2 during cutaneous wound healing, focusing on ﬁndings from genetic animal models, but extending to the potential use of chemical activators of the NRF2 pathway for the promotion of wound healing in humans. 4. Consequences of NRF2 Loss-of-Function during Wound Healing In vitro, skin ﬁbroblasts deﬁcient in NRF2 showed slightly increased levels of ROS, however, proliferation was similar to control cells [43]. In line with other NRF2 loss-of-function studies, mice with ﬁbroblast-speciﬁc deletion of NRF2 also showed no diﬀerences in wound healing kinetics compared to control animals [43]. These mice did feature an altered immune cell content in unwounded skin, but these diﬀerences disappeared upon wounding [43]. Similar to global NRF2 KO mice, this suggests that NRF2 may have some inﬂuence over immune cell recruitment, however, the eﬀect may be diluted in the context of a healing wound, where many other immune-regulatory mechanisms are at play. The prolonged wound inﬂammation in NRF2-deﬁcient mice suggested a possible role for NRF2 in immune cells. Immune cells of the myeloid lineage have important functions during wound healing in ﬁghting oﬀinvading bacteria through the production of ROS and in the production of healing-promoting factors [3]. In light of these observations, mice lacking NRF2 in myeloid cells 5 of 13 Int. J. Mol. Sci. 2019, 20, 3856 were generated and subjected to full-thickness excisional wounding [44]. In wounds of wild-type mice, macrophages, and to an even greater extent neutrophils, were shown to strongly express Nrf2 and cytoprotective NRF2 target genes (e.g., Nqo1). Nevertheless, and similar to other cell types, myeloid-speciﬁc NRF2 KO mice showed no diﬀerence in wound healing as revealed by morphometric analysis of histological wound sections and analysis of the wound immune cell content [44]. One caveat to consider is that wound healing studies conducted on laboratory mice are typically performed in very clean conditions. In situations that feature greater exposure to an abundant variety of bacterial or other environmental challenges, it is possible that NRF2 deﬁciency would prove to be more detrimental. Furthermore, the majority of these studies have focused primarily on examination of wound closure with, to date, less attention given to the eﬀect of NRF2 deﬁciency on scarring. Future work will likely address these open questions further, however the bulk of the evidence thus far strongly suggests that, while NRF2 may have important roles to play, its function is often redundant, making it dispensable for wound healing in healthy mice that do not face additional challenges. g y g In contrast to normal wound healing, pathological wound healing represents a situation where the normal wound healing process has failed, leading to both chronic wounds, ulcers, or hypertrophic scars and keloids. 4. Consequences of NRF2 Loss-of-Function during Wound Healing Due to the increased challenge faced by the skin in these scenarios [1,2], it is possible that the loss of NRF2 would have a more important impact. This hypothesis is supported by experiments performed using a diabetic mouse model of delayed wound healing [45]. Mice injected with streptozotocin develop type I diabetes and exhibit signiﬁcantly delayed wound closure compared to control mice. Interestingly, wound closure was delayed even further in streptozotocin-induced diabetic NRF2 KO mice [45], suggesting that unlike normal wound healing, NRF2 may indeed have a more crucial role in facilitating wound closure in pathological situations, such as diabetes. This hypothesis is further supported by the ﬁnding that the NRF2 pathway is functionally insuﬃcient in bone marrow-derived multipotent stromal cells from diabetic mice [46]. The consequences of NRF2 loss- or gain-of-function on the wound healing process are summarized in Table 1. Table 1. NRF2 gain- or loss-of-function phenotypes during wound healing. Cell Type Loss-of-Function Phenotype Gain-of-Function Phenotype Global No change in wound closure rates [30] Delayed induction of cytokines [30] Prolonged wound inﬂammation [30] Reduced collagen deposition [30] Delayed diabetic wound healing [45] Improved diabetic wound healing [45] Increased keratinocyte proliferation [45] Restoration of normal transforming growth factor β 1 (TGF-β1) and matrix metalloproteinase (MMP)9 levels [45] Keratinocytes No change in wound closure rates [39] Accelerated wound closure/re-epithelialization [47] Increased expansion of pilosebaceous cells [47] No change in keratinocyte proliferation within the wound [47] Fibroblasts No change in wound closure rates [43] Possible alterations in immune cell proﬁle [43] Accelerated wound closure/re-epithelialization [43] Increased onset of ﬁbroblast senescence/senescence-associated secretory phenotype (SASP) [43] Increased keratinocyte proliferation [43] Myeloid cells No changes in wound closure rates [44] No changes in wound closure rates [44] Table 1. NRF2 gain- or loss-of-function phenotypes during wound healing. 5. Consequences of NRF2 Activation during Wound Healing 5. Consequences of NRF2 Activation during Wound Healing Typically, it is in pathological scenarios where NRF2 activators continue to gain attention as possible therapeutics due to their antioxidant properties. As shown in diabetic mice, NRF2 activity appears to be insuﬃcient during impaired wound healing, resulting in excessive ROS levels, which can lead to severe tissue damage and uncontrolled inﬂammation. Consistent with this assumption, delayed wound healing observed in mice that are diabetic as a consequence of a mutation in the leptin receptor (Leprdb/db mice) and in streptozotocin-induced diabetic mice was signiﬁcantly improved through the use of NRF2 activating compounds when applied either systemically or topically [32,45]. Administration of such compounds also led to the restoration of normal TGF-β1 and matrix metalloproteinase (MMP)9 6 of 13 Int. J. Mol. Sci. 2019, 20, 3856 levels in the skin and reduced the oxidative burden [32,45]. Furthermore, NRF2 activating compounds promoted proliferation and migration of the immortalized HaCaT keratinocyte cell line in vitro under hyperglycaemic conditions [45]. It has also been shown that drugs used to treat diabetes can activate the NRF2 pathway [48]. Interestingly, these same drugs have shown eﬃcacy in promoting diabetic wound healing, although in this instance, activation of the NRF2 pathway may not be the principle mechanism of action [49]. Taken together, these data point to activation of the NRF2 pathway as a promising strategy to promote wound healing under stress conditions (Figure 2). Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 6 of 13 used to treat diabetes can activate the NRF2 pathway [48]. Interestingly, these same drugs have shown efficacy in promoting diabetic wound healing, although in this instance, activation of the NRF2 pathway may not be the principle mechanism of action [49]. Taken together, these data point to activation of the NRF2 pathway as a promising strategy to promote wound healing under stress conditions (Figure 2). Figure 2. Characteristic features of chronic wounds, which may be improved by treatment with NRF2 activating compounds. NRF2 activation reduces oxidative stress, thereby enhancing production of TGF-β1, which is important for granulation tissue formation and matrix production. Reduction of ROS also suppresses the chronic inflammation and the excessive production of MMP9. Expansion of pilosebaceous cells by activated NRF2 may promote re-epithelialization of the wound. Activation of NRF2 in fibroblasts may promote senescence and associated production of a SASP, which can further promote wound re-epithelialization. 5. Consequences of NRF2 Activation during Wound Healing Arrows pointing to the top indicate upregulation and arrows pointing to the bottom indicate downregulation. Figure 2. Characteristic features of chronic wounds, which may be improved by treatment with NRF2 activating compounds. NRF2 activation reduces oxidative stress, thereby enhancing production of TGF-β1, which is important for granulation tissue formation and matrix production. Reduction of ROS also suppresses the chronic inﬂammation and the excessive production of MMP9. Expansion of pilosebaceous cells by activated NRF2 may promote re-epithelialization of the wound. Activation of NRF2 in ﬁbroblasts may promote senescence and associated production of a SASP, which can further promote wound re-epithelialization. Arrows pointing to the top indicate upregulation and arrows pointing to the bottom indicate downregulation. Figure 2. Characteristic features of chronic wounds, which may be improved by treatment with NRF2 activating compounds. NRF2 activation reduces oxidative stress, thereby enhancing production of TGF-β1, which is important for granulation tissue formation and matrix production. Reduction of ROS also suppresses the chronic inflammation and the excessive production of MMP9. Expansion of pilosebaceous cells by activated NRF2 may promote re-epithelialization of the wound. Activation of NRF2 in fibroblasts may promote senescence and associated production of a SASP, which can further promote wound re-epithelialization. Arrows pointing to the top indicate upregulation and arrows pointing to the bottom indicate downregulation. Figure 2. Characteristic features of chronic wounds, which may be improved by treatment with NRF2 activating compounds. NRF2 activation reduces oxidative stress, thereby enhancing production of TGF-β1, which is important for granulation tissue formation and matrix production. Reduction of ROS also suppresses the chronic inﬂammation and the excessive production of MMP9. Expansion of pilosebaceous cells by activated NRF2 may promote re-epithelialization of the wound. Activation of NRF2 in ﬁbroblasts may promote senescence and associated production of a SASP, which can further promote wound re-epithelialization. Arrows pointing to the top indicate upregulation and arrows pointing to the bottom indicate downregulation. As mentioned, one downside of using NRF2 activators to elucidate the role of NRF2 in vivo is their likely multiple modes of action, which make the precise role of NRF2 difficult to determine. Alternatively, genetic approaches in rodents have been used to study the consequences of NRF2 activation. In one study, exosomes produced by adipose-derived stem cells overexpressing NRF2 were shown to improve the ulceration of foot wounds in diabetic rats [50]. 5. Consequences of NRF2 Activation during Wound Healing 2019, 20, 3856 7 of 13 in bone marrow-derived multipotent stromal cells of diabetic mice through knock-down of KEAP1 restored their multipotent cell properties and promoted wound healing in diabetic mice [46]. Due to the early lethality of KEAP1 KO mice, and because KEAP1 also targets other factors in the cell besides NRF2 [55–57], an alternative approach for achieving constitutive NRF2 activation is cell-speciﬁc expression of a constitutively active NRF2 (caNRF2) mutant [58,59]. The caNRF2 mutant lacks the KEAP1 binding domain and therefore remains constitutively active in the nucleus without aﬀecting interactions between KEAP1 and other proteins. g p Mice expressing low levels of caNRF2 in keratinocytes only exhibit mild hyperkeratosis in the tail skin, but they are protected from UVB-induced cell death [58]. However, stronger expression of caNRF2 that reﬂects the activation level achieved after activation of endogenous NRF2 by chemical activators, caused obvious skin abnormalities, including mild inﬂammation, hyperkeratosis, sebaceous gland hypertrophy, and defects in epidermal barrier function [60]. Interestingly, these mice showed enhanced re-epithelialization during wound healing, leading to signiﬁcantly faster wound closure [47]. While improved wound healing in these mice is in agreement with results observed in diabetic mice treated with NRF2 activating compounds, caNRF2 expression did not lead to diﬀerences in keratinocyte migration, and resulted in no change in keratinocyte proliferation in the wound epidermis. Instead, caNRF2 increased proliferation of cells within hair follicles and sebaceous glands at the wound periphery that expressed keratinocyte stem cell markers [47]. Stem cell subpopulations in the hair follicle bulge, the junctional zone and the upper isthmus were previously shown to contribute to wound re-epithelialization [61] and caNRF2 speciﬁcally stimulated the proliferation of cells with markers of junctional zone and upper isthmus stem cells [47]. Therefore, the additional pool of cells that are generated at the wound edge most likely via stem cell expansion seems to function as an increased reservoir of cells available to migrate into and re-populate the wound. The expansion of these stem cell populations is plausibly driven by NRF2-mediated expression of epigen in these cells. This epidermal growth factor family member, which is encoded by a direct target gene of NRF2 [60], has been identiﬁed as a strong promoter of hair follicle stem cell proliferation in vivo [62]. 5. Consequences of NRF2 Activation during Wound Healing Furthermore, closure of ear holes was accelerated in mice with a gain of function mutation in the gene encoding rhomboid family protein RHBDF2, and this was associated with activation of the NRF2 pathway [51]. However, the specific contribution of NRF2 in the beneficial effect of RHBDF2 is not fully clear. To more specifically address the effect of activated NRF2 in vivo, KEAP1 KO mice were generated [52]. As the principle NRF2 antagonist, KEAP1 deletion results in constitutive activation of NRF2 in the cell without the need for any additional stimulus. However, global KEAP1 KO mice do not survive longer than 2 to 3 weeks, most likely due to irregular cornification and hyperkeratosis in the esophagus, resulting in gastric obstruction [52]. When diabetic mice were wounded and treated with KEAP1 siRNA, wound healing was significantly improved, featuring faster wound closure, elevated granulation tissue formation, and reduced ROS-mediated formation of 8-oxo-2’-deoxyguanosine As mentioned, one downside of using NRF2 activators to elucidate the role of NRF2 in vivo is their likely multiple modes of action, which make the precise role of NRF2 diﬃcult to determine. Alternatively, genetic approaches in rodents have been used to study the consequences of NRF2 activation. In one study, exosomes produced by adipose-derived stem cells overexpressing NRF2 were shown to improve the ulceration of foot wounds in diabetic rats [50]. Furthermore, closure of ear holes was accelerated in mice with a gain of function mutation in the gene encoding rhomboid family protein RHBDF2, and this was associated with activation of the NRF2 pathway [51]. However, the speciﬁc contribution of NRF2 in the beneﬁcial eﬀect of RHBDF2 is not fully clear. To more speciﬁcally address the eﬀect of activated NRF2 in vivo, KEAP1 KO mice were generated [52]. As the principle NRF2 antagonist, KEAP1 deletion results in constitutive activation of NRF2 in the cell without the need for any additional stimulus. However, global KEAP1 KO mice do not survive longer than 2 to 3 weeks, most likely due to irregular corniﬁcation and hyperkeratosis in the esophagus, resulting in gastric obstruction [52]. When diabetic mice were wounded and treated with KEAP1 siRNA, wound healing was signiﬁcantly improved, featuring faster wound closure, elevated granulation tissue formation, and reduced ROS-mediated formation of 8-oxo-2’-deoxyguanosine [53,54]. Furthermore, NRF2 activation Int. J. Mol. Sci. 5. Consequences of NRF2 Activation during Wound Healing Interestingly, faster wound closure in caNRF2-transgenic mice required a suﬃcient degree of NRF2 activation, as mice expressing lower levels of the caNRF2 transgene failed to show diﬀerences in wound closure, despite a slight elevation in the expression of NRF2 target genes [47]. These data reinforce the idea that NRF2 activation can have beneﬁts for wound healing and that the degree of NRF2 activation may be important to consider when evaluating its eﬀectiveness. The consequences of NRF2 activation in ﬁbroblasts have also been investigated. Fibroblasts in the skin play critical roles during wound healing, including the release of cytokines and growth factors, deposition of ECM, and wound contraction [2,63]. Remarkably, activating NRF2 in ﬁbroblasts lead to ﬁbroblast senescence both in vitro and during wound healing in vivo [43]. This was shown to be driven largely by the NRF2-mediated deposition of an altered matrisome featuring elevated levels of the senescence-promoting factor plasminogen activator inhibitor-1 (PAI-1, serpine1). Accelerated senescence was observed both in caNRF2-expressing ﬁbroblasts and in wild-type ﬁbroblasts treated with the NRF2 activator tert-butyl-hydroquinone, and occurred despite reduced levels of intracellular ROS and reduced DNA damage [43]. This is of relevance during tissue repair as senescent cells provide important molecular cues to neighboring cells via a growth-promoting secretome referred to as the senescence-associated secretory phenotype (SASP). Furthermore, senescent cells have been shown to be important for normal wound healing [64]. caNRF2 ﬁbroblasts do indeed display a SASP and release factors capable of directly promoting keratinocyte proliferation [43]. Accordingly, mice expressing caNRF2 in ﬁbroblasts showed signiﬁcantly faster wound closure, featuring enhanced re-epithelialization as a result of increased keratinocyte proliferation in the wound epidermis. Unlike keratinocytes and ﬁbroblasts, the impact of NRF2 activation in myeloid cells on wound healing has proven to be relatively little by comparison. Mice expressing caNRF2 in myeloid cells showed no signiﬁcant diﬀerence in wound closure rates and even failed to show increased expression of NRF2 target genes to a signiﬁcant degree [44]. A likely explanation for this is that these cells, Int. J. Mol. Sci. 2019, 20, 3856 8 of 13 particularly neutrophils, display very high levels of NRF2 expression and activation even during homeostasis and in the circulation, thereby dwarﬁng any additional eﬀect of the caNRF2 transgene. 6. Cancer and the Dark Side of NRF2 For many years, researchers have observed important parallels between the wound healing process and cancer [67]. This is also evident when considering the potential beneﬁts of NRF2 on wound healing, actions which may prove unwelcome during tumorigenesis [27,68]. This stands to reason given that activating cellular defense pathways tend to promote cell survival, which is undesirable in tumor cells and in conﬂict with the actions of chemotherapeutic drugs and with radiotherapy [68]. In humans, multiple lines of evidence support both pro- and anti-tumorigenic roles for NRF2 including during skin cancer development and progression [69], a phenomenon that can also be observed in gain- and loss-of-function animal models. For example, hyperactivation of NRF2 as a result of KEAP1 loss-of-function protected from UV-induced skin carcinogenesis [70,71], while mice expressing low levels of caNRF2 in keratinocytes showed increased tumor incidence and multiplicity in a genetic model of Human Papilloma Virus 8-induced skin cancer, resulting primarily from increased survival of keratinocytes during the early stages of the transformation process [72]. In contrast, these same mice expressing caNRF2 in keratinocytes showed mildly reduced tumor incidence and multiplicity in a chemically-induced model of skin cancer, where NRF2-mediated detoxiﬁcation of the mutagen and of ROS induced during the treatment overruled the pro-tumorigenic activity of NRF2 [72]. A beneﬁcial aspect of a basal activity of NRF2 in cancer development is supported by experiments performed in mice with NRF2 loss-of-function in keratinocytes. Here, the same chemically-induced skin carcinogenesis protocol lead to signiﬁcantly greater tumor multiplicity in mice lacking a functional NRF2, suggesting an important role for NRF2 in cancer prevention [39,73]. This speaks to the importance of context, as NRF2 activation in healthy cells will indeed lead to reduced cellular stress, preventing DNA damage and cancer-causing mutations, an action that becomes detrimental, however, once malignant transformation has occurred and once NRF2 becomes hyperactivated. Separate from its classical role in detoxiﬁcation of ROS, NRF2 can also inﬂuence PI3K signaling, and has been shown to activate transcription of the gene coding for mechanistic Target of Rapamycin (mTOR), a common promoter of tumor growth [74]. Interestingly, mTOR signaling may also have important implications for wound healing [75–77], however the role of NRF2-mediated mTOR activation in this context remains to be determined. The pro-tumorigenic actions of NRF2 can also be extended beyond the cancer cells. 5. Consequences of NRF2 Activation during Wound Healing This stands in contrast to NRF2 activation in myeloid cells in other organs, where genetic deletion of KEAP1 resulted in protective eﬀects in mouse models of sepsis and ischemia/reperfusion injury in the liver [65,66]. Nevertheless, activation of NRF2 in the wound tissue seems to predominantly aﬀect non-immune cells. Taken together, the bulk of the evidence supports activation of NRF2 as a promising strategy to promote wound repair, particularly in situations where healing is negatively aﬀected by enhanced ROS levels (Figure 2). 6. Cancer and the Dark Side of NRF2 Mice expressing caNRF2 in ﬁbroblasts have been shown to express a cancer-associated ﬁbroblast (CAF) gene expression signature, capable of promoting tumor growth in vivo in a xenograft model of squamous cell carcinoma (SCC) [43]. Here, SCC cells were co-injected with caNRF2-expressing ﬁbroblasts into the skin of immunocompromised mice. The activated NRF2 induced a CAF phenotype in these cells and led to the SCC cells forming signiﬁcantly larger tumors compared to those co-injected with control ﬁbroblasts [43]. This suggests that in addition to promoting cancer cell survival, NRF2 activation in other cell types (e.g., CAFs or others) may have additional impacts on cancer development, and that NRF2 activation can have multiple consequences beyond cytoprotection. 7. Summary and Future Perspectives The last three decades have seen an explosion of interest in NRF2 and its antioxidant role, cementing its importance in maintaining the cellular redox status. During this time, many researchers 9 of 13 Int. J. Mol. Sci. 2019, 20, 3856 have investigated the use of NRF2 activating compounds as a means of cancer prevention and for preventing, or even treating, chronic inﬂammatory and degenerative diseases [26–28]. However, the antioxidant activity also results in survival of cancer cells under stress conditions and, together with NRF2’s important function in drug detoxiﬁcation, makes them chemo- and radioresistant [27]. Furthermore, recent advances using genetic animal models have uncovered additional and unexpected consequences of NRF2 activation beyond cytoprotection. In the skin, this included promotion of keratinocyte stem cell proliferation and of ﬁbroblast senescence, impairment of epidermal barrier function, induction of sebaceous gland hyperplasia, and deposition of an abnormal ECM [43,47]. Interestingly, most of these activities are controlled by cell type-speciﬁc NRF2 target genes that are not conventional NRF2 targets under homeostatic conditions. In addition to the promising eﬀects on wound healing observed in mice, many of these ﬁndings may be translated to humans (Figure 2). NRF2 and its target genes were shown to be activated in the hyperthickened epidermis of human diabetic ulcers, likely as a consequence of elevated ROS levels and reﬂective of the severe oxidative stress that occurs in these wounds [45]. This may well protect keratinocytes from oxidative stress to a certain extent and this protection may be further promoted by application of NRF2 activating compounds. Indeed, and as mentioned above, human keratinocytes (HaCaT cell line) are stimulated to proliferate when treated with NRF2 activators under hyperglycaemic conditions, similar to observations in diabetic mice treated with these same compounds [45]. In the future it will be important to determine if NRF2 is also activated in ﬁbroblasts of chronic human wounds, which could contribute to the ﬁbroblast senescence that is frequently seen in skin ulcers [78]. Indeed, human ﬁbroblasts treated with NRF2 activating compounds, or with CRISPR/Cas-mediated deletion of KEAP1, also undergo senescence at an accelerated rate compared to untreated cells [43]. However, it remains to be determined if these cells are still able to produce a healing promoting SASP in chronic wounds/ulcers as seen in acute mouse wounds [43]. 7. Summary and Future Perspectives For other eﬀects of NRF2 activation in mouse wounds, such as expansion of stem cells within hair follicles [47], the impact on human wounds is unclear, as human skin typically contains far fewer hair follicles than mouse skin. As such, these eﬀects may be more relevant to speciﬁc areas of the skin with a higher hair follicle density. Together, these ﬁndings demonstrate the multifaceted actions of NRF2 during tissue repair, and have helped uncover important mechanisms that may beneﬁt humans suﬀering from chronic wounds. However, it is also evident from multiple studies that pro-tumorigenic actions and other potentially detrimental activities should remain an important consideration prior to employing NRF2 activating compounds to wounds in the clinic. Therefore, application of such compounds should be limited to short treatment periods and restricted to patients in which malignancy at the wound site is excluded. Future work aimed at unravelling beneﬁcial vs. deleterious actions of NRF2 and identiﬁcation of the relevant target genes and their regulation will be of particular importance. A better understanding of these matters will potentially lead to the development of more potent and safer NRF2 activators that may reliably be used to promote wound healing in patients. Funding: Research on NRF2 in our laboratory was/is supported by grants from the Swiss National Science Foundation (grant 31003A_169204 to S.W.), the Wilhelm-Sander Stiftung (grant No. 2-70941-18 to S.W.) and a Banting postdoctoral fellowship (to P.H.). Conﬂicts of Interest: The authors declare no conﬂict of interest. 1. Gurtner, G.C.; Werner, S.; Barrandon, Y.; Longaker, M.T. Wound repair and regeneration. Nature 2008, 453, 314–321. [CrossRef] [PubMed] 2. Eming, S.A.; Martin, P.; Tomic-Canic, M. Wound repair and regeneration: Mechanisms, signaling, and translation. Sci. Transl. Med. 2014, 6, 265–266. [CrossRef] [PubMed] 3. Martin, P.; Leibovich, S.J. Inﬂammatory cells during wound repair: The good, the bad and the ugly. Trends Cell Biol. 2005, 15, 599–607. [CrossRef] [PubMed] References 1. Gurtner, G.C.; Werner, S.; Barrandon, Y.; Longaker, M.T. Wound repair and regeneration. Nature 2008, 453, 314–321. [CrossRef] [PubMed] 3. Martin, P.; Leibovich, S.J. Inﬂammatory cells during wound repair: The good, the bad and the ugly. Trends Cell Biol. 2005, 15, 599–607. [CrossRef] [PubMed] 10 of 13 Int. J. Mol. Sci. 2019, 20, 3856 4. Eming, S.A.; Krieg, T.; Davidson, J.M. Inﬂammation in wound repair: Molecular and cellular mechanisms. J. Investig. Dermatol. 2007, 127, 514–525. [CrossRef] [PubMed] 5. Park, S.; Gonzalez, D.G.; Guirao, B.; Boucher, J.D.; Cockburn, K.; Marsh, E.D.; Mesa, K.R.; Brown, S.; Rompolas, P.; Haberman, A.M.; et al. Tissue-scale coordination of cellular behaviour promotes epidermal wound repair in live mice. Nat. Cell Biol. 2017, 19, 155–163. [CrossRef] [PubMed] p 6. Hinz, B. The role of myoﬁbroblasts in wound healing. Curr. Res. Transl. Med. 2016, 64, 171–177. [CrossRef] 6. Hinz, B. The role of myoﬁbroblasts in wound healing. Curr. Res. Transl. Med. 2016, 64, 171–177. [CrossRef] 7. Opalenik, S.R.; Davidson, J.M. Fibroblast diﬀerentiation of bone marrow-derived cells during wound repair. FASEB J. 2005, 19, 1561–1563. [CrossRef] y g 7. Opalenik, S.R.; Davidson, J.M. Fibroblast diﬀerentiation of bone marrow-derived cells during wound repair. FASEB J. 2005, 19, 1561–1563. [CrossRef] 8. Guerrero-Juarez, C.F.; Dedhia, P.H.; Jin, S.; Ruiz-Vega, R.; Ma, D.; Liu, Y.; Yamaga, K.; Shestova, O.; Gay, D.L.; Yang, Z.; et al. Single-cell analysis reveals ﬁbroblast heterogeneity and myeloid-derived adipocyte progenitors in murine skin wounds. Nat. Commun. 2019, 10, 650. [CrossRef] 9. Shook, B.A.; Wasko, R.R.; Rivera-Gonzalez, G.C.; Salazar-Gatzimas, E.; Lopez-Giraldez, F.; Dash, B.C.; Munoz-Rojas, A.R.; Aultman, K.D.; Zwick, R.K.; Lei, V.; et al. Myoﬁbroblast proliferation and heterogeneity are supported by macrophages during skin repair. Science 2018, 362. [CrossRef] 10. Martin, P. Wound healing–aiming for perfect skin regeneration. Science 1997, 276, 75–81. [CrossRef 11. Grose, R.; Werner, S. Wound healing studies in transgenic and knockout mice. A review. Methods Mol. Med. 2003, 78, 191–216. [CrossRef] 12. Sen, C.K.; Gordillo, G.M.; Roy, S.; Kirsner, R.; Lambert, L.; Hunt, T.K.; Gottrup, F.; Gurtner, G.C.; Longaker, M.T. Human skin wounds: A major and snowballing threat to public health and the economy. Wound Repair Regen. 2009, 17, 763–771. [CrossRef] 13. Darwin, E.; Tomic-Canic, M. Healing Chronic Wounds: Current Challenges and Potential Solutions. Curr. Dermatol. Rep. 2018, 7, 296–302. [CrossRef] p 14. Berman, B.; Maderal, A.; Raphael, B. Keloids and Hypertrophic Scars: Pathophysiology, Classiﬁcation, and Treatment. Dermatol. Surg. 2017, 43 (Suppl. 1), S3–S18. [CrossRef] 15. References [CrossRef] 28. Cuadrado, A.; Rojo, A.I.; Wells, G.; Hayes, J.D.; Cousin, S.P.; Rumsey, W.L.; Attucks, O.C.; Franklin, S.; Levonen, A.L.; Kensler, T.W.; et al. Therapeutic targeting of the NRF2 and KEAP1 partnership in chronic diseases. Nat. Rev. Drug Discov. 2019, 18, 295–317. [CrossRef] g 29. Niethammer, P. Wound redox gradients revisited. Semin. Cell Dev. Biol. 2018, 80, 13–16. [CrossRef] 30. Braun, S.; Hanselmann, C.; Gassmann, M.G.; auf dem Keller, U.; Born-Berclaz, C.; Chan, K.; Kan, Y.W.; Werner, S. Nrf2 Transcription Factor, a Novel Target of Keratinocyte Growth Factor Action Which Regulates Gene Expression and Inﬂammation in the Healing Skin Wound. Mol. Cell. Biol. 2002, 22, 5492–5505. [CrossRef] 31. Eichenﬁeld, D.Z.; Troutman, T.D.; Link, V.M.; Lam, M.T.; Cho, H.; Gosselin, D.; Spann, N.J.; Lesch, H.P.; Tao, J.; Muto, J.; et al. Tissue damage drives co-localization of NF-kappaB, Smad3, and Nrf2 to direct Rev-erb sensitive wound repair in mouse macrophages. Elife 2016, 5. [CrossRef] 32. Rabbani, P.S.; Ellison, T.; Waqas, B.; Sultan, D.; Abdou, S.; David, J.A.; Cohen, J.M.; Gomez-Viso, A.; Lam, G.; Kim, C.; et al. Targeted Nrf2 activation therapy with RTA 408 enhances regenerative capacity of diabetic wounds. Diabetes Res. Clin. Pract. 2018, 139, 11–23. [CrossRef] 33. Hozzein, W.N.; Badr, G.; Badr, B.M.; Allam, A.; Ghamdi, A.A.; Al-Wadaan, M.A.; Al-Waili, N.S. Bee venom improves diabetic wound healing by protecting functional macrophages from apoptosis and enhancing Nrf2, Ang-1 and Tie-2 signaling. Mol. Immunol. 2018, 103, 322–335. [CrossRef] 34. Schanuel, F.S.; Saguie, B.O.; Monte-Alto-Costa, A. Olive oil promotes wound healing of mice pressure injuries through NOS-2 and Nrf2. Appl. Physiol. Nutr. Metab. 2019. [CrossRef] 35. Weller, R.; Finnen, M.J. The eﬀects of topical treatment with acidiﬁed nitrite on wound healing in normal and diabetic mice. Nitric Oxide 2006, 15, 395–399. [CrossRef] 36. Masters, K.S.; Leibovich, S.J.; Belem, P.; West, J.L.; Poole-Warren, L.A. Eﬀects of nitric oxide releasing poly (vinyl alcohol) hydrogel dressings on dermal wound healing in diabetic mice. Wound Repair Regen. 2002, 10, 286–294. [CrossRef] 37. Edmonds, M.E.; Bodansky, H.J.; Boulton, A.J.M.; Chadwick, P.J.; Dang, C.N.; D’Costa, R.; Johnston, A.; Kennon, B.; Leese, G.; Rajbhandari, S.M.; et al. Multicenter, randomized controlled, observer-blinded study of a nitric oxide generating treatment in foot ulcers of patients with diabetes-ProNOx1 study. Wound Repair Regen. 2018, 26, 228–237. [CrossRef] 38. Senger, D.R.; Cao, S. Diabetic Wound Healing and Activation of Nrf2 by Herbal Medicine. J. Nat. Sci. 2016, 2, e247. 39. References Trace, A.P.; Enos, C.W.; Mantel, A.; Harvey, V.M. Keloids and Hypertrophic Scars: A Spectrum of Clinical Challenges. Am. J. Clin. Dermatol. 2016, 17, 201–223. [CrossRef] 16. Schafer, M.; Werner, S. Oxidative stress in normal and impaired wound repair. Pharmacol. Res. 2008, 58, 165–171. [CrossRef] 17. Haertel, E.; Werner, S.; Schafer, M. Transcriptional regulation of wound inﬂammation. Semin. Immunol. 2014, 26, 321–328. [CrossRef] 18. Sykiotis, G.P.; Bohmann, D. Stress-activated cap’n’collar transcription factors in aging and human disease. Sci. Signal. 2010, 3, re3. [CrossRef] 19. Kansanen, E.; Kuosmanen, S.M.; Leinonen, H.; Levonen, A.L. The Keap1-Nrf2 pathway: Mechanisms of activation and dysregulation in cancer. Redox. Biol. 2013, 1, 45–49. [CrossRef] 20. Taguchi, K.; Motohashi, H.; Yamamoto, M. Molecular mechanisms of the Keap1-Nrf2 pathway in stress response and cancer evolution. Genes Cells 2011, 16, 123–140. [CrossRef] 21. Suzuki, T.; Yamamoto, M. Stress-sensing mechanisms and th during cellular stress. J. Biol. Chem. 2017. [CrossRef] 21. Suzuki, T.; Yamamoto, M. Stress-sensing mechanisms and the physiological roles of the Keap1-Nrf2 system during cellular stress. J. Biol. Chem. 2017. [CrossRef] 22. Malhotra, D.; Portales-Casamar, E.; Singh, A.; Srivastava, S.; Arenillas, D.; Happel, C.; Shyr, C.; Wakabayashi, N.; Kensler, T.W.; Wasserman, W.W.; et al. Global mapping of binding sites for Nrf2 identiﬁes novel targets in cell survival response through ChIP-Seq proﬁling and network analysis. Nucleic Acids Res. 2010, 38, 5718–5734. [CrossRef] 23. Chorley, B.N.; Campbell, M.R.; Wang, X.; Karaca, M.; Sambandan, D.; Bangura, F.; Xue, P.; Pi, J.; Kleeberger, S.R.; Bell, D.A. Identiﬁcation of novel NRF2-regulated genes by ChIP-Seq: Inﬂuence on retinoid X receptor alpha. Nucleic Acids Res. 2012, 40, 7416–7429. [CrossRef] 24. Nault, R.; Doskey, C.M.; Fader, K.A.; Rockwell, C.E.; Zacharewski, T. Comparison of Hepatic NRF2 and Aryl Hydrocarbon Receptor Binding in 2,3,7,8-Tetrachlorodibenzo-p-dioxin-Treated Mice Demonstrates NRF2-Independent PKM2 Induction. Mol. Pharmacol. 2018, 94, 876–884. [CrossRef] 25. Kobayashi, E.H.; Suzuki, T.; Funayama, R.; Nagashima, T.; Hayashi, M.; Sekine, H.; Tanaka, N.; Moriguchi, T.; Motohashi, H.; Nakayama, K.; et al. Nrf2 suppresses macrophage inﬂammatory response by blocking proinﬂammatory cytokine transcription. Nat. Commun. 2016, 7, 11624. [CrossRef] 26. Liby, K.T.; Sporn, M.B. Synthetic oleanane triterpenoids: Multifunctional drugs with a broad range of applications for prevention and treatment of chronic disease. Pharmacol. Rev. 2012, 64, 972–1003. [CrossRef] Int. J. Mol. Sci. 2019, 20, 3856 11 of 13 11 of 13 27. Sporn, M.B.; Liby, K.T. NRF2 and cancer: The good, the bad and the importance of context. Nat. Rev. Cancer 2012, 12, 564–571. References NRF2 activation by antioxidant antidiabetic agents accelerates tumor metastasis. Sci. Transl. Med. 2016, 8, 334ra351. [CrossRef] 49. Long, M.; Cai, L.; Li, W.; Zhang, L.; Guo, S.; Zhang, R.; Zheng, Y.; Liu, X.; Wang, M.; Zhou, X.; et al. DPP-4 Inhibitors Improve Diabetic Wound Healing via Direct and Indirect Promotion of Epithelial-Mesenchymal Transition and Reduction of Scarring. Diabetes 2018, 67, 518–531. [CrossRef] 50. Li, X.; Xie, X.; Lian, W.; Shi, R.; Han, S.; Zhang, H.; Lu, L.; Li, M. Exosomes from adipose-derived stem cells overexpressing Nrf2 accelerate cutaneous wound healing by promoting vascularization in a diabetic foot ulcer rat model. Exp. Mol. Med. 2018, 50, 29. [CrossRef] 51. Hosur, V.; Burzenski, L.M.; Stearns, T.M.; Farley, M.L.; Sundberg, J.P.; Wiles, M.V.; Shultz, L.D. Early induction of NRF2 antioxidant pathway by RHBDF2 mediates rapid cutaneous wound healing. Exp. Mol. Pathol. 2017, 102, 337–346. [CrossRef] 52. Wakabayashi, N.; Itoh, K.; Wakabayashi, J.; Motohashi, H.; Noda, S.; Takahashi, S.; Imakado, S.; Kotsuji, T.; Otsuka, F.; Roop, D.R.; et al. Keap1-null mutation leads to postnatal lethality due to constitutive Nrf2 activation. Nat. Genet. 2003, 35, 238–245. [CrossRef] 53. Soares, M.A.; Cohen, O.D.; Low, Y.C.; Sartor, R.A.; Ellison, T.; Anil, U.; Anzai, L.; Chang, J.B.; Saadeh, P.B.; Rabbani, P.S.; et al. Restoration of Nrf2 Signaling Normalizes the Regenerative Niche. Diabetes 2016, 65, 633–646. [CrossRef] 54. Rabbani, P.S.; Zhou, A.; Borab, Z.M.; Frezzo, J.A.; Srivastava, N.; More, H.T.; Rifkin, W.J.; David, J.A.; Berens, S.J.; Chen, R.; et al. Novel lipoproteoplex delivers Keap1 siRNA based gene therapy to accelerate diabetic wound healing. Biomaterials 2017, 132, 1–15. [CrossRef] 55. Lee, D.F.; Kuo, H.P.; Liu, M.; Chou, C.K.; Xia, W.; Du, Y.; Shen, J.; Chen, C.T.; Huo, L.; Hsu, M.C.; et al. KEAP1 E3 ligase-mediated downregulation of NF-kappaB signaling by targeting IKKbeta. Mol. Cell 2009, 36, 131–140. [CrossRef] 56. Mulvaney, K.M.; Matson, J.P.; Siesser, P.F.; Tamir, T.Y.; Goldfarb, D.; Jacobs, T.M.; Cloer, E.W.; Harrison, J.S.; Vaziri, C.; Cook, J.G.; et al. Identiﬁcation and Characterization of MCM3 as a Kelch-like ECH-associated Protein 1 (KEAP1) Substrate. J. Biol. Chem. 2016, 291, 23719–23733. [CrossRef] 57. Orthwein, A.; Noordermeer, S.M.; Wilson, M.D.; Landry, S.; Enchev, R.I.; Sherker, A.; Munro, M.; Pinder, J.; Salsman, J.; Dellaire, G.; et al. A mechanism for the suppression of homologous recombination in G1 cells. Nature 2015, 528, 422–426. [CrossRef] 58. Schafer, M.; Dutsch, S.; auf dem Keller, U.; Navid, F.; Schwarz, A.; Johnson, D.A.; Johnson, J.A.; Werner, S. References Auf dem Keller, U.; Huber, M.; Beyer, T.A.; Kumin, A.; Siemes, C.; Braun, S.; Bugnon, P.; Mitropoulos, V.; Johnson, D.A.; Johnson, J.A.; et al. Nrf transcription factors in keratinocytes are essential for skin tumor prevention but not for wound healing. Mol. Cell. Biol. 2006, 26, 3773–3784. [CrossRef] 40. Telorack, M.; Meyer, M.; Ingold, I.; Conrad, M.; Bloch, W.; Werner, S.A. Glutathione-Nrf2-Thioredoxin Cross-Talk Ensures Keratinocyte Survival and Eﬃcient Wound Repair. PLoS Genet. 2016, 12, e1005800. [CrossRef] 41. Siegenthaler, B.; Deﬁla, C.; Muzumdar, S.; Beer, H.D.; Meyer, M.; Tanner, S.; Bloch, W.; Blank, V.; Schafer, M.; Werner, S. Nrf3 promotes UV-induced keratinocyte apoptosis through suppression of cell adhesion. Cell Death Diﬀer. 2018, 25, 1749–1765. [CrossRef] 42. Steiling, H.; Munz, B.; Werner, S.; Brauchle, M. Diﬀerent types of ROS-scavenging enzymes are expressed during cutaneous wound repair. Exp. Cell Res. 1999, 247, 484–494. [CrossRef] 43. Hiebert, P.; Wietecha, M.S.; Cangkrama, M.; Haertel, E.; Mavrogonatou, E.; Stumpe, M.; Steenbock, H.; Grossi, S.; Beer, H.D.; Angel, P.; et al. Nrf2-Mediated Fibroblast Reprogramming Drives Cellular Senescence by Targeting the Matrisome. Dev. Cell 2018, 46, 145–161. [CrossRef] 44. Joshi, N.; Werner, S. Nrf2 is highly expressed in neutrophils, but myeloid cell-derived Nrf2 is dispensable for wound healing in mice. PLoS ONE 2017, 12, e0187162. [CrossRef] g 45. Long, M.; Rojo de la Vega, M.; Wen, Q.; Bharara, M.; Jiang, T.; Zhang, R.; Zhou, S.; Wong, P.K.; Wondrak, G.T.; Zh H t l A E ti l R l f NRF2 i Di b ti W d H li Di b t 2016 65 780 793 [C R f] 45. Long, M.; Rojo de la Vega, M.; Wen, Q.; Bharara, M.; Jiang, T.; Zhang, R.; Zhou, S.; Wong, P.K.; Wondrak, G.T.; Zheng, H.; et al. An Essential Role of NRF2 in Diabetic Wound Healing. Diabetes 2016, 65, 780–793. [CrossRef] 46. Rabbani, P.S.; Soares, M.A.; Hameedi, S.G.; Kadle, R.L.; Mubasher, A.; Kowzun, M.; Ceradini, D.J. Dysregulation of Nrf2/Keap1 Redox Pathway in Diabetes Aﬀects Multipotency of Stromal Cells. Diabetes 2019, 68, 141–155. [CrossRef] 12 of 13 Int. J. Mol. Sci. 2019, 20, 3856 12 of 13 47. Muzumdar, S.; Hiebert, H.; Haertel, E.; Ben-Yehuda Greenwald, M.; Bloch, W.; Werner, S.; Schafer, M. Nrf2-Mediated Expansion of Pilosebaceous Cells Accelerates Cutaneous Wound Healing. Am. J. Pathol. 2019, 189, 568–579. [CrossRef] 48. Wang, H.; Liu, X.; Long, M.; Huang, Y.; Zhang, L.; Zhang, R.; Zheng, Y.; Liao, X.; Wang, Y.; Liao, Q.; et al. References Rojo de la Vega, M.; Chapman, E.; Zhang, D.D. NRF2 and the Hallmarks of Cancer. Cancer Cell 2018, 34, 21–43. [CrossRef] 70. Knatko, E.V.; Ibbotson, S.H.; Zhang, Y.; Higgins, M.; Fahey, J.W.; Talalay, P.; Dawe, R.S.; Ferguson, J.; Huang, J.T.; Clarke, R.; et al. Nrf2 Activation Protects against Solar-Simulated Ultraviolet Radiation in Mice and Humans. Cancer Prev. Res. 2015, 8, 475–486. [CrossRef] 71. Knatko, E.V.; Higgins, M.; Fahey, J.W.; Dinkova-Kostova, A.T. Loss of Nrf2 abrogates the protective eﬀect of Keap1 downregulation in a preclinical model of cutaneous squamous cell carcinoma. Sci. Rep. 2016, 6, 25804. [CrossRef] 72. Rolfs, F.; Huber, M.; Kuehne, A.; Kramer, S.; Haertel, E.; Muzumdar, S.; Wagner, J.; Tanner, Y.; Bohm, F.; Smola, S.; et al. Nrf2 Activation Promotes Keratinocyte Survival during Early Skin Carcinogenesis via Metabolic Alterations. Cancer Res. 2015, 75, 4817–4829. [CrossRef] 73. Xu, C.; Huang, M.T.; Shen, G.; Yuan, X.; Lin, W.; Khor, T.O.; Conney, A.H.; Kong, A.N. Inhibition of 7,12-dimethylbenz(a)anthracene-induced skin tumorigenesis in C57BL/6 mice by sulforaphane is mediated by nuclear factor E2-related factor 2. Cancer Res. 2006, 66, 8293–8296. [CrossRef] 74. Bendavit, G.; Aboulkassim, T.; Hilmi, K.; Shah, S.; Batist, G. Nrf2 Transcription Factor Can Directly Regulate mTOR: Linking cytoprotective gene expression to a major metabolic regulator that generates redox activity. J. Biol. Chem. 2016, 291, 25476–25488. [CrossRef] 75. Squarize, C.H.; Castilho, R.M.; Bugge, T.H.; Gutkind, J.S. Accelerated wound healing by mTOR activation in genetically deﬁned mouse models. PLoS ONE 2010, 5, e10643. [CrossRef] 76. Feldmeyer, L.; Hofbauer, G.F.; Boni, T.; French, L.E.; Hafner, J. Mammalian target of rapamycin (mTOR) inhibitors slow skin carcinogenesis, but impair wound healing. Br. J. Dermatol. 2012, 166, 422–424. [CrossRef] 77. Huang, H.; Cui, W.; Qiu, W.; Zhu, M.; Zhao, R.; Zeng, D.; Dong, C.; Wang, X.; Guo, W.; Xing, W.; et al. 76. Feldmeyer, L.; Hofbauer, G.F.; Boni, T.; French, L.E.; Hafner, J. Mammalian target of rapamycin (mTOR) inhibitors slow skin carcinogenesis, but impair wound healing. Br. J. Dermatol. 2012, 166, 422–424. [CrossRef] 77. Huang, H.; Cui, W.; Qiu, W.; Zhu, M.; Zhao, R.; Zeng, D.; Dong, C.; Wang, X.; Guo, W.; Xing, W.; et al. Impaired wound healing results from the dysfunction of the Akt/mTOR pathway in diabetic rats. J. Dermatol. Sci. 2015, 79, 241–251. [CrossRef] 78. Sindrilaru, A.; Peters, T.; Wieschalka, S.; Baican, C.; Baican, A.; Peter, H.; Hainzl, A.; Schatz, S.; Qi, Y.; Schlecht, A.; et al. References Nrf2 establishes a glutathione-mediated gradient of UVB cytoprotection in the epidermis. Genes Dev. 2010, 24, 1045–1058. [CrossRef] 59. Schafer, M.; Farwanah, H.; Willrodt, A.H.; Huebner, A.J.; Sandhoﬀ, K.; Roop, D.; Hohl, D.; Bloch, W.; Werner, S. Nrf2 links epidermal barrier function with antioxidant defense. EMBO Mol. Med. 2012, 4, 364–379. [CrossRef] 60. Schafer, M.; Willrodt, A.H.; Kurinna, S.; Link, A.S.; Farwanah, H.; Geusau, A.; Gruber, F.; Sorg, O.; Huebner, A.J.; Roop, D.R.; et al. Activation of Nrf2 in keratinocytes causes chloracne (MADISH)-like skin disease in mice. EMBO Mol. Med. 2014, 6, 442–457. [CrossRef] 61. Ito, M.; Liu, Y.; Yang, Z.; Nguyen, J.; Liang, F.; Morris, R.J.; Cotsarelis, G. Stem cells in the hair follicle bulge contribute to wound repair but not to homeostasis of the epidermis. Nat. Med. 2005, 11, 1351–1354. [CrossRef] 62. Dahlhoﬀ, M.; Frances, D.; Kloepper, J.E.; Paus, R.; Schafer, M.; Niemann, C.; Schneider, M.R. Overexpression of epigen during embryonic development induces reversible, epidermal growth factor receptor-dependent sebaceous gland hyperplasia. Mol. Cell. Biol. 2014, 34, 3086–3095. [CrossRef] 63. Werner, S.; Grose, R. Regulation of wound healing by growth factors and cytokines. Physiol. Rev. 2003, 83, 835–870. [CrossRef] 64. Demaria, M.; Ohtani, N.; Youssef, S.A.; Rodier, F.; Toussaint, W.; Mitchell, J.R.; Laberge, R.-M.; Vijg, J.; Van Steeg, H.; Dollé, M.E.; et al. An Essential Role for Senescent Cells in Optimal Wound Healing through Secretion of PDGF-AA. Dev. Cell 2014, 31, 722–733. [CrossRef] 13 of 13 Int. J. Mol. Sci. 2019, 20, 3856 65. Lee, L.Y.; Harberg, C.; Matkowskyj, K.A.; Cook, S.; Roenneburg, D.; Werner, S.; Johnson, D.A.; Johnson, J.A.; Foley, D.P. Cell-speciﬁc overactivation of nuclear erythroid 2 p45-related factor 2-mediated gene expression in myeloid cells decreases hepatic ischemia/reperfusion injury. Liver Transpl. 2016, 22, 1115–1128. [CrossRef] 66. Kong, X.; Thimmulappa, R.; Craciun, F.; Harvey, C.; Singh, A.; Kombairaju, P.; Reddy, S.P.; Remick, D.; Biswal, S. Enhancing Nrf2 pathway by disruption of Keap1 in myeloid leukocytes protects against sepsis. Am. J. Respir. Crit. Care Med. 2011, 184, 928–938. [CrossRef] 67. Schafer, M.; Werner, S. Cancer as an overhealing wound: An old hypothesis revisited. Nat. Rev. Mol. Cell Biol. 2008, 9, 628–638. [CrossRef] 68. Wang, X.J.; Sun, Z.; Villeneuve, N.F.; Zhang, S.; Zhao, F.; Li, Y.; Chen, W.; Yi, X.; Zheng, W.; Wondrak, G.T.; et al. Nrf2 enhances resistance of cancer cells to chemotherapeutic drugs, the dark side of Nrf2. Carcinogenesis 2008, 29, 1235–1243. [CrossRef] 69. References An unrestrained proinﬂammatory M1 macrophage population induced by iron impairs wound healing in humans and mice. J. Clin. Investig. 2011, 121, 985–997. [CrossRef] © 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/W4295786918	https://www.researchsquare.com/article/rs-2053542/latest.pdf	English	null	Synthesis and Biological Evaluation of Novel Uracil Derivatives as Thymidylate Synthase Inhibitors	Research Square (Research Square)	2,022	cc-by	9,390	Synthesis and Biological Evaluation of Novel Uracil Derivatives as Thymidylate Synthase Inhibitors Manzoor Ahmad Mir (  drmanzoor@kashmiruniversity.ac.in ) University of Kashmir https://orcid.org/0000-0003-3297-1402 Research Article Keywords: Breast cancer, drug discovery, thymidylate synthase, uracil derivatives, one-pot synthesis, Cell cycle Posted Date: September 14th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-2053542/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Synthesis and Biological Evaluation of Novel Uracil Derivatives as Thymidylate Synthase Inhibitors Manzoor Ahmad Mir (  drmanzoor@kashmiruniversity.ac.in ) University of Kashmir https://orcid.org/0000-0003-3297-1402 Research Article Keywords: Breast cancer, drug discovery, thymidylate synthase, uracil derivatives, one-pot synthesis, Cell cycle Posted Date: September 14th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-2053542/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Abstract To synthesize novel uracil derivatives targeting thymidylate synthase for the treatment of high-grade malignancies such as breast cancer. Results BC patients showed high levels of TYMS, and high expression of TYMS was found associated with poor prognosis. In silico studies indicated that synthesized uracil derivatives have a high affinity for TYMS. Notably, the uracil derivatives dramatically inhibited the proliferation and colonization potential of BC cells in vitro. Background Cell division is driven by nucleic acid metabolism, and thymidylate synthase (TYMS) catalyses a rate- limiting step in nucleotide synthesis. As a result, TYMS has emerged as a critical target in chemotherapy. 5-fluorouracil (5-FU) is currently being used to treat a wide range of cancers, including breast, pancreatic, head and neck, colorectal, ovarian, and gastric cancers. Methods One-pot organic synthesis processes using a single solvent were used for the synthesis of drug analogues of Uracil. Integrated bioinformatics using GEPIA2, UALCAN and KM plotter were utilized to study the expression pattern and prognostic significance of TYMS, the key target gene of 5-fluorouracil in breast cancer patients. Cell viability, cell proliferation, and colony formation assays were used as in-vitro methods to validate the in-silico lead obtained. Objectives The objective of this study was to establish a new methodology for the low-cost, one-pot synthesis of uracil derivatives (UD-1 to UD-5) and to evaluate their therapeutic potential in BC cells. Research Article Keywords: Breast cancer, drug discovery, thymidylate synthase, uracil derivatives, one-pot synthesis, Cell cycle Posted Date: September 14th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-2053542/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/25 1. Introduction Due to the long-standing anticancer potency of 5-Fluorouracil, uracil have been a top priority for many researchers, notably in the field of drug discovery and synthetic accessibility (23, 31). Uracil derivatives exhibit bactericidal, herbicidal, and insecticidal activity, with antiviral and anticancer activity being particularly notable (33, 14). Their antiviral activity is based on inhibiting a critical stage in the viral replication pathway, which results in robust effectiveness against HIV, hepatitis B and C, and herpes viruses, among others (40, 32). 5-Fluorouracil and 5-Chlorouracil were the first uracil derivatives to exhibit pharmacological activity and approved for use in the treatment of several ailments (43, 3). Recently, scientists have focused increasing attention on uracil derivatives; several of the most clinically significant compounds are illustrated in Figure. S1 (Supplementary Information) (29). Numerous changes to uracil have been made to address toxicity concerns in the synthesis of derivatives with improved pharmacological and pharmacokinetic features (increased bioactivity, selectivity, metabolic stability, absorption, and decreased toxicity) (29, 23, 14). The development of novel uracil derivatives and fused uracil derivatives as bioactive agents is dependent on substituent changes at various places on the pyrimidine ring. One-pot organic synthesis processes using a single solvent have sparked considerable attention among researchers due to their low cost, environmental friendliness, the potential for high yield products, and insights into green chemistry (8). Green chemistry techniques are crucial because they minimize waste, byproducts, energy use, and expense (8). The capacity to execute multistep reactions in a single pot could significantly increase their efficiency, both economically and environmentally. Due to its immense range of applications in synthetic chemistry, the synthesis of novel heterocyclic molecules has always been a subject of significant interest. Because heterocyclic compounds are abundant in nature and are required for life to exist. Among them, heterocyclic compounds containing the uracil moiety are of interest because they exhibit a variety of pharmacological and biological properties. Uracil and its derivatives are critical starting materials for the synthesis of a wide variety of chemical compounds, pharmaceutical drugs, and intermediates (7, 15, 16). For example, Tegafur-uracil, a pyrimidine derivative is a chemotherapeutic agent used in cancer treatment, most commonly colon cancer (1). Thus, developing novel uracil derivatives is a critical and essential endeavor in organic chemistry. In addition to the aspects discussed in the literature, attempts were made to establish a new methodology for the low-cost, one-pot synthesis of uracil derivatives. Conclusion In conclusion, our study identified novel uracil derivatives as promising therapeutic options for breast cancer patients expressing the augmented levels of TYMS. Page 2/25 Page 2/25 2. Results And Discussion 2..1 Chemistry 1. Introduction Globally, breast cancer (BC) is currently the most prevalent malignancy among women and the second leading cause of cancer-related death (36, 35, 21). Current treatment methods include breast ectomy, radiation therapy (RT), chemotherapy therapy (CT), hormone therapy (HT), and immunological therapy (11, 42, 25, 22, 17). Treatments are decided by each patient's clinicopathologic and tumor grade features (25, 28). Despite advances in diagnostics and therapy, BC mortalities are highest owing to metastatic tumors, tumor relapse, and the development of chemoresistance resulting in poor prognosis (20). Thus, there is an urgent need to develop and explore novel therapeutic regimens for BC patients, particularly for triple-negative breast cancer (TNBC) patients, as chemotherapy is the mainstay of therapeutic protocol Page 3/25 Page 3/25 for these patients (18, 19). Due to the high proliferative nature of tumor cells, raw material for the synthesis of macromolecules viz proteins and DNA and vastly required (34). The rate-limiting step in DNA synthesis is catalyzed by thymidylate synthase (TS), which is the only de novo cellular source of dTTP (9). As a result, TYMS has emerged as a critical target in cancer chemotherapy, especially breast cancer (30). 5-fluorouracil (5-FU) targeting TYMS is presently used in the treatment of several malignancies including breast, pancreatic, head, and neck, colorectal, ovarian, and gastric cancers (37, 44, 26). Despite a favorable initial response to 5-FU, BC patients frequently develop chemoresistance due to the inherent nature of tumor cells to mutate, highlighting an urgent need to investigate new therapeutic options targeting TYMS (45, 24). In the present study, we synthesized novel uracil derivatives and evaluated their anticancer activity using in silico and in vitro approaches. We further explored the expression pattern and prognostic significance of TYMS in BC patients, key target gene of 5-fluorouracil. In silico analysis showed high affinity of uracil derivatives in contrast to 5-FU. Additionally, in vitro studies demonstrated that synthesized derivatives exhibited substantial cytotoxic activity and the potential to impede colonization of breast tumor cells. Taken together, the findings suggest that new uracil analogues may be promising therapeutics for breast cancer in combination with current treatment regimens. Heptyl-4-(1-methyl-3-(4-nitrobenzyl)-2,6-dioxohexahydropyrimidin-4-yl)piperazine-1 carboxylate (UD-3) Yield: 59%, mp; 197–198˚C; White solid powder; 1H-NMR: δ = 0.86–0.88 ( t, 3 H, -CH3), 1.21–1.24 (broad, 10 H, -CH2), 1.45–1.57 (m, 3 H, -CH2), 2.80 (broad, 4H, -CH2), 3.23 (s, 3 H, -N-CH3), 3.47–3.56 (broad, 4 H, - CH2), 3.99–4.03 (t, 2 H, -O-CH2), 5.13 ( s, 2 H, Ar-CH2), 5.33 (s, 1 H, -CH), 7.25–7.34 (d, 2 H, ArH) 8.11– 8.13(d, 2 H, ArH) ppm. Mass: (C24H33N5O6) m/z = 487.24, M + H = 488. 1-(4-Fluorobenzyl)-3-methyl-6-(piperidin-1-yl)dihydropyrimidine-2,4(1H,3H)-dione (UD-4) One-Pot Synthesis of Uracil derivatives (UD-1 to UD-5) One-Pot Synthesis of Uracil derivatives (UD-1 to UD-5) Initially, charged 6-chlorouracil (7.75 g, 0.05 M) and potassium carbonate (9.14 g, 0.06 M) were mixed in 50 mL DMF in 250 mL RBF, followed by halogenated of 1-(bromomethyl) benzene (10 g, 0.05 M), the reaction mixture was stirred for 8 h at RT. Then, with further addition of potassium carbonate (7.31 g, 0.0529 M) and methyl iodide (15.02 g, 0.10 M) the reaction mixture was further stirred overnight at room temperature. After that, the charged piperazine (4.52 g, 0.05 M) or piperidine or 3-aminopiperidine (9.10 g, 0.05 M) was added to the reaction mixture and heated at 80 ˚C for 8 h to get compounds 3, UD-4, and 5 respectively. Finally, the reaction mixture of compound 3 was separately treated with acetone, water, and potassium carbonate with different chloroformates to give UD-1, UD-2, and UD-3. In another reaction, the Moc-L-Valine (9.22g, 0.05 M) was added to the reaction mixture (5) in presence of coupling reagent HATU (40.23 g, 0.10 M) and triethylamine (10.71 g, 0.10M) and stirred the mixture at RT for 25 min. After the completion of the reaction, water was added and the product was extracted in ethyl acetate, the organic layer was dried over sodium sulfate, and the solvent was removed under reduced pressure. The desired product was purified by column chromatography to get the title compound (UD-5). The steps for the synthesis of the uracil derivatives are displayed in Fig. 1 and the chemical structures of the final products are given in Fig. 2. Heptyl-4-(3-(4-fluorobenzyl)-1-methyl-2,6-dioxohexahydropyrimidin-4-yl)piperazine-1-carboxylate (UD-1) Page 4/25 Page 4/25 Yield: 55%, mp; 194–195˚C; White solid powder;1H-NMR: δ = 0.85–0.88 ( t, 3 H, -CH3), 1.27–1.31 (broad, 8 H, -CH2), 1.55–1.63 (m, 2 H, -CH2), 2.80 (broad, 4H, -CH2), 3.32 (s, 3 H, -N-CH3), 3.49–3.63 (broad, 3 H, - CH2), 4.05–4.08 (t, 2 H, -O-CH2), 5.16 ( s, 2 H, Ar-CH2-), 5.33 (s, 1 H, -CH), 7.02–7.07 (t, 1 H, ArH) 7.09– 7.13(m, 2 H, ArH), 7.23–7.28 (m, 1 H, ArH) ppm. Mass: (C24H33FN4O4) m/z = 460.25, M + H = 461.1. One-Pot Synthesis of Uracil derivatives (UD-1 to UD-5) Yield: 55%, mp; 194–195˚C; White solid powder;1H-NMR: δ = 0.85–0.88 ( t, 3 H, -CH3), 1.27–1.31 (broad, 8 H, -CH2), 1.55–1.63 (m, 2 H, -CH2), 2.80 (broad, 4H, -CH2), 3.32 (s, 3 H, -N-CH3), 3.49–3.63 (broad, 3 H, - CH2), 4.05–4.08 (t, 2 H, -O-CH2), 5.16 ( s, 2 H, Ar-CH2-), 5.33 (s, 1 H, -CH), 7.02–7.07 (t, 1 H, ArH) 7.09– 7.13(m, 2 H, ArH), 7.23–7.28 (m, 1 H, ArH) ppm. Mass: (C24H33FN4O4) m/z = 460.25, M + H = 461.1. Octyl-4-(3-(4-fluorobenzyl)-1-methyl-2,6-dioxohexahydropyrimidin-4-yl)piperazine-1-carboxylate (UD2) Yield: 57%, mp; 180–191˚C; White solid powder; 1H-NMR: δ = 0.85–0.88 ( t, 3 H, -CH3), 1.02–1.40 (m, 12 H, -CH2), 1.59–1.80 (t, 3 H, -CH2), 2.82 (broad, 4H, -CH2), 3.31 (s, 3 H, -N-CH3), 3.42–3.52(broad, 4 H, -CH2), 4.05–4.09 (t, 2 H, -O-CH2), 5.08 ( s, 2 H, Ar-CH2-), 5.34 (s, 1 H, -CH), 6.89–6.94 (q, 1 H, ArH) 6.96–6.98 (d, 2 H, ArH), 7.26–7.31(q, 1 H, ArH) ppm. Mass: (C25H35FN4O4) m/z = 474.26, M + H = 475.1. Heptyl-4-(1-methyl-3-(4-nitrobenzyl)-2,6-dioxohexahydropyrimidin-4-yl)piperazine-1 carboxylate (UD-3) 1-(4-Fluorobenzyl)-3-methyl-6-(piperidin-1-yl)dihydropyrimidine-2,4(1H,3H)-dione (UD-4) Yield: 57%, mp; 175–176˚C; White solid powder;1H-NMR: δ = 1.63 (m, 6 H,-CH2), 2.86 (s; 4 H, -CH2), 5.02 (s, 2 H, -CH2), 5.32 (s, 1 H, -CH), 6.98 (t, 2 H, Ar-H), 7.22 (m, 3 H, Ar-H) ppm. 13C NMR δ = 23.77, (-CH2), 25.33 (-CH2), 27.84 (-CH3), 47.22 (-CH2), 52.44 (-CH2), 89.78 (-CH), 115 (Ar-C), 128.95 (Ar-C), 132.17 (Ar-C), 152 (-N-CO-N-), 160.37 (-C-), 163.36 (-N-CO-C-) ppm. Mass (C17H22FN3O2) m/z = 319.17 Methyl-1-1-(3-(2-cyanobenzyl)-1-methyl-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl)piperidin-3-ylamino)-3- methyl-1-oxobutan-2-ylcarbamate (UD-5) Yield: 57%, mp; 184–185˚C; White solid powder;1H-NMR: δ = 0.71 (dd, 6 H,-CH3), 1.29 (m; 2 H, -CH2), 1.74– 1.79 (m, 3 H, -CH2), 2.3 (m, 1 H, -CH2), 2.46 (s, 3 H, CH2), 2.59 (m, 1 H, -CH2), 2.8 (d, 1 H, -CH2), 3.08 (s, 3 H, - N-CH3), 3.48 (s, 3 H, -O-CH3), 3.71 (t, 2 H, -CH2), 4.92 (q, 2 H, -CH2), 5.25 ( s, 1 H, -CH), 6.97 (m, 3 H, Ar-H), 7.09 (d, 1 H, Ar-H), 7.29 (d, 1 H, Ar-H), 7.95 (d, 1 H, -NH). Mass (C25H32N6O5) m/z = 496.56, M + H = 497.2481. It is highly noted that in the available literature, generally in benzylation and methylation reactions base like sodium hydride in dimethylformamide have been used which is highly dangerous in the context of safety, environment pollutant and it also results in poor yield, impure compounds. In our simple protocol, we used low-cost, ecofriendly, and safe inorganic base potassium carbonate in dimethylformamide solvent in one pot during four steps which is one of the major advantages of our protocol. In addition, we got higher percentage yields of products. Initially, we have synthesized N-substituted uracil analogs in It is highly noted that in the available literature, generally in benzylation and methylation reactions base like sodium hydride in dimethylformamide have been used which is highly dangerous in the context of safety, environment pollutant and it also results in poor yield, impure compounds. In our simple protocol, we used low-cost, ecofriendly, and safe inorganic base potassium carbonate in dimethylformamide solvent in one pot during four steps which is one of the major advantages of our protocol. In addition, we got higher percentage yields of products. Initially, we have synthesized N-substituted uracil analogs in Page 5/25 Page 5/25 high yields by simple N-benzylation of 6-chlorouracil with different halogenated benzyl halides and N- methylation by methyl iodide respectively, under catalyst-free conditions in DMF solvent. 1-(4-Fluorobenzyl)-3-methyl-6-(piperidin-1-yl)dihydropyrimidine-2,4(1H,3H)-dione (UD-4) Then the chlorine atoms of N-substituted uracil derivatives were substituted with piperazine, piperidine, and 3- aminopiperidine, respectively. In the final step, the N-substitution of the piperazine ring was carried out by treating it with different alkyl containing chloroformates in the same pot under similar conditions for the formation of compounds UD-1-UD-4. Simultaneously, under similar conditions in the same solvent amino acid coupling reaction was done in presence of coupling reagent HATU (1-bis(dimethylamino)methylene- 1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxid hexafluorophosphate) for the formation of target compounds UD-5. The whole synthesis has been done in one solvent in one pot that results in the reduction of industrial waste, affluent generation, and overall good yields (55–59%) of final products. A reasonable mechanism for the formation of uracil derivatives is shown in Fig. 3. The reaction conditions were optimized using various solvents, bases. The yields ranged from 55 to 59%. A perusal of these indicates that the DMF and inorganic base was the best one; resulting in higher yield and best conversion. During the optimization of organic and inorganic bases were used. It was observed that during organic bases, the reaction was completed at a faster rate, but less conversion was observed. The percentage of yield was increased while using inorganic bases under DMF solvent. Hence, throughout the reaction, potassium carbonate as base and DMF as solvent were used. The proposed structures of uracil derivatives (UD-1 to UD-5) were well supported by analytical and spectroscopic data. All the derivatives were found as solids, stable to air, had good yields, and quite soluble in ethyl acetate, methanol, ethanol, chloroform, DMSO, DMF. The characteristic signal from the CH3N- group in the 1H NMR spectrum was observed as a singlet at ~ 2.90–3.10 ppm, methine proton of uracil ring CH- was observed at 5.00 -5.35 ppm, benzylic proton -CH at 4.86–5.37 ppm, aromatic protons Ar-H appeared in the range of 7.00–8.10 ppm, piperazine protons resonates at 1.50–3.80 ppm, methyl protons appeared in the range of 2.90–3.40 ppm, and methoxy protons -OCH3 appeared in the range of 3.40–3.75 ppm. The 1H NMR spectra of uracil derivatives (UD-1 to UD-5) are shown in Figure S2-S6 (Supplementary Information). Elemental analyses and ESI-MS spectra illustrated the compositions of synthesized compounds. The mass spectra of UD-1 to UD-5 showed the peaks at m/z values of 460.25, 474.26, 487.24, 319.17 and 496.56 corresponding to UD- 1, UD-2, UD-3, UD-4 and UD-5. Drug-likeness Page 6/25 Oral bioavailability (a measure of drug-likeness) is one of the major parameters for developing bioactive molecules as effective pharmaceutical agents. Parameters like good intestinal absorption, reduced molecular flexibility, low polar surface area, or total hydrogen bond count, are essential factors for the prediction of good oral bioavailability (41). Drug properties like membrane permeability and bioavailability are associated with some basic molecular descriptors: molecular mass, hydrogen bond acceptor and donor count, and log P (partition coefficient) in a molecule (12). These properties were originally used by Lipinski (12) to formulate his well-known “Rule of five”. The rule stands, most molecules with good membrane permeability have molecular mass < 500 Da, number of hydrogen bond acceptors < 10, number of hydrogen bond donors < 5, and log P < 5. This rule is commonly used to Page 6/25 evaluate the suitability of a drug for oral administration. This rule is widely used as a filter for the screening of molecules with drug-like properties. Generally, a compound that fulfills at least three points out of five is said to follow Lipinski’s rule. A poor permeation or absorption is more likely to occur when there are more than 5 H-bond donors and 10 H-bond acceptors. Therefore, all these molecular properties of the mentioned uracil derivatives were calculated using MarvinSketch and are displayed in Table 1, showing all uracil derivatives have molecular masses lower than 500, contain hydrogen bond acceptors < 10 and number of hydrogen bond donors < 5. Moreover, the uracil derivatives have lower log P, molar refractivity, and polar surface areas than the values recommended by Lipinski’s rule. Thus, the synthesized uracil derivatives fully meet all the five criteria of Lipinski’s rule, hence having good oral bioavailability. Table 1 Molecular properties of uracil derivatives show their oral bioavailability and drug-likeness. Derivative Molecular mass/u Log P Hydrogen bond acceptors Hydrogen bond donors Molar refractivity/ (m3 M− 1) Polar surface area/ ˚A2 UD1 462.56 -3.80 4 0 122.75 73.40 UD2 476.5 -4.24 4 0 127.35 73.40 UD3 487.4 -4.04 6 0 133.45 116.54 UD4 319.38 -2.30 3 0 85.27 43.86 UD5 498.5 -1.62 6 2 131.15 135.08 Table 1 erivatives show their oral bioavailability and drug-likeness. 2.2 Molecular Docking Studies Uracil derivatives show a high docking affinity for thymidylate synthase CB-Dock by default predicts five binding cavities and based on experimental information of the binding site of the co-crystallized substrate of thymidylate synthase, we proved that the correct binding site is among the predicted cavities (Fig. 4A). Pyrimidine analog namely 5-Fluorouracil (5-FU) has been approved by FDA for a variety of cancers including gastric adenocarcinoma and breast cancer (13). Metabolite of 5-FU by name fluorodeoxyuridine monophosphate (FdUMP) competes with normal CB-Dock by default predicts five binding cavities and based on experimental information of the binding site of the co-crystallized substrate of thymidylate synthase, we proved that the correct binding site is among the predicted cavities (Fig. 4A). Pyrimidine analog namely 5-Fluorouracil (5-FU) has been approved by FDA for a variety of cancers including gastric adenocarcinoma and breast cancer (13). Metabolite of 5-FU by name fluorodeoxyuridine monophosphate (FdUMP) competes with normal substrate dUMP and thus prevents its binding with thymidylate synthase (13). As it is clear that FdUMP but not 5-FU binds with thymidylate synthase thus we used the former rather than the latter as a positive control in the docking study and compared the binding affinity of six novel molecules with FdUMP. All the newly tested molecules showed higher binding affinity than FdUMP (− 6.7 kcal/mol). However, two molecules namely UD4 and UD5 exhibited the highest inclination towards the predefined site of thymidylate synthase. While UD- showed a binding affinity value of − 8.3 kcal/mol, UD4 showed a binding affinity of − 8.0 kcal/mol (Fig. 4B). The ligand-receptor interaction is more favorable when the binding Page 7/25 Page 7/25 affinity values are more negative. Thus, the binding affinity value is more negative for UD5 and UD4 suggests its more inclination towards thymidylate synthase compared to FdUMP. The interaction profile of the five UDs in the docked state with thymidylate synthase was generated. Expectedly, the two most potent molecules showed relatively more interactions than the third strong binder against thymidylate synthase. UD4 formed four hydrophobic interactions (ILE 108, TRP 109, LEU 192, PHE 225) and two hydrogen bonds with amino acid residues GLN 214 and ASP 218 of binding groove. UD5 which was predicted by flexible docking as the most powerful molecule against the binding groove of thymidylate synthase also showed an extensive interaction profile. It displayed three hydrophobic interactions, two with LEU 192 and one with ASP 218. TYMS is upregulated in BC Patients. Using the TCGA BrCa dataset of the Gepia2 online portal, the expression of TYMS was analyzed, and it was revealed that TYMS is highly overexpressed in breast cancer patients in contrast to normal women. TYMS was found to be upregulated with a log2 FC of 2.223, (p-value of 1.58e-179) Fig. 5A. Also, we analyzed the TYMS expression within BC subclasses and women with different ethnicities using the UALCAN portal and it was found that among different subclasses of BC, TYMS was highly upregulated in TNBC and women with African-American ethnicity Fig. 5B, C, and D. High expression of TYMS affects the prognosis of BC patients. The prognostic significance of the TYMS expression was analyzed using the online open database, the Kaplan-Meier Plotter. The KM plots demonstrated that BC patients with high expression of TYMS had worse relapse-free survival and overall survival (p < 0.05) Fig. 5E, F. Also, BC patients with reduced levels of TYMS had better relapse-free survival and overall survival. These results indicate that targeting TYMS may prolong the survival of breast cancer patients, in particular the TNBC patients who solely rely on chemotherapeutic regimens. 2.2 Molecular Docking Studies Furthermore, three hydrogen bonds were seen between ligand 5 and three residues (ARG 50, ASP 218, and TYR 258) of thymidylate synthase (Fig. 4A). Our results align well with the study where enhanced ligand efficiency has been attributed to hydrophobic interactions (8). All the novel molecules and the known binder FdUMP bind at the experimentally certified binding site of dUMP, the substrate of thymidylate synthase, which further authenticates the reliability of our findings Figure S7 (Supplementary Information). 2.3. Expression pattern & prognostic significance of TYMS in BC patients 2.3. Expression pattern & prognostic significance of TYMS in BC patients TYMS is upregulated in BC Patients. 2.4. In vitro anti-cancer activity Uracil derivatives possess potent anti-proliferative activity To examine whether UDs, possessed anti-cancer activity, we performed a preliminary assay of cell viability on MDA-MB-231 cells. UD-1, UD-2, UD-3, UD-4 and UD-5 were given at 50 µM conc. and cells were Page 8/25 Page 8/25 incubated with drugs for 72 h. MTT assay analysis revealed that UD-1, UD-2, and UD-4 possessed high anti-cancer activity compared to UD-3, and UD-5 Fig. 6A. The three derivatives UD-1, UD-2, and UD-4 were selected for further analysis in a panel of breast cancer cell lines. incubated with drugs for 72 h. MTT assay analysis revealed that UD-1, UD-2, and UD-4 possessed high anti-cancer activity compared to UD-3, and UD-5 Fig. 6A. The three derivatives UD-1, UD-2, and UD-4 were selected for further analysis in a panel of breast cancer cell lines. To investigate the effect of UD-1, UD-2, and UD-4, BC cell lines were treated with different concentrations of the selected UDs. The effects of UDs on the survival of BC cells were assessed by MTT assay. As shown in Fig. 6B, C, D & Fig. 7, UDs inhibited cell proliferation in a time- and concentration-dependent manner. UD1 exhibited a log IC50 value of 1.86 µM, 1.89 µM, 2.0 µM, and 1.89 µM in MDA-MB-231, MDA- MB-468, MCF-7, and 4T1 cells respectively. UD2 exhibited a log IC50 value of 1.51 µM, 1.85 µM, 1.86 µM, and 1.83 µM in MDA-MB-231, MDA-MB-468, MCF-7, and 4T1 cells respectively. UD4 exhibited a log IC50 value of 1.3 µM, 1.7 µM, 1.8 µM, and 1.6 µM in MDA-MB-231, MDA-MB-468, MCF-7, and 4T1 cells respectively Table S8 (Supplementary Information). Among the three potent UDs, UD-4 demonstrated high anti-proliferative activity in vitro, with the lowest IC50 compared to UD-1 & UD-2. Among different BC cell lines, the TNBC cell lines showed more sensitivity towards UDs and had lower IC50 compared to ER + cell line MCF-7. Effect of Uracil derivatives on colony formation potential of MDA-MB-231 cells To further validate the anti-cancer activity of UDs, a colony formation assay was performed in BC cell line MDA-MB-231 Fig. 8. Treatment with UDs showed dose-dependent inhibition of colony formation potential of breast tumor cells. Among the UDs, UD-4 showed the highest reduction in colony formation potential of tumor cells. 2.4. In vitro anti-cancer activity UD-1 and UD-2 had less effect on colony formation in MDA-MB-231 cells, however, the small size of colonies in the treated wells of UD1 and UD2 demonstrates that UD1 and UD2 restrict the growth of tumor cells. These results demonstrate that UDs can limit the growth of breast tumor metastases and may be a promising approach in reducing the colonization of tumor cells at metastatic niches. 3. Conclusion In summary, we presented a new efficient, low-cost, eco-friendly highly scalable, and one-pot method for the synthesis of five uracil derivatives from readily available 6-chlorouracil and their biological activity in TNBC. The key steps of our synthetic efforts rely on 1) N-benzylation, 2) N-methylation, 3) substitution and 4) amino acid coupling. The target compounds were obtained in overall good yields (55–57%). Spectroscopic and analytical techniques fully characterized the target compounds. 5-FU is an FDA- approved drug for the treatment of several malignancies including BC. Our study identified UD4, the most potent derivative with micromolar potency against TYMS. Also, UD4 showed a good drug-like profile (e.g., aqueous solubility and ligand efficiency metric), along with an in vitro pharmacokinetic profile. Further exploration of the potency of UD4 in mouse mammary TNBC models is required. Overall, the study identified novel uracil derivatives as potent drug candidates for BC patients with high expression of TYMS which indicates their quite bright future in the field of drug discovery. Page 9/25 4. Materials And Methods All the reagents and chemicals used are of analytical and molecular biology grade and were obtained from the highest grade available from Sigma Aldrich, Acros organics, Spectrochem, Loba Chem, Survival Technology, and Rankem Laboratories, and were used without purification. TLC was performed on Merck All the reagents and chemicals used are of analytical and molecular biology grade and were obtained from the highest grade available from Sigma Aldrich, Acros organics, Spectrochem, Loba Chem, Survival Technology, and Rankem Laboratories, and were used without purification. TLC was performed on Merck TLC Silica gel 60 F254 plates eluting with specific solvents and samples were made visual with a UV lamp, Silica gel (60–120 mesh) was used for column chromatography. Melting points were measured on an Electrothermal 9100 apparatus. High-resolution mass spectra were measured were Bruker. The measurement was run in positive ion mode. 1H NMR was obtained using Bruker (400 MHz) spectrometer in DMSO and CDCl3 with tetramethylsilane as an internal standard. All NMR spectra at room temperature (RT) were determined in DMSO-d6 and CDCl3. Chemical shifts are reported in parts per million (δ) downfield from an internal tetramethylsilane reference. Coupling constants (J values) are reported in hertz (Hz), and spin multiplicities are indicated by the following symbols: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet). Signals related to DMSO and residual ethanol, are indicated on the spectra. 4.1.5. Synthesis of Uracil Derivative (UD-4) Charged 6-Chloro-1-(4-fluorobenzyl)-3-methyldihydropyrimidine-2,4(1H,3H)-dione (9.0 g, 0.0334 moles) followed by 45 ml DMF and potassium carbonate (9.25 g, 0.0669 moles) was added then piperidine ( 3.60 g, 0.0417 moles) then the reaction mass was heated to 70–80°C, then the reaction mass stirred until completion of reaction then cooled to RT water was added into it then MDC was added, the organic layer was separated, the organic layer dried with sodium sulfate solvents was removed under reduced pressure to gives the title compound. Weight: 7.50 g (79.0%). g p g g ( ) 4.1.6. Synthesis of 2-((6-(3-aminopiperidin-1-yl)-3-methyl- 2,4-dioxotetrahydropyrimidin-1(2H)-yl)methyl)benzonitrile (5) (5) Charged 2 (9.0 g, 0.03 M) was dissolved in 45 mL DMF followed by the addition of potassium carbonate (9.25 g, 0.06 M). Then piperidin-3-amine (6.83 g, 0.039 M) was added, and the reaction mixture was heated at 80°C, until the completion of the reaction. Then the reaction mixture was cooled to RT and water was added to it and extracted with dichloromethane. The organic layer was separated and was dried over sodium sulfate. Finally, the solvent was removed under reduced pressure for the formation of compound 5 (9.81 g, 87%). 4.1 Chemistry Charged 6-Chlorouracil (10 g, 0.06824 moles) and diisopropylethylamine (18.52 g, 0.1432 moles) followed by addition of 50 ml DMF this mixture was stirred 10 min. at RT then 1-(bromomethyl)-4- fluorobenzene (12.90 g, 0.06824 moles) was added to it, this reaction mass stirred 8 h at room temperature, after completion of reaction 150 ml water was added into it, the title product was isolated b filtration. Weight: 15.65g (90%). Page 10/25 Page 10/25 completion of reaction then cooled to RT water was added into it then MDC was added, the organic layer was separated, the organic layer dried with sodium sulfate solvents was removed under reduced pressure to give the title compound. Weight: 9.50 g (89.0%). 4.1.4. Synthesis of Uracil Derivatives (UD-1 to UD-3) 4.1.4. Synthesis of Uracil Derivatives (UD-1 to UD-3) To the solution of 1-(4-fluorobenzyl)-3-methyl-6-(piperazin-1-yl)dihydropyrimidine-2,4(1H,3H)-dione ( 10.0 g, 0.0314 moles) in 100 ml acetone and 5 ml water, (8.68 g, 0.0628 moles) of potassium carbonate was added and the reaction mass was stirred for 10 min. at room temperature followed by the addition of 1.20 mole equivalents of chloroformates separately. After that, the reaction mixture was again stirred for 2 h at RT followed by the addition of water, product was extracted with ethyl acetate, purified by column chromatography to afford the UD-1 & UD-2. For the preparation of UD-3, 1-(4-nitrobenzyl)-3-methyl-6- (piperazin-1-yl) dihydropyrimidine-2,4(1H,3H)-dione was used instead of 1-(4-fluorobenzyl)-3-methyl-6- (piperazin-1-yl)dihydropyrimidine-2,4(1H,3H)-dione. 4.1.7. Synthesis of Uracil Derivative (UD-5) Charged 5 (9.0 g, 0.028 M) was initially dissolved in 90 mL dichloromethane, then HATU (16.12 g, 0.042 moles) was added to the solution followed by Moc-L-Valine (5.20 g, 0.029 moles) then TEA (5.72 g, 0.056 moles), the resulting solution was stirred RT till completion of the reaction. Finally, 20 mL of water was added into it an organic layer was separated and dried over sodium sulfate and the left solvent was removed under reduced pressure to give derivative UD-5. Page 11/25 4.2. Molecular docking and Integrated Bioinformatic Analysis 4.2.1 In silico evaluation of Uracil derivatives with Thymidylate Synthase (TYMS) Human thymidylate synthase crystal structure was fetched from Protein Data Bank (PDB ID: 5X66) (2). Chimera software (version 1.11.2) was used to expunge the useless items from the PDB file (27). Following this, the structure was checked for any missing residues using the free Maestro 11.2 (5). Coordinates of 2'-deoxyuridine 5'-monophosphate (dUMP) and 5-fluoro-2'-deoxyuridine-5'- monophosphate (FdUMP) were retrieved from ChemSpider with ChemSpider ID: 58574 and 26330107 respectively ( http://www.chemspider.com/Chemical-Structure.1906.html). Structures of other ligands were drawn in ChemSketch and were exported in the desired format for future use. A web server namely CB-Dock was used for performing molecular docking. CB-Dock first identifies the putative binding sites automatically and then performs cavity sorting (38). According to the ligand to be docked it customizes the size of the docking (grid) box and finally performs molecular docking using AutoDock Vina (flexible docking tool) (6). The ligand poses are re-ranked as per docking score and the best binding pose is ascribed to the first confirmation. We used the knowledge from biology to select the most appropriate conformation. Further, before performing molecular docking we validated CB-Dock for its ability to detect active sites correctly through the self-docking protocol. Once it was assured that CB- Dock can reproduce the experimental binding site of the ligand we did our docking experiments using this web-based tool only. During docking of newly synthesized molecules against the 2'-deoxyuridine-5'- monophosphate (dUMP) binding site of thymidylate synthase, FdUMP was used as control. Ligands interact with protein receptors through various interactions comprising hydrophobic interactions, hydrogen bonds, salt bridges, and so on. For generating the interaction profile of biologically meaningful poses of various ligands individually in the docked state with thymidylate synthase, the respective files were prepared according to the desired format. A protein-ligand interaction profiler (PLIP) was used for exploring ligand-thymidylate synthase interactions. This web service has the potential to detect seven types of interactions existing between the ligand and receptor in docked state. These interactions encompass hydrophobic contacts, hydrogen bonds, pi-cation interactions, halogen bonds, salt bridges, pi- stacking, and water bridges. 4.2.3 Kaplan Meier Analysis. The Kaplan-Meier plotter (https://kmplot.com/), is a web resource with gene expression data and information regarding the survival of BC patients (10). The mRNA gene chip BC dataset of the KM Plotter has RFS data for 4929 and OS data for 1879 patients. The patients were grouped into two cohorts based on the median expression of the gene. The effect of the TYMS on OS and RFS of the two defined groups was analyzed by KM- survival curves and the hazard ratio intervals and log-rank P-value was calculated using the KM plotter. 4.3 Cell Culture MCF-7, MDA-MB-231, and MDA-MB-468 human breast cancer cell lines were obtained from the National Centre for Cell Science (NCCS) in Pune. Prof Annapoorni Rangarajan, IISC, Bangalore, kindly supplied the murine cell line 4T1. Cell morphology was used to confirm the identification of these cells regularly. Dulbecco's Modified Eagle's Medium (DMEM; Thermo Fisher Scientific, Waltham, MA, United States) was used to culture MCF-7, MDA-MB-231, and MDA-MB-468 cells. 4T1 cells were cultured in RPMI1640 (Thermo Fisher Scientific, Waltham, MA, United States). The media was supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, United States) and 1% penicillin-streptomycin (Thermo Fisher Scientific, Waltham, MA, United States). The cells were cultured in a CO2 incubator (5%) at 37°C. 4.2.2. Gene expression analysis of Thymidylate Synthase in breast cancer. 4.2.2. Gene expression analysis of Thymidylate Synthase in breast cancer. The Gepia2 database (http://gepia2.cancer-pku.cn/) is a web-based online data mining platform for exploring the gene expression data of normal samples and tumors (39). The database has 1085 breast tumor samples and 291 GTEx samples. The GEPIA2 was used to study the expression of TYMS in breast Page 12/25 tumor tissues. The UALCAN database (4), a comprehensive web resource for analyzing cancer OMICS data was further used to analyze the expression of TYMS in BC subclasses, age groups and ethnicities. 4.3.1. Cell Viability Assay MCF-7, MDA-MB-231, MDA-MB-468, and murine 4T1 human breast cancer cell lines were seeded in 96- well plates at 3x103 cells/well. Following seeding, the cells were treated with uracil derivatives, which demonstrated substantial anticancer activity in early testing. The cells were treated with uracil derivatives in a dose - and time-dependent manner. The medium was replaced with MTT solution after 72 h of incubation, and the absorption of each well was then assessed at 490 nm. The inhibition of the production of formazan crystals was used as a cytotoxicity activity indicator. Non-linear regression analysis with Graph Pad software (v8) was performed to estimate the IC50 values on the cancer cells. Declarations Funding: The work was supported by Research Grant sanctioned to Manzoor Ahmad Mir by Jammu Kashmir Science Technology and Innovation Council (JKST&IC), Department of Science and Technology, Govt of J&K vide Grant NO. JKST&IC/SRE/885-87 and Ramalinga Swami Fellowship, Grant No. BT/HRD/35/02/2006, sanctioned to Nissar Ahmad Wani from the Department of Biotechnology (DBT), Ministry of Science & Technology, New Delhi. Acknowledgments: Umar Mehraj is a recipient of a junior research fellowship (JRF) from UGC-CSIR, Govt. of India. Nissar Ahmad Wani is the recipient of the Ramanujan Fellowship from the Science & Engineering Research Board (SERB), Department of Science and Technology, Govt. of India, New Delhi. Conflict of Interest: All the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Author contributions: Conceived and designed the experiments: MAM, MAZ, & NAW. In silico analysis: SAG, MAM & UM. Performed the experiments: MNL, & UM. Analyzed the data: MNL, MAM, SG, &, UM. Wrote the manuscript. MNL, MAM & UM. Revived and edited the manuscript NAW, SS, MAZ & MAM. Author contributions: Conceived and designed the experiments: MAM, MAZ, & NAW. In silico analysis: SAG, MAM & UM. Performed the experiments: MNL, & UM. Analyzed the data: MNL, MAM, SG, &, UM. Wrote the manuscript. MNL, MAM & UM. Revived and edited the manuscript NAW, SS, MAZ & MAM. 4.3.2. Colony Formation Assay Cells were seeded onto six-well plates at a density of 1,000 cells per well and left to adhere for 24 hours. Cells were subsequently treated for 12 to 15 days with a continuous dosage of medicines in DMSO (final concentration 1%). The media was replaced after every 3 days and the wells were observed for colonies using an inverted microscope. After the colonies had grown large enough, the cells were fixed using 3.7 percent paraformaldehyde (in PBS). After that, cells were stained with a 0.05 percent crystal violet solution and photographed and analyzed with ImageJ software. For each cell type and therapeutic concentration, the experiment was carried out in biological triplicates. Page 13/25 Page 13/25 Page 13/25 References (2019) Design, synthesis and biological evaluation of novel uracil derivatives bearing 1, 2, 3-triazole moiety as thymidylate synthase (TS) inhibitors and as potential antitumor drugs. Eur. J. Med. Chem., 171, 282–296. 15. Ma, H., Jiang, W.-R., Robledo, N., Leveque, V., Ali, S., Lara-Jaime, T., Masjedizadeh, M., Smith, D. B., Cammack, N. and Klumpp, K. (2007) Characterization of the metabolic activation of hepatitis C virus nucleoside inhibitor β-d-2′-deoxy-2′-fluoro-2′-C-methylcytidine (PSI-6130) and identification of a novel active 5′-triphosphate species. J. Biol. Chem., 282, 29812–29820. Jiang, W.-R., Robledo, N., Leveque, V., Ali, S., Lara-Jaime, T., Masjedizadeh, M., Smith, D. B., Cammack, N. and Klumpp, K. (2007) Characterization of the metabolic activation of hepatitis C virus nucleoside inhibitor β-d-2′-deoxy-2′-fluoro-2′-C-methylcytidine (PSI-6130) and identification of a novel active 5′-triphosphate species. J. Biol. Chem., 282, 29812–29820. cKeage, K. (2015) Trelagliptin: first global approval. Drugs, 75, 1161–1164. 16. McKeage, K. (2015) Trelagliptin: first global approval. Drugs, 75, 1161–1164. 17. Mehraj, U., Aisha, S., Sofi, S. and Mir, M. A. (2022) Expression pattern and prognostic significance of baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5) in breast cancer: A comprehensive analysis. Advances in Cancer Biology-Metastasis, 100037. 17. Mehraj, U., Aisha, S., Sofi, S. and Mir, M. A. (2022) Expression pattern and prognostic significance of baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5) in breast cancer: A comprehensive analysis. Advances in Cancer Biology-Metastasis, 100037. 18. Mehraj, U., Dar, A. H., Wani, N. A. and Mir, M. A. (2021) Tumor microenvironment promotes breast cancer chemoresistance. Cancer chemotherapy and pharmacology, 1–12. 18. Mehraj, U., Dar, A. H., Wani, N. A. and Mir, M. A. (2021) Tumor microenvironment promotes breast cancer chemoresistance. Cancer chemotherapy and pharmacology, 1–12. 19. Mehraj, U., Ganai, R. A., Macha, M. A., Hamid, A., Zargar, M. A., Bhat, A. A., Nasser, M. W., Haris, M., Batra, S. K. and Alshehri, B. (2021) The tumor microenvironment as driver of stemness and therapeutic resistance in breast cancer: New challenges and therapeutic opportunities. Cell. Oncol., 1–21. 20. Mehraj, U., Qayoom, H. and Mir, M. A. (2021) Prognostic significance and targeting tumor-associated macrophages in cancer: new insights and future perspectives. Breast Cancer, 1–17. 20. Mehraj, U., Qayoom, H. and Mir, M. A. (2021) Prognostic significance and targeting tumor-associated macrophages in cancer: new insights and future perspectives. Breast Cancer, 1–17. 21. Mehraj, U., Wani, N. A., Hamid, A., Alkhanani, M., Almilaibary, A. and Mir, M. A. References 1. Bergstrom, D. E. (1982) Organometallic intermediates in the synthesis of nucleoside analogs. Nucleosides Nucleotides, 1, 1–34. 1. Bergstrom, D. E. (1982) Organometallic intermediates in the synthesis of nucleoside analogs. Nucleosides Nucleotides, 1, 1–34. 2. Berman, H., Henrick, K. and Nakamura, H. (2003) Announcing the worldwide protein data bank. Nat. Struct. Mol. Biol., 10, 980–980. 3. Brown, D. J. (2009) The pyrimidines. ed. John Wiley & Sons. 4. Chandrashekar, D. S., Bashel, B., Balasubramanya, S. A. H., Creighton, C. J., Ponce-Rodriguez, I., Chakravarthi, B. V. S. K. and Varambally, S. (2017) UALCAN: a portal for facilitating tumor subgroup gene expression and survival analyses. Neoplasia, 19, 649–658. 5. Duan, J., Dixon, S. L., Lowrie, J. F. and Sherman, W. (2010) Analysis and comparison of 2D fingerprints: insights into database screening performance using eight fingerprint methods. J. Mol. Graph. Model., 29, 157–170. 6. Eberhardt, J., Santos-Martins, D., Tillack, A. and Forli, S. (2021) AutoDock Vina 1.2. 0: new docking methods, expanded force field, and Python bindings. 7. Feng, J., Zhang, Z., Wallace, M. B., Stafford, J. A., Kaldor, S. W., Kassel, D. B., Navre, M., Shi, L., Skene, R. J. and Asakawa, T. (2007) Discovery of alogliptin: a potent, selective, bioavailable, and efficacious inhibitor of dipeptidyl peptidase IV. J. Med. Chem., 50, 2297–2300. 8. Hayashi, Y. (2016) Pot economy and one-pot synthesis. Chemical science, 7, 866–880. 9. Huang, M. and Graves, L. M. (2003) De novo synthesis of pyrimidine nucleotides; emerging interfaces with signal transduction pathways. Cellular and Molecular Life Sciences CMLS, 60, 321– Page 14/25 336. 10. Lánczky, A. and Győrffy, B. (2021) Web-based survival analysis tool tailored for medical research (KMplot): development and implementation. J. Med. Internet Res., 23, e27633. 11. Lebert, J. M., Lester, R., Powell, E., Seal, M. and McCarthy, J. (2018) Advances in the systemic treatment of triple-negative breast cancer. Current oncology, 25, 142–150. 12. Lipinski, C. A., Lombardo, F., Dominy, B. W. and Feeney, P. J. (1997) Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Advanced drug delivery reviews, 23, 3–25. 13. Longley, D. B., Harkin, D. P. and Johnston, P. G. (2003) 5-fluorouracil: mechanisms of action and clinical strategies. Nature reviews cancer, 3, 330–338. 14. Lu, G.-q., Li, X.-y., Wang, D. and Meng, F.-h. References (2022) Adapalene inhibits the growth of triple-negative breast cancer cells by S-phase arrest and potentiates the antitumor efficacy of GDC-0941. Frontiers in Pharmacology, 13, 958443. 22. Mir, M. A., Qayoom, H., Mehraj, U., Nisar, S., Bhat, B. and Wani, N. A. (2020) Targeting different pathways using novel combination therapy in triple negative breast Cancer. Curr. Cancer Drug Targets, 20, 586–602. 23. Pałasz, A. and Cież, D. (2015) In search of uracil derivatives as bioactive agents. Uracils and fused uracils: Synthesis, biological activity and applications. Eur. J. Med. Chem., 97, 582–611. 23. Pałasz, A. and Cież, D. (2015) In search of uracil derivatives as bioactive agents. Uracils and fused uracils: Synthesis, biological activity and applications. Eur. J. Med. Chem., 97, 582–611. 23. Pałasz, A. and Cież, D. (2015) In search of uracil derivatives as bioactive agents. Uracils and fused uracils: Synthesis, biological activity and applications. Eur. J. Med. Chem., 97, 582–611. Page 15/25 Page 15/25 24. Paschall, A. V., Yang, D., Lu, C., Choi, J.-H., Li, X., Liu, F., Figueroa, M., Oberlies, N. H., Pearce, C. and Bollag, W. B. (2015) H3K9 trimethylation silences Fas expression to confer colon carcinoma immune escape and 5-fluorouracil chemoresistance. The Journal of Immunology, 195, 1868–1882. 25. Pasha, N. and Turner, N. C. (2021) Understanding and overcoming tumor heterogeneity in metastatic breast cancer treatment. Nature Cancer, 1–13. 26. Pereira, M. A., Ramos, M. F. K. P., Dias, A. R., Faraj, S. F., dos Santos Cirqueira, C., de Mello, E. S., Zilberstein, B., Alves, V. A. F. and Ribeiro Jr, U. (2018) Immunohistochemical expression of thymidylate synthase and prognosis in gastric cancer patients submitted to fluoropyrimidine-based chemotherapy. Chinese Journal of Cancer Research, 30, 526. 27. Pettersen, E. F., Goddard, T. D., Huang, C. C., Couch, G. S., Greenblatt, D. M., Meng, E. C. and Ferrin, T. E. (2004) UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem., 25, 1605–1612. 28. Qayoom, H., Wani, N. A., Alshehri, B. and Mir, M. A. (2021) An insight into the cancer stem cell survival pathways involved in chemoresistance in triple-negative breast cancer. Future Oncol. 29. Ramesh, D., Vijayakumar, B. G. and Kannan, T. (2020) Therapeutic potential of uracil and its derivatives in countering pathogenic and physiological disorders. Eur. J. Med. Chem., 112801. 30. Rose, M. G., Farrell, M. P. and Schmitz, J. C. (2002) Thymidylate synthase: a critical target for cancer chemotherapy. Clin. Colorectal Cancer, 1, 220–229. 31. References Shah, A., Nosheen, E., Zafar, F., Dionysiou, D. D., Badshah, A. and Khan, G. S. (2012) Photochemistry and electrochemistry of anticancer uracils. Journal of Photochemistry and Photobiology B: Biology, 117, 269–277. 32. Shi, H., Han, Z., Liu, J., Xue, J., Zhang, S., Zhu, Z., Xia, J. and Huang, M. (2017) Comparing efficacy of lamivudine, adefovir dipivoxil, telbivudine, and entecavir in treating nucleoside analogues naive for HBeAg-negative hepatitis B with medium hepatitis B virus (HBV) DNA levels. Medical science monitor: international medical journal of experimental and clinical research, 23, 5230. 33. Singh, A., Biot, C., Viljoen, A., Dupont, C., Kremer, L., Kumar, K. and Kumar, V. (2017) 1H-1, 2, 3‐triazole‐ tethered uracil‐ferrocene and uracil‐ferrocenylchalcone conjugates: Synthesis and antitubercular evaluation. Chem. Biol. Drug Des., 89, 856–861. 34. Sofi, S., Mehraj, U., Qayoom, H., Aisha, S., Almilaibary, A., Alkhanani, M. and Mir, M. A. (2022) Targeting cyclin-dependent kinase 1 (CDK1) in cancer: molecular docking and dynamic simulations of potential CDK1 inhibitors. Medical Oncology, 39, 1–15. 35. Sofi, S., Mehraj, U., Qayoom, H., Aisha, S., Asdaq, S. M. B., Almilaibary, A. and Mir, M. A. (2022) Cyclin- dependent kinases in breast cancer: expression pattern and therapeutic implications. Medical Oncology, 39, 1–16. 36. Sung, H., Ferlay, J., Siegel, R. L., Laversanne, M., Soerjomataram, I., Jemal, A. and Bray, F. (2021) Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin., 71, 209–249. Page 16/25 Page 16/25 37. Takemura, Y. and Jackman, A. L. (1997) Folate-based thymidylate synthase inhibitors in cancer chemotherapy. Anticancer Drugs, 8, 3–16. 38. Tan, H.-X., Gong, W.-Z., Zhou, K., Xiao, Z.-G., Hou, F.-T., Huang, T., Zhang, L., Dong, H.-Y., Zhang, W.-L. and Liu, Y. (2020) CXCR4/TGF-β1 mediated hepatic stellate cells differentiation into carcinoma- associated fibroblasts and promoted liver metastasis of colon cancer. Cancer Biology & Therapy, 21, 258–268. 39. Tang, Z., Li, C., Kang, B., Gao, G., Li, C. and Zhang, Z. (2017) GEPIA: a web server for cancer and normal gene expression profiling and interactive analyses. Nucleic Acids Res., 45, W98-W102. 40. Thomson, J. M. and Lamont, I. L. (2019) Nucleoside analogues as antibacterial agents. Front. Microbiol., 10, 952. 41. Veber, D. F., Johnson, S. R., Cheng, H.-Y., Smith, B. R., Ward, K. W. and Kopple, K. D. (2002) Molecular properties that influence the oral bioavailability of drug candidates. J. Med. Chem., 45, 2615–2623. 42. Wahba, H. A. References and El-Hadaad, H. A. (2015) Current approaches in treatment of triple-negative breast cancer. Cancer biology & medicine, 12, 106. 43. Wu, C.-H., Wu, C.-C. and Ho, Y.-S. (2007) Antitumor activity of combination treatment of Lentinus edodes mycelium extracts with 5-fluorouracil against human colon cancer cells xenografted in nude mice. J Cancer Mol, 3, 15–22. 44. Xie, P., Mo, J.-L., Liu, J.-H., Li, X., Tan, L.-M., Zhang, W., Zhou, H.-H. and Liu, Z.-Q. (2020) Pharmacogenomics of 5-fluorouracil in colorectal cancer: review and update. Cell. Oncol., 43, 989– 1001. 45. Zhang, W., Feng, M., Zheng, G., Chen, Y., Wang, X., Pen, B., Yin, J., Yu, Y. and He, Z. (2012) Chemoresistance to 5-fluorouracil induces epithelial–mesenchymal transition via up-regulation of Snail in MCF7 human breast cancer cells. Biochem. Biophys. Res. Commun., 417, 679–685. 45. Zhang, W., Feng, M., Zheng, G., Chen, Y., Wang, X., Pen, B., Yin, J., Yu, Y. and He, Z. (2012) Chemoresistance to 5-fluorouracil induces epithelial–mesenchymal transition via up-regulation of Snail in MCF7 human breast cancer cells. Biochem. Biophys. Res. Commun., 417, 679–685. Figures Page 17/25 Page 17/25 Page 17/25 Figure 1 Schematic for the synthesis of novel uracil derivatives. Schematic for the synthesis of novel uracil derivatives. Schematic for the synthesis of novel uracil derivatives. Page 18/25 Figure 2 Figure 2 The chemical structures of synthesized uracil derivatives. Page 19/25 Figure 3 Scheme for the proposed mechanism for the synthesis of uracil derivatives. Figure 3 Figure 3 Figure 3 Scheme for the proposed mechanism for the synthesis of uracil derivatives. Page 20/25 Figure 4 In silico evaluation of the binding affinity of novel UDs with thymidylate synthase, A. CB-Dock serve predict active site correctly. The green region shows the thymidylate synthase enzyme and the yellow Figure 4 Page 21/25 In silico evaluation of the binding affinity of novel UDs with thymidylate synthase, A. CB-Dock server can predict active site correctly. The green region shows the thymidylate synthase enzyme and the yellow- colored molecule (ball and stick) indicates its co-crystallized ligand. We separated this co-crystallized ligand from thymidylate synthase and then redocked it with this synthase using the CB-Dock server. The redocked ligand (red-colored) and co-crystallized ligand (yellow) colocalized at the same place. This Page 21/25 shows that the docking algorithm can predict the binding site correctly. We also analyzed the interaction profile of UDs with thymidylate synthase in 2D view. While 6 interactions were observed both in the case of UD4 and UD6. Relatively more interactions were observed with UD4 and UD5 in contrast to other UDs. B. Binding affinity values of newly synthesized five molecules against human thymidylate synthase. 5- FdUMP (FdUMP) (5-FU metabolite), the known binder of thymidylate synthase was put as control. The more negative value of binding affinity signifies stronger binding. Among the tested molecules, UD5 followed by UD4 and UD2 proved to be strong binders of thymidylate synthase as indicated by the binding affinity values. Further, all the novel molecules proved to be relatively more effective binders than the known metabolite of 5FU namely FdUMP. Figure 5 Expression pattern and prognostic significance of thymidylate synthase (TYMS) in breast cancer. A. TYMS was found high upregulated in BC patients compared to normal control or solid normal samples. B. Among breast cancer subtypes, TYMS showed high expression in aggressive subtypes viz TNBC followed by luminal and HER2 enriched. C. Enhanced expression of TYMS was observed among women under the age group of 21-40 yrs and, D.African-American women. E. High expression of TYMS was found associated with worse relapse-free survival (RFS) and F. overall survival (OS). Figure 5 Expression pattern and prognostic significance of thymidylate synthase (T prognostic significance of thymidylate synthase (TYMS) in breast cancer. A Expression pattern and prognostic significance of thymidylate synthase (TYMS) in breast cancer. A. TYMS was found high upregulated in BC patients compared to normal control or solid normal samples. B. Among breast cancer subtypes, TYMS showed high expression in aggressive subtypes viz TNBC followed by luminal and HER2 enriched. C. Enhanced expression of TYMS was observed among women under the age group of 21-40 yrs and, D.African-American women. E. Figure 4 High expression of TYMS was found associated with worse relapse-free survival (RFS) and F. overall survival (OS). Expression pattern and prognostic significance of thymidylate synthase (TYMS) in breast cancer. A. TYMS was found high upregulated in BC patients compared to normal control or solid normal samples. B. Among breast cancer subtypes, TYMS showed high expression in aggressive subtypes viz TNBC followed by luminal and HER2 enriched. C. Enhanced expression of TYMS was observed among women under the age group of 21-40 yrs and, D.African-American women. E. High expression of TYMS was found associated with worse relapse-free survival (RFS) and F. overall survival (OS). Page 22/25 Figure 6 Uracil derivatives reduce the cell viability of breast cancer cell lines in a dose-dependent manner. A. Reduced cell viability of MDA-MB-231 cells treated with 50 µM of UDs for 72 hrs. Among the screened UDs, UD1, UD2 and UD4 showed high potency in reducing cell viability at 50 µM conc. One-way Annova was used for statistical analysis. p < 0.0001 (, significantly different from DMSO treated controls. B, UD1, C, UD2, and D. UD4 were analyzed for potency in a panel of BC cell lines and showed an enhanced reduction in cell viability in a dose-dependent manner. The IC50 values were calculated using non-linear regression (curve fit) in Graphpad Prism V8. Among the three potent UDs, UD4 showed the highest reduction in cell viability and lowest IC50 value. Figure 6 Figure 6 Uracil derivatives reduce the cell viability of breast cancer cell lines in a dose-dependent manner. A. Reduced cell viability of MDA-MB-231 cells treated with 50 µM of UDs for 72 hrs Among the screened Uracil derivatives reduce the cell viability of breast cancer cell lines in a dose-dependent manner. A. Reduced cell viability of MDA-MB-231 cells treated with 50 µM of UDs for 72 hrs. Among the screened UDs, UD1, UD2 and UD4 showed high potency in reducing cell viability at 50 µM conc. One-way Annova was used for statistical analysis p < 0 0001 (* significantly different from DMSO treated controls B Uracil derivatives reduce the cell viability of breast cancer cell lines in a dos U ac de at es educe t e ce ab ty o b east ca ce ce es a dose depe de t a e . . Reduced cell viability of MDA-MB-231 cells treated with 50 µM of UDs for 72 hrs. Among the screened UDs, UD1, UD2 and UD4 showed high potency in reducing cell viability at 50 µM conc. One-way Annova was used for statistical analysis. p < 0.0001 (*, significantly different from DMSO treated controls. B, UD1, C, UD2, and D. UD4 were analyzed for potency in a panel of BC cell lines and showed an enhanced reduction in cell viability in a dose-dependent manner. The IC50 values were calculated using non-linear regression (curve fit) in Graphpad Prism V8. Among the three potent UDs, UD4 showed the highest reduction in cell viability and lowest IC50 value. A-MB-231 cells treated with 50 µM of UDs for 72 hrs. Among the screened wed high potency in reducing cell viability at 50 µM conc. One-way Annova Page 23/25 Page 23/25 Page 23/25 Figure 7 Uracil derivatives reduced the proliferation of breast cancer cell lines in a time-dependent manner. We next evaluated the effect of potent UDs, viz UD1, UD2, and UD2 on BC cell lines in a time-dependent manner, and the statistical significance increased with time. A. MDA-MB-231 cells, B. MCF-7 cells, C. MDA-MB-468 cells, and D. 4T1 cells demonstrated a reduction in cell viability with time upon treatment with UDs. Two-way annova was used for statistical analysis followed by Dunnett's multiple comparisons test. p > 0.01 (ns), p < 0.01 (), p < 0.001 (), p < 0.005 (), p < 0.0001 (**). Figure 6 Figure 7 Uracil derivatives reduced the proliferation of breast cancer cell lines in a time-dependent manner. We next evaluated the effect of potent UDs, viz UD1, UD2, and UD2 on BC cell lines in a time-dependent manner, and the statistical significance increased with time. A. MDA-MB-231 cells, B. MCF-7 cells, C. MDA-MB-468 cells, and D. 4T1 cells demonstrated a reduction in cell viability with time upon treatment with UDs. Two-way annova was used for statistical analysis followed by Dunnett's multiple comparisons test. p > 0.01 (ns), p < 0.01 (), p < 0.001 (), p < 0.005 (), p < 0.0001 (**). Page 24/25 Page 24/25 Page 24/25 Figure 8 Uracil derivatives reduced colony formation potential of MDA-MB-231 cells. The effect of UDs on colony formation was evaluated and a significant reduction in colony formation was observed at conc lower than IC50 values. Among UDs, UD4 showed an enhanced reduction in cell colonies at 17 µM conc. One- way annova was used for statistical analysis followed by Dunnett's multiple comparisons test. p < 0.01 (), p < 0.001 (), p < 0.005 (), p < 0.0001 (**). Figure 8 Uracil derivatives reduced colony formation potential of MDA-MB-231 cells. The effect of UDs on colony formation was evaluated and a significant reduction in colony formation was observed at conc lower than IC50 values. Among UDs, UD4 showed an enhanced reduction in cell colonies at 17 µM conc. One- way annova was used for statistical analysis followed by Dunnett's multiple comparisons test. p < 0.01 (), p < 0.001 (), p < 0.005 (), p < 0.0001 (**). Uracil derivatives reduced colony formation potential of MDA-MB-231 cells. The effect of UDs on colony formation was evaluated and a significant reduction in colony formation was observed at conc lower than IC50 values. Among UDs, UD4 showed an enhanced reduction in cell colonies at 17 µM conc. One- way annova was used for statistical analysis followed by Dunnett's multiple comparisons test. p < 0.01 (), p < 0.001 (), p < 0.005 (), p < 0.0001 (***). Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. GraphicalAbstract.jpg SupplementaryData.pdf CompoundCharacterizationChecklist.pdf Page 25/25
https://openalex.org/W2141223520	https://zenodo.org/records/1262953/files/article.pdf	English	null	Computation of LFT uncertainty bounds with repeated parametric uncertainties	null	1,998	public-domain	4,472	elphia, Pennsylvanla June 1998 COMPUTATION OF LFT UNCERTAINTY BOUNDS WITH REPEATED PARAMETRIC UNCERTAINTIES K. B. Lim: D. P. Giesyt NASA Langley Research Center, MS 161, Hampton, Virginia, 29681-0001 K. B. Lim: D. P. Giesyt K. B. Lim: D. P. Giesyt y NASA Langley Research Center, MS 161, Hampton, Virginia, 29681-0001 y NASA Langley Research Center, MS 161, Hampton, Virginia, 29681-0001 y+----u plant measurement noise Figure 1: Block diagram for uncertainty identification Abstract A new methodology in which linear fractional transfor- mation uncertainty bounds are directly constructed for use in robust control design and analysis is proposed. Existence conditions for model validating solutions with or without repeated scalar uncertainty are given. The approach is based on minimax formulation to deal with multiple non-repeated structured uncertainty components subject to fixed levels of repeated scalar uncertainties. Input directional dependence and variations with different experiments are addressed by maximizing uncertainty levels over multiple experimental data sets. Preliminary results show that reasonable uncertainty bounds on structured non-repeated uncertainties can be iden- tified directly from measurement data by assuming reasonable levels of repeated scalar uncertainties. Figure 1: Block diagram for uncertainty identification Proceedings of the American Control Conference Philadelphia, Pennsylvanla * June 1998 Proceedings of the American Control Conference Philadelphia, Pennsylvanla * June 1998 (1) A = blk-diag(A1,. . .,AT); Aj E CmaXn’, i E TT (1) where TT := (1, . . . , T ) . Consider a set of measurements from ne independent experiments whose inputs and outputs are denoted by ( ~ ( j ) , for the j t h experiment. Define the set where TT := (1, . . . , T ) . Consider a set of measurements from ne independent experiments whose inputs and outputs are denoted by ( ~ ( j ) , for the j t h experiment. Define the set T ne := { u(j 1, y(j) : j = 1, . , . , ne} (2) (2) Henceforth, consider input and output pairs that satisfy (U, y) E Tn,. At each frequency, the signals and 17 depend on input vector U and in particular its direction so that a ratio of their norms will also be dependent on the particular input direction. To account for this dependence, consider satisfying model validating conditions [l] - [5] for all available experiments: where @ := blk-diag(SIIn,, . . .,&In,.) and 6j E F and Ai are defined in equation (1). Given a model validating set, consider choosing a “small- est” size uncertainty in terms of I = AV (3) 17 = P l l t + P l Z U (4) ey = !/ - P215 - PZZU = 0, V(% y) E Tne ( 5 ) ey = !/ - P215 - PZZU = 0, V(% y) E Tne ( 5 ) i.e., minimum over maximum over all components. A po- tential hazard with this viewpoint is the numerical values of the component uncertainties may not be of equal physical relevance. This is particularly true for more complex systems where there is usually a mixture of parametric (repeated or not) and nonparametric uncertainties including unmodeled dynamics. In practice, more uncertainty parameters are used (as compared to the “true” set of uncertain parameters) to sufficiently satisfy existence of model validating solutions. This means that robust identification problems generally lead to underdetermined problems, which appears to require the selection of physically significant uncertainty configurations and levels from all model validating sets. For the above reasons, we treat the parametric scalar uncertainties which i.e., minimum over maximum over all components. A po- tential hazard with this viewpoint is the numerical values of the component uncertainties may not be of equal physical relevance. ‘Research Engineer, Guidance & Control Branch, Flight Dy- namics & Control Division, k . b . limelarc .nasa. gov U.S. Government Work Not Protected by U.S. Copyright 1 Problem Definition Of course fictitious signals e and 17 cannot be measured so it is necessary to look at their dependence on real signals U and y and their transmission through the system P. We consider a constant matrix problem; i.e., the supremum is taken at each frequency and is due to the directional dependence of ratio of 2-norms. IT) T) Figure 1 shows measured input, U E CnU and output, y E C”., across a true plant. These inputs and outputs are assumed to be discrete Fourier transforms obtained from discrete time records. The corresponding output, @, from an upper linear fractional transformation model, F,(P, A), depends on a given augmented plant, P, and an assumed structured uncertainty of the form Let col(E1, . . . , IT) and col(q1, . . . , vT) be the partition- ing of the vectors I and 7 which conform to the block diagonal partition of A in equation (1). For convenience, assume that all repeated scalar blocks are grouped into the first r blocks in A. Then, equation (3) becomes: (1) A = blk-diag(A1,. . .,AT); Aj E CmaXn’, i E TT (1) A = blk-diag(A1,. . .,AT); Aj E CmaXn’, i E TT (1) A = blk-diag(A1,. . .,AT); Aj E CmaXn’, i E TT This is particularly true for more complex systems where there is usually a mixture of parametric (repeated or not) and nonparametric uncertainties including unmodeled dynamics. In practice, more uncertainty parameters are used (as compared to the “true” set of uncertain parameters) to sufficiently satisfy existence of model validating solutions. This means that robust identification problems generally lead to underdetermined problems, which appears to require the selection of physically significant uncertainty configurations and levels from all model validating sets. For the above reasons, we treat the parametric scalar uncertainties which The basic problem we address is to determine smallest bounds among all uncertainties which are model validating with re- spect to available input/output measurements. From equation (3), the uncertainty bound can be written as a ratio of %norms The basic problem we address is to determine smallest bounds among all uncertainties which are model validating with re- spect to available input/output measurements. From equation (3), the uncertainty bound can be written as a ratio of %norms ‘Research Engineer, Guidance & Control Branch, Flight Dy- namics & Control Division, k . b . limelarc .nasa. gov ‘Research Engineer, Guidance & Control Branch, Flight Dy- namics & Control Division, k . b . limelarc .nasa. gov t Mathematician, Guidance & Control Branch, Flight Dynamics & Control Division, d . p . giesyalarc . nasa .gov 1018 Th 2 ( i h d l bl k) are often repeated separately from component unstructured uncertainties. are often repeated separately from component unstructured uncertainties. Theorem 2 (with repeated scalar block) Suppose the condition in Theorem 1 holds. Let (U, y) E Tn,. Then, a model validating set <, Q, and A exists if and only if there exists 61,. . .,&, and $ such that Theorem 2 (with repeated scalar block) Suppose the condition in Theorem 1 holds. Let (U, y) E Tn,. Then, a model validating set <, Q, and A exists if and only if there exists 61,. . .,&, and $ such that Theorem 1 (no repeated scalar block) Notice that si, in equation (20) is the smallest 6i making equation (21) true for all $. A violation of inequality (21) implies that 6i is not an upper bound on the ratio of signal norms, i.e., it fails as an uncertainty bound. For large 6a, equation (21) is more likely to be satisfied, which makes intu- itive sense. Finally, notice that in equation (20), $ generally influences all components ti, vi, i E T,. This motivates the minimax formulation. Let ( U , y) E Tne. Then a corresponding model validating uncertainty set, (, r ] , and A, satisfying equations (3) to (5) exists if and only if e; E Im(P21). 0 If A contains repeated scalar blocks, then the condition in Theorem 1 becomes only a necessary condition for model validation. So, with repeated scalar blocks we ask, “Among all pairs (‘, q), satisfying equation (12), does a pair exist that also satisfies equation (3)?” Given any pair (c, v), A,,,, . . ., A, always exist, so we need to consider only the existence of the first r blocks, ~ I I ~ ~ , . . ., &In7. Let us denote 6 := ni = m := ET=1 mi and define 2 Existencle of [, q For each experiment, consider the existence of ( which is an unconstrained by the structure in the uncertainty block and satisfying equation (5) which is rewritten as Theorem 2 is a constraint on 4 to limit 5 and 17 that satisfy the repeated scalar structure implicit in equation (7). Note that 2 has full row rank independent of the set 61,. . .,6,, so that Lemma 1: For each ( u , ~ ) E Tne, a E exists which satisfies equation (5) if and only if dim(Ker(2)) = n + m - 6 (17) (17) Note that although the rank of 2 does not change as long as 6 j # 0, V i E T,, the bases changes with Si, i E T,, due to the repeated scalar uncertainty structure. Theorem 2 results in either a yes or no answer to the existence question. However, as a test, it is complicated because Z is dependent on the values of the set 61,. . .,fir, which is as yet undetermined. cy” E Im(P2i) 0 (10) cy” E Im(P2i) 0 cy” E Im(P2i) 0 (10) Lemma 1 is a test which gives a yes or no answer. If the test indicates that a solution exists, then all ( satisfying equation (5) can be written as (11) = + Nzid (11) = + Nzid 3.1 Uncertainty Bound Inequality From equation (8), it is of interest to note that Consequently we state the following result which was also obtained earlier in [4] in a more general context. The asterisk in equation 20 refers to the minimum 2-norm ratio with respect to $. To avoid a ratio of square root of quadratics in I ) , consider the condition Theorem 1 (no repeated scalar block) 3.1 Uncertainty Bound Inequality From equation (8), it is of interest to note that < sup - I I<: I I 2 = s;? /2 I I Aid I I2 := @(Ai), i E T, (18) llrlilla - 7: 11171112 1117.11 Lemma 1 defines the conditions under which the output nominal error e; can be cancelled by the fictitious signals (4). Lemma 1 defines the conditions under which the output nominal error e; can be cancelled by the fictitious signals Pz1( from the uncert,ainty block. The Lemma presupposes that the fictitious signal ( is unconstrained by the structure in the uncertainty block. Note that the null freedom $ is an independent variable. In fact, any choice of $ for a given experiment gives a corresponding pair of‘ and r] which satisfy equations (4) and (5). (4). With physical signals, the best we can expect to do is to estimate the supremum by a maximum over all available experiments, i.e., With physical signals, the best we can expect to do is to estimate the supremum by a maximum over all available experiments, i.e., q ( ) ( ) If the condition in Lemma 1 holds, the fictitious signals satisfying equations (4) and (5) can be written as For each experiment, the free null parameter 4 (and therefore ( and 11) can be chosen that minimizes the ratio of 2-norms in equation (19). The maximum over all experiments in equation (19) can be determined after solving for minimum ratio with respect to $ for each experiment, independently. Hence, consider first the following minimization problem for each experiment: For each experiment, the free null parameter 4 (and therefore ( and 11) can be chosen that minimizes the ratio of 2-norms in equation (19). The maximum over all experiments in equation (19) can be determined after solving for minimum ratio with respect to $ for each experiment, independently. Hence, consider first the following minimization problem for each experiment: {;I>={ :}+[ p;1]N21* where It turns out that for general complex block diagonal uncer- tainty structure with no repeated scalar blocks, for given [ and r], a A satisfying equation (3) always exists [I]. Consequently we state the following result which was also obtained earlier in [4] in a more general context. It turns out that for general complex block diagonal uncer- tainty structure with no repeated scalar blocks, for given [ and r], a A satisfying equation (3) always exists [I]. 3 Uncertainty Bound where e; is determined by the particular experiment using equation (9), N21 is chosen so that its columns span Ker(Pzl), and 4 E Cn* is arbitrary. Given any E from equation (ll), the complementary signal r] can be computed from equation (4) 3.1 Uncertainty Bound Inequality From equation (8), it is of interest to note that 3.2 Minimax problem With the remaining freedom in the restricted model validating set, we seek a smallest uncertainty bound for the remaining nonre- peated uncertainty components (i = T + 1,. . . , T ) . 4 Illustrative Example The dimensions of the equations are as follows: equation (22) consists of fi complex equalities, equation (23) consists of T real inequalities, equation (25) consists of T - T real inequalities, and equation (26) consists of T - T real inequal- ities. Equation (22) defines the admissible set of fictitious signals parameterized by ?I, that satisfies the repeated scalar uncertainty structure. Equation (25) defines the dependence of nonrepeated uncertainty bounds on the above admissible set of fictitious signals. Finally, condition (23) specifies the bound on repeated scalar uncertainties while condition (26) is used to define a bound on the maximum nonrepeated uncertainty component for minimax optimization purpose. Consider a simple dyamical system given in [5] but with two force inputs and two outputs. The system consists of two structural modes resulting in four state variables. The system is discretized at 50 Hertz assuming a zero order hold at both has the form inputs. The fourth order discrete A matrix for this system A = blk-diag(A1, A2) (30) A = blk-diag(A1, A2) (30) where Ai is a 2 by 2 matrix of the form Re(zi) -Im(zj) 1 Ai := [ Im(zi) Re(za) For numerical solutions, we suggest the following steps: , f -&?Ii"llha, + ~ 1 1 , , ~ 2 1 4 I I ; + Il%7l, + N21,4ll; 50, i = T + l , . . . , T (25) (25) 6; -y 5 0, i = T + I,. . . , f 3.2 Minimax problem The problem for cash experiment then becomes: find the smallest bound set, Si, i E T,, such that inequality (21) is satisfied and subject to model validation constraints on ‘ j and 9 s s 1019 1019 Note from condition (25) that for any fixed $, the smallest 8: holds when condition (25) becomes an equality, i.e., 77;. The signal $ is a free parameter from excess uncertainty degrees of freedom that can be judiciously chosen for each experiment to minimize Jj for all i. The multiple objective nature is addressed by formulating it as a particular type of minimax problem. p Using Theorem 2, we can parameterize the set of all signal pairs, ( E , 7) that satisfies model validation for each experiment. This set corresponds to all $, and 61,. . .,& that satisfies equation (16) which can be rewritten as The OP2 problem for each experiment reduces to the follow- ing: The OP2 problem for each experiment reduces to the follow- ing: op3 miny (28) dJ ,T op3 miny dJ ,T (28) subject to and (.)A denotes the first fi rows of (.). Notice from equation (22) that it depends only on the repeated scalar uncertainties. and (.)A denotes the first fi rows of (.). Notice from equation (22) that it depends only on the repeated scalar uncertainties. In our approach, we choose to restrict a model validating set parameterized by the set $, and 51,. . .,6, that is also bounded by and (.)A denotes the first fi rows of (.). Notice from equation (22) that it depends only on the repeated scalar uncertainties. In our approach, we choose to restrict a model validating set parameterized by the set $, and 51,. . .,6, that is also bounded by ( ) p y p In our approach, we choose to restrict a model validating set parameterized by the set $, and 51,. . .,6, that is also bounded by In OP3, the null freedom vector $ is used to minimize the maximum uncertainty bound. Note that for each experiment (or ne = 1 case), OP3 is equivalent to the earlier results using a minimum norm model validating (MNMV) formulation as given in [1]-[2]. where E R which denotes the ith repeated scalar uncer- tainty bound as chosen a priori by an engineer. For numerical solutions, we suggest the following steps: For numerical solutions, we suggest the following steps: Step 1: Choose repeated scalar bounds 6i, i = 1, . . . , T. Step 2: Solve optimization problem OP2 for each exper- Step 1: Choose repeated scalar bounds 6i, i = 1, . . . , T. Step 2: Solve optimization problem OP2 for each exper- If OP2 results in an infeasible domain, increase appro- priate &. Redo Step 2 until solution exists. Step 3: Compute worst bounds for nonrepeated uncer- iment. tainty components over all experiments. subject to equations (22), (B), and subject to equations (22), (B), and If OP2 results in an infeasible domain, increase appro- & iment. -@;i211bn, + P1ln,N21$11; + I l ~ m , 4- N21,,41E I O 1 i = T + l , ..., T pp priate &. Redo Step 2 until solution exists. b d f d p p Step 3: Compute worst bounds for nonrepeated uncer- tainty components over all experiments. where (.)n, and refers to the ith block of rows associated with Ai E Cmzxna of dimensions nj and mi respectively for matrix (.). The possible non-smoothness in the minimax problem in OP1 can be avoided by recasting the problem for each experiment as where (.)n, and refers to the ith block of rows associated with Ai E Cmzxna of dimensions nj and mi respectively for matrix (.). The possible non-smoothness in the minimax problem in OP1 can be avoided by recasting the problem for each experiment as - - Sa = maxSj, i = T + 1,. . . , T T n c Fit uncertainty components using stable low Step 4: order rational functions over all frequencies. subject to equations 22 23 and Note that in Step 4, an interactive utility program such as drawmag, a p-Tools command [6] can be used. Analytical sensitivities for all constraint functions are derived. and used but not shown due to lack of space. All complex variables and equations are expanded out in real variable form to obtain the derivatives. subject to equations 22,23, and -&?Ii"llha, + ~ 1 1 , , ~ 2 1 4 I I ; + Il%7l, + N21,4ll; 50, 6; -y 5 0, i = T + l , . . . , T (25) (26) i = T + I,. . . 3.4 Compuational Algorithm p y p ( , , ) We summarize the problem for each experiment as For numerical solutions, we suggest the following steps: min y subject to conditions (25) and (26). at 1. ..,sa ,dj 3 7 I . . 61 62 True .005 .01 Case 1 .001 .001 Case 2 .01 .01 Case 3 .1 .1 02x2, and E2 = [0, -1; l , O ] . 61 62 True .005 .01 Case 1 .001 .001 Case 2 .01 .01 Case 3 .1 .1 [ , ; , ] Figure 3 shows ithe interconnection structure of the parametric uncertainty with an additive uncertainty from two inputs to two outputs. Figure 4 shows the frequency 5 1 5, 4.1 Nominal aind Uncertainty Structure 2 E % 2 -1 00 a --zoo 2 -400 a -200 -600 1 oo 1 o2 1 oo 1 o2 with parametric pertuicbations set to 61 = 62 = 0. Hence, the Consider a nominal model consisting of only the first mode nominal model contains both parametric error (in the first mode) and unmodeled dynamics (truncated second mode). Figure 2 shows the poles of the true plant corresponding to the two structural modes and an approximation of the first pole by the nominal model. This parametric error is represented 3.3 No repeated scalar block To establish a truth model which is then used to simulate measurements, we perturb the first structural mode by the following ] To establish a truth model which is then used to simulate measurements, we perturb the first structural mode by the following ] For the case when there are no repeated scalar blocks (T = O), OP2 reduces to A 1 = A ; + 6 1 [ ; ; ] + 6 2 [ 0 1 -1 ] 0 min y subject to conditions (25) and (26). at 1. ..,sa ,dj 3 7 I . . min y subject to conditions (25) and (26). at 1. ..,sa ,dj 3 7 I . . 1020 1020 The first error peak is due to parametric error in the first mode and the second due to truncated second mode. where 61 and 62 corresponds to the perturbations in the real and imaginary components of the first discrete eigenvalue, respectively. The perturbation matrices for this choice of parametric uncertainties are of rank two, i.e., repeated mul- tiplicity of order 2 for each scalar parameter. Of course in order to preserve the (complex conjugate pair property of the set of perturbed plantar, both 61 and 62 must be real numbers. For the simulated measurements, a small level of independent Gaussian white noise was included at both outputs. A single time record was divided into two segments for use as two independent experiments (ne = 2). where 61 and 62 corresponds to the perturbations in the real and imaginary components of the first discrete eigenvalue, respectively. The perturbation matrices for this choice of parametric uncertainties are of rank two, i.e., repeated mul- tiplicity of order 2 for each scalar parameter. Of course in order to preserve the (complex conjugate pair property of the set of perturbed plantar, both 61 and 62 must be real numbers. For the simulated measurements, a small level of independent Gaussian white noise was included at both outputs. A single time record was divided into two segments for use as two independent experiments (ne = 2). loo - ,- - .s io-' (II 2 E % 2 -1 00 a --zoo 2 -400 a -200 -600 1 oo 1 o2 1 oo 1 o2 loo - ,- - .s io-' (II loo - ,- - .s io-' (II Figures 5 to 7 show the identified uncertainty bounds for un- modeled dynamics ( 0 ) while figure 8 shows the corresponding curved-fitted stable low order rational functions for additive uncertainties in all three cases. It appears that when the assumed parametric uncertainty bound is very small relative to true values (Case 1, figure 5 ) , the additive uncertainty tries to capture a large error incurred by the optimistic level of as- 2 J IO’ Frequency (radlsec) 1 o2 Figure 7: Case 3: Additive unc bounds (0) and SV frequency responses - True( -), Nominal( --), True- Nominal(. . .) is consistent with the expectation that in general, parametric changes for a structural mode do not significantly affect other structural modes. 2 J IO’ Frequency (radlsec) 1 o2 structural modes. Finally, note that from a more detailed parametric study, a smaller level of parametric uncertainty level could be de- duced. This “minimal” parametric uncertainty level corre- sponds to a threshold level whereby a further decrease in the parametric uncertainty level will give a large increase in unmodeled dynamics uncertainty level. Of course, a priori physical knowledge of the system under investigation will generally help in determining this optimal tradeoff point. T O 1 m 2 10-~1 1 o1 1 o2 Frequency (rad/sec) Figure 5: Case 1: Additive unc bounds (0) and SV frequency responses - True( -), Nominal( --), True- Nominal(. . .) T O 1 m 2 10-~1 1 o1 1 o2 Frequency (rad/sec) Frequency (radlsec) Figure 7: Case 3: Additive unc bounds (0) and SV frequency responses - True( -), Nominal( --), True- Nominal(. . .) ’ I 0 - ~ 1 I 1 o1 1 0‘ Frequency (rawsec) Figure 8: Fitted additive weighting function for Case 1 (. . .), Case 2(--), Case 3(-.) ’ Frequency (rad/sec) Figure 5: Case 1: Additive unc bounds (0) and SV frequency responses - True( -), Nominal( --), True- Nominal(. . .) m $Io1 2 10-~1 I 1 o1 1 0’ Frequency (radlsec) Figure 6: Case 2: Additive unc bounds ( 0 ) and SV frequency responses - True( -), Nominal( --), True- Nominal(. . .) m $Io1 2 10-~1 I 1 o1 1 0’ Frequency (radlsec) Figure 8: Fitted additive weighting function for Case 1 (. . .), Case 2(--), Case 3(-.) References Lim, K.B., Balas, G. J., and Anthony, T.C., “Minimum- norm model validating identification for robust control,” AIAA Paper No. 96-3717. Lim, K.B., “Closed form solution for minimum norm model validating uncertainty,” 1997 American Control Con feren ce, Albuquerque, NM . Frequency (radlsec) Frequency (radlsec) Lim, K.B., Cox, D.E., Balas, G.J., and Juang, J-N., “Val- idation of an experimentally derived uncertainty model,” AIAA Journal of Guidance, Control, and Dynamics, v.21, no.2, 1998. Figure 6: Case 2: Additive unc bounds ( 0 ) and SV frequency responses - True( -), Nominal( --), True- Nominal(. . .) Smith, R.S., Doyle, J.C., “Model Validation: A connec- tion between robust control and identification,” IEEE Transactions on Automatic Control, vol37, No 7, July 1992, pp.942-952. Kumar, A., and Balas, G.J., “A scaling approach to model validation in the /I framework,” 1995 American Control Conference, pp.693-697. 5 Conclusion Balas, G.J., Doyle, J.C., Glover, K., Packard, A., and Smith, R., p-Analysis and Synthesis Toolbox, MUSYN Inc., Minneapolis, MN, 1994. A new algorithm is presented for identifying both non para- metric uncertainties and repeated scalar uncertainties. The algorithm uses a minimax search which is constrained to all fictitious input/output uncertainty signal sets that zero out output errors for a nominal model. This development extends an earlier method involving a minimum norm model validating approach. Morton, B., and McAfoos, R., “A Mu-test for real param- eter variations,” 1985 American Control Conference, Boston, MA. Branch, M.A., and A. Grace, Optimization Toolbox, The Mathworks, Inc., Natick, MA, December 1996. 2 1022
https://openalex.org/W2948057274	https://europepmc.org/articles/pmc6600244?pdf=render	English	null	Improved Carotenoid Processing with Sustainable Solvents Utilizing Z-Isomerization-Induced Alteration in Physicochemical Properties: A Review and Future Directions	Molecules/Molecules online/Molecules annual	2,019	cc-by	13,288	Improved Carotenoid Processing with Sustainable Solvents Utilizing Z-Isomerization-Induced Alteration in Physicochemical Properties: A Review and Future Directions Masaki Honda 1,* , Hakuto Kageyama 1 , Takashi Hibino 1, Yelin Zhang 2, Wahyu Diono 2 , Hideki Kanda 2 , Ryusei Yamaguchi 3, Ryota Takemura 4, Tetsuya Fukaya 4,5,* and Motonobu Goto 2,* 1 Faculty of Science & Technology, Meijo University, Shiogamaguchi, Tempaku-ku, Nagoya 468-8502, Japan; kageyama@meijo-u.ac.jp (H.K.); hibino@meijo-u.ac.jp (T.H.) 1 Faculty of Science & Technology, Meijo University, Shiogamaguchi, Tempaku-ku, Nagoya 468-8502 k @ ij j (H K ) hibi @ ij j (T H ) 1 Faculty of Science & Technology, Meijo University, Shiogamaguchi, Tempaku-ku, Nagoya 468-8502, Japan; kageyama@meijo-u.ac.jp (H.K.); hibino@meijo-u.ac.jp (T.H.) 2 Department of Materials Process Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan; zhang.yelin@c.mbox.nagoya-u.ac.jp (Y.Z.); wahyudiono@b.mbox.nagoya-u.ac.jp (W.D.); kanda.hideki@material.nagoya-u.ac.jp (H.K.) 3 Technical Center, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan; yamaguchi.ryusei@i.mbox.nagoya-u.ac.jp 4 Innovation Division, Kagome Company, Limited, Nishitomiyama, Nasushiobara 329-2762, Japan; Ryota_Takemura@kagome.co.jp 5 Institutes of Innovation for Future Society, Nagoya University, Furo-cho, Chikusa-ku, N 464 8603 J 5 Institutes of Innovation for Future Society, Nagoya University, Furo-cho, Chikusa-ku Nagoya 464-8603, Japan * Correspondence: honda@meijo-u.ac.jp (M.H.); Tetsuya_Fukaya@kagome.co.jp (T.F.); goto.motonobu@material.nagoya-u.ac.jp (M.G.); Tel.: +81-52-838-2284 (M.H.); +81-287-36-2935 (T.F.); +81-52-789-3392 (M.G.) Academic Editor: Monica Nardi Academic Editor: Monica Nardi Received: 8 April 2019; Accepted: 5 June 2019; Published: 7 June 2019 ved: 8 April 2019; Accepted: 5 June 2019; Published: 7 Abstract: Carotenoids—natural fat-soluble pigments—have attracted considerable attention because of their potential to prevent of various diseases, such as cancer and arteriosclerosis, and their strong antioxidant capacity. They have many geometric isomers due to the presence of numerous conjugated double bonds in the molecule. However, in plants, most carotenoids are present in the all-E-conﬁguration. (all-E)-Carotenoids are characterized by high crystallinity as well as low solubility in safe and sustainable solvents, such as ethanol and supercritical CO2 (SC-CO2). Thus, these properties result in the decreased eﬃciency of carotenoid processing, such as extraction and emulsiﬁcation, using such sustainable solvents. On the other hand, Z-isomerization of carotenoids induces alteration in physicochemical properties, i.e., the solubility of carotenoids dramatically improves and they change from a “crystalline state” to an “oily (amorphous) state”. For example, the solubility in ethanol of lycopene Z-isomers is more than 4000 times higher than the all-E-isomer. Recently, improvement of carotenoid processing eﬃciency utilizing these changes has attracted attention. Namely, it is possible to markedly improve carotenoid processing using safe and sustainable solvents, which had previously been diﬃcult to put into practical use due to the low eﬃciency. molecules molecules 1. Introduction 1. Introduction Chemical structures of (A) (all-E)-lycopene, (B) (all-E)-β-carotene, (C) (all-E)-lutein, (D) (all- E)-astaxanthin, (E) (13Z)-astaxanthin, and (F) (9Z)-astaxanthin. Figure 1. Chemical structures of (A) (all-E)-lycopene, (B) (all-E)-β-carotene, (C) (all-E)-lutein, (D) (all-E)-astaxanthin, (E) (13Z)-astaxanthin, and (F) (9Z)-astaxanthin. Commercially available carotenoids are obtained by chemical syntheses or extraction from plants, photosynthetic bacteria, and microalgae. Generally, these carotenoids are in the all-E- configuration and the isomers are characterized by high crystallinity and low solubility in solvents [13,14]. Most carotenoid processing, such as extraction, micronization, and emulsification, employs a mediator—an organic solvent—to increase the processing efficiency. However, because of the physicochemical properties of (all-E)-carotenoids, processing efficiencies are low. Moreover, in recent years, there has been increased demand for the use of safe and sustainable solvents such as ethanol and supercritical CO2 (SC-CO2) for the processing of food components including carotenoids, i.e., environmentally benign processing using sustainable solvents is a topic of growing interest in both the research community and the food industry because of the growing awareness of the impact of solvents on energy usage, pollution, and their contribution to climate change and air quality [15–17]. However, since (all-E)-carotenoids have very low solubility in ethanol and SC-CO2 [18–21], toxic organic solvents are used in many cases. Very recently, several studies demonstrated that Z- isomerization of carotenoids induces alteration in physicochemical properties, such as crystallinity and solubility. Namely, solubility in solvents including SC-CO2 was dramatically improved and crystallinity was reduced by Z-isomerization. In addition, application of these alterations in Commercially available carotenoids are obtained by chemical syntheses or extraction from plants, photosynthetic bacteria, and microalgae. Generally, these carotenoids are in the all-E-conﬁguration and the isomers are characterized by high crystallinity and low solubility in solvents [13,14]. Most carotenoid processing, such as extraction, micronization, and emulsiﬁcation, employs a mediator—an organic solvent—to increase the processing eﬃciency. However, because of the physicochemical properties of (all-E)-carotenoids, processing eﬃciencies are low. Moreover, in recent years, there has been increased demand for the use of safe and sustainable solvents such as ethanol and supercritical CO2 (SC-CO2) for the processing of food components including carotenoids, i.e., environmentally benign processing using sustainable solvents is a topic of growing interest in both the research community and the food industry because of the growing awareness of the impact of solvents on energy usage, pollution, and their contribution to climate change and air quality [15–17]. 1. Introduction 1. Introduction Carotenoids are a class of lipid-soluble pigments responsible for the colors of plants, animals, and microorganisms [1–4]. Since the ﬁrst structural elucidation of β-carotene by Kuhn and Karrer in the 1930s, approximately 1100 natural carotenoids have been reported so far [5]. Carotenoids can be classiﬁed into the following two groups based on their chemical composition: (1) carotenes, nonoxygenated molecules such as lycopene and β-carotene and (2) xanthophylls, molecules containing oxygen such as lutein and astaxanthin. (Figure 1) [4,6]. The daily consumption of carotenoid-rich foods, such as fruits and vegetables, is considered to be beneﬁcial for human health because of their high antioxidant, anticancer, and antiatherosclerotic activities [7–9]. As carotenoids contain multiple conjugated double bonds, numerous geometric isomers are theoretically possible. While carotenoids in plants are accumulated predominantly as the all-E-conﬁguration (Figure 1A–D), Z-isomers of carotenoids (Figure 1E,F) exist in abundance in the human body and in processed foods. For example, more than 50 and 30% of total lycopene are present as Z-isomers in human blood plasma and processed tomato products such as tomato sauce and tomato soup, respectively [10–12]. Carotenoids are a class of lipid-soluble pigments responsible for the colors of plants, animals, and microorganisms [1–4]. Since the first structural elucidation of β-carotene by Kuhn and Karrer in the 1930s, approximately 1100 natural carotenoids have been reported so far [5]. Carotenoids can be classified into the following two groups based on their chemical composition: (1) carotenes, nonoxygenated molecules such as lycopene and β-carotene and (2) xanthophylls, molecules containing oxygen such as lutein and astaxanthin. (Figure 1) [4,6]. The daily consumption of carotenoid-rich foods, such as fruits and vegetables, is considered to be beneficial for human health because of their high antioxidant, anticancer, and antiatherosclerotic activities [7–9]. As carotenoids contain multiple conjugated double bonds, numerous geometric isomers are theoretically possible. While carotenoids in plants are accumulated predominantly as the all-E-configuration (Figure 1A– D), Z-isomers of carotenoids (Figure 1E,F) exist in abundance in the human body and in processed foods. For example, more than 50 and 30% of total lycopene are present as Z-isomers in human blood plasma and processed tomato products such as tomato sauce and tomato soup, respectively [10–12]. Figure 1. Chemical structures of (A) (all-E)-lycopene, (B) (all-E)-β-carotene, (C) (all-E)-lutein, (D) (all- E)-astaxanthin, (E) (13Z)-astaxanthin, and (F) (9Z)-astaxanthin. Figure 1. Chemical structures of (A) (all-E)-lycopene, (B) (all-E)-β-carotene, (C) (all-E)-lutein, (D) (all-E)-astaxanthin, (E) (13Z)-astaxanthin, and (F) (9Z)-astaxanthin. Figure 1. Improved Carotenoid Processing with Sustainable Solvents Utilizing Z-Isomerization-Induced Alteration in Physicochemical Properties: A Review and Future Directions The objective of this paper is to review the eﬀect of Z-isomerization on the physicochemical properties of carotenoids and its application to carotenoid processing, such as extraction, micronization, and emulsiﬁcation, using sustainable solvents. Moreover, aspects of Z-isomerization methods for carotenoids and functional diﬀerence, such as bioavailability and antioxidant capacity, between isomers are also included in this review. Keywords: lycopene; β-carotene; astaxanthin; E/Z-isomerization; solubility; crystallinity; extraction; emulsiﬁcation; micronization; supercritical CO2 Molecules 2019, 24, 2149; doi:10.3390/molecules24112149 www.mdpi.com/journal/molecules Molecules 2019, 24, 2149 M l l 2019 24 2 of 19 2 of 19 2. Typical Methods for Z-Isomerization of Carotenoids As the method for Z-isomerization of carotenoids, heat treatment, microwave treatment, light irradiation, electrolysis treatment, and catalytic treatment have been well documented to date (Table 1). (all-E)-Carotenoids, e.g., lycopene and astaxanthin, were eﬃciently isomerized to the Z-isomers by heating in organic solvents, especially alkyl halides such as dichloromethane (CH2Cl2) and dibromomethane (CH2Br2) [26–28]. The all-E-isomers were also thermally Z-isomerized in the presence of vegetable oils, animal fats, and SC-CO2 [12,21]. These results indicate that for Z-isomerization of (all-E)-carotenoids, it is important that the carotenoid is in a dissolved state. Microwave heating also promoted Z-isomerization [29–32], and several studies indicated the increased eﬃciency compared to conventional heating [29,30]. In the microwave treatment of (all-E)-lycopene-rich tomato oleoresin, the total Z-isomer content reached 65.9 ± 2.7% with 4-min irradiation at 2450 MHz frequency and 500 W power, and the temperature of the oleoresin reached 136.7 ± 6.6 ◦C at that time, while the total Z-isomer content with conventional oil bath heating at 140 ◦C for 5 min was 50.8 ± 3.2% [29]. Light irradiation also caused E/Z-isomerization of carotenoids. When carotenoids were directly exposed to light, Z-isomers of carotenoids isomerized to the all-E-isomers [33,34]. On the other hand, when light irradiation was carried out with photosensitizers, such as chlorophyll a and erythrosine, Z-isomerization of (all-E)-carotenoids was promoted [35,36]. For example, when puriﬁed (all-E)-lycopene dissolved in hexane in the presence of erythrosine was irradiated at 480–600 nm for 1 h, the proportion of lycopene Z-isomers reached over 80% [36]. A few studies have demonstrated that electrolysis treatment promoted Z-isomerization of (all-E)-carotenoids such as β-carotene and canthaxanthin [37,38]. This electrochemical method shows very high eﬃciency and can prevent thermal degradation of carotenoids, e.g., approximately 50% of (all-E)-canthaxanthin was converted to the Z-isomers during 4–6 min of bulk electrolysis at 4 ◦C [37]. Catalytic Z-isomerization of (all-E)-carotenoids have been traditionally conducted using iodine [39–41]. More recently, it was reported that disulﬁde compounds [29,42,43], isothiocyanates, carbon disulﬁde [42], iron(III) chloride [44], titanium tetrachloride [45], and iodine-doped titanium dioxide [46] induced Z-isomerization of carotenoids. For example, when iron(III) chloride, which is usually used as a food additive for iron fortiﬁcation, was used, greater isomerization (79.9%) could be attained by a 3-h reaction at 60 ◦C, with almost no degradation of lycopene [44]. 1. Introduction 1. Introduction However, since (all-E)-carotenoids have very low solubility in ethanol and SC-CO2 [18–21], toxic organic solvents are used in many cases. Very recently, several studies demonstrated that Z-isomerization of carotenoids induces alteration in physicochemical properties, such as crystallinity and solubility. Namely, solubility in solvents including SC-CO2 was dramatically improved and crystallinity was reduced by Z-isomerization. In addition, application of these alterations in carotenoid processing using the above safe and sustainable solvents has attracted Molecules 2019, 24, 2149 3 of 19 attention. For example, Z-isomerization pretreatment signiﬁcantly improved the extraction eﬃciency of lycopene contained in tomatoes and gac (Momordica cochinchinensis Spreng.) aril using organic solvents and SC-CO2 [18,19]. In this review, the eﬀect of Z-isomerization on the physicochemical properties of carotenoids and recent researches on carotenoid processing technology exploiting these characteristics are summarized and discussed. In addition, we also outline the typical methods for Z-isomerization of carotenoids and alterations in the bioavailability and functionality of carotenoids by Z-isomerization. Ample studies have demonstrated that Z-isomerization of carotenoids results in changes to bioavailability and antioxidant capacity, e.g., Z-isomers of lycopene and astaxanthin have greater bioavailability and show a higher antioxidant capacity than the all-E-isomers [22–25]. Thus, it is important to have a thorough understanding of the impact of E/Z-isomerization on functional changes of carotenoids. 2. Typical Methods for Z-Isomerization of Carotenoids Although catalyst utilization for carotenoid isomerization is a very eﬃcient method, most catalysts, such as iodine and heavy metals, exert a negative impact on the human body and the environment. Hence, in industrial view, the discovery and use of low toxicity and environmentally sustainable catalysts, e.g., plant-derived natural catalysts such as disulﬁde compounds and isothiocyanates, will be of great importance [29,42,43,47–49]. As shown in Table 1, each Z-isomerization method has several advantages and disadvantages; consequently, it is necessary to select the appropriate Z-isomerization method according to the circumstances. 4 of 19 Molecules 2019, 24, 2149 Table 1. Summary of representative methods for Z-isomerization of carotenoids and their advantages and disadvantages. Method Reported Carotenoid Advantage Disadvantage Reference Heat treatment Lycopene, β-carotene, astaxanthin, lutein, etc. • Simple and conventional method • Requires minimal amount of additive • Can cause thermal degradation [12,21,26–28] Microwave treatment Lycopene, β-carotene, astaxanthin, lutein, etc. • Simple and fast (few minutes) method • Requires minimal amount of additive • Can cause thermal degradation • Diﬃcult to control ﬁnal product quality • High cost of instrumentation [29–32] Light irradiation Lycopene, β-carotene, lutein, etc. • Rapid method • Non-thermal process • Low energy usage • Can cause light degradation • Need to add photosensitizers • Need to remove photosensitizers if toxic ones used • High cost of some photosensitizers [26,33–36] Electrolysis treatment β-Carotene, 8′-apo-β-caroten-8′-al, canthaxanthin • Simple and highly eﬃcient method • Non-thermal process • Can prevent degradation during processing • High cost of instrumentation • Need to remove electrolyte substances if toxic ones used [37,38] Catalytic treatment Lycopene, β-carotene, astaxanthin, zeaxanthin, etc. • Simple and highly eﬃcient method • Can prevent degradation during the processing • Low energy usage • Need to remove catalysts if toxic ones used • Can promote degradation in some catalysts • High cost of some catalysts [29,39–46] . Eﬀect of Z-Isomerization on Bioavailability and Functionality of Carotenoids Table 1. Summary of representative methods for Z-isomerization of carotenoids and their advantages and disadvantages. 3. Eﬀect of Z-Isomerization on Bioavailability and Functionality of Carotenoids ﬀect of Z-Isomerization on Bioavailability and Functionality of Carotenoids . Eﬀect of Z-Isomerization on Bioavailability and Functionality of Carotenoids Ample studies have addressed the alterations in bioavailability and functionality, such as antioxidant, anticancer, and antiatherosclerotic activities, of carotenoids by Z-isomerization (Table 2). Further, the alterations diﬀered among carotenoids. Z-Isomers of lycopene and astaxanthin showed greater bioavailability than the all-E-isomers [22,23,25,50–53]. For example, investigation of the eﬀect of red tomato juice (90% all-E-isomer lycopene) and tangerine tomato juice (94% Z-isomer lycopene) ingestion on plasma lycopene concentrations revealed that lycopene from tangerine tomato juice showed approximately 8.5-fold greater bioavailability than lycopene from red tomato juice [22]. On the other hand, some carotenoid Z-isomers, such as β-carotene and lutein, may be less bioavailable than the all-E-isomers [54–62]. In general, the bioavailability of carotenoids is very low because they are strongly bound to the food matrix and because of their physicochemical characteristics, such as low solubility, high crystallinity, and lipophilicity [4,13,14]. Thus, to improve the bioavailability of carotenoids contained in fruits and vegetables, physical treatments, such as high-pressure homogenization and ultrasound treatment, have been traditionally studied [63]. In some carotenoids, such as lycopene and astaxanthin, by combining chemical approaches such as Z-isomerization treatment and the above physical approaches, it may be possible to further improve the bioavailability. Depending on the assay method, many studies have reported that Z-isomers of carotenoids have equal or higher antioxidant capacity compared with the all-E-isomers [24,25,46,63–70]. Böhm et al. (2002) [64] reported that Z-isomers of lycopene exhibited approximately 1.2 times higher antioxidant capacities than the all-E-isomer in the Trolox equivalent antioxidant capacity (TEAC) assay. In heme-induced peroxidation of linoleic acid in mildly acidic emulsions, which mimics postprandial lipid oxidation in the gastric compartment (MbFeIII-LP) assay, (5Z)-lycopene showed approximately 3 times higher antioxidant capacity than the all-E-isomer [24]. In contrast, when antioxidant capacity 5 of 19 Molecules 2019, 24, 2149 was evaluated by the TEAC assay, (9Z)-zeaxanthin exhibited lower capacity than the all-E-isomer [64]. The degree of antioxidant capacity varied among Z-isomers, e.g., that of lutein isomers was higher in the order of 13Z-isomer > 9Z-isomer > all-E-isomer with the ferric reducing antioxidant power (FRAP) assay [62]. Carotenoid Z-isomers are likely to be superior to the all-E-isomers in preventative eﬀects on atherosclerosis, cancer, and inﬂammatory [71–76]. For example, (9Z)-β-carotene contained in the microalgae Dunaliella sp. showed higher antiatherogenic [71] and antiatherosclerotic [72,73] activities than the all-E-isomer in mouse experiments. 3. Eﬀect of Z-Isomerization on Bioavailability and Functionality of Carotenoids (9Z)-Canthaxanthin isolated from Dietzia sp. exhibited higher proapoptotic activity than the all-E-isomer in THP-1 macrophages [74]. Nakazawa et al. (2009) [75] reported that Z-isomers of fucoxanthin had greater anticancer activity than the all-E-isomer in human promyelocytic leukemia (HL-60) and colon cancer (Caco-2) cells. Very recently, Yang et al. (2019) [76] showed that Z-isomers of astaxanthin, especially the 9Z-isomer, exhibited greater antiinﬂammatory eﬀect than the all-E-isomer by downregulating proinﬂammatory cytokines COX-2 and TNF-α gene expression, which was evaluated in a Caco-2 cell monolayer model. As another notable functional change by carotenoid Z-isomerization, Z-isomers of β-carotene, which is a very important retinol precursor with a high conversion rate, showed lower conversion eﬃciencies to retinol than the all-E-isomer [77,78]. The antiaging activity would also diﬀer among astaxanthin isomers. Namely, the median lifespan of Caenorhabditis elegans fed with 9-Z-, 13-Z-, and all-E-isomers was observed to increase by 59.39%, 24.99%, and 30.43%, respectively [79]. Moreover, Fenni et al. (2019) [80] reported that lycopene isomers exert similar biological functions in adipocytes, linked to their ability to transactivate PPARγ. Since Z-isomerization had “positive” or “negative” eﬀects on the bioavailability and functionality of carotenoids (Table 2), it is important to have a detailed understanding of the impact of E/Z-isomerization on corresponding functional changes. 6 of 19 Molecules 2019, 24, 2149 Table 2. Comparison of the bioavailability and functionality of all-E- and Z-isomers of carotenoids. 3. Eﬀect of Z-Isomerization on Bioavailability and Functionality of Carotenoids Carotenoid Bioavailability Antioxidant Capacity Other Functionality Lycopene • E a < Z b (Oral study in humans) [22,50] • E < Z (Oral study in ferrets) [51] • E < Z (Caco-2 cell model) [52] • E < Z (Diﬀusion model) [53] • E ≤Z (TEAC assay) [24,64] • E < Z (LPSC assay) [24] • E ≤Z (MbFeIII-LP assay) [24] • E ≈Z (FRAP assay) [24] Antiobesity activity: • E ≈5Z (Adipocyte model) [80] α-Carotene – • 13′Z > E ≈9′Z > 9Z ≈13Z (TEAC assay) [64] – β-Carotene • E > 9Z (Oral study in humans) [54–58] • E > Z (Oral study in ferrets) [59] • E > Z (Oral study in gerbils) [60] • E > Z (Caco-2 cell model) [61] • E < Z (Digestion model) [81] • E < Z (Oral study in rats) [65] • E ≈Z (TEAC assay) [64] • E ≈9Z ≈13Z > 15Z (TEAC assay) [66] • E ≈Z (FRAP assay) [66] • E ≈9Z ≈13Z > 15Z (CL assay) [66] Antiatherogenesis activity: • E < 9Z (Oral study in mice) [71] Antiatherosclerosis activity: • E < 9Z (Oral study in mice) [72,73] Astaxanthin • E ≤Z (Oral study in humans) [23] • E < Z (Caco-2 cell model) [25] • E < Z (Digestion model) [25] • E < Z (DPPH assay) [46,67] • E < Z (ORAC assay) [46] • E < Z (PLC assay) [46] • E < Z (Enzyme activity assay) [25] • E < Z (Lipid- peroxidation assay) [67] Antiinﬂammatory activity: • E < Z (Caco-2 cell model) [76] Antiaging activity: • 9Z > E > 13Z (Caenorhabditis elegans model) [79] Canthaxanthin – • E < 9Z (DPPH assay) [68] • E < 9Z (Fluorescence assay) [68] Proapoptotic activity: • E < 9Z (THP-1 macrophage model) [74] Fucoxanthin – • E < Z (DPPH assay) [69] • 13Z and 13′Z > E > 9′Z (DPPH assay) [70] • 13Z and 13′Z > E > 9′Z (Superoxide-detection assay) [70] • 9′Z > E > 13Z and 13′Z (ABTS assay) [70] • 9′Z > E > 13Z and 13′Z (Hydroxyl radical-scavenging assay) [70] Anticancer activity: • E < Z (Caco-2 cell model) [75] • E < Z (HL-60 cell model) [75] Lutein • E > Z (Caco-2 cell model) [62] • E < Z (Digestion model) [62] • E < Z (FRAP assay) [62] • 13′Z > E ≈9Z (DPPH assay) [62] • 13′Z > E ≈9Z (ORAC assay) [62] • E ≈Z (CAA assay) [62] – Zeaxanthin – • E ≈13Z > 9Z (TEAC assay) [64] – a all-E-isomer of carotenoid. 4. Eﬀect of Z-Isomerization on Physicochemical Properties of Carotenoids The Z-isomerization of (all-E)-carotenoids induces change in physicochemical properties such as color, solubility, crystallinity, melting point, and stability. Z-Isomerization of carotenoids resulted in a shift in absorption to a shorter wavelength and a reduction in the molar extinction coeﬃcient and color value [27,46,82,83]. For example, Jing et al. (2012) [83] reported that maximum absorption wavelengths of (all-E)-, (9Z)-, and (13Z)-β-carotene were 451.4, 446.4, and 439.1 nm, respectively. The molar extinction coeﬃcients of (all-E)-, (9Z)-, and (13Z)-lycopene at the maximum absorption wavelengths were 182 × 103, 164 × 103, and 137 × 103 M−1 cm−1, respectively [27]. In fact, tomatoes rich in (all-E)-lycopene show a red color, whereas tomatoes rich in the Z-isomers, known as tangerine tomatoes, show an orange color [22]. Several studies reported that Z-isomers of carotenoids had much higher solubility than the all-E-isomers in organic solvents, oils, and SC-CO2 [13,14,18–21,84,85]. Although the solubility of (all-E)-lycopene in ethanol, acetone, ethyl acetate, and hexane was 0.6, 42.7, 145.3, and 25.6 mg/mL, respectively, that of lycopene containing 75.6% Z-isomers was 2401.7, 3702.9, 3961.1, and 3765.2 mg/mL, respectively [13]. Namely, in the case of ethanol, which is frequently used for food processing such as extraction and puriﬁcation, the solubility of lycopene Z-isomers was over 4000 times higher than that of the all-E-isomer. Also, in SC-CO2, the solubility of (9Z)-β-carotene was nearly four times higher than that of the all-E-isomer [84], and lycopene Z-isomers also showed higher solubility than the all-E-isomer [19,21]. The increased solubility of carotenoids by Z-isomerization is likely to be associated with changes in bioavailability. Generally, carotenoids are absorbed from the duodenum and prior to the absorption they are incorporated into bile acid micelles [86]. Thus, since carotenoid Z-isomers may have higher solubility in bile acid than all-E-isomers, they are preferentially incorporated into enterocytes and show higher bioavailability [51,87]. On the other hand, Z-isomers of β-carotene exhibit lower bioavailability in humans than the all-E-isomer [54–58]. Several proteins, which are temporarily present at the apical membrane of the duodenum, mediate selective carotenoid uptake [86]. Therefore, β-carotene Z-isomers may be eﬃciently incorporated into bile acid micelles due to their high solubility, but may have lower transport eﬃciency in the duodenum than the all-E-isomer. In vitro experiments using Caco-2 cells strongly support the above hypothesis. Namely, Z-isomers of lycopene and astaxanthin showed higher cellular uptake eﬃciency than the all-E-isomers [25,52], while the opposite result was obtained for β-carotene [61]. Similarly, Yang et al. 3. Eﬀect of Z-Isomerization on Bioavailability and Functionality of Carotenoids b Z-isomer of carotenoid. able 2. Comparison of the bioavailability and functionality of all-E- and Z-isomers of carotenoids. Molecules 2019, 24, 2149 7 of 19 4. Eﬀect of Z-Isomerization on Physicochemical Properties of Carotenoids (2018) [62] reported that in vitro experiments using a digestion model shown higher bioaccessibility of lutein Z-isomers than the all-E-isomer, while a Caco-2 cell monolayer model revealed lower bioavailability. y y Z-Isomerization of carotenoids aﬀects the crystallinity. Murakami et al. (2017) [13] and Honda et al. (2018) [14] experimentally revealed that increases in the Z-isomer content of lycopene, β-carotene, and astaxanthin was related to a reduction in crystallinity, i.e., scanning electron microscopy (SEM), diﬀerential scanning calorimetry (DSC), and powder X-ray diﬀraction (XRD) analyses clearly demonstrated that (all-E)-carotenoids were present in a crystal state, while Z-isomers were present in an amorphous state. Carotenoids have multiple conjugated double bonds in the molecule, resulting in strong π–π stacking interactions between molecules. For this reason, carotenoids have high crystallinity. However, the presence of Z-isomers is suggested to lead to enormous steric hindrance and decrease the potential attractive π–π forces, thus aﬀecting the crystallinity [13,88]. Generally, carotenoids in fresh plants occur predominantly in the (all-E)-conﬁguration, and (all-E)-carotenoids are present in the crystal state. On the other hand, some plants, such as tangerine tomato and peach palm (Bactris gasipaes Kunth), contain high amounts of carotenoid Z-isomers that are present in an oily aggregate form [22,89]. Similarly, 9Z-isomer-rich β-carotene contained in Dunaliella was in the oily form [90]. The melting point of carotenoids was altered by Z-isomerization, i.e., increases in the Z-isomer content were associated with a lower melting point [13,14,85,91]. For example, the melting point of (all-E)-lycopene and lycopene containing 23.8, 46.9, and 75.6% Z-isomers was 174.4, 173.7, 170.0, and 162.3 ◦C, respectively, as measured by DSC [13]. Molecules 2019, 24, 2149 8 of 19 The stability of carotenoids varies among isomers, i.e., (all-E)-carotenoids had higher stability than the Z-isomers. Several studies investigated the stability of carotenoid isomers using a Gaussian program and revealed that Gibbs free energy diﬀered among the isomers [82,92–94]. For example, Takehara et al. (2015) [93] reported that the relative stability of lycopene isomers was in the following order; all-E-isomer ≈5Z-isomer > 9Z-isomer > 13Z-isomer > 15Z-isomer, and Guo et al. (2008) [94] reported that the relative stability of β-carotene isomers was in the following order; all-E-isomer > 9Z-isomer > 13Z-isomer > 15Z-isomer > 7Z-isomer ≈11Z-isomer. Murakami et al. (2018) [33] experimentally conﬁrmed the above for lycopene. 5. Improvement of Carotenoid Processing Eﬃciency by Z-Isomerization In recent years, due to the discovery of altered physicochemical properties of carotenoids by Z-isomerization, eﬀorts to improve the eﬃciency of carotenoid processing by exploiting these alterations has attracted attention. In particular, carotenoid processing using a safe and sustainable solvent—SC-SO2—as a mediator is being actively studied. Since natural carotenoids, the all-E-isomer, exhibit very low solubility in SC-SO2, there is a high hurdle for its industrial use in carotenoid processing. However, utilizing alterations in the physical properties by Z-isomerization represents a breakthrough. In this section, we introduce recent studies of carotenoid processing (extraction, micronization, and emulsiﬁcation) utilizing alterations in solubility and crystallinity of carotenoids by Z-isomerization. 4. Eﬀect of Z-Isomerization on Physicochemical Properties of Carotenoids Furthermore, they investigated the stability of lycopene isomers against light irradiation, and the stability was in the following order; all-E-isomer ≈ 5Z-isomer > 9Z-isomer > 13Z-isomer > multi-Z-isomers. As for lycopene Z-isomers, the 5Z-isomer showed the highest stability against heat and light. In addition, (5Z)-lycopene would have higher antioxidant capacity [24] and bioavailability [95] compared with the all-E-isomer and possibly the 9Z- and 13Z-isomers. Therefore, it is important to develop a facile procedure for lycopene isomerization from the all-E-isomer to the 5Z-isomer. The diﬀerences in physicochemical properties between (all-E)-carotenoids and Z-isomers are summarized in Table 3. A systematic understanding of these carotenoid properties is likely to be important in the analysis, processing, and so on. Table 3. Diﬀerences in physicochemical properties between (all-E)-carotenoids and Z-isomers. Color Value Solubility Crystallinity Melting Point Stability E a > Z b E < Z E > Z E > Z E > Z a all-E-isomer of carotenoid. b Z-isomer of carotenoid. Table 3. Diﬀerences in physicochemical properties between (all-E)-carotenoids and Z-isomers. Color Value Solubility Crystallinity Melting Point Stability E a > Z b E < Z E > Z E > Z E > Z a all-E-isomer of carotenoid. b Z-isomer of carotenoid. Table 3. Diﬀerences in physicochemical properties between (all-E)-carotenoids and Z-isomers. 5.1. Improvement of Carotenoid Extraction Generally, commercially available natural carotenoids, which are obtained from plants and microorganisms by solvent extraction and utilized for supplements, food colorants, and cosmetics, are very expensive [96–99]. This is because carotenoids in plants and microorganisms accumulate predominantly in the all-E-conﬁguration, whose isomers have low solubility in solvents, resulting in very low extraction eﬃciencies. For example, extraction of lycopene from tomato pulp with ethanol and SC-CO2 showed a recovery of only 6.3 and 6.5%, respectively [19]. However, when the extractions were conducted after Z-isomerization treatment, the recovery was notably improved to 75.9 and 27.6%, respectively [19]. More speciﬁcally, the total Z-isomer content of lycopene in tomato pulp was 8.8%, whereas it increased to 75.7% by heating at 150 ◦C for 1 h with a small amount (1 wt%) of olive oil. After ethanol extraction of lycopene from the Z-isomer-rich tomato pulp, the obtained extract had a very high Z-isomer content (93.5%), while almost all lycopene in the extraction residue was the all-E-isomer. These results strongly indicated that lycopene Z-isomers have higher solubility in solvents than the all-E-isomer; thus, the extraction eﬃciency was improved. In addition, since the Z-isomer content of carotenoids in the obtained extract was improved by Z-isomerization pretreatment, 9 of 19 Molecules 2019, 24, 2149 the treatment is eﬀective not only for the production of carotenoid concentrates but also for increasing the bioavailability and functionality of carotenoids (Figure 2). The improved extraction eﬃciency was also conﬁrmed in gac (M. cochinchinensis Spreng.) aril [18]. Gac is a tropical vine originating from South and South-East Asia and belongs to the Cucurbitaceae family, and the aril (seed membrane) contains a very high amount of lycopene [100,101]. Since gac aril contains a large amount of oil (18–34% of dry weight) rich in lycopene, lycopene is often obtained by press extraction with the oil [102]. Although more than 90% of lycopene exists as the all-E-isomer in gac aril, the total Z-isomer content increased by 58.5% with microwave irradiation at 1050 W for 60 s. When lycopene was obtained by press extraction with gac oil from non-microwave pretreated and treated gac aril, lycopene contents in the obtained oils were 160.6 and 1365.9 mg/100 g, respectively. Thus, Z-isomers of carotenoids show higher solubility in oils than the all-E-isomer. Moreover, Z-isomerization pretreatment of gac aril was also eﬀective for lycopene extraction using ethanol and SC-CO2. 5.1. Improvement of Carotenoid Extraction For example, when lycopene was extracted using SC-CO2 from the non-treated gac aril, the lycopene content in the extract was only 76.6 mg/100 g, whereas Z-isomerization pretreatment by microwave irradiation resulted in a lycopene content of 342.0 mg/100 g. As the extraction eﬃciency of carotenoids is improved by Z-isomerization pretreatment, the development of eﬃcient Z-isomerization methods for carotenoids in plants is very important in the future. On the other hand, several plants and microalgae such as tangerine tomato and Dunaliella contain a high amount of carotenoid Z-isomers [22,71,72]. Thus, carotenoids should be eﬃciently extracted using these raw materials. In fact, Gamlieli-Bonshtein et al. (2002) [84] reported that (9Z)-β-carotene in Dunaliella exhibited nearly 4 times higher extraction eﬃciency by SC-CO2 than the all-E-isomer. Pretreatments of samples by physical and chemical approaches such as grinding, osmotic shock, bead-beating, high-pressure homogenization, and enzymatic treatment are eﬀective in releasing carotenoids from complex matrices, and have been performed in basic and applied studies [103,104]. On the other hand, Z-isomerization pretreatment is a new technology reported very recently. By combing traditional physical and chemical pretreatments and Z-isomerization pretreatment, further improvement of carotenoid extraction can be expected. In addition, when the Z-isomerization pretreatment is used in combination with several extraction technique, such as pulsed electric ﬁeld-assisted extraction, microwave-assisted extraction, and ultrasonic-assisted extraction, synergistic eﬀects are expected [105–109]. Molecules 2019, 24, x 9 of 19 contains a very high amount of lycopene [100,101]. Since gac aril contains a large amount of oil (18– 34% of dry weight) rich in lycopene, lycopene is often obtained by press extraction with the oil [102]. Although more than 90% of lycopene exists as the all-E-isomer in gac aril, the total Z-isomer content increased by 58.5% with microwave irradiation at 1050 W for 60 s. When lycopene was obtained by press extraction with gac oil from non-microwave pretreated and treated gac aril, lycopene contents in the obtained oils were 160.6 and 1365.9 mg/100 g, respectively. Thus, Z-isomers of carotenoids show higher solubility in oils than the all-E-isomer. Moreover, Z-isomerization pretreatment of gac aril was also effective for lycopene extraction using ethanol and SC-CO2. For example, when lycopene was extracted using SC-CO2 from the non-treated gac aril, the lycopene content in the extract was only 76.6 mg/100 g, whereas Z-isomerization pretreatment by microwave irradiation resulted in a lycopene content of 342.0 mg/100 g. 5.1. Improvement of Carotenoid Extraction As the extraction efficiency of carotenoids is improved by Z- isomerization pretreatment, the development of efficient Z-isomerization methods for carotenoids in plants is very important in the future. On the other hand, several plants and microalgae such as tangerine tomato and Dunaliella contain a high amount of carotenoid Z-isomers [22,71,72]. Thus, carotenoids should be efficiently extracted using these raw materials. In fact, Gamlieli-Bonshtein et al. (2002) [84] reported that (9Z)-β-carotene in Dunaliella exhibited nearly 4 times higher extraction efficiency by SC-CO2 than the all-E-isomer. Pretreatments of samples by physical and chemical approaches such as grinding, osmotic shock, bead-beating, high-pressure homogenization, and enzymatic treatment are effective in releasing carotenoids from complex matrices, and have been performed in basic and applied studies [103,104]. On the other hand, Z-isomerization pretreatment is a new technology reported very recently. By combing traditional physical and chemical pretreatments and Z-isomerization pretreatment, further improvement of carotenoid extraction can be expected. In addition, when the Z-isomerization pretreatment is used in combination with several extraction technique, such as pulsed electric field-assisted extraction, microwave-assisted extraction, and ultrasonic-assisted extraction, synergistic effects are expected [105–109] y g p Figure 2. Schematic chart showing extraction of lycopene from plant material by solvents in the case of using (all-E)-lycopene and lycopene Z-isomers as the raw materials [18,19]. Figure 2. Schematic chart showing extraction of lycopene from plant material by solvents in the case of using (all-E)-lycopene and lycopene Z-isomers as the raw materials [18,19]. Figure 2. Schematic chart showing extraction of lycopene from plant material by solvents in the case of using (all-E)-lycopene and lycopene Z-isomers as the raw materials [18,19]. Figure 2. Schematic chart showing extraction of lycopene from plant material by solvents in the case of using (all-E)-lycopene and lycopene Z-isomers as the raw materials [18,19]. 5.2. Improvement of Carotenoid Micronization 5.2. Improvement of Carotenoid Micronization Ample studies have reported that micronization of carotenoids results in their increased bioavailability [110,111]. Generally, carotenoid micronization is conducted by milling, grinding, and chemical precipitation [112–114]. However, there are some concerns regarding the above conventional methods, as carotenoids are easily decomposed by friction heat and oxygen contact. In addition, when using chemical processes, toxic organic solvents may remain. Thus, in recent years, micronization of carotenoids using SC-CO2 has attracted increasing attention. Since CO2 is nontoxic Ample studies have reported that micronization of carotenoids results in their increased bioavailability [110,111]. Generally, carotenoid micronization is conducted by milling, grinding, and chemical precipitation [112–114]. However, there are some concerns regarding the above conventional methods, as carotenoids are easily decomposed by friction heat and oxygen contact. In addition, when using chemical processes, toxic organic solvents may remain. Thus, in recent years, micronization of carotenoids using SC-CO2 has attracted increasing attention. Since CO2 is 10 of 19 Molecules 2019, 24, 2149 nontoxic and has a low critical temperature (Tc = 31.1 ◦C), it is suitable for heat-sensitive materials such as carotenoids, and SC-CO2 is easily separated from the products along with the toxic organic solvent [115,116]. To the best of our knowledge, improved micronization eﬃciency of carotenoids utilizing alterations in the physicochemical properties by Z-isomerization has been reported only for the method using SC-CO2 [117]. Particle micronization techniques using SC-CO2, supercritical antisolvent (SAS), solution-enhanced dispersion by supercritical ﬂuids (SEDS), rapid expansion of supercritical solutions (RESS), gas antisolvent (GAS), supercritical ﬂuid extraction of emulsions (SFEE), and particles from gas saturated solutions (PGSS) have been well-documented [118–122]. Several studies have examined the micronization of carotenoids using the above techniques; however, there was diﬃculty in obtaining nano-sized carotenoid particles [123–125]. For example, Tavares-Cardoso et al. (2009) [125] conducted micronization of (all-E)-β-carotene using a SAS process under various conditions; however, nano-sized β-carotene particles could not be obtained. This is likely because of the high crystallinity of carotenoids. On the other hand, Kodama et al. (2018) [117] successfully prepared nano-sized lycopene by SEDS precipitation using lycopene Z-isomers as the raw material. Namely, when using (all-E)-lycopene as the raw material, particles having an average size of 3.6 µm were obtained, whereas when using lycopene containing 97.8% Z-isomers, uniformly sized particles of an average size of 75 nm were obtained (Figure 3). The reason why nanoparticles were successfully formed from Z-isomers is due to the low crystallinity compared with the all-E-isomer. 5.2. Improvement of Carotenoid Micronization 5.2. Improvement of Carotenoid Micronization In addition, little has been reported on carotenoid micronization using RESS precipitation: the substance, which must be reduced in size, is dissolved in pure SC-CO2 and then the solution is suddenly depressurized through a nozzle and expands inside a chamber under lower pressure. This would be because carotenoids have extremely low solubility in pure SC-CO2. However, as Z-isomers of carotenoids have relatively high solubility in SC-CO2 [18,19,21,84], the Z-isomers would successfully form nano-sized particles by RESS precipitation, representing a micronization method without the use of organic solvents. Molecules 2019, 24, x 10 of 19 supercritical solutions (RESS), gas antisolvent (GAS), supercritical fluid extraction of emulsions (SFEE), and particles from gas saturated solutions (PGSS) have been well-documented [118−122]. Several studies have examined the micronization of carotenoids using the above techniques; however, there was difficulty in obtaining nano-sized carotenoid particles [123−125]. For example, Tavares-Cardoso et al. (2009) [125] conducted micronization of (all-E)-β-carotene using a SAS process under various conditions; however, nano-sized β-carotene particles could not be obtained. This is likely because of the high crystallinity of carotenoids. On the other hand, Kodama et al. (2018) [117] successfully prepared nano-sized lycopene by SEDS precipitation using lycopene Z-isomers as the raw material. Namely, when using (all-E)-lycopene as the raw material, particles having an average size of 3.6 μm were obtained, whereas when using lycopene containing 97.8% Z-isomers, uniformly sized particles of an average size of 75 nm were obtained (Figure 3). The reason why nanoparticles were successfully formed from Z-isomers is due to the low crystallinity compared with the all-E- isomer. In addition, little has been reported on carotenoid micronization using RESS precipitation: the substance, which must be reduced in size, is dissolved in pure SC-CO2 and then the solution is suddenly depressurized through a nozzle and expands inside a chamber under lower pressure. This would be because carotenoids have extremely low solubility in pure SC-CO2. However, as Z-isomers of carotenoids have relatively high solubility in SC-CO2 [18,19,21,84], the Z-isomers would successfully form nano-sized particles by RESS precipitation, representing a micronization method without the use of organic solvents. Figure 3. Schematic chart showing preparation of lycopene particles with supercritical CO2 (solution-enhanced dispersion by supercritical fluids), using (all-E)-lycopene and lycopene Z-isomers as the raw materials [117]. 5 3 I f C id E l ifi i Figure 3. 5.2. Improvement of Carotenoid Micronization 5.2. Improvement of Carotenoid Micronization Schematic chart showing preparation of lycopene particles with supercritical CO2 (solution-enhanced dispersion by supercritical ﬂuids), using (all-E)-lycopene and lycopene Z-isomers as the raw materials [117]. ure 3. Schematic chart showing preparation of lycopene particles with supercritical CO2 (solution-enhan ersion by supercritical fluids), using (all-E)-lycopene and lycopene Z-isomers as the raw materials [117 I t f C t id E l ifi ti Figure 3. Schematic chart showing preparation of lycopene particles with supercritical CO2 (solution-enhanced dispersion by supercritical ﬂuids), using (all-E)-lycopene and lycopene Z-isomers as the raw materials [117]. 5.3. Improvement of Carotenoid Emulsification I t t id f 5.3. Improvement of Carotenoid Emulsiﬁcation 5.3. Improvement of Carotenoid Emulsification I t t id f 5.3. Improvement of Carotenoid Emulsiﬁcation In recent years, as carotenoids are safe natural pigments that have health enhancing effects, their demand by the food industry is continuously increasing [126,127]. However, the low water solubility of carotenoids has made their use problematic for food formulations, limiting the favorable effects of carotenoids. Furthermore, the low water solubility of carotenoids reduces their bioavailability [128,129]. Therefore, improved dispersibility in water by emulsification is very important for the food industry and acts to increase their bioavailability. It is preferred that the suspended preparation contains nano-sized particles for higher dispersibility and bioavailability [111,130]. To obtain nanosuspensions of carotenoids, the following emulsification–evaporation technique is frequently used [131−133]: (1) Dissolution of carotenoids in an organic phase; (2) Distribution processing of the solution with water containing an emulsifier; (3) Solvent evaporation under reduced pressure. In this In recent years, as carotenoids are safe natural pigments that have health enhancing eﬀects, their demand by the food industry is continuously increasing [126,127]. However, the low water solubility of carotenoids has made their use problematic for food formulations, limiting the favorable eﬀects of carotenoids. Furthermore, the low water solubility of carotenoids reduces their bioavailability [128,129]. Therefore, improved dispersibility in water by emulsiﬁcation is very important for the food industry and acts to increase their bioavailability. It is preferred that the suspended preparation contains nano-sized particles for higher dispersibility and bioavailability [111,130]. To obtain nanosuspensions of carotenoids, the following emulsiﬁcation–evaporation technique is frequently used [131–133]: (1) Dissolution of carotenoids in an organic phase; (2) Distribution processing of the solution with 11 of 19 Molecules 2019, 24, 2149 water containing an emulsiﬁer; (3) Solvent evaporation under reduced pressure. In this technique, it is important to select an appropriate distribution processing method, e.g., ultrasound treatment, high-speed homogenization, high-pressure homogenization, and microﬂuidizer treatment [131–136]. In addition, the selection of a solvent that can dissolve the target carotenoid is also a very important factor to eﬃciently produce carotenoid emulsions. However, since the degree of carotenoid solubility in safe and sustainable solvents, such as ethanol and supercritical SC-CO2, is very low [14,18,19,21,84], toxic solvents are used in many cases. To improve the emulsiﬁcation eﬃciency of carotenoids using the sustainable solvent SC-CO2, Ono et al. (2018) [20] focused on increased carotenoid solubility in solvents by Z-isomerization. Namely, they investigated the impact of Z-isomer content on the production of β-carotene suspensions by the emulsiﬁcation–evaporation technique. As the organic phase, they used SC-CO2 (Figure 4). 5.3. Improvement of Carotenoid Emulsification I t t id f 5.3. Improvement of Carotenoid Emulsiﬁcation When β-carotene rich in Z-isomers (79.1% of total β-carotene) was used as the raw material, the encapsulated β-carotene content was notably increased compared with the all-E-isomer. For example, the encapsulated β-carotene content was 21.2 times higher after emulsiﬁcation treatment by ultrasound at 45 kHz for 60 min. In addition, when (all-E)-β-carotene was used as the raw material, the mean particle size of the obtained suspension was approximately 700 nm, whereas that of β-carotene rich in Z-isomers was approximately 100 nm. Thus, Z-isomerization treatment before distributed processing is eﬀective for the preparation of carotenoid suspensions by the emulsiﬁcation–evaporation technique. However, the storage stability of a Z-isomer-rich β-carotene suspension was lower than that of all-E-isomer-rich one, possibly due to increases in the contact area with oxygen as the particle size decreased [20]. For practical application of this suspension preparation technique, establishment of a method to increase the storage stability of carotenoid Z-isomers is essential. Molecules 2019, 24, x 11 of 19 efficiency of carotenoids using the sustainable solvent SC-CO2, Ono et al. (2018) [20] focused on increased carotenoid solubility in solvents by Z-isomerization. Namely, they investigated the impact of Z-isomer content on the production of β-carotene suspensions by the emulsification–evaporation technique. As the organic phase, they used SC-CO2 (Figure 4). When β-carotene rich in Z-isomers (79.1% of total β-carotene) was used as the raw material, the encapsulated β-carotene content was notably increased compared with the all-E-isomer. For example, the encapsulated β-carotene content was 21.2 times higher after emulsification treatment by ultrasound at 45 kHz for 60 min. In addition, when (all-E)-β-carotene was used as the raw material, the mean particle size of the obtained suspension was approximately 700 nm, whereas that of β-carotene rich in Z-isomers was approximately 100 nm. Thus, Z-isomerization treatment before distributed processing is effective for the preparation of carotenoid suspensions by the emulsification–evaporation technique. However, the storage stability of a Z-isomer-rich β-carotene suspension was lower than that of all-E-isomer- rich one, possibly due to increases in the contact area with oxygen as the particle size decreased [20]. For practical application of this suspension preparation technique, establishment of a method to increase the storage stability of carotenoid Z-isomers is essential. igure 4. Schematic chart showing preparation of β-carotene suspensions by emulsification–evaporation echnique with SC-CO2, using (all-E)-β-carotene and β-carotene Z-isomers as the raw materials [20]. Figure 4. 5.3. Improvement of Carotenoid Emulsification I t t id f 5.3. Improvement of Carotenoid Emulsiﬁcation Schematic chart showing preparation of β-carotene suspensions by emulsiﬁcation–evaporation technique with SC-CO2, using (all-E)-β-carotene and β-carotene Z-isomers as the raw materials [20]. re 4. Schematic chart showing preparation of β-carotene suspensions by emulsification–evaporation nique with SC-CO2, using (all-E)-β-carotene and β-carotene Z-isomers as the raw materials [20]. Figure 4. Schematic chart showing preparation of β-carotene suspensions by emulsiﬁcation–evaporation technique with SC-CO2, using (all-E)-β-carotene and β-carotene Z-isomers as the raw materials [20]. 6 Conclusion and Future Perspectives 6. Conclusions and Future Perspectives p This review summarizes alterations in the physicochemical properties (color value, solubility, crystallinity, melting point, and stability) of carotenoids by Z-isomerization and their application for carotenoid processing (extraction, micronization, and emulsification), specifically using a green and sustainable solvent—SC-CO2—and presents typical Z-isomerization methods and the effect of Z- isomerization on the bioavailability and functionality of carotenoids. As the method for Z- isomerization of carotenoids, heat treatment, microwave treatment, light irradiation, electrolysis treatment, and catalytic treatment have been well reported. Since these Z-isomerization methods have several advantages and disadvantages, it is necessary to select the appropriate Z-isomerization method according to the circumstances. Ample studies have demonstrated that Z-isomerization of This review summarizes alterations in the physicochemical properties (color value, solubility, crystallinity, melting point, and stability) of carotenoids by Z-isomerization and their application for carotenoid processing (extraction, micronization, and emulsiﬁcation), speciﬁcally using a green and sustainable solvent—SC-CO2—and presents typical Z-isomerization methods and the eﬀect of Z-isomerization on the bioavailability and functionality of carotenoids. As the method for Z-isomerization of carotenoids, heat treatment, microwave treatment, light irradiation, electrolysis treatment, and catalytic treatment have been well reported. Since these Z-isomerization methods have several advantages and disadvantages, it is necessary to select the appropriate Z-isomerization method according to the circumstances. Ample studies have demonstrated that Z-isomerization of carotenoid 12 of 19 Molecules 2019, 24, 2149 aﬀected the bioavailability, antioxidant capacity, and functionalities such as anticancer activity and antiinﬂammatory activity and often oﬀered positive impacts on human. The Z-isomerization also induces changes in the physicochemical properties of carotenoids, such as solubility and crystallinity. Namely, the solubility in organic solvents, SC-CO2, and oils dramatically is enhanced and crystallinity is reduced by Z-isomerization. Since the (all-E)-carotenoid, which is a predominant isomer in plants and synthetic ingredients, has very low solubility in SC-CO2, its industrial use in carotenoid processing faces a very high hurdle. However, it is highly expected that this impediment could be improved by utilizing the alterations in physicochemical properties of carotenoids by Z-isomerization. Carotenoid processing utilizing Z-isomerization and the expected application of Z-isomer-rich carotenoid materials are summarized in Figure 5. Plants and microalgae rich in carotenoid Z-isomers would be applicable as raw materials for the eﬃcient extraction of carotenoids using solvents such as SC-CO2, for use in health foods, food colorants, and animal feed. 6 Conclusion and Future Perspectives 6. Conclusions and Future Perspectives The obtained extract rich in carotenoid Z-isomers is expected to be applied to the production of supplements and food colorants with high carotenoid bioavailability and functionality. When safe and sustainable extraction solvents, such as ethanol and supercritical CO2, are employed, the value of the extract is anticipated to increase. Furthermore, utilization of carotenoid Z-isomer-rich extracts as the raw material is expected to increase the production and quality of nano-sized carotenoids and carotenoid emulsions. The obtained nano-sized carotenoids and carotenoid emulsions rich in Z-isomers are expected to be utilized as supplements, food colorants, and cosmetics. In addition, alterations in the physicochemical properties of carotenoids by Z-isomerization may be beneﬁcial for the production of microcapsules prepared using carotenoid-containing liposomes. The studies on increasing eﬃciency of carotenoid processing by Z-isomerization pretreatment has just started in recent years. Thus, there is still considerable room for the development of this research ﬁeld. Fundamental study of this technology will be actively conducted in the future and practical applications are expected. Molecules 2019, 24, x 12 of 19 this impediment could be improved by utilizing the alterations in physicochemical properties of carotenoids by Z-isomerization. Carotenoid processing utilizing Z-isomerization and the expected application of Z-isomer-rich carotenoid materials are summarized in Figure 5. Plants and microalgae rich in carotenoid Z-isomers would be applicable as raw materials for the efficient extraction of carotenoids using solvents such as SC-CO2, for use in health foods, food colorants, and animal feed. The obtained extract rich in carotenoid Z-isomers is expected to be applied to the production of supplements and food colorants with high carotenoid bioavailability and functionality. When safe and sustainable extraction solvents, such as ethanol and supercritical CO2, are employed, the value of the extract is anticipated to increase. Furthermore, utilization of carotenoid Z-isomer-rich extracts as the raw material is expected to increase the production and quality of nano-sized carotenoids and carotenoid emulsions. The obtained nano-sized carotenoids and carotenoid emulsions rich in Z- isomers are expected to be utilized as supplements, food colorants, and cosmetics. In addition, alterations in the physicochemical properties of carotenoids by Z-isomerization may be beneficial for the production of microcapsules prepared using carotenoid-containing liposomes. The studies on increasing efficiency of carotenoid processing by Z-isomerization pretreatment has just started in recent years. Thus, there is still considerable room for the development of this research field. Acknowledgments: The authors are grateful to Dr. Tsutomu Kumagai, Dr. Chitoshi Kitamura, Dr. Yoshinori Inoue, and Dr. Munenori Takehara (Department of Materials Science, The University of Shiga Prefecture), and Mr. Hiroyuki Ueda, Mr. Takuma Higashiura, and Mr. Kohei Ichihashi (Innovation Division, Kagome Co., Ltd.) f th i ki d h l d t ti ti Acknowledgments: The authors are grateful to Tsutomu Kumagai, Chitoshi Kitamura, Yoshinori Inoue, and Munenori Takehara (Department of Materials Science, The University of Shiga Prefecture), and Hiroyuki Ueda, Takuma Higashiura, and Kohei Ichihashi (Innovation Division, Kagome Co., Ltd.) for their kind help and constructive suggestions. References 1. Maoka, T. Recent progress in structural studies of carotenoids in animals and plants. Arch. Biochem. Biophys. 2009, 483, 191–195. [CrossRef] [PubMed] 1. Maoka, T. Recent progress in structural studies of carotenoids in animals and plants. Arch. Biochem. Biophys. 2009, 483, 191–195. [CrossRef] [PubMed] 2. Maoka, T. Carotenoids in marine animals. Mar. Drugs 2011, 9, 278–293. [CrossRef] [PubMed] 2. Maoka, T. Carotenoids in marine animals. Mar. Drugs 2011, 9, 278–293. [CrossRef] [PubMed] 3. Pods˛edek, A. Natural antioxidants and antioxidant capacity of Brassica vegetables: A review. LWT-Food Sci. Technol. 2007, 40, 1–11. [CrossRef] 3. Pods˛edek, A. Natural antioxidants and antioxidant capacity of Brassica vegetables: A review. LWT-Food Sci. Technol. 2007, 40, 1–11. [CrossRef] 3. Pods˛edek, A. Natural antioxidants and antioxidant capacity of Brassica vegetables: A review. LWT-Food Sci. Technol. 2007, 40, 1–11. [CrossRef] 4. Rodriguez-Concepcion, M.; Avalos, J.; Bonet, M.L.; Boronat, A.; Gomez-Gomez, L.; Hornero-Mendez, D.; Limon, M.C.; Meléndez-Martínez, A.J.; Olmedilla-Alonso, B.; Palou, A.; et al. A global perspective on carotenoids: Metabolism, biotechnology, and beneﬁts for nutrition and health. Prog. Lipid Res. 2018, 70, 62–93. [CrossRef] [PubMed] 5. Yabuzaki, J. Carotenoids database: Structures, chemical ﬁngerprints and distribution among organisms. Database 2017, 2017, bax004. [CrossRef] [PubMed] 6. Amorim-Carrilho, K.T.; Cepeda, A.; Fente, C.; Regal, P. Review of methods for analysis of carotenoids. TrAC Trends Anal. Chem. 2014, 56, 49–73. [CrossRef] 7. Krinsky, N.I.; Johnson, E.J. Carotenoid actions and their relation to health and disease. Mol. Asp. Med. 2005, 26, 459–516. [CrossRef] 8. Ouchi, A.; Aizawa, K.; Iwasaki, Y.; Inakuma, T.; Terao, J.; Nagaoka, S.; Mukai, K. Kinetic study of the quenching reaction of singlet oxygen by carotenoids and food extracts in solution. Development of a singlet oxygen absorption capacity (SOAC) assay method. J. Agric. Food Chem. 2010, 58, 9967–9978. [CrossRef] 9. Xu, X.R.; Zou, Z.Y.; Huang, Y.M.; Xiao, X.; Ma, L.; Lin, X.M. Serum carotenoids in relation to risk factors for development of atherosclerosis. Clin. Biochem. 2012, 45, 1357–1361. [CrossRef] 10. Schierle, J.; Bretzel, W.; Bühler, I.; Faccin, N.; Hess, D.; Steiner, K.; Schüep, W. Content and isomeric ratio of lycopene in food and human blood plasma. Food Chem. 1997, 59, 459–465. [CrossRef] 11. Van Breemen, R.B.; Xu, X.; Viana, M.A.; Chen, L.; Stacewicz-Sapuntzakis, M.; Duncan, C.; Bowen, P.E.; Shariﬁ, R. Liquid chromatography-mass spectrometry of cis-and all-trans-lycopene in human serum and prostate tissue after dietary supplementation with tomato sauce. J. Agric. Food Chem. 2002, 50, 2214–2219. [CrossRef] 12. 6 Conclusion and Future Perspectives 6. Conclusions and Future Perspectives Fundamental study of this technology will be actively conducted in the future and practical applications are expected. Figure 5. Increased efficiency of carotenoids processing by Z-isomerization and applications of Z- isomer-rich carotenoids materials. Figure 5. Increased eﬃciency of carotenoids processing by Z-isomerization and applications of Z-isomer-rich carotenoids materials. Figure 5. Increased efficiency of carotenoids processing by Z-isomerization and applications of Z- isomer-rich carotenoids materials. Figure 5. Increased eﬃciency of carotenoids processing by Z-isomerization and applications of Z-isomer-rich carotenoids materials. Author Contributions: Conceptualization, M.H., T.F., and M.G.; Writing—Original Draft Preparation, M.H.; Writing—Review & Editing, M.H., H.K., T.H., T.F., and M.G.; Revisions & Final editing, M.H., H.K., T.H., Y.Z., W., H.K., R.Y., R.T. T.F., and M.G. Author Contributions: Conceptualization, M.H., T.F., and M.G.; Writing—Original Draft Preparation, M.H.; Writing—Review & Editing, M.H., H.K., T.H., T.F., and M.G.; Revisions & Final editing, M.H., H.K., T.H., Y.Z., W.D., H.K., R.Y., R.T., T.F., and M.G. Author Contributions: Conceptualization, M.H., T.F., and M.G.; Writing Original Draft Preparation, M.H.; Writing—Review & Editing, M.H., H.K., T.H., T.F., and M.G.; Revisions & Final editing, M.H., H.K., T.H., Y.Z., W., H.K., R.Y., R.T. T.F., and M.G. Author Contributions: Conceptualization, M.H., T.F., and M.G.; Writing—Original Draft Preparation, M.H.; Writing—Review & Editing, M.H., H.K., T.H., T.F., and M.G.; Revisions & Final editing, M.H., H.K., T.H., Y.Z., W.D., H.K., R.Y., R.T., T.F., and M.G. Acknowledgments: The authors are grateful to Dr. Tsutomu Kumagai, Dr. Chitoshi Kitamura, Dr. Yoshinori Inoue, and Dr. Munenori Takehara (Department of Materials Science, The University of Shiga Prefecture), and Mr. Hiroyuki Ueda, Mr. Takuma Higashiura, and Mr. Kohei Ichihashi (Innovation Division, Kagome Co., Ltd.) f th i ki d h l d t ti ti Acknowledgments: The authors are grateful to Tsutomu Kumagai, Chitoshi Kitamura, Yoshinori Inoue, and Munenori Takehara (Department of Materials Science, The University of Shiga Prefecture), and Hiroyuki Ueda, Takuma Higashiura, and Kohei Ichihashi (Innovation Division, Kagome Co., Ltd.) for their kind help and constructive suggestions. 13 of 19 Molecules 2019, 24, 2149 Conﬂicts of Interest: The authors declare no conﬂicts of interest. References Honda, M.; Murakami, K.; Watanabe, Y.; Higashiura, T.; Fukaya, T.; Wahyudiono; Kanda, H.; Goto, M. The E/Z isomer ratio of lycopene in foods and eﬀect of heating with edible oils and fats on isomerization of (all-E)-lycopene. Eur. J. Lipid Sci. Technol. 2017, 119, 1600389. [CrossRef] 13. Murakami, K.; Honda, M.; Takemura, R.; Fukaya, T.; Kubota, M.; Wahyudiono; Kanda, H.; Goto, M. The thermal Z-isomerization-induced change in solubility and physical properties of (all-E)-lycopene. Biochem. Biophys. Res. Commun. 2017, 491, 317–322. [CrossRef] 14. Honda, M.; Kodama, T.; Kageyama, H.; Hibino, T.; Wahyudiono; Kanda, H.; Goto, M. Enhanced solubility and reduced crystallinity of carotenoids, β-carotene and astaxanthin, by Z-Isomerization. Eur. J. Lipid Sci. Technol. 2018, 120, 1800191. [CrossRef] 15. Clarke, C.J.; Tu, W.C.; Levers, O.; Bröhl, A.; Hallett, J.P. Green and sustainable solvents in chemical processes. Chem. Rev. 2018, 118, 747–800. [CrossRef] 16. Procopio, A.; Alcaro, S.; Nardi, M.; Oliverio, M.; Ortuso, F.; Sacchetta, P.; Pieragostino, D.; Sindona, G. Synthesis, biological evaluation, and molecular modeling of oleuropein and its semisynthetic derivatives as cyclooxygenase inhibitors. J. Agric. Food Chem. 2009, 57, 11161–11167. [CrossRef] 17. Nardi, M.; Sindona, G.; Costanzo, P.; Oliverio, M.; Procopio, A. Eco-friendly stereoselective reduction of α,β-unsaturated carbonyl compounds by Er(OTf)3/NaBH4 in 2-MeTHF. Tetrahedron 2015, 71, 1132–1135. [CrossRef] 18. Honda, M.; Watanabe, Y.; Murakami, K.; Hoang, N.N.; Wahyudiono; Kanda, H.; Goto, M. Enhanced lycopene extraction from gac (Momordica cochinchinensis Spreng.) by the Z-isomerization induced with microwave irradiation pre-treatment. Eur. J. Lipid Sci. Technol. 2018, 120, 1700293. [CrossRef] 19. Honda, M.; Watanabe, Y.; Murakami, K.; Takemura, R.; Fukaya, T.; Wahyudiono; Kanda, H.; Goto, M. Thermal isomerization pre-treatment to improve lycopene extraction from tomato pulp. LWT-Food Sci. Technol. 2017, 86, 69–75. [CrossRef] Molecules 2019, 24, 2149 14 of 19 20. Ono, M.; Honda, M.; Wahyudiono; Yasuda, K.; Kanda, H.; Goto, M. Production of β-carotene nanosuspensions using supercritical CO2 and improvement of its eﬃciency by Z-isomerization pre-treatment. J. Supercrit. Fluids 2018, 138, 124–131. [CrossRef] 21. Watanabe, Y.; Honda, M.; Higashiura, T.; Fukaya, T.; Machmudah, S.; Wahyudiono; Kanda, H.; Goto, M. Rapid and selective concentration of lycopene Z-isomers from tomato pulp by supercritical CO2 with co-solvents. Solvent Extr. Res. Dev. 2018, 25, 47–57. [CrossRef] 22. Cooperstone, J.L.; Ralston, R.A.; Riedl, K.M.; Haufe, T.C.; Schweiggert, R.M.; King, S.A.; Timmers, C.D.; Francis, D.M.; Lesinski, G.B.; Clinton, S.K.; et al. References Enhanced bioavailability of lycopene when consumed as cis-isomers from tangerine compared to red tomato juice, a randomized, cross-over clinical trial. Mol. Nutr. Food Res. 2015, 59, 658–669. [CrossRef] 23. Østerlie, M.; Bjerkeng, B.; Liaaen-Jensen, S. Plasma appearance and distribution of astaxanthin E/Z and R/S isomers in plasma lipoproteins of men after single dose administration of astaxanthin. J. Nutr. Biochem. 2000, 11, 482–490. [CrossRef] 24. Müller, L.; Goupy, P.; Fröhlich, K.; Dangles, O.; Caris-Veyrat, C.; Böhm, V. Comparative study on antioxidant activity of lycopene (Z)-isomers in diﬀerent assays. J. Agric. Food Chem. 2011, 59, 4504–4511. [CrossRef] 25. Yang, C.; Zhang, H.; Liu, R.; Zhu, H.; Zhang, L.; Tsao, R. Bioaccessibility, cellular uptake, and transport of astaxanthin isomers and their antioxidative eﬀects in human intestinal epithelial Caco-2 cells. J. Agric. Food Chem. 2017, 65, 10223–10232. [CrossRef] 26. Aman, R.; Schieber, A.; Carle, R. Eﬀects of heating and illumination on trans-cis isomerization and degradation of β-carotene and lutein in isolated spinach chloroplasts. J. Agric. Food Chem. 2005, 53, 9512–9518. [CrossRef] 27. Honda, M.; Takahashi, N.; Kuwa, T.; Takehara, M.; Inoue, Y.; Kumagai, T. Spectral characterisation of Z-isomers of lycopene formed during heat treatment and solvent eﬀects on the E/Z isomerisation process. Food Chem. 2015, 17, 323–329. [CrossRef] 28. Yuan, J.P.; Chen, F. Isomerization of trans-astaxanthin to cis-isomers in organic solvents. J. Agric. Food Chem. 1999, 47, 3656–3660. [CrossRef] 29. Honda, M.; Sato, H.; Takehara, M.; Inoue, Y.; Kitamura, C.; Takemura, R.; Fukaya, T.; Wahyudiono; Kanda, H.; Goto, M. Microwave-accelerated Z-isomerization of (all-E)-lycopene in tomato oleoresin and enhancement of the conversion by vegetable oils containing disulﬁde compounds. Eur. J. Lipid Sci. Technol. 2018, 120, 180060. [CrossRef] 30. Kessy, H.N.; Zhang, L.; Zhang, H. Lycopene (Z)-isomers enrichment and separation. Int. J. Food Sci. Technol. 2013, 48, 2050–2056. [CrossRef] 31. Zhao, L.; Zhao, G.; Chen, F.; Wang, Z.; Wu, J.; Hu, X. Diﬀerent eﬀects of microwave and ultrasound on the stability of (all-E)-astaxanthin. J. Agric. Food Chem. 2006, 54, 8346–8351. [CrossRef] 32. Chen, B.H.; Chen, Y.Y. Stability of chlorophylls and carotenoids in sweet potato leaves during microwave cooking. J. Agric. Food Chem. 1993, 41, 1315–1320. [CrossRef] 33. Murakami, K.; Honda, M.; Takemura, R.; Fukaya, T.; Wahyudiono; Kanda, H.; Goto, M. Eﬀect of thermal treatment and light irradiation on the stability of lycopene with high Z-isomers content. Food Chem. 2018, 250, 253–258. [CrossRef] 34. Kuki, M.; Koyama, Y.; Nagae, H. References Triplet-sensitized and thermal isomerization of all-trans, 7-cis, 9-cis, 13-cis and 15-cis isomers of β-carotene: Conﬁgurational dependence of the quantum yield of isomerization via the T1 state. J. Phys. Chem. 1991, 95, 7171–7180. [CrossRef] 35. Jensen, N.H.; Nielsen, A.B.; Wilbrandt, R. Chlorophyll a-sensitized trans-cis photoisomerization of all-trans-β-carotene. J. Am. Chem. Soc. 1982, 104, 6117–6119. [CrossRef] 36. Honda, M.; Igami, H.; Kawana, T.; Hayashi, K.; Takehara, M.; Inoue, Y.; Kitamura, C. Photosensitized E/Z isomerization of (all-E)-lycopene aiming at practical applications. J. Agric. Food Chem. 2014, 62, 11353–11356. [CrossRef] 37. Wei, C.C.; Gao, G.; Kispert, L.D. Selected cis/trans isomers of carotenoids formed by bulk electrolysis and iron(III) chloride oxidation. J. Chem. Soc. Perkin Trans. 1997, 2, 783–786. [CrossRef] 38. Gao, G.; Wei, C.C.; Jeevarajan, A.S.; Kispert, L.D. Geometrical isomerization of carotenoids mediated by cation radical/dication formation. J. Phys. Chem. 1996, 100, 5362–5366. [CrossRef] 39. Wyman, G.M. The cis-trans isomerization of conjugated compounds. Chem. Rev. 1955, 55, 625–657. [CrossRef] 40 Zechmeister, L Cis-trans isomerization and stereochemistry of carotenoids and diphenyl-polyenes Chem. Rev. 39. Wyman, G.M. The cis-trans isomerization of conjugated compounds. Chem. Rev. 1955, 55, 625–657. [CrossRef] 39. Wyman, G.M. The cis-trans isomerization of conjugated compounds. Chem. Rev. 1955, 55, 625–657. [CrossRef] 40. Zechmeister, L. Cis-trans isomerization and stereochemistry of carotenoids and diphenyl-polyenes. Chem. Rev. 1944, 34, 267–344. [CrossRef] 40. Zechmeister, L. Cis-trans isomerization and stereochemistry of carotenoids and diphenyl-polyenes. Chem. Rev. 1944, 34, 267–344. [CrossRef] 15 of 19 Molecules 2019, 24, 2149 15 of 19 41. Molnár, P. Research of the (E/Z)-isomerization of carotenoids in Pécs since the 1970s. Arch. Biochem. Biophys. 2009, 483, 156–164. [CrossRef] 42. Honda, M.; Kageyama, H.; Hibino, T.; Takemura, R.; Goto, M.; Fukaya, T. Enhanced Z-isomerization of tomato lycopene through the optimal combination of food ingredients. Sci. Rep. 2019, 9, 7979. [CrossRef] 43. Yu, J.; Gleize, B.; Zhang, L.; Caris-Veyrat, C.; Renard, C.M.G.C. Heating tomato puree in the presence of lipids and onion: The impact of onion on lycopene isomerization. Food Chem. 2019, 296, 9–16. [CrossRef] 44. Honda, M.; Kawana, T.; Takehara, M.; Inoue, Y. Enhanced E/Z isomerization of (all-E)-lycopene by employing iron(III) chloride as a catalyst. J. Food Sci. 2015, 80, C1453–C1459. [CrossRef] 45. Rajendran, V.; Chen, B.H. Isomerization of β-carotene by titanium tetrachloride catalyst. J. Chem. Sci. 2007, 119, 253–258. [CrossRef] 46. Yang, C.; Zhang, L.; Zhang, H.; Sun, Q.; Liu, R.; Li, J.; Wu, L.; Tsao, R. References Rapid and eﬃcient conversion of all-E-astaxanthin to 9Z-and 13Z-isomers and assessment of their stability and antioxidant activities. J. Agric. Food Chem. 2017, 65, 818–826. [CrossRef] 47. Hechelski, M.; Ghinet, A.; Louvel, B.; Dufrénoy, P.; Rigo, B.; Daïch, A.; Waterlot, C. From conventional Lewis acids to heterogeneous montmorillonite K10: Eco-friendly plant-based catalysts used as green Lewis acids. ChemSusChem 2018, 11, 1249–1277. [CrossRef] 48. Nardi, M.; Bonacci, S.; Cariati, L.; Costanzo, P.; Oliverio, M.; Sindona, G.; Procopio, A. Synthesis and antioxidant evaluation of lipophilic oleuropein aglycone derivatives. Food Funct. 2017, 8, 4684–4692. [CrossRef] 49. Oliverio, M.; Nardi, M.; Costanzo, P.; Di Gioia, M.; Procopio, A. Erbium salts as non-toxic catalysts compatible with alternative reaction media. Sustainability 2018, 10, 721. [CrossRef] 50. Unlu, N.Z.; Bohn, T.; Francis, D.M.; Nagaraja, H.N.; Clinton, S.K.; Schwartz, S.J. Lycopene from heat-induced cis-isomer-rich tomato sauce is more bioavailable than from all-trans-rich tomato sauce in human subjects. Br. J. Nutr. 2007, 98, 140–146. [CrossRef] 51. Boileau, A.C.; Merchen, N.R.; Wasson, K.; Atkinson, C.A.; Erdman, J.W., Jr. Cis-lycopene is more bioavailable than trans-lycopene in vitro and in vivo in lymph-cannulated ferrets. J. Nutr. 1999, 129, 1176–1181. [CrossRef] 52. Failla, M.L.; Chitchumroonchokchai, C.; Ishida, B.K. In vitro micellarization and intestinal cell uptake of cis isomers of lycopene exceed those of all-trans lycopene. J. Nutr. 2008, 138, 482–486. [CrossRef] 53. Sun, Q.; Yang, C.; Li, J.; Raza, H.; Zhang, L. Lycopene: Heterogeneous catalytic E/Z isomerization and in vitro bioaccessibility assessment using a diﬀusion model. J. Food Sci. 2016, 81, C2381–C2389. [CrossRef] 54. Gaziano, J.M.; Johnson, E.J.; Russell, R.M.; Manson, J.E.; Stampfer, M.J.; Ridker, P.M.; Frei, B.; Hennekens, C.H.; Krinsky, N.I. Discrimination in absorption or transport of β-carotene isomers after oral supplementation with either all-trans- or 9-cis-β-carotene. Am. J. Clin. Nutr. 1995, 61, 1248–1252. [CrossRef] 55. Johnson, E.J.; Krinsky, N.I.; Russell, R.M. Serum response of all-trans and 9-cis isomers of β-carotene in humans. J. Am. Coll. Nutr. 1996, 15, 620–624. [CrossRef] 56. Stahl, W.; Schwarz, W.; von Laar, J.; Sies, H. All-trans β-carotene preferentially accumulates in human chylomicrons and very low density lipoproteins compared with the 9-cis geometrical isomer. J. Nutr. 1995, 125, 2128–2133. [CrossRef] 57. Stahl, W.; Schwarz, W.; Sies, H. Human serum concentrations of all-trans β- and α-carotene but not 9-cis β-carotene increase upon ingestion of a natural isomer mixture obtained from Dunaliella salina (Betatene). J. Nutr. 1993, 123, 847–851. [CrossRef] 58. References Anticholinesterase and antioxidant activities of fucoxanthin puriﬁed from the microalga Phaeodactylum tricornutum. Nat. Prod. Commun. 2013, 8, 1381–1386. [CrossRef] 70. Zhang, Y.; Fang, H.; Xie, Q.; Sun, J.; Liu, R.; Hong, Z.; Yi, R.; Wu, H. Comparative evaluation of the radical-scavenging activities of fucoxanthin and its stereoisomers. Molecules 2014, 19, 2100–2113. [CrossRef] 71. Harari, A.; Harats, D.; Marko, D.; Cohen, H.; Barshack, I.; Kamari, Y.; Gonen, A.; Gerber, Y.; Ben-Amotz, A.; Shaish, A. A 9-cis β-carotene–enriched diet inhibits atherogenesis and fatty liver formation in LDL receptor knockout mice. J. Nutr. 2008, 138, 1923–1930. [CrossRef] [PubMed] 72. Harari, A.; Abecassis, R.; Relevi, N.; Levi, Z.; Ben-Amotz, A.; Kamari, Y.; Harats, A.; Shaish, A. Prevention of atherosclerosis progression by 9-cis-β-carotene rich alga Dunaliella in apoE-deﬁcient mice. Biomed. Res. Int. 2013, 2013, 169517. [CrossRef] [PubMed] 73. Relevy, N.Z.; Rühl, R.; Harari, A.; Grosskopf, I.; Barshack, I.; Ben-Amotz, A.; Nir, U.; Gottlieb, H.; Kamari, Y.; Harats, D.; et al. 9-cis β-Carotene inhibits atherosclerosis development in female LDLR-/- mice. Funct. Foods Health Dis. 2015, 5, 67–79. 74. Venugopalan, V.; Verma, N.; Gautam, H.K.; Saradhi, P.P.; Das, R.H. 9-cis-Canthaxanthin exhibits higher pro-apoptotic activity than all-trans-canthaxanthin isomer in THP-1 macrophage cells. Free Radic. Res. 2009, 43, 100–105. [CrossRef] [PubMed] 75. Nakazawa, Y.; Sashima, T.; Hosokawa, M.; Miyashita, K. Comparative evaluation of growth inhibitory eﬀect of stereoisomers of fucoxanthin in human cancer cell lines. J Funct. Foods 2009, 1, 88–97. [CrossRef] 76. Yang, C.; Hassan, Y.I.; Liu, R.; Zhang, H.; Chen, Y.; Zhang, L.; Tsao, R. Anti-inﬂammatory eﬀects of diﬀerent astaxanthin isomers and the roles of lipid transporters in the cellular transport of astaxanthin isomers in Caco-2 cell monolayers. J. Agric. Food Chem. 2019, 67, 6222–6231. [CrossRef] y g 77. Nagao, A.; Olson, J.A. Enzymatic formation of 9-cis, 13-cis, and all-trans retinals from isomers of β-carotene. FASEB J. 1994, 8, 968–973. [CrossRef] 78. Schieber, A.; Carle, R. Occurrence of carotenoid cis-isomers in food: Technological, analytical, and nutritional implications. Trends Food Sci. Technol. 2005, 16, 416–422. [CrossRef] 79. Liu, X.; Chen, X.; Liu, H.; Cao, Y. Antioxidation and anti-aging activities of astaxanthin geometrical isomers and molecular mechanism involved in Caenorhabditis elegans. J. Funct. Foods 2018, 44, 127–136. [CrossRef] 80. Fenni, S.; Astier, J.; Bonnet, L.; Karkeni, E.; Gouranton, E.; Mounien, L.; Couturier, C.; Tourniaire, F.; Böhm, V.; Hammou, H.; et al. (all-E)-and (5Z)-Lycopene display similar biological eﬀects on adipocytes. Mol. Nutr. Food Res. 2019, 63, 1800788. [CrossRef] 81. References Tamai, H.; Morinobu, T.; Murata, T.; Manago, M.; Mino, M. 9-cis β-Carotene in human plasma and blood cells after ingestion of β-carotene. Lipids 1995, 30, 493–498. [CrossRef] 59. Erdman Jr, J.W.; Thatcher, A.J.; Hofmann, N.E.; Lederman, J.D.; Block, S.S.; Lee, C.M.; Mokady, S. All-trans β-carotene is absorbed preferentially to 9-cis β-carotene, but the latter accumulates in the tissues of domestic ferrets (Mustela putorius puro). J. Nutr. 1998, 128, 2009–2013. [CrossRef] 60. Deming, D.M.; Teixeira, S.R.; Erdman Jr, J.W. All-trans β-carotene appears to be more bioavailable than 9-cis or 13-cis β-carotene in gerbils given single oral doses of each isomer. J. Nutr. 2002, 132, 2700–2708. [CrossRef] 61. During, A.; Hussain, M.M.; Morel, D.W.; Harrison, E.H. Carotenoid uptake and secretion by CaCo-2 cells: β-carotene isomer selectivity and carotenoid interactions. J. Lipid Res. 2002, 43, 1086–1095. [CrossRef] [PubMed] 61. During, A.; Hussain, M.M.; Morel, D.W.; Harrison, E.H. Carotenoid uptake and secretion by CaCo-2 cells: β-carotene isomer selectivity and carotenoid interactions. J. Lipid Res. 2002, 43, 1086–1095. [CrossRef] [PubMed] 16 of 19 Molecules 2019, 24, 2149 16 of 19 62. Yang, C.; Fischer, M.; Kirby, C.; Liu, R.; Zhu, H.; Zhang, H.; Chen, Y.; Sun, Y.; Zhang, L.; Tsao, R. Bioaccessibility, cellular uptake and transport of luteins and assessment of their antioxidant activities. Food Chem. 2018, 249, 66–76. [CrossRef] 3. Kopec, R.E.; Failla, M.L. Recent advances in the bioaccessibility and bioavailability of carotenoids and eﬀ of other dietary lipophiles. J. Food Compos. Anal. 2018, 68, 16–30. [CrossRef] 64. Böhm, V.; Puspitasari-Nienaber, N.L.; Ferruzzi, M.G.; Schwartz, S.J. Trolox equivalent antioxidant capacity of diﬀerent geometrical isomers of α-carotene, β-carotene, lycopene, and zeaxanthin. J. Agric. Food Chem. 2002, 50, 221–226. [CrossRef] [PubMed] 65. Levin, G.; Yeshurun, M.; Mokady, S. In vivo antiperoxidative eﬀect of 9-cis β-carotene compared with that of the all-trans isomer. Nutr. Cancer 1997, 27, 293–297. [CrossRef] [PubMed] 66. Mueller, L.; Boehm, V. Antioxidant activity of β-carotene compounds in diﬀerent in vitro assays. Molecules 2011, 16, 1055–1069. [CrossRef] [PubMed] 67. Liu, X.; Osawa, T. Cis astaxanthin and especially 9-cis astaxanthin exhibits a higher antioxidant activity in vitro compared to the all-trans isomer. Biochem. Biophys. Res. Commun. 2007, 357, 187–193. [CrossRef] [PubMed] 68. Venugopalan, V.; Tripathi, S.K.; Nahar, P.; Saradhi, P.P.; Das, R.H.; Gautam, H.K. Characterization of canthaxanthin isomers isolated from a new soil Dietzia sp. and their antioxidant activities. J. Microbiol. Biotechnol. 2013, 23, 237–245. [CrossRef] [PubMed] 69. Kawee-ai, A.; Kuntiya, A.; Kim, S.M. References Ferruzzi, M.G.; Lumpkin, J.L.; Schwartz, S.J.; Failla, M. Digestive stability, micellarization, and uptake of β-carotene isomers by Caco-2 human intestinal cells. J. Agric. Food Chem. 2006, 54, 2780–2785. [CrossRef] [PubMed] 17 of 19 Molecules 2019, 24, 2149 82. Honda, M.; Kudo, T.; Kuwa, T.; Higashiura, T.; Fukaya, T.; Inoue, Y.; Kitamura, C.; Takehara, M. Isolation and spectral characterization of thermally generated multi-Z-isomers of lycopene and the theoretically preferred pathway to di-Z-isomers. Biosci. Biotechnol. Biochem. 2017, 81, 365–371. [CrossRef] [PubMed] 3. Jing, C.; Qun, X.; Rohrer, J. HPLC separation of all-trans-β-carotene and its iodine-induced isomers usi C30 column. Thermo Sci. 2012, 187, 1–5. 84. Gamlieli-Bonshtein, I.; Korin, E.; Cohen, S. Selective separation of cis-trans geometrical isomers of β-carotene via CO2 supercritical ﬂuid extraction. Biotechnol. Bioeng. 2002, 80, 169–174. [CrossRef] [PubMed] 85. Murakami, K.; Honda, M.; Wahyudiono; Kanda, H.; Goto, M. Thermal isomerization of (all-E)-lycopene and separation of the Z-isomers by using a low boiling solvent: Dimethyl ether. Sep. Sci. Technol. 2017, 52, 2573–2582. [CrossRef] 86. Desmarchelier, C.; Borel, P. Overview of carotenoid bioavailability determinants: From dietary factors to host genetic variations. Trends Food Sci. Technol. 2017, 69, 270–280. [CrossRef] 87. Boileau, T.W.M.; Boileau, A.C.; Erdman, J.W., Jr. Bioavailability of all-trans and cis-isomers of lycopene. Exp. Biol. Med. 2002, 227, 914–919. [CrossRef] 88. Hempel, J.; Schädle, C.N.; Leptihn, S.; Carle, R.; Schweiggert, R.M. Structure related aggregation behavior of carotenoids and carotenoid esters. J. Photochem. Photobiol. A Chem. 2016, 317, 161–174. [CrossRef] 89. Hempel, J.; Amrehn, E.; Quesada, S.; Esquivel, P.; Jiménez, V.M.; Heller, A.; Carle, R.; Schweiggert, R.M. Lipid-dissolved γ-carotene, β-carotene, and lycopene in globular chromoplasts of peach palm (Bactris gasipaes Kunth) fruits. Planta 2014, 240, 1037–1050. [CrossRef] 90. Ben-Amotz, A.; Lers, A.; Avron, M. Stereoisomers of β-carotene and phytoene in the alga Dunaliella bardawil. Plant Physiol. 1988, 86, 1286–1291. [CrossRef] 91. Takehara, M.; Nishimura, M.; Kuwa, T.; Inoue, Y.; Kitamura, C.; Kumagai, T.; Honda, M. Characterization and thermal isomerization of (all-E)-lycopene. J. Agric. Food Chem. 2014, 62, 264–269. [CrossRef] [PubMed] 91. Takehara, M.; Nishimura, M.; Kuwa, T.; Inoue, Y.; Kitamura, C.; Kumagai, T.; Honda, M. Characterization and thermal isomerization of (all-E)-lycopene. J. Agric. Food Chem. 2014, 62, 264–269. [CrossRef] [PubMed] 92. Chasse, G.A.; Mak, M.L.; Deretey, E.; Farkas, I.; Torday, L.L.; Papp, J.G.; Sarma, D.S.R.; Agarwal, A.; 92. Chasse, G.A.; Mak, M.L.; Deretey, E.; Farkas, I.; Torday, L.L.; Papp, J.G.; Sarma, D.S.R.; Agarwal, A.; Chakravarthi, S.; Agarwal, S.; et al. References An ab initio computational study on selected lycopene isomers. J. Mol. Struct. THEOCHEM 2001, 571, 27–37. [CrossRef] 93. Takehara, M.; Kuwa, T.; Inoue, Y.; Kitamura, C.; Honda, M. Isolation and characterization of (15Z)-lycopene thermally generated from a natural source. Biochem. Biophys. Res. Commun. 2015, 467, 58–62. [CrossRef] [PubMed] 94. Guo, W.H.; Tu, C.Y.; Hu, C.H. Cis-trans isomerizations of β-carotene and lycopene: A theoretical study. J. Phys. Chem. B 2008, 112, 12158–12167. [CrossRef] [PubMed] 95. Richelle, M.; Lambelet, P.; Rytz, A.; Tavazzi, I.; Mermoud, A.F.; Juhel, C.; Borel, P.; Bortlik, K. The proportion of lycopene isomers in human plasma is modulated by lycopene isomer proﬁle in the meal but not by lycopene preparation. Br. J. Nutr. 2012, 107, 1482–1488. [CrossRef] [PubMed] 96. Mohan, S.; Rao, P.R.; Hemachandran, H.; Pullela, P.K.; Tayubi, I.A.; Subramanian, B.; Gothandam, K.M.; Singh, P.; Ramamoorthy, S. Prospects and progress in the production of valuable carotenoids: Insights from metabolic engineering, synthetic biology, and computational approaches. J. Biotechnol. 2018, 266, 89–101. 97. Mussagy, C.U.; Winterburn, J.; Santos-Ebinuma, V.C.; Pereira, J.F.B. Production and extraction of carotenoids produced by microorganisms. Appl. Microbiol. Biotechnol. 2019, 103, 1095–1114. [CrossRef] 98. Rammuni, M.N.; Ariyadasa, T.U.; Nimarshana, P.H.V.; Attalage, R.A. Comparative assessment on the extraction of carotenoids from microalgal sources: Astaxanthin from H. pluvialis and β-carotene from D. salina. Food Chem. 2019, 277, 128–134. [CrossRef] 99. Sathasivam, R.; Ki, J.S. A review of the biological activities of microalgal carotenoids and their potential use in healthcare and cosmetic industries. Mar. Drugs 2018, 16, 26. [CrossRef] 100. Chuyen, H.V.; Nguyen, M.H.; Roach, P.D.; Golding, J.B.; Parks, S.E. Gac fruit (Momordica cochinchinensis Spreng.): A rich source of bioactive compounds and its potential health beneﬁts. Int. J. Food Sci. Technol. 2015, 50, 567–577. [CrossRef] 101. Vuong, L.T.; Franke, A.A.; Custer, L.J.; Murphy, S.P. Momordica cochinchinensis Spreng. (gac) fruit carotenoids reevaluated. J. Food Compos. Anal. 2006, 19, 664–668. [CrossRef] 102. Kha, T.C.; Nguyen, M.H.; Roach, P.D.; Stathopoulos, C.E. Eﬀects of Gac aril microwave processing conditions on oil extraction eﬃciency, and β-carotene and lycopene contents. J. Food Eng. 2013, 117, 486–491. [CrossRef] 102. Kha, T.C.; Nguyen, M.H.; Roach, P.D.; Stathopoulos, C.E. Eﬀects of Gac aril microwave processing conditions on oil extraction eﬃciency, and β-carotene and lycopene contents. J. Food Eng. 2013, 117, 486–491. [CrossRef] 103. Martins, N.; Ferreira, I.C. Wastes and by-products: Upcoming sources of carotenoids for biotechnological 103. Martins, N.; Ferreira, I.C. References Wastes and by-products: Upcoming sources of carotenoids for biotechnological purposes and health-related applications. Trends Food Sci. Technol. 2017, 62, 33–48. [CrossRef] 18 of 19 Molecules 2019, 24, 2149 18 of 19 104. Saini, R.K.; Keum, Y.S. Carotenoid extraction methods: A review of recent developments. Food Chem. 2018, 240, 90–103. [CrossRef] [PubMed] 105. Luengo, E.; Álvarez, I.; Raso, J. Improving carotenoid extraction from tomato waste by pulsed electric ﬁelds. Front. Nutr. 2014, 1, 12. [CrossRef] [PubMed] 106. Jaeschke, D.P.; Menegol, T.; Rech, R.; Mercali, G.D.; Marczak, L.D.F. Carotenoid and lipid extraction from Heterochlorella luteoviridis using moderate electric ﬁeld and ethanol. Proc. Biochem. 2016, 51, 1636–1643. [CrossRef] 107. Altemimi, A.; Lakhssassi, N.; Baharlouei, A.; Watson, D.; Lightfoot, D. Phytochemicals: Extraction, isolation, and identiﬁcation of bioactive compounds from plant extracts. Plants 2017, 6, 42. [CrossRef] [PubMed] 108. Chuyen, H.V.; Nguyen, M.H.; Roach, P.D.; Golding, J.B.; Parks, S.E. Microwave-assisted extraction and ultrasound-assisted extraction for recovering carotenoids from Gac peel and their eﬀects on antioxidant capacity of the extracts. Food Sci. Nutr. 2018, 6, 189–196. [CrossRef] [PubMed] 109. Song, J.; Yang, Q.; Huang, W.; Xiao, Y.; Li, D.; Liu, C. Optimization of trans lutein from pumpkin (Cucurbita moschata) peel by ultrasound-assisted extraction. Food Bioprod. Proc. 2018, 107, 104–112. [CrossRef] 110. Aﬀandi, M.M.M.; Julianto, T.; Majeed, A.B.A. Enhanced oral bioavailability of astaxanthin with droplet size reduction. Food Sci. Technol. Res. 2012, 18, 549–554. [CrossRef] 111 Vishwanathan R ; Wilson T A ; Nicolosi R J Bioavailability of a nanoemulsion of lutein is greater than a 110. Aﬀandi, M.M.M.; Julianto, T.; Majeed, A.B.A. Enhanced oral bioavailability of astaxanthin with drop reduction. Food Sci. Technol. Res. 2012, 18, 549–554. [CrossRef] 11. Vishwanathan, R.; Wilson, T.A.; Nicolosi, R.J. Bioavailability of a nanoemulsion of lutein is greater th lutein supplement. Nano Biomed. Eng. 2009, 1, 57–73. [CrossRef] 112. Karam, M.C.; Petit, J.; Zimmer, D.; Djantou, E.B.; Scher, J. Eﬀects of drying and grinding in production of fruit and vegetable powders: A review. J. Food Eng. 2016, 188, 32–49. [CrossRef] 113. De Paz, E.; Martín, Á.; Estrella, A.; Rodríguez-Rojo, S.; Matias, A.A.; Duarte, C.M.; Cocero, M.J. Formulation of β-carotene by precipitation from pressurized ethyl acetate-on-water emulsions for application as natural colorant. Food Hydrocoll. 2012, 26, 17–27. [CrossRef] 114. An, Y.; Sun, Y.; Zhang, M.; Adhikari, B.; Li, Z. Eﬀect of ball milling time on physicochemical properties of Cordyceps militaris ultraﬁne particles. J. Food Proc. Eng. 2019, e13065. [CrossRef] 115. References Sahena, F.; Zaidul, I.S.M.; Jinap, S.; Karim, A.A.; Abbas, K.A.; Norulaini, N.A.N.; Omar, A.K.M. Application of supercritical CO2 in lipid extraction—A review. J. Food Eng. 2009, 95, 240–253. [CrossRef] 116. Zuknik, M.H.; Norulaini, N.N.; Omar, A.M. Supercritical carbon dioxide extraction of lycopene: A J. Food Eng. 2012, 112, 253–262. [CrossRef] 117. Kodama, T.; Honda, M.; Takemura, R.; Fukaya, T.; Uemori, C.; Wahyudiono; Kanda, H.; Goto, M. Eﬀect of the Z-isomer content on nanoparticle production of lycopene using solution-enhanced dispersion by supercritical ﬂuids (SEDS). J. Supercrit. Fluids 2018, 133, 291–296. [CrossRef] 118. Martín, A.; Cocero, M.J. Micronization processes with supercritical ﬂuids: Fundamentals and mechanisms. Adv. Drug Del. Rev. 2008, 60, 339–350. 119. Mattea, F.; Martín, Á.; Cocero, M.J. Carotenoid processing with supercritical ﬂuids. J. Food Eng. 2009, 93, 255–265. [CrossRef] 120. Kodama, T.; Honda, M.; Machmudah, S.; Wahyudiono; Kanda, H.; Goto, M. Crystallization of all trans-β-carotene by supercritical carbon dioxide antisolvent via co-axial nozzle. Eng. J. 2018, 22, 25–38. [CrossRef] 121. Kaga, K.; Honda, M.; Adachi, T.; Honjo, M.; Wahyudiono; Kanda, H.; Goto, M. Nanoparticle formation of PVP/astaxanthin inclusion complex by solution-enhanced dispersion by supercritical ﬂuids (SEDS): Eﬀect of PVP and astaxanthin Z-isomer content. J. Supercrit. Fluids 2018, 136, 44–51. [CrossRef] 122. Nerome, H.; Machmudah, S.; Wahyudiono; Fukuzato, R.; Higashiura, T.; Youn, Y.S.; Lee, Y.W.; Goto, M. Nanoparticle formation of lycopene/β-cyclodextrin inclusion complex using supercritical antisolvent precipitation. J. Supercrit. Fluids 2013, 83, 97–103. [CrossRef] 123. Boonnoun, P.; Nerome, H.; Machmudah, S.; Goto, M.; Shotipruk, A. Supercritical anti-solvent micronization of chromatography puriﬁed marigold lutein using hexane and ethyl acetate solvent mixture. J. Supercrit. Fluids 2013, 80, 15–22. [CrossRef] 124. Miguel, F.; Martin, A.; Gamse, T.; Cocero, M.J. Supercritical anti solvent precipitation of lycopene: Eﬀect of the operating parameters. J. Supercrit. Fluids 2006, 36, 225–235. [CrossRef] 125. Tavares-Cardoso, M.A.; Antunes, S.; van Keulen, F.; Ferreira, B.S.; Geraldes, A.; Cabral, J.; Palavra, A.M. Supercritical antisolvent micronisation of synthetic all-trans-β-carotene with tetrahydrofuran as solvent and carbon dioxide as antisolvent. J. Chem. Technol. Biotechnol. 2009, 84, 215–222. [CrossRef] 19 of 19 Molecules 2019, 24, 2149 126. Leong, H.Y.; Show, P.L.; Lim, M.H.; Ooi, C.W.; Ling, T.C. Natural red pigments from plants and their health beneﬁts: A review. Food Rev. Int. 2018, 34, 463–482. [CrossRef] 127. Coultate, T.; Blackburn, R.S. Food colorants: Their past, present and future. Color. Technol. 2018, 134, 165–186. [CrossRef] 128. Anarjan, N.; Tan, C.P. © 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/). References Eﬀects of selected polysorbate and sucrose ester emulsiﬁers on the physicochemical properties of astaxanthin nanodispersions. Molecules 2013, 18, 768–777. [CrossRef] 129. Spernath, A.; Aserin, A. Microemulsions as carriers for drugs and nutraceuticals. Adv. Colloid Interface Sci. 2006, 128, 47–64. [CrossRef] 130. Salvia-Trujillo, L.; Qian, C.; Martín-Belloso, O.; McClements, D.J. Inﬂuence of particle size on lipid digestion and β-carotene bioaccessibility in emulsions and nanoemulsions. Food Chem. 2013, 141, 1472–1480. [CrossRef] 131. Chu, B.S.; Ichikawa, S.; Kanafusa, S.; Nakajima, M. Preparation of protein-stabilized β-carotene nanodispersions by emulsiﬁcation–evaporation method. J. Am. Oil Chem. Soc. 2007, 84, 1053–1062. [CrossRef] 132. De Paz, E.; Martín, Á.; Mateos, E.; Cocero, M.J. Production of water-soluble β-carotene micellar formulations by novel emulsion techniques. Chem. Eng. Process. Process Intensif. 2013, 74, 90–96. [CrossRef] 133. Tan, C.P.; Nakajima, M. β-Carotene nanodispersions: Preparation, characterization and stability evaluation. Food Chem. 2005, 92, 661–671. [CrossRef] 134. Lakshmi, P.; Kumar, G.A. Nanosuspension technology: A review. Int. J. Pharm. Sci 2010, 2, 35–40. 134. Lakshmi, P.; Kumar, G.A. Nanosuspension technology: A review. Int. J. Pharm. Sci 2010, 2, 35–40. 135. Ezhilarasi, P.N.; Karthik, P.; Chhanwal, N.; Anandharamakrishnan, C. Nanoencapsulation techniques for food bioactive components: A review. Food Bioprocess. Technol. 2013, 6, 628–647. [CrossRef] 135. Ezhilarasi, P.N.; Karthik, P.; Chhanwal, N.; Anandharamakrishnan, C. Nanoencapsulation techniques for food bioactive components: A review. Food Bioprocess. Technol. 2013, 6, 628–647. [CrossRef] 136. Silva, H.D.; Cerqueira, M.A.; Souza, B.W.S.; Ribeiro, C.; Avides, M.C.; Quintas, M.A.C.; Coimbra, J.S.R.; Carneiro-da-Cunha, M.G.; Vicente, A.A. Nanoemulsions of β-carotene using a high-energy emulsiﬁcation–evaporation technique. J. Food Eng. 2011, 102, 130–135. [CrossRef] © 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/W2123119133	https://bmcgastroenterol.biomedcentral.com/counter/pdf/10.1186/1471-230X-14-75	English	null	Antibiotic exposure in pregnancy and risk of coeliac disease in offspring: a cohort study	BMC gastroenterology	2,014	cc-by	6,616	Karl Mårild1,2, Johnny Ludvigsson3, Yolanda Sanz4 and Jonas F Ludvigsson1,5 Karl Mårild1,2, Johnny Ludvigsson3, Yolanda Sanz4 and Jonas F Ludvigsson1,5 * Correspondence: karlmarild@gmail.com 1Dept. Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden 2Astrid Lindgren Children’s Hospital, Karolinska University Hospital, Solna, Sweden Full list of author information is available at the end of the article © 2014 Mårild et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Mårild et al. BMC Gastroenterology 2014, 14:75 http://www.biomedcentral.com/1471-230X/14/75 Mårild et al. BMC Gastroenterology 2014, 14:75 http://www.biomedcentral.com/1471-230X/14/75 Open Access Abstract Background: The infant microbiota may play a pathogenic role in coeliac disease (CD). Antibiotic treatment in pregnancy is common and could significantly impact the infant microbiota. In this study, we aimed to investigate the association between antibiotic exposure during pregnancy and CD in offspring. Methods: Prospective questionnaire data on antibiotic exposure in pregnancy were available in 8729 children participating in the All Babies in Southeast Sweden (ABIS) cohort study, and of these 46 developed CD until December 2006. Cox regression estimated hazard ratios (HRs) for CD in the offspring among mothers exposed to antibiotics during pregnancy, with adjustment for parent-reported diary data on breastfeeding, age at gluten introduction and number of infections in the child’s first year of life. Results: Of the 1836 children exposed to antibiotics during pregnancy, 12 (0.7%) children developed CD as compared with 34/6893 (0.5%) unexposed children (HR = 1.33; 95% CI = 0.69-2.56). Risk estimates remained unchanged after adjustment for breastfeeding, age at gluten introduction and infection load in the child’s first year of life (HR = 1.28; 95% CI = 0.66-2.48). Conclusions: We found no statistically significant association between antibiotic exposure during pregnancy and CD in offspring. This lack of association may either be true or the result of limited statistical power. Keywords: Antibiotics, Celiac disease, Microbiota, Pregnancy Background of the intestinal immune system, the establishment of oral tolerance and the mucosal barrier function [8]. Likewise, imbalances in the intestinal microbiota (dysbiosis) are frequently related to immune dysregulation and development of immune-mediated diseases and vice versa. Several studies have found dysbiosis in individuals with CD as compared with healthy controls, suggesting that the gut microbiota may play a pathogenic role in CD [9]. The infant gut colonization begins perinatally and is strongly influenced by the maternal microbiota, the mode of delivery and subsequently by the infant feeding practice [10]. Some data also indicate that maternal antibiotic intake in pregnancy influences the gut microbiota in the offspring, [11] and that perturbations caused by antibiotics in the infant [12] and adult microbiota [13,14] may persist for several years. Antibiotics are in fact one of the main environmental stressors that lead to the replacement of symbiotic bacteria by otherwise under-represented potentially pathogenic bacteria [13,15]. Coeliac disease (CD) is a life-long autoimmune disease occurring in 1-2% of children and adults living in Western countries [1,2]. In CD, genetically predisposed individuals develop small-intestinal villous atrophy in response to dietary gluten intake [3]. In the last decades the prevalence of CD has more than doubled, [4] suggesting that environmental factors other than gluten-exposure may have a significant influence on CD development [5]. Earlier research has in particular emphasized the importance of environmental factors early in life, including pregnancy and the perinatal period, [6,7] for the development of CD. The microbial colonization of the infant’s gut is considered to be critical for the appropriate development * Correspondence: karlmarild@gmail.com 1Dept. Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden 2Astrid Lindgren Children’s Hospital, Karolinska University Hospital, Solna, Sweden Full list of author information is available at the end of the article Mårild et al. BMC Gastroenterology 2014, 14:75 http://www.biomedcentral.com/1471-230X/14/75 Mårild et al. BMC Gastroenterology 2014, 14:75 http://www.biomedcentral.com/1471-230X/14/75 Page 2 of 7 Page 2 of 7 Today, some 10-25% [16,17] of pregnant women use antibiotics. Recent data have shown a positive association between antibiotic use during pregnancy and offspring asthma, [18] a disease that shares potential etiological and epidemiological traits with CD [19-21]. We have recently shown a positive association between antibiotic use in early childhood and subsequent CD [22]. However, there are few data whether antibiotic exposure in pregnancy influences the risk of CD in offspring. Methods Data on CD were collected through contact with all paediatric departments (n = 8) in the ABIS study area. The majority of children with CD were identified through a study published in 2004 [24]. In 2007–2008, we again contacted the same paediatric departments and asked them to report additional ABIS children with a biopsy-verified CD (villous atrophy) diagnosed until December 1st 2006. In addition to a biopsy suggestive of CD (Marsh grade III), children with CD were required either to have CD-specific antibody markers or CD-consistent symptoms that resolved after introduction of gluten-free diet [24]. In the current study, date of CD diagnosis equals date of first positive small-intestinal biopsy. The ABIS population was not actively screened for CD and therefore the children with CD were investigated due to clinical manifestations of possible CD. In this prospective population-based cohort study we used data from the ABIS cohort (All Babies in Southeast Sweden) in order to examine the association between antibiotic exposure in pregnancy and CD in the offspring. Background main analysis we included 8729 individuals with data on use of systemic antibiotics during pregnancy and with complete diary data for duration of breastfeeding and time of gluten introduction (see flow chart, Figure 1). The parents of the study participants were slightly more often born in Sweden and with a higher level of education, as compared with the source population of southeast Sweden [23]. Additional background data of the ABIS cohort have been described elsewhere [24,25]. Study population Between October 1997 and October 1999, all parents to babies born in southeast Sweden were invited to participate in the ABIS cohort. Of the 21,700 babies born during the study period, the parents of 17,055 children (78.6%) gave their informed consent to participate. In the maternity ward (at childbirth), parents to 16,285 children completed a questionnaire that included questions on antibiotics use in pregnancy, maternal education level, heath status and first-degree heredity for CD, type 1 diabetes mellitus and other autoimmune diseases. Parents were asked to complete a structured study diary during the child’s first year of life reporting infectious diseases and feeding practice, including duration of breastfeeding and age at gluten introduction. The diaries were completed prospectively at home and collected when the child was one year of age. In our Statistical analyses We used Cox regression to estimate Hazard Ratios (HRs) and 95% confidence intervals (CIs) for the risk of childhood CD according to maternal antibiotic exposure in pregnancy. To increase the statistical power of the study, we performed a post-hoc analysis including all 14,942 ABIS children with available data on antibiotic exposure in pregnancy. In this post-hoc analysis we used multiple imputation to replace missing values for infant nutrition data. However, since the statistics software SPSS cannot handle survival analyses with imputed data (the command "selection variable: rule: imputation > =1" only runs in linear regression and logistic regression), we first examined the odds ratio (OR) for future CD in our original dataset adjusting for infant nutrition (according to the covariates in Model A) before using multiple imputation to adjust infant feeding. In our main analysis (Model A, n = 8729) we used diary data to adjust for duration of breastfeeding (0–2; 3–4; 5–6; 7–8; 9–10 and ≥11 months) and age at gluten introduction (0–2; 3–4; 5–6; 7–8; 9–10 and 11–12 months of age) as potential confounders for CD development [25-27]. In a second analysis (Model B, n = 8698) we also adjusted for maternal education level (≤12 vs. >12 years) and number of any parent-reported infectious disease in the child’s first year of life, categorized into the following months of age: 0–2; 3–4; 5–6; 7–8; 9–10 and 11–12 months of age. Education level has been associated with antibiotic use [28] and may influence the risk of CD diagnosis [29]. Also infectious load in early childhood may be associated with subsequent CD [30]. Follow-up began at child’s birth and ended at time of CD diagnosis or December 1st 2006 (end of follow-up). However, because CD only develop after gluten introduction, which often occurs during the first year of life, we also performed a Cox regression in which the time scale began at one year of age until CD diagnosis or December 1st 2006 (end of follow-up). In a final post-hoc analysis we adjusted for the children’s use of antibiotics during their first year of life. Data on anti- biotic use was collected through a questionnaire when the child was one year old. The number of antibiotic courses used during the child’s first year of life was classified into the following categories: no use; 1–2; 3–5; ≥6 courses of antibiotics. Ethics This study was as part of the ABIS study approved by the Research Ethics Committees of Linköping University (Li 287–96) and Lund University (Lu 83–97). Mothers gave their written informed consent after careful written as well as oral information and information via videotape. Statistical significance was defined as 95% CIs not including 1.0. We used SPSS version 20.0 (SPSS, Inc, Chicago, IL) for the statistical analyses. Statistical analyses Regrettably, we largely lacked data on type of antibiotic agent used by the children. Maternal antibiotic exposure in pregnancy p p g y We collected questionnaire data on use of any systemic antibiotics during pregnancy. The mothers exposed to antibiotics were asked to specify the name on the type of antibiotics. Mothers who were uncertain of their antibiotic n=21,700 Babies born in southeast Sweden October 1997- October 1999 n=16,285 participants with completed at-birth questionnaire INCLUDED n=8729 Available data on use of systemic antibiotics during pregnancy and data on duration of breastfeeding and age at gluten introduction EXCLUDED n=7556 NO data on use of systemic antibiotics during pregnancy or data on duration of breastfeeding and age at gluten introduction n=4645 declined n=770 participants without questionnaire data Figure 1 Flow chart study participants. n=21,700 Babies born in southeast Sweden October 1997- October 1999 n=4645 declined Figure 1 Flow chart study participants. Page 3 of 7 Mårild et al. BMC Gastroenterology 2014, 14:75 http://www.biomedcentral.com/1471-230X/14/75 use in pregnancy (n = 27) or who had only used non-systemic antibiotics (n = 2) were excluded from the study. questionnaire we defined heredity for an autoimmune disease as presence of at least one first-degree relative with any of the following conditions: Goitre with hypothyroidism or hyperthyroidism, Grave `s disease, per- nicious anaemia, systemic lupus erythematosus, Addison’s disease, any diabetes mellitus, gestational diabetes, CD, inflammatory bowel disease or rheumatoid arthritis. Power calculation At a significance level of 0.05 we had an 80% power to detect a relative risk of 2.45 for CD in offspring to mothers treated with antibiotics during pregnancy. Mode of delivery and gestational age pertain a great impact on the infant gut colonization [10,31-33] and have been associated with an increased risk of CD in offspring [6,34]. In two subanalyses we therefore restricted our data to term deliveries and children born through vaginal delivery, respectively. We also performed stratified analyses by infant sex and heredity for CD or type 1 diabetes mellitus. Post-hoc analyses Out of the 8729 children included in the study, 1836 (21%) had been exposed to antibiotics during pregnancy (unexposed: 6893; Table 1). Type of antibiotics was only listed in a minority of children (n = 235), with penicillin V being the most common type of antibiotic (n = 147). The children were followed up to December 1st 2006, corresponding to an average age of eight years. At end of follow-up, 46 out of 8729 children were diagnosed with CD, yielding a baseline CD prevalence of 0.5%. Girls made up half of the children, and the average maternal age at delivery was close to 30 years (Table 1). As opposed to short duration of breastfeeding, no breastfeeding at all can have a different effect on gut colonization. In a post-hoc analysis we therefore chose to adjust for duration of breastfeeding splitting the first time category (0–2 months) into: 0–6 days and 7 days-2 months (keeping 3–4 months; 5–6 months etc.). In a second post-hoc analysis we estimated the risk of CD in offspring according to antibiotic exposure in pregnancy excluding children with a first-degree relative with autoimmune disease. According to data from the at-birth Mårild et al. BMC Gastroenterology 2014, 14:75 http://www.biomedcentral.com/1471-230X/14/75 Page 4 of 7 Table 1 Descriptive characteristics of individuals according to antibiotic exposure in pregnancy Antibiotics No antibiotics TotalA (%) 1836 (21.0) 6893 (79.0) Infant sex Girls, n (%) 904 (49.2) 3342 (48.5) Boys, n (%) 932 (50.8) 3551 (51.5) HeredityB Coeliac disease, n (%) 30 (1.6) 84 (1.2) Type 1 diabetes mellitus, n (%) 56 (3.1) 155 (2.2) Maternal characteristics Age at delivery; mean +/−SD (years) 30.3 +/−4.4 29.7 +/−4.5 Maternal university educationC, n (%) 715 (39.1) 2272 (33.1) AIndividuals with complete data on breastfeeding and gluten introduction. BFirst-degree relative with coeliac disease/type 1 diabetes mellitus. C>12 years of education at time of delivery. Table 1 Descriptive characteristics of individuals according to antibiotic exposure in pregnancy Some 316 children had a first-degree relative with either CD (n = 105) or type 1 diabetes mellitus (n = 202) or with both CD and type 1 diabetes (n = 9). Restricting our data to children without heredity for CD and type 1 diabetes did not change our risk estimates (adjusted HR = 1.22; 95% CI = 0.58-2.59). Post-hoc analyses Neither did the risk estimates change appreciably when we restricted our data to children born full-term (n = 8270 [94.7%]) or with a vaginal delivery (n = 7647 [87,6%]) (Table 2). Because CD only develops after gluten introduction (typically occurring within the first year of life), we also performed a number of pre-planned Cox regression analyses in which the time scale began at one year of age, thereby excluding six children diagnosed with CD before that age. Antibiotic exposure during pregnancy was not significantly associated with CD in offspring diagnosed after age one year (adjusted HR = 1.42; 95% CI = 0.71-2.83). The risk estimates for CD diagnosed after age one year stratified by sex and restricted to children with no heredity for CD and type 1 diabetes are presented in Table 3. AIndividuals with complete data on breastfeeding and gluten introduction. BFirst-degree relative with coeliac disease/type 1 diabetes mellitus. C>12 years of education at time of delivery. AIndividuals with complete data on breastfeeding and gluten introduction. BFirst-degree relative with coeliac disease/type 1 diabetes mellitus. C>12 years of education at time of delivery. Twelve (0.7%) exposed and 34 (0.5%) unexposed children developed CD, corresponding to a HR of 1.33 (95% CI = 0.69-2.56). Risk estimates did not change more than marginally after adjustment for duration of breastfeeding and age at gluten introduction (Model A: adjusted HR = 1.32, 95% CI = 0.69-2.56) or when adding first-year childhood infections and maternal education level (Model B: adjusted HR = 1.28, 95% CI = 0.66-2.48). We found unchanged HR:s with follow-up time starting at time of gluten introduction (data not shown). The relative risk estimates for CD after antibiotic exposure during pregnancy were similar in boys and girls (Boys: adjusted HR = 1.28; 95% CI = 0.41-3.96; Girls: adjusted HR = 1.36; 95% CI = 0.60-3.05). Moreover, antibiotic exposure during pregnancy did not seem to favour an earlier appearance of CD (median age at CD diagnosis was some 2 years and 9 months among both children exposed and unexposed to antibiotics during pregnancy). th a first-degree relative with celiac disease (n = 105), type 1 diabetes mellitus (n = 202) or both celiac disease and type 1 diabetes (n = al weeks. Post-hoc analyses I i l In our main analysis we identified 64 children with less than 7 days of breastfeeding. In a post-hoc analysis adjust- ing for no breastfeeding (duration <7 days), age at gluten introduction, infectious load in the first year of life and maternal education level we found largely unchanged risk estimates (adjusted HR = 1.29; 95% CI = 0.66-2.49). Finally, we excluded children with first-degree relatives with any autoimmune disease (remaining n = 7785 [89,2%]; HR = 1.40; 95% CI = 0.65-2.99). To address the issue of low statistical power we per- formed a post-hoc analysis including all 14,942 children with available data on antibiotic exposure in pregnancy. We used multiple imputation to replace missing values for infant nutrition data. However, since the statistics software coeliac disease in offspring (follow-up from birth) according to antibiotic exposure in pregnancy Table 2 Risk of coeliac disease in offspring (follow-up from birth) according to antibiotic exposure Table 2 Risk of coeliac disease in offspring (follow-up from birth) according to antibiotic exposure in pregnancy Antibiotics (%) No Antibiotics (%) Crude HR; 95% CI Adjusted HR; 95% CIA Adjusted HR; 95% CIB All 12/1836 (0.7) 34/6893 (0.5) 1.33; 0.69-2.56 1.32; 0.69-2.56 1.28; 0.66-2.48 Sex Boys 4/932 (0.4) 12/3551 (0.3) 1.27; 0.41-3.94 1.28; 0.41-3.96 1.37; 0.44-4.27 Girls 8/904 (0.9) 22/3342 (0.7) 1.35; 0.60-3.02 1.36; 0.60-3.05 1.21; 0.53-2.73 Subgroups No heredityC 9/1753 (0.5) 28/6660 (0.4) 1.22; 0.58-2.59 1.22; 0.58-2.59 1.16; 0.55-2.47 Term deliveriesD 12/1739 (0.7) 32/6531 (0.5) 1.41; 0.73-2.74 1.41; 0.73-2.74 1.37; 0.70-2.66 Vaginal delivery 10/1584 (0.6) 30/6063 (0.5) 1.28; 0.63-2.61 1.28; 0.63-2.62 1.25; 0.61-2.56 AIn Model A we adjusted for duration of breastfeeding and age at gluten introduction. Children with complete data on breastfeeding and gluten introduction were included in the analyses (n varied between 7647 and 8729). BIn Model B we adjusted for any parent-reported infection during the child’s first year of life, maternal education level as well as duration of breastfeeding and age at gluten introduction. Children with complete data on breastfeeding, gluten introduction as well as education level were included in the analyses (n varied between 7622 and 8698). CExcluding individuals with a first-degree relative with celiac disease (n = 105), type 1 diabetes mellitus (n = 202) or both celiac disease and type 1 diabetes (n = 9). DFull-term, ≥37 gestational weeks. Hazard ratios (HR) estimated through Cox regression. Follow-up from birth. Mårild et al. Post-hoc analyses I i l BMC Gastroenterology 2014, 14:75 http://www.biomedcentral.com/1471-230X/14/75 Page 5 of 7 Table 3 Risk of coeliac disease in offspring (follow-up from one year of age) according to antibiotic exposure in pregnancy Antibiotics (%) No Antibiotics (%) Crude HR; 95% CI Adjusted HR; 95% CIA Adjusted HR; 95% CIB All 11/1835 (0.6) 29/6888 (0.4) 1.43; 0.71-2.86 1.42; 0.71-2.83 1.37; 0.68-2.76 Sex Boys 4/932 (0.4) 8/3547 (0.2) 1.90; 0.57-6.32 1.91; 0.58-6.35 2.00; 0.60-6.66 Girls 7/903 (0.8) 21/3341 (0.6) 1.24; 0.53-2.91 1.24; 0.53-2.92 1.13; 0.48-2.67 Subgroups No heredityC 8/1752 (0.5) 24/6656 (0.4) 1.27; 0.57-2.82 1.26; 0.57-2.81 1.21;0.54-2.70 Term deliveriesD 11/1738 (0.6) 27/6526 (0.4) 1.53; 0.76-3.09 1.52; 0.76-3.07 1.48; 0.73-2.99 Vaginal delivery 9/1583 (0.6) 25/6058 (0.4) 1.38; 0.64-2.96 1.37; 0.64-2.94 1.34; 0.62-2.88 AIn Model A we adjusted for duration of breastfeeding and age at gluten introduction. Children with complete data on breastfeeding and gluten introduction were included in the analyses (n varied between 7641 and 8723). BIn Model B we adjusted for any parent-reported infection during the child’s first year of life, education level as well as duration of breastfeeding and age at gluten introduction. Children with complete data on breastfeeding, gluten introduction as well as education level were included in the analyses (n varied between 7616 and 8692). CExcluding individuals with a first-degree relative with celiac disease (n = 105), type 1 diabetes mellitus (n = 202) or both celiac disease and type 1 diabetes (n = 9). DFull-term, ≥37 gestational weeks. Hazard ratios (HR) estimated through Cox regression. Follow-up from one year of age. Table 3 Risk of coeliac disease in offspring (follow-up from one year of age) according to antibiotic exposure in Table 3 Risk of coeliac disease in offspring (follow-up from one year of age) according to a pregnancy n offspring (follow-up from one year of age) according to antibiotic exposure in Swedish infants with minor infections do not seek medical advice. Although, the infections were not recorded using standardized self-reporting criteria we do not suspect this potential risk of misclassification to be differential between children exposed/unexposed to antibiotics in pregnancy. SPSS cannot handle survival analyses with imputed data, we first examined the OR for future CD in our original dataset adjusting for infant nutrition (maternal antibiotics: OR for offspring CD = 1.32; 95% CI = 0.68-2.56). Using multiple imputation when adjusting for infant feeding did not change the risk estimate more than marginally (OR = 1.10; 95% CI = 0.64-1.89). Post-hoc analyses I i l Similar to other birth-cohort studies [36,37], the current study suffers from attrition. Half of the population entering at birth completed the 1-year-diaries, and there was a slight underrepresentation of parents with foreign origin and low education [38]. However, this should not influence the risk estimates if the association between maternal antibiotic exposure and risk of offspring CD is independent of education status. We are not aware of any study indicating that education modifies the risk factors of CD, even if socioeconomic status per se may be linked to the risk of CD [39,40]. In a post-hoc analysis, including 6773 children with complete diary data and available data on use of antibiotics during the first year of life, we found only marginally changed risk estimates for CD (adjusted HR = 1.65; 95% CI = 0.83-3.28). The association between CD and use of antibiotics during the child’s first year of life has been presented in Additional file 1, Table A1. Discussion In this cohort study, we examined the association between maternal use of antibiotics in pregnancy and risk of CD in the offspring. The rationale for this study was the increasing amount of data indicating that the maternal intestinal microbiota in pregnancy influences the foetal and infant immune system development [35]. However, we found no statistically significant association between antibiotic exposure in pregnancy and CD in the offspring. Although this may be reassuring, our study does not rule out a modestly excess risk of CD in children to mothers who were treated with antibiotics during pregnancy. Of greater concern is the limited statistical power of this study and the correspondingly increased risk of type 2 error (i.e. to erroneously accept a false null hypothesis). Additionally, we have not been able to screen the ABIS cohort for CD, and our patients consist of children with clinically diagnosed CD. The baseline CD preva- lence of 0.5% (46 out of 8729 children) is therefore considerably lower than the estimated 2% prevalence of screening-detected CD in Swedish children [41] (in our study corresponding to 175 children). However, false-negative CD in our control group (<2% [41]) should not affect our risk estimates. Further, we do not expect risk factors to differ between children with or without symptoms (meriting investigation for CD). Unfortunately we lack data on clinical phenotype and were unable to examine if intrauterine antibiotic exposure is linked to a certain type of CD. A strength of this study is its prospective design, where data on antibiotic exposure in pregnancy were collected before CD diagnosis, eliminating the risk of recall bias. We also used prospectively collected data on infant feeding and infectious diseases during the child’s first year of life. Infections were measured through concurrent parent- reporting, and not through patient charts, because most Page 6 of 7 Mårild et al. BMC Gastroenterology 2014, 14:75 http://www.biomedcentral.com/1471-230X/14/75 Data on antibiotics during pregnancy were collected retrospectively (at childbirth) and we did not have data on the specific date of the antibiotic treatment or on intrapar- tum antibiotics. Speculatively, antibiotic treatment during the perinatal period may have a more profound impact on infant gut colonization, as compared with antibiotic treat- ment early in pregnancy [11]. References 1. Walker MM, Murray JA, Ronkainen J, Aro P, Storskrubb T, D'Amato M, Lahr B, Talley NJ, Agreus L: Detection of celiac disease and lymphocytic enteropathy by parallel serology and histopathology in a population-based study. Gastroenterology 2010, 139:112–119. Gastroenterology 2010, 139:112 119. 2. Dube C, Rostom A, Sy R, Cranney A, Saloojee N, Garritty C, Sampson M, Zhang L, Yazdi F, Mamaladze V, Pan I, Macneil J, Mack D, Patel D, Moher D: The prevalence of celiac disease in average-risk and at-risk Western European populations: a systematic review. Gastroenterology 2005, 128:S57–S67. 3. Di Sabatino A, Corazza GR: Coeliac disease. Lancet 2009, 373:1480–1493. 4. Rubio-Tapia A, Kyle RA, Kaplan EL, Johnson DR, Page W, Erdtmann F, Brantner TL, Kim WR, Phelps TK, Lahr BD, Zinsmeister AR, Melton LJ 3rd, Murray JA: Increased prevalence and mortality in undiagnosed celiac disease. Gastroenterology 2009, 137:88–93 gy , 2. Dube C, Rostom A, Sy R, Cranney A, Saloojee N, Garritty C, Sampson M, Zhang L, Yazdi F, Mamaladze V, Pan I, Macneil J, Mack D, Patel D, Moher D: The prevalence of celiac disease in average-risk and at-risk Western European populations: a systematic review. Gastroenterology 2005, 128:S57–S67. 3. Di Sabatino A, Corazza GR: Coeliac disease. Lancet 2009, 373:1480–1493. 4. Rubio-Tapia A, Kyle RA, Kaplan EL, Johnson DR, Page W, Erdtmann F, Brantner TL, Kim WR, Phelps TK, Lahr BD, Zinsmeister AR, Melton LJ 3rd, Murray JA: Increased prevalence and mortality in undiagnosed celiac disease Gastroenterology 2009 137:88–93 gy 2. Dube C, Rostom A, Sy R, Cranney A, Saloojee N, Garritty C, Sampson M, Zhang L, Yazdi F, Mamaladze V, Pan I, Macneil J, Mack D, Patel D, Moher D: The prevalence of celiac disease in average-risk and at-risk Western European populations: a systematic review. Gastroenterology 2005, 128:S57–S67. 3 Di S b ti A C GR C li di L t 2009 373 1480 1493 3. Di Sabatino A, Corazza GR: Coeliac disease. Lancet 2009, 373:1480 1493. 4. Rubio-Tapia A, Kyle RA, Kaplan EL, Johnson DR, Page W, Erdtmann F, Brantner TL, Kim WR, Phelps TK, Lahr BD, Zinsmeister AR, Melton LJ 3rd, Murray JA: Increased prevalence and mortality in undiagnosed celiac disease. Gastroenterology 2009, 137:88–93. Authors’ contributions ICMJE criteria for authorship read and met: KM, JFL, YS, JL. Agree with the manuscript’s results and conclusions: KM, JFL, YS, JL. Designed the experiments/the study: KM, JFL, JL. Collected data: JFL, JL. Analyzed the data: KM. Wrote the first draft of the paper: KM. Contributed to the writing of the paper: JFL, YS, JL. Contributed to design of study and interpretation of the data analyses: KM, JFL, YS, JL. Interpretation of data; approved the final version of the manuscript: KM, JFL, YS, JL. Responsible for data integrity: KM, JFL. Supervised the project including data analyses: JFL.Obtained funding: JFL, JL. All authors read and approved the final manuscript. Conclusions This study found no statistically significant association between maternal use of antibiotics during pregnancy and CD in the offspring. This lack of association may either be true or due to limited statistical power. 5. Lohi S, Mustalahti K, Kaukinen K, Laurila K, Collin P, Rissanen H, Lohi O, Bravi E, Gasparin M, Reunanen A, Maki M: Increasing prevalence of coeliac disease over time. Aliment Pharmacol Ther 2007, 26:1217–1225. 5. Lohi S, Mustalahti K, Kaukinen K, Laurila K, Collin P, Rissanen H, Lohi O, Bravi E, Gasparin M, Reunanen A, Maki M: Increasing prevalence of coeliac disease over time. Aliment Pharmacol Ther 2007, 26:1217–1225. 6. Marild K, Stephansson O, Montgomery S, Murray JA, Ludvigsson JF: Pregnancy outcome and risk of celiac disease in offspring: a nationwide case–control study. Gastroenterology 2012, 142:39–45. e33. y gy 7. Sandberg-Bennich S, Dahlquist G, Kallen B: Coeliac disease is associated with intrauterine growth and neonatal infections. Acta Paediatr 2002, 91:30–33. Author details 1 1Dept. Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden. 2Astrid Lindgren Children’s Hospital, Karolinska University Hospital, Solna, Sweden. 3Div. of Paediatrics, Department of Clinical and Experimental Medicine, Linköping University, and Östergötland County Council, Linköping, Sweden. 4Microbial Ecology and Nutrition Research Group, Institute of Agrochemistry and Food Technology, National Research Council (IATA-CSIC), Valencia, Spain. 5Department of Paediatrics, Örebro University Hospital, Örebro, Sweden. Postnatal microbial exposure is likely to play a greater role in immune maturation and thereby CD development as compared with prenatal microbial exposure [35]. Still, vast amount of data suggest that pregnancy is a critical time for immune development and epigenetic models of allergic outcomes suggest that foetal immune development may be influenced by epigenetic modifications from microbial products [35]. In addition, several epidemiological studies have shown that prenatal exposures to antibiotics are asso- ciated with the development of childhood asthma, a disease that shares potential etiological traits with CD [19-21]. Received: 10 December 2013 Accepted: 9 April 2014 Published: 14 April 2014 Received: 10 December 2013 Accepted: 9 April 2014 Published: 14 April 2014 Competing interests The authors declare that they have no competing interests. Competing interests The authors declare that they have no competing interests. Discussion Additionally, we largely lack data on type of antibiotic agent, its dosage, duration of treatment as well as the indication for treatment, which all influence the mother’s microbiota, and that of the offspring [42]. The lacking specificity regarding type of antibiotic exposure may also have contributed to an increased risk of a type 2 error. The type of antibiotic used by the mother should also be linked to the type of infections she experi- enced during pregnacy and her inflammatory status that per se may also influence fetal immune programming and risk of autoimmune disease in the offspring. Acknowledgements This work was supported by; KM: The Swedish Society of Medicine, Karolinska Institutet; JL: Swedish Child Diabetes Foundation (Barndiabetesfonden), Research Council for Southeast Sweden, ALF (Swedish Government), Canadian Research Council, Östgöta Brandstodsbolag, Östergötland County Council, EU Research Grants. YS: grants AGL2011-25169 and Consolider Fun-C-Food CSD2007-00063 from the Spanish Ministry of Economy and Competitiveness; JFL: The Swedish Society of Medicine, the Swedish Research Council, the Örebro Society of Medicine, the Karolinska Institutet, and the Swedish Celiac Society. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. It is well-established that the postnatal intestinal micro- biota influences the maturation of the intestinal immune system and that individuals with CD, or with an increased genetic risk of developing CD, have an imbalanced intestinal microbiota, which may potentially enhance the inflammatory response elicited by gluten [9,43-45]. Animal studies suggest that the intestinal dysbiosis associated with CD may, in the presence of gliadin, increase the permeability of the small-intestine [9] and enable epithelial translocation of gliadin that may trigger CD [9,46]. Although the current study lacks evidence for a prenatal association between antibiotics and CD, our results do not refute the hypothesis that the early intestinal microbiota affects CD development. Abbreviations ABIS: All Babies in Southeast Sweden; CD: Celiac disease; CI: Confidence interval; HR: Hazard ratio; OR: Odds ratio. Additional file 1: Table A1. Risk of coeliac disease according to the child’s use of antibiotics in the first year of life. Hazard ratios (HR) estimated through Cox regression. Competing interests Competing interests The authors declare that they have no competing interests. Additional file Decker E, Engelmann G, Findeisen A, Gerner P, Laass M, Ney D, Posovszky C, Hoy L, Hornef MW: Cesarean delivery is associated with celiac disease but not inflammatory bowel disease in children. Pediatrics 2010, 125:e1433–e1440. 13. Dethlefsen L, Relman DA: Incomplete recovery and individualized responses of the human distal gut microbiota to repeated antibiotic perturbation. Proc Natl Acad Sci U S A 2011, 108(Suppl 1):4554–4561. 35. Prescott SL, Clifton V: Asthma and pregnancy: emerging evidence of epigenetic interactions in utero. Curr Opin Allergy Clin Immunol 2009, 9:417–426. 14. Jakobsson HE, Jernberg C, Andersson AF, Sjolund-Karlsson M, Jansson JK, Engstrand L: Short-term antibiotic treatment has differing long-term impacts on the human throat and gut microbiome. PLoS One 2010, 5:e9836. 36. Magnus MC, Haberg SE, Stigum H, Nafstad P, London SJ, Vangen S, Nystad W: Delivery by Cesarean section and early childhood respiratory symptoms and disorders: the Norwegian mother and child cohort study. Am J Epidemiol 2011, 174:1275–1285. 15. Johnson CL, Versalovic J: The human microbiome and its potential importance to pediatrics. Pediatrics 2012, 129:950–960. 37. Andersen AM, Olsen J: The Danish National Birth Cohort: selected scientific contributions within perinatal epidemiology and future perspectives. Scand J Public Health 2011, 39:115–120. 16. Stephansson O, Granath F, Svensson T, Haglund B, Ekbom A, Kieler H: Drug use during pregnancy in Sweden - assessed by the Prescribed Drug Register and the Medical Birth Register. Clin Epidemiol 2011, 3:43–50. 38. Sepa A, Frodi A, Ludvigsson J: Mothers' experiences of serious life events increase the risk of diabetes-related autoimmunity in their children. Diabetes Care 2005, 28:2394–2399. g g p 17. Stalberg K, Haglund B, Stromberg B, Kieler H: Prenatal exposure to medicines and the risk of childhood brain tumor. Cancer Epidemiol 2010, 34:400–404. 18. Stensballe LG, Simonsen J, Jensen SM, Bonnelykke K, Bisgaard H: Use of antibiotics during pregnancy increases the risk of asthma in early childhood. J Pediatr 2013, 162:832–838. e833. 39. Olen O, Bihagen E, Rasmussen F, Ludvigsson JF: Socioeconomic position and education in patients with coeliac disease. Dig Liver Dis 2012, 44:471–476. 40. Wingren CJ, Bjorck S, Lynch KF, Ohlsson H, Agardh D, Merlo J: Coeliac disease in children: a social epidemiological study in Sweden. Acta Paediatr 2012, 101:185–191. 19. Kero J, Gissler M, Hemminki E, Isolauri E: Could TH1 and TH2 diseases coexist? Evaluation of asthma incidence in children with coeliac disease, type 1 diabetes, or rheumatoid arthritis: a register study. Additional file 8. Round JL, Mazmanian SK: The gut microbiota shapes intestinal immune responses during health and disease. Nat Rev Immunol 2009, 9:313–323. 9 S Y D P G L M U li h i b li di 8. Round JL, Mazmanian SK: The gut microbiota shapes intestinal immune responses during health and disease. Nat Rev Immunol 2009, 9:313–323. 9. Sanz Y, De Pama G, Laparra M: Unraveling the ties between celiac disease and intestinal microbiota. Int Rev Immunol 2011, 30:207–218. Additional file 1: Table A1. Risk of coeliac disease according to the child’s use of antibiotics in the first year of life. Hazard ratios (HR) estimated through Cox regression. 9. Sanz Y, De Pama G, Laparra M: Unraveling the ties between celiac disease and intestinal microbiota. Int Rev Immunol 2011, 30:207–218. Page 7 of 7 Mårild et al. BMC Gastroenterology 2014, 14:75 http://www.biomedcentral.com/1471-230X/14/75 Mårild et al. BMC Gastroenterology 2014, 14:75 http://www.biomedcentral.com/1471-230X/14/75 Mårild et al. BMC Gastroenterology 2014, 14:75 http://www.biomedcentral.com/1471-230X/14/75 10. Penders J, Thijs C, Vink C, Stelma FF, Snijders B, Kummeling I, van den Brandt PA, Stobberingh EE: Factors influencing the composition of the intestinal microbiota in early infancy. Pediatrics 2006, 118:511–521. infancy: a prospective follow up study of healthy infants aged 0–6 months. Arch Dis Child Fetal Neonatal Ed 2000, 83:F186–F192. infancy: a prospective follow up study of healthy infants aged 0–6 months. Arch Dis Child Fetal Neonatal Ed 2000, 83:F186–F192. 32. 32. Adlerberth I, Lindberg E, Aberg N, Hesselmar B, Saalman R, Strannegard IL, Wold AE: Reduced enterobacterial and increased staphylococcal colonization of the infantile bowel: an effect of hygienic lifestyle? Pediatr Res 2006, 59:96–101. 11. Fallani M, Young D, Scott J, Norin E, Amarri S, Adam R, Aguilera M, Khanna S, Gil A, Edwards CA, Dore J: Intestinal microbiota of 6-week-old infants across Europe: geographic influence beyond delivery mode, breast-feeding, and antibiotics. J Pediatr Gastroenterol Nutr 2010, 51:77–84. 33. Dominguez-Bello MG, Costello EK, Contreras M, Magris M, Hidalgo G, Fierer N, Knight R: Delivery mode shapes the acquisition and structure of the initial microbiota across multiple body habitats in newborns. Proc Natl Acad Sci U S A 2010, 107:11971–11975. 12. Fouhy F, Guinane CM, Hussey S, Wall R, Ryan CA, Dempsey EM, Murphy B, Ross RP, Fitzgerald GF, Stanton C, Cotter PD: High-throughput sequencing reveals the incomplete, short-term recovery of infant gut microbiota following parenteral antibiotic treatment with ampicillin and gentamicin. Antimicrob Agents Chemother 2012, 56:5811–5820. 34. Additional file J Allergy Clin Immunol 2001, 108:781–783. 41. Ivarsson A, Myleus A, Norstrom F, van der Pals M, Rosen A, Hogberg L, Danielsson L, Halvarsson B, Hammarroth S, Hernell O, Karlsson E, Stenhammar L, Webb C, Sandstrom O, Carlsson A: Prevalence of childhood celiac disease and changes in infant feeding. Pediatrics 2013, 131:e687–e694. 20. Ludvigsson JF, Hemminki K, Wahlstrom J, Almqvist C: Celiac disease confers a 1.6-fold increased risk of asthma: a nationwide population-based cohort study. J Allergy Clin Immunol 2011, 127:1071–1073. 42. Jernberg C, Lofmark S, Edlund C, Jansson JK: Long-term impacts of antibiotic exposure on the human intestinal microbiota. Microbiology 2010, 156:3216–3223. 21. Cho CE, Norman M: Cesarean section and development of the immune system in the offspring. Am J Obstet Gynecol 2013, 208:249–254. 22. Marild K, Ye W, Lebwohl B, Green PH, Blaser MJ, Card T, Ludvigsson JF: Antibiotic exposure and the development of coeliac disease: a nationwide case–control study. BMC Gastroenterol 2013, 13:109. 43. Guarner F, Malagelada JR: Gut flora in health and disease. Lancet 2003, 361:512–519. 44. De Palma G, Cinova J, Stepankova R, Tuckova L, Sanz Y: Pivotal Advance: Bifidobacteria and Gram-negative bacteria differentially influence immune responses in the proinflammatory milieu of celiac disease. J Leukoc Biol 2010, 87:765–778. 23. Sepa A, Wahlberg J, Vaarala O, Frodi A, Ludvigsson J: Psychological stress may induce diabetes-related autoimmunity in infancy. Diabetes Care 2005, 28:290–295. 24. Ludvigsson JF, Ansved P, Falth-Magnusson K, Hammersjo JA, Johansson C, Edvardsson S, Ljungkrantz M, Stenhammar L, Ludvigsson J: Symptoms and signs have changed in Swedish children with coeliac disease. J Pediatr Gastroenterol Nutr 2004, 38:181–186. 45. Palma GD, Capilla A, Nova E, Castillejo G, Varea V, Pozo T, Garrote JA, Polanco I, Lopez A, Ribes-Koninckx C, Marcos A, Garcia-Novo MD, Calvo C, Ortigosa L, Pena-Quintana L, Palau F, Sanz Y: Influence of milk-feeding type and genetic risk of developing coeliac disease on intestinal microbiota of infants: the PROFICEL study. PLoS One 2012, 7:e30791. 25. Welander A, Tjernberg AR, Montgomery SM, Ludvigsson J, Ludvigsson JF: Infectious disease and risk of later celiac disease in childhood. Pediatrics 2010, 125:e530–e536. 46. Cinova J, De Palma G, Stepankova R, Kofronova O, Kverka M, Sanz Y, Tuckova L: Role of intestinal bacteria in gliadin-induced changes in intestinal mucosa: study in germ-free rats. PLoS One 2011, 6:e16169. 26. Additional file Akobeng AK, Ramanan AV, Buchan I, Heller RF: Effect of breast feeding on risk of coeliac disease: a systematic review and meta-analysis of observational studies. Arch Dis Child 2006, 91:39–43. doi:10.1186/1471-230X-14-75 Cite this article as: Mårild et al.: Antibiotic exposure in pregnancy and risk of coeliac disease in offspring: a cohort study. BMC Gastroenterology 2014 14:75. 27. Norris JM, Barriga K, Hoffenberg EJ, Taki I, Miao D, Haas JE, Emery LM, Sokol RJ, Erlich HA, Eisenbarth GS, Rewers M: Risk of celiac disease autoimmunity and timing of gluten introduction in the diet of infants at increased risk of disease. Jama 2005, 293:2343–2351. 28. Huether CA, Ivanovich J, Goodwin BS, Krivchenia EL, Hertzberg VS, Edmonds LD, May DS, Priest JH: Maternal age specific risk rate estimates for Down syndrome among live births in whites and other races from Ohio and metropolitan Atlanta, 1970–1989. J Med Genet 1998, 35:482–490. 29. Kondrashova A, Mustalahti K, Kaukinen K, Viskari H, Volodicheva V, Haapala AM, Ilonen J, Knip M, Maki M, Hyoty H: Lower economic status and inferior hygienic environment may protect against celiac disease. Ann Med 2008, 40:223–231. 30. Stene LC, Honeyman MC, Hoffenberg EJ, Haas JE, Sokol RJ, Emery L, Taki I, Norris JM, Erlich HA, Eisenbarth GS, Rewers M: Rotavirus infection frequency and risk of celiac disease autoimmunity in early childhood: a longitudinal study. Am J Gastroenterol 2006, 101:2333–2340. 31. Gronlund MM, Arvilommi H, Kero P, Lehtonen OP, Isolauri E: Importance of intestinal colonisation in the maturation of humoral immunity in early
https://openalex.org/W3105757594	https://www.epj-conferences.org/articles/epjconf/pdf/2020/21/epjconf_chep2020_02025.pdf	English	null	GTS – Garfield-based Triple-GEM Simulator	EPJ web of conferences	2,020	cc-by	3,698	∗e-mail: rfarinelli@fe.infn.it Riccardo Farinelli1,∗on behalf of the CGEM-IT BESIII working group 1INFN, Sezione di Ferrara, via G. Saragat 1, 44122 Ferrara, Italy Abstract. Triple-GEM detectors are gaseous devices used in high energy physics to measure the path of the particles which cross them. The charac- terisation of triple GEM detectors and the estimation of the performance for real data experiments require a complete comprehension of the mechanisms which transform the passage of one particle in the detector into electric signals, and dedicated MonteCarlo simulations are needed. In this work we will de- scribe GTS (Garﬁeld-based Triple-gem Simulator), a MonteCarlo code which has been developed to simulate the detector response to the passage of parti- cles inside triple GEMs. The software takes into account the processes from the primary ionization up to the signal formation, e.g. avalanche multiplication and the eﬀects of the diﬀusion in the gas volume. It uses a parametrization of the variables of interest meant to reproduce the same detector response (i.e. the charge distribution at the anode) as provided by Garﬁeld++, a well known soft- ware that already performs this kind of simulation more detailed and therefore with a high CPU time consumption. In addition to the detector response, the simulation of the APV-25 electronics is implemented and the output is used to reconstruct the particle position with the Charge Centroid (CC) and the micro- Time Projection Chamber (µTPC) methods. A comparison of the simulated performance and the one collected in testbeams is used to tune the parameters used in GTS. Results in diﬀerent conditions of incident angle will be shown. © 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/). EPJ Web of Conferences 245, 02025 (2020) CHEP 2019 EPJ Web of Conferences 245, 02025 (2020) CHEP 2019 https://doi.org/10.1051/epjconf/202024502025 GTS - Garﬁeld-based Triple-GEM Simulator Riccardo Farinelli1,∗on behalf of the CGEM-IT BESIII working group 1INFN, Sezione di Ferrara, via G. Saragat 1, 44122 Ferrara, Italy 1 Introduction Gaseous Electron Multiplier (GEM) is a well know technology introduced by F. Sauli in 1997 [1] used to amplify the signal generated by ionizing particles interacting with the gas volume of the detector. The GEM foil considered in this paper consists of a copper coated (5 µm) on a 50 µm kapton foil, with holes of 50 µm diameter and 140 µm of pitch. Electrons entering these holes are accelerated and generate an electron cascade. Multiple stages of GEM foils allow to reach gain of about 104 with a very low discharge probability. The full design of a triple-GEM detector is given by a cathode, three GEM foils and an anode readout. Simulation of this technology is useful to understand the processes that play the main role in this detection technique. An existing software Garﬁeld++ [2], a toolkit for the detailed simulation of particle de- tectors based on ionisation measurement in gases and semiconductors, can perform a detailed simulation of a triple-GEM detector. It simulates step by step all the interactions of the ion- izing particle, the interaction between the electrons and the gas mixture, as a function of the electric ﬁeld. Since the simulation steps are small (about few µm) compared to the size of a EPJ Web of Conferences 245, 02025 (2020) CHEP 2019 https://doi.org/10.1051/epjconf/202024502025 triple-GEM (about 1 cm) and the number of electrons involved is large (about 105-106) then the simulation of the space and time distributions of the signal at the anode can become quite expensive in terms of CPU time (10-20 hours per event). Examples of triple-GEM simula- tions performed with Garﬁeld++ can be found in literature [3–5]. triple-GEM (about 1 cm) and the number of electrons involved is large (about 105-106) then the simulation of the space and time distributions of the signal at the anode can become quite expensive in terms of CPU time (10-20 hours per event). Examples of triple-GEM simula- tions performed with Garﬁeld++ can be found in literature [3–5]. A fully parametrized simulation, GTS, is described in this work: it takes into account the primary ionization process, the generation of the avalanche in the GEM holes and the electron drift to the readout plane where the induction takes place. The description of the electronics is also taken into account. The results obtained from the simulation are compared with the experimental ones collected in a testbeam. 1 Introduction GTS is not a Garﬁeld++ competitor but a complementary tool to measure the triple-GEM performance, i.e. the spatial resolution, in a faster way than the one proposed by Garﬁeld++ through the parametrization of the main processes (multiplication and diﬀusion) in order to avoid the step by step simulation performed by Garﬁeld++. 3 Computation of inputs to GTS using the Garﬁeld++ simulation The simulation described in this work, is based on the idea described by Bonivento et al. [9], where the generation of the triple-GEM signal is divided into independent parts: ioniza- tion, GEM ampliﬁcation, electron drift in each gap, induction and electronics readout. These processes have been simulated with Garﬁeld++ and their output have been parametrized and used as input to the GTS simulation. The geometry description of the detector and its electric ﬁeld map were performed with Ansys software [10]. Every process is considered indepen- dent. The Garﬁeld++ results are shown in the following subsections. 2 Setup description The triple-GEM detector under study is deﬁned by ﬁve electrodes (cathode, three GEM foils, and the anode) and four gaps used to deﬁne the drift properties of the electrons: a drift gap between the cathode and the ﬁrst GEM of 5 mm, two transfer gaps between the GEM foils of 2 mm and an induction gap between the third GEM and the anode. A scheme of the detector is shown in Fig. 2 left. g A gas mixture of Ar+10%iC4H10 ﬁlls the detector volume and deﬁnes its primary ion- ization properties, the electron drift and the gain of a GEM foil. The high voltage used on each GEM is 275 V, while the electric ﬁeld in the diﬀerent gaps is 1.5/2.75/2.75/5 kV/cm. The ionizing particles used in the simulation and in the testbeam are 150 GeV/c momentum muons with variable incident angle with respect to the detector surface. The used readout electronics is the APV-25 ASIC [6], a chip that samples the signal every 25 ns with a shaping time of 50 ns. The experimental data [7] were collected in several testbeams at CERN North Area within the RD51 collaboration [8]. 3.1 Ionization Charged particles crossing a gas mixture lose their kinetic energy and generate primary elec- trons. These electrons constitute the signal that a triple-GEM detector measures. The ioniza- tion process is determined by two factors: the kinetic energy of the particle and the gas mix- ture used. The ionization was simulated separately from the subsequent processes. Several muons were shot in a volume with 5 mm gap to extract the following variables: the number of electrons clusters and the number of electrons inside each cluster. The distributions of those 2 EPJ Web of Conferences 245, 02025 (2020) CHEP 2019 https://doi.org/10.1051/epjconf/202024502025 two quantities are shown in Fig. 1. The primary ionization follows a Poissonian statistic and the most probable value was parametrized to be used in GTS. The number of primary electrons inside each electron cluster was tabulated up to 100 electrons per cluster. Figure 1: Left: distribution of the number of primary electrons (i.e. clusters) generated in the ionization created by 150 GeV/c muons in 5 mm Ar + 10%iC4H10; the histogram is ﬁtted with a Poissonian distribution. Right: distribution of the number of electrons per cluster; the histogram is tabulated to be implemented in the simulation. Figure 1: Left: distribution of the number of primary electrons (i.e. clusters) generated in the ionization created by 150 GeV/c muons in 5 mm Ar + 10%iC4H10; the histogram is ﬁtted with a Poissonian distribution. Right: distribution of the number of electrons per cluster; the histogram is tabulated to be implemented in the simulation. Figure 2: Left: eﬀective gain for a triple-GEM. The histogram is the result of the full chain of simulations of the gain, collection and extraction eﬃciencies for the three GEMs. The contribution of zero eﬀective gain is removed from the histogram. Right: sketch of a triple- GEM detector with the cathode, three ampliﬁcation stages and the anode. Each strip measures time and charge and continuous strips are used for CC and µTPC reconstruction methods. Figure 2: Left: eﬀective gain for a triple-GEM. The histogram is the result of the full chain of simulations of the gain, collection and extraction eﬃciencies for the three GEMs. The contribution of zero eﬀective gain is removed from the histogram. Right: sketch of a triple- GEM detector with the cathode, three ampliﬁcation stages and the anode. Each strip measures time and charge and continuous strips are used for CC and µTPC reconstruction methods. 3.2 GEM ampliﬁcation A simulation of a GEM foil was performed in an independent framework to measure the products of the interaction of the electron in the electric ﬁeld inside the hole: here the elec- trons are accelerated so much that they can ionize several times the gas. It is important to measure the number of electrons generated inside each hole but also the number of electrons that can be collected and extracted by a GEM. Some of the electrons drifting inside a GEM do not enter the hole and another fraction of the electrons generated inside the hole do not reach the next gap. The simulation of this portion of detector is used to extract the intrin- sic gain of the GEM and its transparency. This simulation studied the drift of the electrons from 150 µm before the foil up to 150 µm after the ampliﬁcation stage; this has been used 3 EPJ Web of Conferences 245, 02025 (2020) EPJ Web of Conferences 245, 02025 (2020) CHEP 2019 https://doi.org/10.1051/epjconf/202024502025 Figure 3: Left: distribution of the position shift, once an electron drifts in the transfer gap, is shown. Superimposed is the Gaussian ﬁt used to measure the mean value and the sigma. Center and right: the motion of the electrons in the drift gap has been evaluated in these ﬁgures. The position distribution on the ﬁrst GEM has been evaluated for electrons generated at several distances from the cathode. The drift gap has been divided in ten slices and each one of them a distribution has been ﬁtted with a Gaussian. The mean value of the Gaussian functions as a function of the position in the drift gap is shown in the center; while the sigma of these distributions is shown in the right ﬁgure. The three plots have been evaluated with 1 T magnetic ﬁeld. Figure 3: Left: distribution of the position shift, once an electron drifts in the transfer gap, is shown. Superimposed is the Gaussian ﬁt used to measure the mean value and the sigma. Center and right: the motion of the electrons in the drift gap has been evaluated in these ﬁgures. The position distribution on the ﬁrst GEM has been evaluated for electrons generated at several distances from the cathode. The drift gap has been divided in ten slices and each one of them a distribution has been ﬁtted with a Gaussian. 3.2 GEM ampliﬁcation The mean value of the Gaussian functions as a function of the position in the drift gap is shown in the center; while the sigma of these distributions is shown in the right ﬁgure. The three plots have been evaluated with 1 T magnetic ﬁeld. to extract the GEM transparency, as the product of the collection eﬃciency (the probability to enter the hole) and the extraction eﬃciency (the probability to leave it). Together with the transparency, the number of secondary electrons generated by one electron entering the hole was extracted and ﬁtted with a Polya distribution that well describes the ﬂuctuations of the gain of a single GEM. The GEM transparency depends on the electric ﬁelds involved: the one between the previous electrode and the GEM and the one between the GEM and the next electrode. For this reason the transparency of each GEM foil has been measured separately. The product of the transparency and the intrinsic gain gives the extrinsic gain. The full de- tector gain can not be evaluated by the product of the three Polya distribution. Therefore a detailed simulation of the three GEM ampliﬁcation stages was used to generate a distribu- tion that fully describes the multiplication in a triple-GEM detector: one million of electrons have been simulated and for each ampliﬁcation stage an intrinsic gain was sampled from the corresponding Polya distribution and transparency value, as shown in Fig. 2 right. 4 GTS simulation and position reconstruction The processes described in Sec. 3 deﬁne the details of a triple-GEM signal ampliﬁcation and their parametrization from Garﬁeld simulation. These were studied separately because they do not interfere at present setup conditions. Now the description of the GTS simulation is presented together with a simulation of the electronics and the position reconstruction. The simulation of an ionizing particle interacting with a triple-GEM starts from the ion- ization that generates the primary electrons in the drift gap. The inter-primary distance is randomized from an exponential function since the number of primary electrons follows a Poissonian statistics, see Fig. 1 left. For each primary, a certain number of secondary elec- trons are generated according to the distribution shown in Fig. 1 right. The transparency value is used to determine how many electrons survive to the ﬁrst GEM and the eﬀective gain distribution, shown in Fig. 2 left, to calculate the number of secondary electrons generated by each primary. The secondary electrons are randomly smeared in space and time in agreement with a Gaussian distribution whose mean value and sigma are those shown in Fig. 3. At the end, the ﬁnal position of the electrons is used to determine the strip position for the readout and the signal generated by the electronics. Each electron reaching the anode is associated to the proper strip; its arrival time together with its charge is used to deﬁne the induced cur- rent. This is an approximation of the real induction process described by the Ramo [11] and Riegler [12] theorems. The induced current, shown in Fig. 4 top left, was used to determine the signal exiting the ASIC with the simulation of a pre-ampliﬁer that integrates the charge and a shaper with integration time of 50 ns to determine the fraction of charge readout, see Fig. 4 mid-left and mid-right. A Gaussian noise was included for each channel, as well as threshold levels compatible with the testbeam condition. Once the readout signal was generated strip by strip, the charge and time information were used to apply two reconstruction algorithms on clusters of contiguous strips: the charge centroid (CC) and the micro-Time Projection Chamber (µTPC). 3.3 Electron drift Drift properties such as transverse and longitudinal diﬀusion strongly depend on the gas mixture and the electromagnetic ﬁeld. These values are well known in literature and a parametrization was performed based on the results of the Garﬁeld++ simulation. A sim- ulation of the electron drift was run for each gap and the distribution of the space shift was evaluated: i.e. ten thousand electrons were drifted from 150 µm below the ﬁrst GEM up to 150 µm before the second GEM. A Gaussian ﬁt was used to extract the mean value and the sigma of the distribution, see Fig. 3 left. The same procedure was followed for the other transfer gap and the induction gap. The drift in these gaps is performed by each electron gen- erated before the ﬁrst GEM. This method can not be used in the drift gap, where the primary ionization can take place in diﬀerent positions and the path of each electron is diﬀerent from the other one. Here a simulation of the electron drift was performed for diﬀerent distances from the ﬁrst GEM. A Gaussian ﬁt has been used for each point to extract the mean value and the sigma as a function of the distance from the ﬁrst GEM: Fig. 3 center and right show these results. The same studies have been performed for the time information. 4 https://doi.org/10.1051/epjconf/202024502025 EPJ Web of Conferences 245, 02025 (2020) CHEP 2019 4 GTS simulation and position reconstruction The CC performs a weighted average of the strips position with their charge as weight, while the µTPC uses the time information to measure the distance between the particle path and the anode: a bi-dimensional point is associated to each strip and a straight line ﬁt is used to measure the particle position. A sketch of the two methods is shown in Fig. 2 right, while the formulas are described in Eq. 1: xCC = Pcl.size i=0 xhit,i qhit,i Pcl.size i=0 qhit,i ; xµTPC = gap/2−b a (1) (1) where a is the slope of the ﬁtting line, b is the constant and gap is the drift gap thickness. where a is the slope of the ﬁtting line, b is the constant and gap is the drift gap thickness. 5 Tuning with experimental data Despite the detailed simulation performed with Garﬁeld++ used to implement the parameter in GTS, the simulated results diﬀer from the ones measured in the experimental setup. The reason is due to some approximation applied in the GTS simulation and some known prob- lem in the Garﬁeld++ simulations [13, 14]. Four sentinel variables were used to tune the simulations: strip-cluster charge, strip-cluster size, CC and µTPC spatial resolution. A com- parison of these values from the experimental results and the simulation has been performed for diﬀerent incident angles. A χ2 evaluation was used to measure the matching of the values. A set of conﬁgurations was evaluated to minimize the χ2 and the best results were ob- tained using a tuning factor on the gain and on the electron diﬀusion. The results of the tuning are shown in Fig. 5. 5 5 5 EPJ Web of Conferences 245, 02025 (2020) EPJ Web of Conferences 245, 02025 (2020) CHEP 2019 https://doi.org/10.1051/epjconf/202024502025 Figure 4: Top left: induced current on a single strip in a single particle simulation. Top right: shape of the current after the pre-ampliﬁer simulation on a single strip. Bottom left: shape of the current after a shaper simulation with a RC-CR circuit having an integration time of 50 ns. Bottom right: example of the signal measured by a real event from an experimental setup to be compared with the previous picture. Figure 4: Top left: induced current on a single strip in a single particle simulation. Top right: shape of the current after the pre-ampliﬁer simulation on a single strip. Bottom left: shape of the current after a shaper simulation with a RC-CR circuit having an integration time of 50 ns. Bottom right: example of the signal measured by a real event from an experimental setup to be compared with the previous picture. 6 Conclusion A detailed parametrized simulation of a triple-GEM detector has been performed starting from Garﬁeld++ simulation. The simulation takes into account also the eﬀects of the elec- tronics and a tuning procedure of the data. This new approach exploits a parametrization of the Garﬁeld++ output to reduce signiﬁcantly the computational time up to few seconds to generate the charge and time distribution of the electron at the anode. With this parametriza- tion of the distributions obtained with the Garﬁeld++ simulation, tuned to better reproduce the real test beam data, we are able to describe satisfactorily the GEM detector response. Therefore, this tool can be used to quickly emulate the response of other possible detector conﬁgurations. Acknowledgments This work is supported by the Italian Institute of Nuclear Physics (INFN). The research leading to these results has been performed within the FEST Project, funded by the European Commission in the call RISE-MSCA-H2020-2020. 6 6 https://doi.org/10.1051/epjconf/202024502025 EPJ Web of Conferences 245, 02025 (2020) CHEP 2019 Figure 5: Experimental and simulated results for the four sentinel variables as a function of the incident angle: cluster charge (top left), cluster size (top right), CC resolution (bottom left) and µTPC (bottom right). The tuning values have been already applied in the shown set of results. Figure 5: Experimental and simulated results for the four sentinel variables as a function of the incident angle: cluster charge (top left), cluster size (top right), CC resolution (bottom left) and µTPC (bottom right). The tuning values have been already applied in the shown set of results. [12] W. Riegler, Extended theorems for signal induction in particle detectors VCI 2004, Nucl. Instr. and Meth. A 535, 287-293, DOI:10.1016/j.nima.2004.07.129 (2004). [14] A. Sharma, A How-to Approach for a 3d Simulation of Charge Transfer Characteristics in a Gas Electron Multiplier, ICFA Instrum.Bull. 19 (1999) 2 [11] S. Ramo, Currents Induced by Electron Motion, Proc. of the IRE 27(9), 584–585, DOI:10.1109/JRPROC.1939.228757 (1939). References [1] F. Sauli, GEM: A new concept for electron ampliﬁcation in gas detectors, Nucl. Instr. and Meth. A 386, 531–534, DOI:10.1016/S0168-9002(96)01172-2 (1997) [2] R. Veenhof, GARFIELD, recent developments, Nucl. Instr. and Meth. A 419, 726-730, DOI:10.1016/S0168-9002(98)00851-1 (1998) [3] S. Swain et al., A Simulation Model for Triple GEM Detector, ADNHEAP 2017, 223- 229 (2017) [4] D. Baranov et al., Simulation of the GEM detector for BM@N experiment, EPJ Web of Conferences 138, 11004 (2017) [5] V. Brio et al., Simulations of Triple-GEM tracker’s response for experiments at Jeﬀerson Lab, Radiation Eﬀects and Defects in Solids, 174:11-12, 1031-1046 (2019) [6] L. L. Jones et al., The APV25 Deep Submicron Readout Chip for CMS Detectors, Conf. Proc. C 9909201 162-166, DOI:10.5170/CERN-1999-009.162 (1999) [7] M. Alexeev et al., Triple GEM performance in magnetic ﬁeld, 2019 JINST 14 P08018, DOI:10.1088/1748-0221/14/08/P08018 (2019) [7] M. Alexeev et al., Triple GEM performance in ma DOI:10.1088/1748-0221/14/08/P08018 (2019) [8] http://rd51-public.web.cern.ch/rd51-public/ [9] W. Bonivento et al., A Complete Simulation of a Triple-GEM Detector, IEEE Trans. Nucl. Sci. 49(4), DOI:10.1109/TNS.2002.805170 (2002) 7 https://doi.org/10.1051/epjconf/202024502025 EPJ Web of Conferences 245, 02025 (2020) CHEP 2019 [13] Rob Veenhof, private communication [13] Rob Veenhof, private communication [14] A. Sharma, A How-to Approach for a 3d Simulation of Charge Transfer Characteristics in a Gas Electron Multiplier, ICFA Instrum.Bull. 19 (1999) 2 8 8
https://openalex.org/W2032140135	https://www.scielo.br/j/rbh/a/886ZsW8YGYsjtFdqJJ7bCsb/?lang=pt&format=pdf	Portuguese	null	Estratégias populares de sobrevivência: o mutualismo no Rio de Janeiro republicano	Revista brasileira de história	2,009	cc-by	10,588	* Professora Associada do Programa de Pós-Graduação em História da UFJF. Doutora em História Social pela UFRJ. Bolsista de produtividade do CNPq. Laboratório de História Política e Social (LAHPS), Departamento de História/ICH, salas 26 e 27. Campus Universitário (UFJF), Bairro Martelos. 36036-330 Juiz de Fora – MG – Brasil. claudia.viscardi@ufjf.edu.br Estratégias populares de sobrevivência: o mutualismo no Rio de Janeiro republicano1 Popular strategies of survival: mutualism in the republican Rio de Janeiro Popular strategies of survival: mutualism in the republican Rio de Janeiro Cláudia Maria Ribeiro Viscardi* Abstract Como narra a historiografia sobre o tema, especialmente relacio- nada à Europa Ocidental, no período medieval a pobreza era vista como vir- tude daqueles que por ela optavam com o fim de expurgar pressupostos peca- dos. Mais tarde, constituiu-se em indício da presença de vícios, como a preguiça, o desânimo, a imprudência, ou da ausência de caráter. Ao final do século XIX, passou a ser vista como um mal derivado das circunstâncias his- tóricas, e responsabilizaram-se agentes econômicos impessoais, como o mer- cado, os mecanismos desiguais de distribuição de renda, o desemprego e tan- tos outros.2 Tais diferentes olhares sobre a pobreza foram acompanhados de estratégias construídas para a sua superação ou amenização, as quais igualmente variaram no tempo e no espaço. As elites políticas e econômicas, os religiosos, os filan- tropos laicos, os intelectuais e os cientistas, entre outros setores sociais, não se eximiram de opinar sobre o problema ou de agir sobre ele. Tão logo a pobreza foi estigmatizada, deixando de ser uma virtude responsável pela salvação de suas vítimas, os filantropos (laicos ou religiosos) empenharam-se para minorá- la mediante inúmeras ações, que incluíam a doação de alimentos, roupas, re- médios e dinheiro, entre outros itens. Como o pauperismo não regredia, a solução foi investir na construção de abrigos, que assumiram o formato de hospitais, hospícios, albergues, orfanatos ou asilos. Em todo o mundo ociden- tal tais soluções se repetiram, de forma minimamente diferenciada. Em geral, quando se aborda o tema da pobreza, as preocupações se voltam para os não pobres, que lutam por dirimi-la. Pouca atenção é conferida para as estratégias que os próprios pobres engendravam, em sua luta cotidiana pe- la sobrevivência. Quando o tema é assistência ou seguridade social, pouco se fala sobre os incontáveis momentos em que os próprios pobres se ajudavam mutuamente. Mesmo vitimados por mazelas comuns, encontravam meios de expandir laços de solidariedade entre si, responsáveis pelo fortalecimento de elos comunitários, sem os quais dificilmente se manteriam vivos. Ações simples como o cuidado com as crianças dos vizinhos, os pequenos emprésti- mos e o abrigo voluntário a quem dele não dispusesse, entre outras colaborações, são fartamente encontradas nas mais diversas comunidades carentes, desde tempos imemoriais. Por essa razão, Robert Castel chamou-nos a atenção para o personagem que adveio no novo roteiro inaugurado com a implantação do capitalismo no campo: o “desfiliado”. Abstract Trata-se de um estudo sobre o mutualis- mo na cidade do Rio de Janeiro, visto como uma das estratégias escolhidas pe- los trabalhadores para escapar às condi- ções de pobreza em que viviam ou, pelo menos, amenizá-las. O artigo busca ofe- recer um quadro geral do fenômeno na então capital brasileira, levando em con- ta sua dimensão, duração, trajetória e principais características. Ao mesmo tempo, procura realçar quais setores eram excluídos do movimento, enten- dendo que sua identidade era definida com base no estabelecimento de algumas fronteiras delimitadoras entre setores sociais distintos. Busca-se estabelecer um diálogo com os trabalhos produzidos sobre o mutualismo, no Brasil e, sobre- tudo, no exterior. Foram utilizados, co- mo fontes primárias, dois levantamentos inéditos produzidos sobre o fenômeno no Rio de Janeiro, além de informações anteriormente referenciadas por outros autores, às quais tivemos acesso. This article is about friendly societies movement in the city of Rio de Janeiro, seen as one of the strategies chosen by the workers to face poverty and to im- prove life conditions. The article intends to offer a general overview about the mutual movement in the Brazilian capi- tal at that moment, considering differ- ent aspects as its dimensions, lifetime, trajectory, and main characteristics. At the same time, it aims to stress sectors excluded from the movement, whose identity was based on the established frontiers that separated distinct social sectors. The article intents, as well, to promote a dialogue with other research- es about friendly societies in Brazil, and especially the ones produced abroad. We used, as the main sources, two sta- tistic series never studied before, about the phenomenon in Rio de Janeiro, be- sides other group of sources. Keywords: friendly societies; workers; poverty. Palavras-chave: sociedades de auxílio mútuo; trabalhadores; pobreza. * Professora Associada do Programa de Pós-Graduação em História da UFJF. Doutora em História Social pela UFRJ. Bolsista de produtividade do CNPq. Laboratório de História Política e Social (LAHPS), Departamento de História/ICH, salas 26 e 27. Campus Universitário (UFJF), Bairro Martelos. 36036-330 Juiz de Fora – MG – Brasil. claudia.viscardi@ufjf.edu.br Revista Brasileira de História. São Paulo, v. 29, nº 58, p. 291-315 - 2009 Revista Brasileira de História. São Paulo, v. 29, nº 58, p. 291-315 - 2009 Cláudia Maria Ribeiro Viscardi Historicamente, o olhar sobre a pobreza permitiu diferentes abordagens em relação a suas causas, sua contingência real ou ilusória e alternativas de sua superação. Revista Brasileira de História, vol. 29, nº 58 Abstract Para o autor, esse personagem se definia como o trabalhador que, ao não encontrar mais condições de ocupação no Revista Brasileira de História, vol. 29, nº 58 292 Estratégias populares de sobrevivência... campo, em razão das mudanças lá introduzidas, abandonava a sua comunida- de de origem e se lançava numa aventura nova em busca de sobrevivência, normalmente migrando das áreas rurais para as cidades. Tal movimento mi- gratório rompia com os laços de proteção comunitária em que esse personagem se enredava, tornando-o um desfiliado (Castel, 1998, p.43). Os pobres possuem à sua disposição algumas estratégias de superação da pobreza. Mas elas podem ser agrupadas em duas modalidades de escolha, ra- ramente excludentes: o apelo por proteção de alguém que dispõe de bens dis- poníveis a serem doados, e o recurso à ajuda mútua, na qual o próprio grupo se apoia, estabelecendo redes de colaboração entre seus membros. Daí decorrem dois tipos de relação social. No primeiro caso, o outro a quem se recorre pode assumir a face de uma Igreja, do Estado, do cidadão benemérito ou do coronel, e outras tantas variações que se encaixam bem no conceito de paternalismo. Tal relação tende a ter um trajeto verticalizado, no qual o doador estabelece com o receptor uma hierarquia cujo tom, à revelia das intenções ou motivações, será o do poder de quem doa sobre quem recebe. Nessa situação, o receptor se encontrará submetido ao doador, mesmo que este último não se utilize da relação em seu próprio proveito. No segundo caso, o outro é um igual, aquele que compartilha das mesmas necessidades e potencialidades. Juntos, desenvolvem relações de reciprocidade que tendem a ser mais balanceadas. Todos contribuem e todos recebem a contribuição. Re- alçam o ethos da obrigação mútua e a responsabilidade coletiva pelo bem-estar dos outros. Nesse contexto as relações tendem a ser mais horizontalizadas, e as hierarquias, menos definidas. A dependência persiste, mas assume um ca- ráter mútuo. A definição e a mensuração da pobreza sempre se constituíram em pro- blema para os cientistas sociais e, sobretudo, para os historiadores, na medida em que o conceito variou no tempo e no espaço. Para os fins desta pesquisa, optou-se por adotar um conceito mais geral, por ser mais adequado à realidade brasileira nas primeiras décadas do século XX, foco do presente artigo. Abstract Udaya Wagle sugere a junção de três referenciais para que se possa con- ceituar melhor a pobreza: o referencial econômico, o capacitário e o da exclu- são social. Dessa forma, a autora entende a pobreza como a ausência de renda e riqueza (econômico); de educação e saúde, condições que capacitam os in- divíduos a adquirirem renda ou riqueza (capacitário); e a presença da exclusão étnica ou de gênero, o que impede que os indivíduos participem politicamen- te da vida cívica e cultural de uma sociedade (referencial da exclusão social).3 Nesse sentido, os recursos à filantropia e ao mutualismo podem ser lidos 293 Dezembro de 2009 Cláudia Maria Ribeiro Viscardi como duas das inúmeras alternativas disponíveis aos pobres para a superação de determinadas carências, resultantes da situação de pobreza. Graças à filan- tropia era possível obter, em parte, condições para complementar a renda, manter ou melhorar a saúde ou ter acesso à educação. No que tange à estraté- gia mutualista, os trabalhadores poderiam garantir por meio dela uma renda complementar, o acesso ao atendimento médico, ou diminuir os efeitos da exclusão social, na medida em que tais sociedades ofereciam espaços de for- mação de uma cultura cívica e, eventualmente, de participação política. É sobre uma dessas alternativas de enfrentamento da pobreza que preten- demos refletir neste artigo. Para tal, escolhemos o recurso à ajuda mútua, atra- vés da participação dos trabalhadores em associações mutualistas, muito em- bora a filiação a uma mutual estivesse longe de garantir imunidade contra o empobrecimento. Além disso, recorrer à autoajuda ou submeter-se à caridade muitas vezes não derivavam de escolhas racionais dos agentes vitimados pela pobreza, mas de inúmeros outros fatores que os levavam à condição ou de sujeitos de sua própria sobrevivência, ou de objetos da boa vontade alheia, ou mesmo de ambos. Fazem parte do debate sobre o mutualismo as teses que alegam ter sido o movimento composto não por trabalhadores pobres, mas pela “aristocracia operária”.4 Ademais, sabe-se que alguns indivíduos bem aquinhoados poderiam compor uma mutual, embora não se constituíssem na maioria de seus sócios. A despeito de tais ponderações, é inegável que o movimento associativo mu- tualista foi composto, sobretudo, por trabalhadores – assalariados ou não – que por não serem ricos, precisavam garantir a sua sobrevivência e a de seus fami- liares em momentos de infortúnio. Abstract Dessa forma, as mutuais recrutaram seus sócios preferencialmente entre os trabalhadores humildes, que não fossem totalmente destituídos ou marginalizados, mas que delas necessitassem, por não disporem nem da proteção do Estado nem de riquezas acumuladas. Na ausência das mutuais, só lhes restaria a caridade alheia. A proximidade entre tais estratégias de sobrevivência levou grande parte de seus agentes a ter dificuldade no estabelecimento das fronteiras entre mu- tualismo e filantropia. Tanto no ocidente europeu como no Brasil, várias mutuais praticavam caridade, e os legisladores tiveram problemas em definir as sociedades como de socorro mútuo, beneficentes, filantrópicas, seguradoras privadas com fins lucrativos, ou mesmo sindicais. Simon Cordery afirma que no Reino Unido, só a partir de 1860 se estabeleceu uma melhor distinção entre caridade e ajuda mútua, após a ocorrência de mudanças culturais e políticas. 294 Revista Brasileira de História, vol. 29, nº 58 Estratégias populares de sobrevivência... No Brasil, em trabalho anterior, tivemos a oportunidade de refletir sobre a ausência de distinções entre as duas práticas, ao final do século XIX.5 Com o fim de fundamentar as reflexões aqui propostas, escolhemos a cidade do Rio de Janeiro, nas primeiras décadas do século XX. Pretendemos acompanhar de que forma os pobres se ampararam mutuamente, em um pe- ríodo marcado pelo intenso processo de modernização. Em definição mais clássica, a modernização pode ser entendida como um conjunto de mudanças sociais e políticas que acompanharam a industrialização. Ao longo desse processo, ocorreu a expansão da autoridade pública e da cida- dania. Para Bendix, esse intenso movimento de mudança implicou a coexis- tência entre o tradicional e o moderno nos países onde ocorreu, na medida em que a modernidade implantada foi necessariamente pautada pela tradição acu- mulada ao longo dos anos anteriores.6 No Brasil, as primeiras décadas do século XX soaram sob o tom da mo- dernização. A abolição havia retirado as amarras que freavam a expansão da cidadania. E o fim da Monarquia, ao descentralizar as estruturas governamen- tais, permitira maior expansão da autoridade pública. No que diz respeito à coexistência entre o tradicional e o moderno, típica desse período de transição, pode-se afiançar que o recurso à filantropia deno- tava a permanência de relações paternalistas tradicionais, que sedimentavam a cultura política brasileira desde o período colonial. Dezembro de 2009 Abstract Porém, o recurso à ajuda mútua pode ser lido como uma estratégia típica dos contextos nos quais o processo de modernização entrava em curso, na medida em que era capaz de conferir a seus agentes valores muito difundidos pelo discurso liberal dos con- temporâneos, tais como os da respeitabilidade, da autonomia e da liberdade. Não pretendemos com tais relações afiançar que a modernidade, tão logo implantada, teria posto fim às relações de caridade que interligavam pobres e ricos, receptores e doadores de ajuda. Mas pode-se afirmar que o seu avanço, sobretudo nos locais onde ocorreu significativa expansão da cidadania, reduziu as estratégias populares de recorrer-se à filantropia. Abram de Swaan destaca que nos países onde inexistia seguridade social, o apelo para a generosidade das pessoas era muito grande. Após ser instituído o sistema de contribuição compulsória, que garantiu aos pobres a proteção social necessária, retirou-se um pouco da tragédia e também um pouco da magia relativa à religiosidade do homem ocidental.7 Na Europa, o advento do Welfare State foi responsável pela redução sig- nificativa do recurso à caridade, embora não tenha posto um fim definitivo às ações filantrópicas, mesmo em seu território. Mas é incontestável admitir que 295 Cláudia Maria Ribeiro Viscardi tais ações voltaram-se majoritariamente para outros países e internacionaliza- ram as relações de caridade, que se tornaram mais impessoais. Atualmente, as Ongs vêm cumprindo um papel cada vez mais efetivo na institucionalização desse tipo de ajuda. Referindo-se a esse contexto, Godelier afirma que as dá- divas tornaram-se institucionalizadas, e o dom passou a ser um ato a ligar sujeitos abstratos.8 Tendo por alvo a análise de uma das mais frequentes estratégias escolhidas pelos pobres, como meio de suprir suas necessidades mais imediatas – o recur- so à ajuda mútua –, voltaremos agora nosso olhar para o Rio de Janeiro repu- blicano, enfocando esse conjunto de experiências na então capital do Brasil. O mutualismo no Rio de Janeiro sob as lentes dos historiadores Do que temos conhecimento, o mutualismo carioca foi estudado pionei- ramente por Michael Conniff, em 1975.9 O autor teve como objetivo mapear o comportamento associativo do município entre 1870 e 1945. Com esse fim, lançou mão de alguns levantamentos produzidos por encomenda dos governos Imperial e Republicano. Ao analisar as várias modalidades de associações, levou em conta as de caráter mutualista, por ele qualificadas como entidades laicas, inicialmente compostas por trabalhadores qualificados e de renda estável. Mais tarde, o fenômeno teria se tornado mais difuso, ampliando o espectro de as- sociados. O autor confere destaque especial às associações dos funcionários públicos e às associações de trabalhadores por categoria ou empresa. Porém, não trata essas sociedades como mutualistas, embora o fossem em sua maioria. Talvez por essa razão, tenha encontrado tão poucas mutuais no Rio de Janeiro republicano. Em que pese o pouco relevo conferido ao fenômeno, enquanto espaço de representação de interesses, para ele o movimento contribuiu com a explicitação de uma série de conflitos sociais, responsáveis por trazer à tona a chamada ‘questão social’. Mais de dez anos depois, Beatriz Kushnir 10 abordou o tema, embora sua pesquisa não tenha tido como objetivo precípuo estudar o fenômeno mutua- lista de forma global. Seu trabalho, acerca da prostituição e da sexualidade das chamadas ‘polacas’ identificou a existência de cinco sociedades mutualistas nas cidades do Rio de Janeiro, Santos, São Paulo, Buenos Aires e Nova York. Tais sociedades atuavam como mecanismos de proteção das mulheres – em sua maioria judias – em meio às situações adversas em que viviam. Cada uma das 296 Revista Brasileira de História, vol. 29, nº 58 Estratégias populares de sobrevivência... associações tinha uma sede social própria, um cemitério e uma sinagoga, além de todo um aparato administrativo eficiente. Graças às mutuais, aquelas mu- lheres em situação de risco conseguiam reforçar uma identidade afirmativa, amenizando em parte os inconvenientes resultantes de sua marginalidade so- cial. Muito embora o conhecimento sobre a existência de tais sociedades seja relevante para o entendimento do fenômeno mutualista, sabe-se que se cons- tituíram em exceções, na medida em que as mutuais, em sua maioria, eram espaços masculinos. Cláudio Batalha, ao analisar um conjunto variado de fontes, identificou que a maior parte das sociedades de trabalhadores formadas na cidade do Rio de Janeiro entre 1835 e 1899 era de caráter mutualista. Dezembro de 2009 O mutualismo no Rio de Janeiro sob as lentes dos historiadores Em seu trabalho abor- da as relações dessas sociedades com a escravidão, com o republicanismo e com o movimento sindical. Levanta a hipótese de que as mutuais eram os únicos espaços legalmente disponíveis aos trabalhadores manuais que quises- sem se organizar após 1824. Por essa razão, muitas mutuais se utilizavam des- se precedente para atender ao seu objetivo primordial, que era o da defesa profissional. Batalha leva em conta também as relações existentes entre as mu- tuais, irmandades e corporações de ofícios, modalidades diferentes de organi- zação da sociedade civil, mas que compartilhavam uma série de valores, rituais e práticas comuns.11 Mais tarde, o trabalho de Ronaldo de Jesus,12 focado nas relações entre o povo e o monarca, tal como Batalha, utilizou igualmente do conjunto docu- mental disponível no Conselho de Estado. Em sua análise destacou que algumas mutuais recorriam ao Império para requisitar algum tipo de apoio que lhes garantisse a subsistência. Quando conseguiam, podiam usar o prefixo “Impe- rial” em seus nomes. O autor destaca a existência de mutuais compostas ex- clusivamente por escravos, algumas delas formadas para financiar a compra de alforrias. Encontrou outras, de caráter menos popular, que recebiam o apoio de setores da elite, levando-as a travar compromissos políticos que resultaram, na maioria das vezes, em restrição de sua autonomia. Em levantamento posterior realizado sobre a mesma documentação, o mesmo autor encontrou 239 associações funcionando no Rio de Janeiro, entre 1860 e 1889. Delas realçou as compostas por trabalhadores: ourives, tipógrafos, artistas da construção naval, maquinistas, alfaiates e operários fabris, entre outros. Ronaldo de Jesus identificou tais sociedades como espaços de constru- ção de relações de solidariedade horizontais e levantou a hipótese de que, a despeito de as sociedades terem sido marcadas pelos mecanismos tradicionais de dominação política, próprios da sociedade escravista monárquica, suas ex- 297 Dezembro de 2009 Cláudia Maria Ribeiro Viscardi periências contribuíram para a formação de uma cultura emergente, específi- ca dos subordinados.13 Recentemente, Vitor Fonseca14 voltou seu olhar sobre o fenômeno asso- ciativista da cidade do Rio de Janeiro, de forma mais ampla, incluindo em suas análises as mutuais, as associações filantrópicas, recreativas, religiosas, de lazer etc. Revista Brasileira de História, vol. 29, nº 58 O mutualismo no Rio de Janeiro sob as lentes dos historiadores Para a produção de um levantamento inédito do fenômeno associativo na cidade, no período compreendido entre 1903 e 1916, o autor se utilizou prio- ritariamente dos estatutos depositados no Registro Especial de Títulos e Do- cumentos do Rio de Janeiro, bem como de dados referentes a associações en- contrados no Almanaque Laemmert. No trabalho produzido consta um acompanhamento minucioso de todo o processo de regulamentação jurídica do fenômeno associativo no Brasil, realizado tanto no Império como na Re- pública. Destaca-se, igualmente, a análise empreendida pelo autor sobre as sedes e os diplomas de várias associações. Para os fins deste artigo, procuramos acessar uma documentação alterna- tiva e complementar às que foram pesquisadas, muito embora, em projeto anterior, tenhamos trabalhado também com a documentação depositada no Conselho de Estado, da qual igualmente lançaremos mão. Assim, utilizaremos dois levantamentos das associações da cidade do Rio de Janeiro, os quais foram produzidos no início do século XX. O primeiro levantamento foi feito por Ferreira da Rosa, a pedido de Pe- reira Passos.15 O autor publicou um livro ilustrado sobre a cidade do Rio de Janeiro, do qual constam um breve histórico sobre o município e inúmeras informações sobre seu sistema viário, seus serviços de instrução pública, polí- cia, abastecimento de água, sua economia e os serviços de assistência. Ao final da obra há um anexo com 181 associações por ele conhecidas, no ano de 1902. Os dados foram recolhidos de 1815 a 1904 (e o livro foi publicado no ano se- guinte). Por trabalhar com um pequeno número de associações e com critérios mais homogêneos, os dados de Rosa são muito úteis. Na gestão do prefeito Bento Ribeiro (1910-1914), o advogado, escritor e jornalista Ataulfo de Paiva foi incumbido de realizar um segundo levantamen- to estatístico geral de todos os estabelecimentos de assistência pública e priva- da, existentes na capital. Esse levantamento – no qual foram incluídas as asso- ciações mutualistas – buscava produzir um diagnóstico que orientasse o combate à crescente pobreza que avassalava o Rio de Janeiro. A encomenda, iniciada em 1903, só pôde ser concluída em 1922, quando foi publicada em edição comemorativa do centenário de “nascimento” do Brasil.16 Para esse fim, Paiva organizou três tipos diferentes de questionários, que Revista Brasileira de História, vol. 29, nº 58 298 Estratégias populares de sobrevivência... foram enviados às associações de auxílio mútuo, aos asilos e recolhimentos e a hospitais. O mutualismo no Rio de Janeiro sob as lentes dos historiadores Ademais, visitou quase todas as instituições sobre as quais obteve informações através do questionário. Foram recenseadas 624 instituições, que se encontravam em funcionamento entre os anos de 1912 e 1920 no Rio de Janeiro. Com base nessa importante documentação, discutiremos o fenômeno na cidade do Rio de Janeiro sobre três eixos: a composição do movimento, sua dimensão e sua trajetória. Dezembro de 2009 Os incluídos e os excluídos Raramente as mutuais se construíram em bases igualitárias. Muito embo- ra definissem sua identidade a partir da isonomia e da ajuda mútua, eram es- truturas hierarquizadas e excludentes. Para integrar uma mutual era preciso, em primeiro lugar, possuir renda regular, que possibilitasse o pagamento das mensalidades, embora elas fossem baixas. Desse critério já estavam excluídos os desempregados, os miseráveis e os trabalhadores sazonais, entre outros. Mas não era essa a única fronteira que afastava do movimento boa parcela da so- ciedade civil. Havia outros critérios excludentes como os etários, raciais, de gênero, de status ou de região. Tal fenômeno não foi exclusivamente brasilei- ro, mas se repetiu em diversos locais do mundo, com ênfase em um ou outro critério, dependendo do país. Ao se constituírem em espaços de ajuda solidária, tentavam obscurecer as diferenças sociais entre seus membros. Em contraposição, realçavam outras diversidades, tais como as de cor, de gênero e de nacionalidade. Nesse sentido, as mutuais esforçaram-se em negar a importância das diferenças de classe, oferecendo idade, gênero e raça como categorias apropriadas para a organiza- ção de identidades coletivas. Mas enquanto a importância social da instituição mutual é reconhecida, há pouco consenso e menos clareza teórica sobre o seu papel na construção da identidade de classe nesse período. Sua composição multiclassista pode ter facilitado mais uma solidariedade interclassista do que intraclassista. Por essa e por outras razões, alguns historiadores questionam a contribuição dada pelo mutualismo ao processo de formação de uma classe trabalhadora homogênea e igualitária.17 No Brasil percebem-se, ao menos, quatro modalidades de exclusão: por renda ou profissão, por gênero, por idade e por nacionalidade. A de gênero era a mais visível. Mary Clawson afirma que organizações como as corporações de ofício, irmandades, maçonaria, sociedades de socorros mútuos e demais fra- 299 Dezembro de 2009 Cláudia Maria Ribeiro Viscardi ternidades tinham em comum o fato de se constituírem em espaços de reforço de alguns valores. Entre eles destacava-se o da masculinidade, e a identidade masculina era lida como autoridade patriarcal. O fraternalismo servia, entre outras coisas, para delimitar fronteiras de exclusão daqueles que não tivessem o status de homens e adultos (Clawson, 1989, p.45-52). Num contexto em que a maioria das mulheres não se encontrava no mercado de trabalho, a interação social era dificultada pela sua permanência no lar. Revista Brasileira de História, vol. 29, nº 58 Os incluídos e os excluídos Daí sua pouca participação nos espaços de sociabilidade que se multiplicavam nas cidades, a partir do final do século XIX. Em relação à cidade do Rio de Janeiro, o levantamento de Paiva nos oferece dados referentes a mulheres e estrangeiros. Como tais dados não foram obtidos de maneira uniforme, Paiva só apresenta o número deles para o ano de 1912. Não obstante, os resultados são ilustrativos, e é interessante reproduzi-los. Tabela 1 – Composição dos Sócios do Rio de Janeiro em 1912 Tabela 1 – Composição dos Sócios do Rio de Janeiro em 1912 Nacionalidade Homens Mulheres Total Brasileira 98.752 20.888 119.640 Estrangeira 98.108 4.334 102.442 Ignorada 55.213 5.642 60.855 Total 252.073 30.864 282.937 Fonte: Dados obtidos em Prefeitura do Distrito Federal, 1922, p.745. Os dados mostram que as associações eram compostas em sua grande maioria por homens (89%), e, entre eles, 38,92% eram estrangeiros. Tendo sido a cidade um polo assaz atrativo para os imigrantes, e sendo o mutualismo já uma tradição em seus países de origem, era normal que os estrangeiros se associassem em grande número. Ademais, as mutuais funcionavam para eles como espaços de sociabilidade, fortalecimento e conservação de suas identi- dades nacionais, na medida em que reproduziam as divisões de seus respecti- vos países na Europa. Nos Estados Unidos os negros eram proibidos de se filiar a mutuais de brancos. Por essa razão criaram as suas próprias mutuais, que, por sua vez, excluíam os brancos.18 No Brasil havia algumas mutuais de ex-escravos ou de negros, mas em muito menor número. Nos estatutos aos quais tivemos acesso, que compunham a documentação já citada do Conselho de Estado, não en- 300 Revista Brasileira de História, vol. 29, nº 58 Estratégias populares de sobrevivência... contramos muitas restrições à cor. Mas a maior parte das mutuais de imigran- tes impunha restrições a sócios de outras nacionalidades. Em levantamento feito sobre essa documentação, encontramos 21 mutu- ais reconhecidamente de imigrantes num universo de 123 sociedades, no pe- ríodo entre 1860 e 1882. Para compor uma amostra mais homogênea, retiramos do levantamento as associações patronais, filantrópicas, religiosas, recreativas e de lazer, científicas ou literárias, os montepios, as cooperativas e seguradoras, cujos processos também integravam o conjunto documental. Entre elas havia: cinco portuguesas, três francesas, três alemãs, três italianas, duas suíças e cin- co de outras nacionalidades variadas. Tal como no Brasil, no Reino Unido a principal discriminação era de gênero. Os incluídos e os excluídos Embora as mutuais masculinas fossem mais numerosas, havia socie- dades que aceitavam mulheres ou eram compostas só por elas, especialmente do final do século XVIII às primeiras décadas do XIX. Em geral, tinham pou- ca duração e não sobreviviam mais que trinta anos. Em boa parte dessas mu- tuais, as funções administrativas, que demandavam maior conhecimento téc- nico, eram entregues a homens.19 Retomando os dados de Paiva, percebe-se pelo quadro que as mulheres, brasileiras ou estrangeiras, compunham aproximadamente 11% do número total de sócios, e o número de brasileiras era cinco vezes maior que o de es- trangeiras. Ou seja, independentemente da nacionalidade, as mulheres que moravam no Rio foram majoritariamente excluídas do associativismo. Esses números referem-se a todos os tipos de sociedades, não somente as mutuais. Como as mulheres tinham maior participação nas associações de caráter reli- gioso ou filantrópico incluídas na amostra, o percentual de participação femi- nina nas mutuais deve ter sido ainda mais baixo. Dezembro de 2009 As mutuais e seus sócios em números A quantificação das mutuais e de seus sócios é sempre um desafio para os estudiosos. Na maior parte das vezes não se encontra disponível, para um conjunto expressivo de mutuais, a sua documentação específica, como atas, correspondências, relatórios e livros contábeis, entre outras fontes, as quais poderiam garantir acesso ao cotidiano de seu funcionamento. Os trabalhos produzidos têm recorrido à análise de estatutos ou às notícias publicadas na imprensa, documentos que, quando muito, podem apenas fornecer algumas pistas sobre como funcionavam tais instituições. Em geral, os estatutos seguiam 301 Dezembro de 2009 Cláudia Maria Ribeiro Viscardi um padrão minimamente diferenciado entre si, e as notícias, além de esparsas, acabavam por apenas atestar que a sociedade tinha existido. No Brasil tal situação foi agravada em razão das contínuas mudanças da legislação sobre as mutuais. Entre 1860 e 1882 as mutuais da capital registravam- se no Conselho de Estado. No início da República, passaram a registrar-se nas juntas comerciais ou em cartórios das comarcas. Algumas sociedades, sobre- tudo as esportivas e de lazer, eram registradas nas delegacias de polícia. A ausência de um registro único ou mesmo a não obrigatoriedade de registro são fatores que, certamente, levaram a que incontáveis sociedades não deixassem pistas de sua existência. Mesmo em países como a Inglaterra, a existência de um registro único a partir de 1846 não garantiu aos pesquisadores o acesso ao número real de mutuais e de sócios, uma vez que muitas associações não se registravam.20 Diante de tantas incertezas, os levantamentos feitos pelos contemporâneos podem ser ferramentas muito úteis no dimensionamento do fenômeno. Mas apresentam problemas para os quais devemos estar atentos. Ataulfo de Paiva fez seu primeiro levantamento em 1912 e depois o completou com dados co- lhidos entre 1913 e 1920. Mas os critérios utilizados no momento de sistema- tização dos dados não foram uniformes. No primeiro levantamento, relativo ao ano de 1912, o autor listou 421 associações, com 282.937 sócios. No segun- do levantamento (1913-1920), trabalhou com um universo de 319 instituições, com um número total de sócios que variou entre 204.008 (1918) e 205.707 (1915). O gráfico anexo ilustra o “movimento social” do Rio de Janeiro obser- vado nos dois levantamentos. Número de sócios, Rio de Janeiro (1912-1920) 0 50.000 100.000 150.000 200.000 250.000 300.000 1912 1913 1914 1915 1916 1917 1918 1919 1920 302 Revista Brasileira de História, vol. As mutuais e seus sócios em números 29, nº 58 Número de sócios, Rio de Janeiro (1912-1920) Gráfico da Evolução Patrimonial 0 50.000 100.000 150.000 200.000 250.000 300.000 1912 1913 1914 1915 1916 1917 1918 1919 1920 Fonte: Construído com dados obtidos em Prefeitura do Distrito Federal, 1922, p.745, 741. Número de sócios, Rio de Janeiro (1912-1920) Fonte: Construído com dados obtidos em Prefeitura do Distrito Federal, 1922, p.745, 741. Revista Brasileira de História, vol. 29, nº 58 Revista Brasileira de História, vol. 29, nº 58 302 Estratégias populares de sobrevivência... O gráfico nos mostra que houve pouca variação do número de sócios no decorrer da segunda década republicana, o que indica que o fenômeno asso- ciativo manteve-se constante, ao longo do período. O que chama mais a aten- ção é o alto número de sócios, de 645 a 715 por instituição, aproximadamente. (Para a obtenção deste número, calculamos a média entre o primeiro e o se- gundo levantamentos, ano a ano.) No entanto, estudos anteriores já haviam revelado que o Distrito Federal possuía o maior número mutuais do Brasil.21 Se levarmos em conta que o total de homens em 1910, no Distrito Federal, era de 496.284, incluindo-se entre eles as crianças, o número de sócios em 1912 parece, de fato, superestimado (282.937 sócios, 57%), embora não impossível. O número se torna alto apenas quando comparado com outras realidades an- teriormente estudadas no Brasil.22 Além do levantamento de Paiva, o trabalho de Ferreira da Rosa pode nos ajudar a entender melhor a dimensão do fenômeno. Segundo consta em sua obra, havia na cidade do Rio de Janeiro, no ano de 1902, 170 mutuais (retiraram‑se da listagem as associações que não se configuravam como tais). Elas possuíam, no conjunto, 86.241 sócios (incluídos os remidos), o que produz uma média de aproximadamente 510 sócios por instituição. Essa média é um pouco menor que a obtida por Paiva, mas ainda alta em relação à população masculina adulta da cidade. Havia, em 1902, 727.919 habitantes no Distrito Federal, entre homens, mulheres e crianças. Considerando-se que aproximadamente 57% desse total era de homens (incluídos entre eles as crianças), o percentual de associados encontrado por Rosa correspondia a 20,78% desse universo, número que acre- ditamos ser mais próximo da realidade, por sua amostra ser mais homogênea e por coincidir com levantamentos feitos posteriormente. Se compararmos os dados do Rio de Janeiro com os de outras capitais, os números parecerão bastante razoáveis. As mutuais e seus sócios em números Para Buenos Aires, estima-se que em 1914 metade dos trabalhadores encontrava-se filiada a algum tipo de socieda- de de socorro mútuo. Para o caso inglês, estima-se que 60% dos homens adul- tos eram associados a uma mutual que incorporava trabalhadores e setores médios. Os dados para os Estados Unidos, levantados mais recentemente por David Beito, apontam que em 1920, para cada três homens adultos, um per- tencia a mutuais, compostas sobretudo por negros e imigrantes. No Canadá, 30% dos homens trabalhadores eram mutualizados. Na cidade do México, em 1873, havia mais de cem mutuais, e cerca de um quinto de sua população par- ticipava das associações.23 O levantamento de Ferreira da Rosa nos possibilitou uma explicação pa- 303 Dezembro de 2009 Cláudia Maria Ribeiro Viscardi ra o alto número de sócios encontrados no Distrito Federal, quando compa- rado com outras cidades brasileiras. Pudemos perceber que algumas mutuais tinham um número muito grande de membros – tais como as dos comerciários,24 dos ferroviários, dos trabalhadores navais e as de portugueses –, e elas foram responsáveis pela elevação da média. O levantamento de Vitor Fonseca, feito com base no Registro Especial de Títulos e Documentos do Rio de Janeiro, entre os anos de 1815 e 1914, encon- trou 346 mutuais (Fonseca, 2008, p.120). Mas a documentação por ele utiliza- da não lhe fornecia o número de sócios. A despeito desse problema, o seu le- vantamento atesta que o número de mutuais era maior do que o encontrado por Paiva e por Rosa. No ano de 1912, Paiva listou 220 mutuais, e, nos levan- tamentos feitos para o período posterior (1913-1920), trabalhou com um uni- verso de 216 mutuais.25 Como o método utilizado por Paiva foi o envio de questionários, muitas associações podem ter se eximido de lhe responder, o que torna os números encontrados por Fonseca mais próximos da realidade, muito embora devamos admitir a hipótese de que algumas mutuais podem igualmente ter deixado de se registrar. Em que pesem as incertezas em relação aos números, podem-se afiançar algumas conclusões em relação ao tema. A primeira é que a cidade do Rio de Janeiro teve, a partir da segunda metade do século XIX, um número expressi- vo de mutuais, certamente, o maior do país. Esse número se ampliou conside- ravelmente ao longo das duas primeiras décadas do século XX, atingindo o mínimo de 170 e o máximo de 346. Revista Brasileira de História, vol. 29, nº 58 As mutuais e seus sócios em números As mutuais possuíam em média de 510 a 715 sócios, número elevado em relação aos padrões encontrados para outras cidades brasileiras, mas proporcionalmente próximo de algumas capitais no exterior. Esses indicadores apontam para um dinamismo muito grande da sociedade civil, que não se limitava às mutuais. Tal dinamismo é compreensível em razão das especificidades da então capital nacional. Era a maior cidade do país e tornou-se polo atrativo de um grande número de imigrantes. Seu desenvolvimento comercial, industrial e do setor de serviços propiciava a ocupação de diferentes setores de mão de obra qualificada, sócios em potencial das mutuais. Como centro político do país, tinha condições mais propícias à emergência e consolidação de uma cultura associativa. Esse conjunto de características fez que o Rio de Janeiro atingisse níveis expressivos de organização de sua sociedade civil. 304 Revista Brasileira de História, vol. 29, nº 58 Estratégias populares de sobrevivência... Dezembro de 2009 Trajetórias O levantamento de Ferreira da Rosa permitiu que aferíssemos o período de fundação das mutuais por ele listadas. Como seus dados e critérios são mais homogêneos que os de Paiva – o qual também nos indica a data de fundação das sociedades –, são mais confiáveis. Tabela 2 – Período de Fundação das Mutuais do Rio de Janeiro (1815-1904) Fundação Quantidade Percentual Sem data 10 5,89 1815-1824 1 0,60 1825-1834 0 0 1835-1844 2 1,19 1845-1854 4 2,35 1855-1864 12 7,05 1865-1874 8 4,70 1875-1884 38 22,35 1885-1894 29 17,05 1895-1904 66 38,82 Total: 170 100 Fonte: Rosa, 1905, p.194 ss. la 2 – Período de Fundação das Mutuais do Rio de Janeiro (1815-1904) Tabela 2 – Período de Fundação das Mutuais do Rio de Janeiro ( Como se percebe, o número das mutuais teve grande crescimento a partir de 1875, tendência que não foi interrompida nas décadas seguintes, conforme nos aponta o levantamento de Paiva, observado anteriormente. O período de maior crescimento foi ao longo da década de 1890 e até as primeiras décadas do século XX. Os dados obtidos na documentação reunida no Conselho de Estado apon- tam para a mesma realidade. Das 123 mutuais, fundadas entre 1860 e 1882, mais de 70% haviam sido fundadas após 1870. O levantamento de Vitor Fonseca traz resultados muito próximos dos outros citados. Entre as 346 mutuais listadas por ele, quase 76% haviam sido fundadas entre 1875 e 1914. (Em sua listagem só incluímos as definidas por 305 Dezembro de 2009 Cláudia Maria Ribeiro Viscardi ele como entidades de socorros mútuos.) Como os dados avançam até a se- gunda década do século XX, foi possível perceber que o maior crescimento se deu entre 1905 e 1914. Nos Estados Unidos, elas proliferaram igualmente na segunda metade do século XIX, sobretudo após a Guerra de Secessão. No período da depressão, a falência das seguradoras contribuiu para a ampliação do número de mutuais, as quais gozavam de melhor reputação (Cordery, 2003, p.89-90). Na Itália, o maior crescimento também se deu na segunda metade do século XIX. A partir de 1898, o país possuía quase um milhão de associados. Trajetórias Pelo período de emergência e crescimento do mutualismo italiano, percebe-se que foi tardio em relação aos seus congêneres europeus, e próximo cronologi- camente ao mutualismo das Américas.26 Tomando por base os diversos levantamentos anteriormente expostos, perceberemos que as mutuais da cidade do Rio de Janeiro, embora tenham sido criadas na primeira metade do século XIX, conheceram grande expansão nas últimas décadas do século, expansão esta que vigorou até o final da pri- meira década republicana, quando houve uma estabilização do número de sócios, seguida pela desaceleração do fenômeno nas décadas de 1930 e 1940, após a implantação de medidas de proteção social. À medida que as leis pro- tetoras iam surgindo, os socorros oferecidos pelas mutuais tornavam-se dis- pensáveis. Destacam-se, sobretudo, o impacto proporcionado pela Lei de Aci- dentes de Trabalho (1919), as Caixas de Aposentadorias e Pensões (1923 em diante), a Lei de Férias (1925) e o Código de Menores (1927), que muito em- bora não tenham sido iniciativas extensivas a todos os trabalhadores, atingiam boa parte da clientela das mutuais.27 Revista Brasileira de História, vol. 29, nº 58 As modalidades associativas e os socorros oferecidos Com base ainda nos levantamentos de Paiva, separamos as associações listadas por modalidade, para identificarmos o número de mutuais no conjun- to do associativismo do Rio de Janeiro. A Tabela 3 sintetiza os resultados ob- tidos: 306 Revista Brasileira de História, vol. 29, nº 58 Estratégias populares de sobrevivência... Tabela 3 – O Associativismo do Rio de Janeiro por Modalidade (1912-1920) 1912 1913-1920 Modalidade Número Percentual Número Percentual Mutuais 220 52,25 216 67,71 Religiosas 131 31,11 63 19,76 Maçônicas 28 6,65 1 0,31 Filantrópicas 23 5,47 24 7,52 Patronais e Sindicais 13 3,09 9 2,82 Saúde ou hospitalares 2 0,48 2 0,63 Artísticas e Científicas 4 0,95 4 1,25 Total 421 100 319 100 Fonte: Tabela montada a partir de cálculos retirados de Prefeitura do Distrito Federal, 1922, p.765-771, 820-831. la 3 – O Associativismo do Rio de Janeiro por Modalidade (1912-1920) Como se observa, do movimento associativo pesquisado pelo autor para a cidade do Rio de Janeiro, na segunda década republicana, as mutuais repre- sentavam a grande maioria, entre 50 e 70% do geral. É difícil afirmar que a diferença entre os números de mutuais e de sindicatos tenha sido assim tão grande. Entre as associações listadas por Paiva como “beneficentes” estavam incluídos alguns poucos sindicatos que, além de atuarem como organismos de resistência, prestavam também algum tipo de assistência a seus associados e, por essa razão, foram qualificados pelo autor como “sociedades beneficentes”. Os demais sindicatos não foram incluídos em seu levantamento, daí seu pe- queno número. No que diz respeito aos benefícios pagos pelas mutuais aos sócios, constam do levantamento de Ataulfo de Paiva as relações das despesas efetuadas pelas associações ao longo dos anos, bem como o volume de seu patrimônio. Muito embora os dados para 1912 não obedeçam ao mesmo critério utilizado para os anos posteriores, percebe-se que a maior parte dos gastos das associações – retirando-se o item “despesas gerais”, que sempre foi o maior – era realizada com o pagamento de pecúlios ou pensões. Em terceiro lugar, os maiores gastos referiam-se às despesas médicas; por último, constavam as despesas com fu- nerais. Estes representavam, em geral, de um terço à metade dos valores gastos com pecúlios ou pensões. Entre as mutuais dos Estados Unidos predominavam aquelas que ofereciam 307 Dezembro de 2009 Cláudia Maria Ribeiro Viscardi socorros em caso de doenças ou funerais. As modalidades associativas e os socorros oferecidos Em segundo lugar, e com mais ca- pital, constavam as que ofereciam seguros de vida (Beito, 2000, p.1, 14). Já o mutualismo inglês era mais próximo do modelo brasileiro do que o estadunidense. A maior parte dos pecúlios pagos aos sócios era composta por auxílios a funerais – as mutuais mais populares – e, depois, por auxílios a doen- tes. O pagamento de seguros de vida – raros no Brasil e comuns nos Estados Unidos – era propiciado somente pelas grandes mutuais – conhecidas como Ordens –, que se espalhavam por todo o território britânico e também sobre suas colônias e ex-colônias e eram compostas por uma série de sociedades afiliadas (Neave, 1996, p.55). Tais coberturas não resolviam o problema da pobreza, mas garantiam àqueles que pudessem ser incluídos ao menos um recurso adicional, em caso de necessidade. Ter um funeral digno, garantir um seguro para os familiares após a morte ou eximir-se de recorrer à caridade eram motivações suficientes para que um grande número de trabalhadores, em diversas regiões do globo, se empenhasse na edificação e manutenção dessas organizações sociais. Revista Brasileira de História, vol. 29, nº 58 As Insolvências Em vários lugares do mundo onde o mutualismo se desenvolveu, os re- sultados das pesquisas apontam que grande parte das mutuais faliram após alguns anos de existência. As razões para a insolvência de tais sociedades esti- veram ligadas a fatores como a incapacidade ou a impossibilidade de calcular os riscos. Para que tal cálculo fosse feito era indispensável ter acesso às taxas de expectativa média de vida da região onde a mutual se inseria e conhecer as principais causas que levavam a doenças e falecimentos. Com base em tais informações era possível às mutuais calcular devidamente o valor de suas men- salidades e dos benefícios pagos, garantindo seu equilíbrio financeiro. Tais dados raramente estiveram disponíveis para os contemporâneos, o que os levou a agir de forma mais intuitiva do que racionalmente calculada. Ademais, Swaan destaca que muitos sócios pertenciam a grupos homo- gêneos, formados por trabalhadores de uma mesma cidade ou até de uma mesma empresa. Tal conjuntura poderia levá-los a adoecer em um mesmo momento, ou serem vítimas de um mesmo acidente de trabalho ou epidemia, o que levava a mutual a cobrir um custo volumoso de uma só vez, fato que a conduziria inevitavelmente à insolvência. O levantamento de Paiva incluiu análise do patrimônio, das receitas e 308 Revista Brasileira de História, vol. 29, nº 58 Estratégias populares de sobrevivência... Estratégias populares de so despesas de todas as sociedades – não somente as mutuais –, e revela uma constante oscilação. O gráfico anexo ilustra melhor nossas afirmações. Gráfico da Evolução Patrimonial das Associações (1913-1920) 0 20.000 40.000 60.000 80.000 100.000 1913 1914 1915 1916 1917 1918 1919 1920 Fonte: Para a elaboração deste gráfico e do seguinte, utilizamos os dados de Prefeitura do Distrito Federal, 1922, p.881-882. Retiramos os dados relativos a 1912, porque se vinculam a um maior número de associações, o que poderia levar a distorções. Gráfico da Evolução Patrimonial das Associações (1913-1920) 0 20.000 40.000 60.000 80.000 100.000 1913 1914 1915 1916 1917 1918 1919 1920 Fonte: Para a elaboração deste gráfico e do seguinte, utilizamos os dados de Prefeitura do Distrito Federal, 1922, p.881-882. Retiramos os dados relativos a 1912, porque se vinculam a um maior número de associações, o que poderia levar a distorções. Gráfico da Evolução Patrimonial das Associações (1913-1920) Fonte: Para a elaboração deste gráfico e do seguinte, utilizamos os dados de Prefeitura do Distrito Federal, 1922, p.881-882. As Insolvências Retiramos os dados relativos a 1912, porque se vinculam a um maior número de associações, o que poderia levar a distorções. Receitas Despesas Gráfico da relação entre receitas e despesas das associações (1913-1920) 0 5.000 10.000 15.000 20.000 1913 1914 1915 1916 1917 1918 1919 1920 A leitura do gráfico aponta para uma ligeira evolução patrimonial das sociedades até 1915, com abrupta redução nos anos seguintes. A queda se es- tabiliza a partir de 1916 e apresenta leve tendência de crescimento até 1920, mas nunca atinge o nível em que se encontrava antes, o que indica que ao longo do período houve perda patrimonial. Acredita-se que a queda vincula-se ao impacto da Primeira Guerra Mundial sobre a economia brasileira, inicial- mente levando à carestia, ao desemprego e à desaceleração econômica, proble- mas amenizados a posteriori. Pode ser, também, que essa queda fizesse parte da trajetória de insolvência que se anunciava para grande parte das mutuais. Como foram criadas, em sua maioria, ao final do século XIX, no contexto de crise possuíam, em média, de vinte a trinta anos de existência, tempo suficien- te para o envelhecimento de seus primeiros sócios. Esse tempo de vida de uma mutual levava sempre ao aumento de seus gastos, nem sempre compensados pela adesão de novos sócios jovens. Como o levantamento não prossegue nos anos seguintes, não se pode saber se, de fato, tais sociedades tiveram seu patri- mônio totalmente depreciado ao final da década de 1920, como ocorreu em outros lugares no Brasil. A análise das relações entre as receitas e despesas ajuda a explicar as osci- lações do gráfico anterior. Dezembro de 2009 309 Cláudia Maria Ribeiro Viscardi Receitas Despesas Gráfico da relação entre receitas e despesas das associações (1913-1920) 0 5.000 10.000 15.000 20.000 1913 1914 1915 1916 1917 1918 1919 1920 Receitas Despesas Gráfico da relação entre receitas e despesas das associações (1913-1920) 0 5.000 10.000 15.000 20.000 1913 1914 1915 1916 1917 1918 1919 1920 Nas relações entre receita e despesa percebe-se que, pelos levantamentos de Ataulfo de Paiva, no conjunto, as sociedades não foram deficitárias em suas contas correntes ao longo do período, muito embora o gráfico anterior nos mostre que elas abriram mão de seu patrimônio para manter tal situação, em períodos de crise. Pelo gráfico nota-se, igualmente, que os anos mais difíceis foram os de 1916, 1917 e 1918, por indicarem uma descapitalização das sociedades. As Insolvências Há queda na arrecadação nos anos de 1915 e 1916. Essa situação só começa a se alterar a partir de 1919, apontando para uma tendência de crescimento que se refletiu, também, na evolução patrimonial das associações, conforme apon- tou o gráfico. A situação financeira das mutuais era agravada pelo hábito de não cobra- rem mensalidades mais altas para os idosos, que naturalmente apresentavam maior possibilidade de adoecer. Ao mesmo tempo, as mutuais não se esforça- vam muito em atrair sócios mais jovens. Tal situação foi destacada por um estudioso do mutualismo irlandês, o qual alegou que muitas mutuais preferiam filiar os mais idosos, porque acreditavam que o momento da morte dependia menos da idade e mais da vontade de Deus (Campbell, 1996, p.69). Dessa forma, não se pode caracterizar tais comportamentos como economicamente irracionais, pois encontravam suas justificativas no ambiente cultural que os principais agentes compartilhavam. No que tange a modalidades dos Estados Unidos, as primeiras a falir foram as afro-americanas. As étnicas não conseguiram atrair a segunda e terceira gerações, já nascidas no país, o que levou a seu esvaziamento progressivo. Na década de 1920 houve considerável queda no número de mutuais, e em 1930 o movimento perdeu 378 mil membros. Porém, no Rio Grande do Sul e em Minas Gerais as entidades de imigrantes foram as mais duradouras.28 310 Revista Brasileira de História, vol. 29, nº 58 Estratégias populares de sobrevivência... Com o tempo, o equilíbrio entre sociabilidade e seguridade – dois prin- cípios que expressavam a identidade das mutuais – foi se tornando um pro- blema para elas. Tão logo a falência se anunciava, os gastos com a sociabilida- de iam sendo diminuídos, muito a contragosto dos gestores, para que a seguridade pudesse ser mantida. Tais decisões sempre representavam conflitos para os pares, os quais muitas vezes sentiam que os valores que haviam origi- nado a associação estavam sendo postos em segundo plano, em prol de outros como os do lucro e da competição. Tais conflitos expressavam bem as contra- dições de um período de modernização. Dezembro de 2009 Do privado ao público Em vários países da Europa ocidental a desaceleração do mutualismo coin- cidiu com a expansão das companhias de seguros, mais capazes de calcular ris- cos e gerenciar seus lucros. Em que pesem tantas habilidades, elas igualmente faliram diante da enorme demanda por socorros e os baixos valores das contri- buições pagas, assim mantidas para que novos sócios fossem conquistados. Segundo análises existentes para a Inglaterra, os trabalhadores, que antes apostavam nas mutuais como a melhor estratégia para escapar da pobreza, perceberam que após a falência do mutualismo e das iniciativas privadas re- presentadas pelas seguradoras só lhes restava recorrer ao Estado. Tal decisão não foi fácil, já que tinham de abrir mão de valores que deram origem à cria ção das mutuais, tais como os de voluntarismo, autonomia e respeitabilidade. Mas no contexto de adversidade o cálculo racional levou-os a aderir a propos- tas de inúmeros ‘reformadores’ que defendiam que o Estado assumisse o ônus pela proteção social.29 Na França, tal como na Inglaterra, as mutuais rejeitaram inicialmente a contribuição compulsória, pois viam com desconfiança a intervenção do Es- tado sobre os princípios da livre escolha. Essa realidade foi alterada após o repentino aumento das demandas por proteção social, em razão da Primeira Guerra Mundial. A mudança de conjuntura levou as mutuais a apoiarem a intervenção do Estado, desde que fossem envolvidas no processo de implan- tação das políticas sociais.30 No Brasil, pouco se sabe acerca do papel que as mutuais tiveram na pro- posição das políticas de amparo social. Pesquisas nesse campo apontam para o estabelecimento de alguns benefícios para os trabalhadores, sobretudo a par- tir da década de 1920, e, muito mais tarde, da Previdência Social. Se tais ini- ciativas resultaram da ação dos reformadores, das conquistas dos trabalhado- 311 Cláudia Maria Ribeiro Viscardi res organizados ou das concessões do Estado, a historiografia brasileira tem discutido. Mas o papel das mutuais nesse processo ainda requer investigação. Coloca-se aí uma nova pergunta, que ainda aguarda por resposta, entre tantas outras referentes ao tema. A opção pelo mutualismo pode ter significado, para aqueles que a fizeram, uma estratégia fundamental de manutenção de um importante valor compar- tilhado pelos trabalhadores, o da respeitabilidade. Os benefícios recebidos nos momentos de necessidade eram tidos como direitos e não como favores. O mesmo não acontecia com aqueles que dependiam da caridade alheia. Do privado ao público É pra- ticamente impossível identificar se os pobres recorreram mais às mutuais do que à caridade, pois se os números são incertos para a primeira opção, são ainda mais fluidos em relação à segunda. O que é patente, no entanto, é que antes do estabelecimento das políticas protecionistas, os pobres recorreram a diferentes estratégias de sobrevivência, mais ou menos dignas. Além disso, a permanência da filantropia no Brasil, em larga escala até os dias de hoje, nos remete à distância em que nos encontramos em relação às experiências mais igualitárias de outras nações, uma vez que a extensão da cidadania não acom- panhou a expansão de nossa autoridade pública. NOTAS 1 Este artigo é parte dos resultados de pesquisas que contaram com financiamento das agências de fomento Capes, CNPq e Fapemig. 1 Este artigo é parte dos resultados de pesquisas que contaram com financiamento das agências de fomento Capes, CNPq e Fapemig. 1 Este artigo é parte dos resultados de pesquisas que contaram com financiamento das agências de fomento Capes, CNPq e Fapemig. 1 Este artigo é parte dos resultados de pesquisas que contaram com financiamento das agências de fomento Capes, CNPq e Fapemig. 2 Entre as inúmeras análises sobre o tema, destacamos: GEREMECK, Bronislaw. Os filhos de Caim. Trad. Henryk Siewierski. São Paulo: Companhia das Letras, 1995; ROBERTS, S. Contexts of charity in the Middle Ages: religious, social, and civics. In: SCHNEEWIND, J. B. (Ed.) Giving: western ideas of philanthropy. Indiana: Indiana University Press, 1996; CASTEL, Robert. As metamorfoses da questão social: uma crônica do salário. Trad. Iraci D. Poleti. 3.ed. Petrópolis: Vozes, 1998. 3 WAGLE, Udaya. Rethinking poverty: definition and measurement. International Social Science Journal, London: Blackwell, v.54, n.171, 2002. p.163. 4 Ver a respeito: Hobsbawm, Eric J. Artisan or labour aristocrat? The Economic History Review, New Series, Blackwell Publishing on behalf of the Economic History Society, v.37, n.3, p.355-372, Aug. 1984; KIRK, Neville. The growth of working-class reformism in mid- Victorian England. Champaign: University of Illinois Press, 1985. 5 CORDERY, Simon. British friendly societies, 1750-1914. New York: Palgrave Macmillan, 2003, p.108; VISCARDI, Cláudia M. R. Experiências da prática associativa no Brasil (1860- 312 Revista Brasileira de História vol 29 nº 58 5 CORDERY, Simon. British friendly societies, 1750-1914. New York: Palgrave Macmillan, 2003, p.108; VISCARDI, Cláudia M. R. Experiências da prática associativa no Brasil (1860- 312 Revista Brasileira de História, vol. 29, nº 58 Estratégias populares de sobrevivência... 1880). Topoi, Revista de História, Rio de Janeiro: PPGHIS-UFRJ; 7 Letras, v.9, n.16, p.117- 136, 2008. 6 BENDIX, Reinhard. Construção nacional e cidadania. Trad. Mary Amazonas Leite de Barros. São Paulo: Edusp, 1996, p.39. 7 SWAAN, Abram de. In care of the state: health care, education and welfare in Europe and the USA in the modern era. Cambridge: Polity Press, 1988. p.10. 8 GODELIER, Maurice. O enigma do dom. Trad. Eliana Aguiar. Rio de Janeiro: Civilização Brasileira, 2001. p.25. 9 CONNIFF, Michael. Voluntary associations in Rio, 1870-1945. A new approach to urban social dynamics. Journal of Interamerican Studies and World Affairs, v.17, n.1, 1975. 10 KUSHNIR, Beatriz. NOTAS Baile de máscaras: mulheres judias e prostituição. As polacas e suas associações de ajuda mútua. Rio de Janeiro: Imago, 1996. 11 Batalha baseou-se em documentos disponíveis na Secretaria de Negócios do Conselho de Estado, compostos de estatutos e algumas atas que para lá eram endereçados pelas mutu- ais, com o fim de obterem permissão para o seu funcionamento, em obediência à lei n. 1083 e ao decreto 2.711, ambos de 1860. Além desses, utilizou dados de almanaques e de- mais levantamentos estatísticos feitos por contemporâneos do fenômeno. BATALHA, Cláudio H. M. Sociedades de trabalhadores do Rio de Janeiro do século XIX: algumas re- flexões em torno da formação da classe operária. Cadernos do AEL: Sociedades operárias e mutualismo, Campinas: Unicamp/IFCH, v.6, n.10/11, 1999. 12 JESUS, Ronaldo P. de. O povo e a monarquia: a apropriação da imagem do imperador e do regime monárquico entre a gente comum da corte (1870-1889). Tese (Doutorado em História) – USP. São Paulo, 2001. Cap. 3. 13 JESUS, Ronaldo P. de. História e historiografia do fenômeno associativo no Brasil mo- nárquico (1860-1887). In: ALMEIDA, Carla M. C. de; OLIVEIRA, Mônica R. de. Nomes e números: alternativas metodológicas para a história econômica e social. Juiz de Fora: Ed. UFJF, 2006. 14 FONSECA, Vítor M. M. da. No gozo dos direitos civis: associativismo no Rio de Janeiro, 1903-1916. Rio de Janeiro: Faperj-Muiraquitã, 2008. 15 ROSA, Ferreira da. Rio de Janeiro. Rio de Janeiro: Edição Oficial da Prefeitura, 1905. 16 PREFEITURA DO DISTRITO FEDERAL. Assistência pública e privada no Rio de Janei- ro: história e estatística. Rio de Janeiro: Typografia do Annuario do Brasil, 1922. 17 Esse debate pode ser acompanhado em CLAWSON, Mary. Constructing brotherhood: class, gender, and fraternalism. New Jersey: Princeton University Press, 1989. Parte 2, cap. 3. 18 CORDERY, Simon. Fraternal orders in the United States: a question for protection and identity. In: LINDEN, Marcel V. D. (Ed.). Social security mutualism: the comparative his- tory of mutual benefit societies. Bern, Berlin: Peter Lang, 1996. p.87. 313 Dezembro de 2009 Cláudia Maria Ribeiro Viscardi 19 NEAVE, David. Friendly societies in Great Britain. In: LINDEN, 1996, p.45. 20 GOSDEN, P. H. J. H. Self-help: voluntary associations in XIX century Britain. London: B. T. Batsford, 1973, p.66-67. 21 SILVA JR., Adhemar L. da. As sociedades de socorros mútuos: estratégias privadas e públicas. Estudo centrado no Rio Grande do Sul – Brasil, 1854-1940. Tese (Doutorado em História) – PUC/RS. NOTAS Porto Alegre, 2005. p.53. Segundo os dados apresentados, em 1882 o DF possuía 56,07% das mutuais do país e, em 1917, 23,28%. Em ambas as ocasiões, esse foi o maior percentual do país. 21 SILVA JR., Adhemar L. da. As sociedades de socorros mútuos: estratégias privadas e públicas. Estudo centrado no Rio Grande do Sul – Brasil, 1854-1940. Tese (Doutorado em História) – PUC/RS. Porto Alegre, 2005. p.53. Segundo os dados apresentados, em 1882 o DF possuía 56,07% das mutuais do país e, em 1917, 23,28%. Em ambas as ocasiões, esse foi o maior percentual do país. 22 Os dados populacionais foram obtidos em: MINISTÉRIO da Indústria e Comércio. Di- retoria Geral de Estatística, Anuário Estatístico do Brasil, 1908-1912, Território e Popula- ção. Rio de Janeiro: Tipografia da Estatística, 1916. v.1, p.310. Para o Rio Grande do Sul, Silva Junior afirma que nos anos de 1917 e 1924 havia 148 mutuais no estado. SILVA JR., 2005, p.83. Os dados que encontramos para Minas, em 1923, apontam para a existência de 178 sociedades mutuais e sindicais, com uma filiação de 2,4% dos homens adultos do esta- do e com uma média de 202 sócios por associação. VISCARDI, Cláudia M. R.; JESUS, Ronaldo P. de. A experiência mutualista e a formação da classe trabalhadora no Brasil. In: FERREIRA, Jorge; REIS, Daniel A. (Org.). As esquerdas no Brasil: a formação das tradições (1889-1945). Rio de Janeiro: Civilização Brasileira, 2008. v.1, cap. 1, p.41-42. Tânia de Lu- ca, em seu estudo sobre o mutualismo paulista, afirma que na cidade de São Paulo, no início do século XX, 8% da população da cidade era associada, número nunca igualado depois. LUCA, Tânia R. de. Mutualism in Brazil (São Paulo). In: LINDEN, 1996, p.617. 22 Os dados populacionais foram obtidos em: MINISTÉRIO da Indústria e Comércio. Di- retoria Geral de Estatística, Anuário Estatístico do Brasil, 1908-1912, Território e Popula- ção. Rio de Janeiro: Tipografia da Estatística, 1916. v.1, p.310. Para o Rio Grande do Sul, Silva Junior afirma que nos anos de 1917 e 1924 havia 148 mutuais no estado. SILVA JR., 2005, p.83. Os dados que encontramos para Minas, em 1923, apontam para a existência de 178 sociedades mutuais e sindicais, com uma filiação de 2,4% dos homens adultos do esta- do e com uma média de 202 sócios por associação. VISCARDI, Cláudia M. R.; JESUS, Ronaldo P. de. NOTAS A experiência mutualista e a formação da classe trabalhadora no Brasil. In: FERREIRA, Jorge; REIS, Daniel A. (Org.). As esquerdas no Brasil: a formação das tradições (1889-1945). Rio de Janeiro: Civilização Brasileira, 2008. v.1, cap. 1, p.41-42. Tânia de Lu- ca, em seu estudo sobre o mutualismo paulista, afirma que na cidade de São Paulo, no início do século XX, 8% da população da cidade era associada, número nunca igualado depois. LUCA, Tânia R. de. Mutualism in Brazil (São Paulo). In: LINDEN, 1996, p.617. 23 Argentina: MUNCK, Ronaldo. Mutual Benefit Societies in Argentina: workers, nationa- lity, social security and trade-unionism. Journal of Latin-American Studies, v.30, p.573, 1998; Reino Unido: KIDD, Alan. State, society and the poor in 19th Century England. Lon- don: Macmillan Press, 1999. p.120-121; CORDERY, 2003, p.68. Estados Unidos: BEITO, David. From mutual aid to the welfare state: fraternal societies and social services, 1890- 1967. The University of North Caroline Press, 2000. p.2. Canadá: PALMER, Bryan. Mutu- ality and the masking/making of difference: mutual benefit societies in Canadá – 1850- 1950. In: LINDEN, 1996, p.112. México: HART, John M. Mexican mutualism in historical perspective. In: LINDEN, 1996, p.595. 24 Na relação de Rosa havia três mutuais que reuniam os comerciários: a Sociedade dos Empregados do Comércio do Rio de Janeiro, fundada em 1880, tinha 13.542 sócios; a So- ciedade Protetora dos Empregados no Comércio, fundada em 1902, tinha 2.569 sócios; a Sociedade Beneficente dos Empregados do Comércio, fundada em 1903, tinha 510 sócios no ano de sua fundação (os demais dados referem-se ao ano de 1902). ROSA, 1905, p.194. 24 Na relação de Rosa havia três mutuais que reuniam os comerciários: a Sociedade dos Empregados do Comércio do Rio de Janeiro, fundada em 1880, tinha 13.542 sócios; a So- ciedade Protetora dos Empregados no Comércio, fundada em 1902, tinha 2.569 sócios; a Sociedade Beneficente dos Empregados do Comércio, fundada em 1903, tinha 510 sócios no ano de sua fundação (os demais dados referem-se ao ano de 1902). ROSA, 1905, p.194. 25 Dados retirados de PREFEITURA DO DISTRITO FEDERAL, 1922, p.765-771 e 820- 831. 25 Dados retirados de PREFEITURA DO DISTRITO FEDERAL, 1922, p.765-771 e 820- 831. 25 Dados retirados de PREFEITURA DO DISTRITO FEDERAL, 1922, p.765-771 e 820- 831. 26 Para o caso italiano ver: TOMASSINI, Luigi. Mutual benefit societies in Italy, 1861-1922. In: LINDEN, 1996, p.228-231. Revista Brasileira de História, vol. 29, nº 58 NOTAS A contribuição do mutualismo italiano para o processo de formação de uma cultura cívica no país pode ser observado em: PUTNAM, Robert D. Co- munidade e democracia: a experiência da Itália Moderna. 3.ed. Rio de Janeiro: FGV, 2002. 314 Revista Brasileira de História, vol. 29, nº 58 Estratégias populares de sobrevivência... 27 Tânia de Luca destaca o declínio das sociedades paulistas a partir da Lei Elói Chaves (1923), que originou os primeiros fundos de aposentadoria e pensões no Brasil. A partir dessa lei, as mutuais passaram a agir de forma complementar, preenchendo as lacunas deixadas pela ausência do seu cumprimento integral (LUCA, 1996, p.620). Silva Jr. de- monstra a desaceleração do fenômeno no Rio Grande do Sul a partir da década de 1940, mas considera essa informação uma provável distorção de registros (SILVA JR., 2005, p.81). Para o fenômeno brasileiro geral ver VISCARDI; JESUS, 2008, p.40-43. 29 SWAAN, 1988, p.150; CORDERY, 2003, p.154-173; GILBERT, Bentley B. The decay of Nineteenth-century provident institutions and the coming of old age pensions in Great Britain. The Economic History Review, New series, v.17, n.3, p.551-563, 1965. 29 SWAAN, 1988, p.150; CORDERY, 2003, p.154-173; GILBERT, Bentley B. The decay of Nineteenth-century provident institutions and the coming of old age pensions in Great Britain. The Economic History Review, New series, v.17, n.3, p.551-563, 1965. 30 Para o caso francês ver: DREYFUS, Michel. Mutual benefit societies in France. In: LINDEN, 1996, p.214-216. Artigo recebido em agosto de 2009. Aprovado em outubro de 2009. Artigo recebido em agosto de 2009. Aprovado em outubro de 2009. Artigo recebido em agosto de 2009. Aprovado em outubro de 2009. 315 Dezembro de 2009 Dezembro de 2009
https://openalex.org/W2889883155	https://europepmc.org/articles/pmc6125288?pdf=render	English	null	A novel prognostic marker and immunogenic membrane antigen: prohibitin (PHB) in pancreatic cancer	Clinical and translational gastroenterology	2,018	cc-by	7,439	© The Author(s) 2018 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/. A R T I C L E O p e n A c c e s s O p e n A c c e s s Abstract Background: Previous study, using immunoblotting with IgG and membrane proteins, identiﬁed prohibitin (PHB) as a potential immunogenic membrane antigen. Now, investigate PHB expression and biological functions in pancreatic cancer. Methods: PHB expression was analysed in PDAC cell lines, normal pancreas tissues, cancer tissues, PDAC patient sera and healthy volunteer sera using QRT-PCR, Western blotting, IHC, and ELISA, and a survival analysis and a COX regression analysis were performed. Low and high PHB expression levels were accomplished using RNA interference technology and gene transfer techniques. Cell proliferation, migration, and invasion, apoptosis-related proteins were assessed 48 h after transfection. Results: PHB was generally expressed in the 8 tested PDAC cell lines. PHB was signiﬁcantly increased in PDAC tissues and negatively correlated with overall survival (p < 0.01). PHB was an independent prognostic factor in PDAC (p < 0.01). PHB was increased in PDAC patient sera (p < 0.01). siRNA-PHB decreased cell growth, migration and invasion. However, PHB overexpression resulted in the opposite effects. Western blotting and Flow cytometric analysis revealed apoptosis inhibition in siRNA-PHB PDAC cells. Conclusions: PHB plays a key role in modulating the malignant phenotype and apoptosis induction, which may be a novel prognostic predictor and a candidate for targeted therapy against PDAC. Weibin Wang, MD1, Lai Xu, MD1, Yu Yang, MD2, LiangBo Dong, MD1, BangBo Zhao, MD1, Jun Lu, MD1, Taiping Zhang, MD1 and Yupei Zhao, MD1 Weibin Wang, MD1, Lai Xu, MD1, Yu Yang, MD2, LiangBo Dong, MD1, BangBo Zhao, MD1, Jun Lu, MD1, Taiping Zhang, MD1 and Yupei Zhao, MD1 Weibin Wang, MD1, Lai Xu, MD1, Yu Yang, MD2, LiangBo Dong, MD1, BangBo Zhao, MD1, Jun Lu, MD1, Taiping Zhang, MD1 and Yupei Zhao, MD1 Wang et al. Clinical and Translational Gastroenterology (2018) 9:178 DOI 10.1038/s41424-018-0044-1 Wang et al. Clinical and Translational Gastroenterology (2018) 9:178 DOI 10.1038/s41424-018-0044-1 Clinical and Translational Gastroenterology Ofﬁcial journal of the American College of Gastroenterology Introduction occur because more than one-half of cases are diagnosed at a distant stage1. Chemotherapy, such as Gemcitabine, also does not contribute a signiﬁcant survival beneﬁt2–4. Therefore, there is strong demand for novel speciﬁc markers in pancreatic cancer patients for early diagnosis, targeted therapy and prognosis. Pancreatic cancer is one of the most aggressive clinical malignancies, and it remains the fourth leading cause of cancer-related deaths. In contrast to the steady increase in survival for most cancers, advances have been slow for pancreatic cancers, and the 5-year relative survival is currently 8%. These low survival rates partially Autoimmunity was demonstrated against several pro- teins, including MUC1, p53, and Rad51, in pancreatic cancer. MUC1 autoantibodies were observed in sera from patients with a variety of different tumours and have been associated with a favourable prognosis in pancreatic cancer5. The presence of p53 autoantibodies has been observed in 18.2% of patients with pancreatic Correspondence: Yupei Zhao (zhao8028@263.net) 1Department of General Surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Science & Peking Union Medical College, 1 Shuai Fu Yuan Hu Tong, Beijing 100730, China 2Department of General Surgery, Beijing Tsinghua Chang Gung Hospital, Tsinghua University, 168 soup road, Changping District, Beijing 102218, China Correspondence: Yupei Zhao (zhao8028@263.net) 1Department of General Surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Science & Peking Union Medical College, 1 Shuai Fu Yuan Hu Tong, Beijing 100730, China 2Department of General Surgery, Beijing Tsinghua Chang Gung Hospital, Tsinghua University, 168 soup road, Changping District, Beijing 102218, China © The Author(s) 2018 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/. Ofﬁcial journal of the American College of Gastroenterology Page 2 of 12 Wang et al. Clinical and Translational Gastroenterology (2018) 9:178 (See legend on next page ) Fig. 1 (See legend on next page.) Fig. Western blot analysis Proteins were extracted from the cells and subjected to SDS-PAGE. The proteins were transferred to poly- vinylidene diﬂuoride (PVDF) membranes (Millipore, USA). A polyclone rabbit anti-prohibitin antibody was purchased from Abcam (ab70672). Polyclonal rabbit anti- human PARP antibody, polyclonal rabbit anti-human caspase-3 antibody, polyclonal rabbit anti-human cleaved caspase-3 antibody, polyclonal rabbit anti-human cas- pase-9 antibody, polyclonal rabbit anti-human Bcl-2 antibody and polyclone rabbit anti-β-actin antibody were obtained from Cell Signaling Technology. The mem- branes were probed with all primary antibodies at a dilution of 1:1000, and secondary peroxidase-conjugated antibodies were used at a dilution of 1:2000. Bands were imaged using a chemiluminescence method (Amersham Biosciences, Sweden). Anti-β-actin antibody was used as an internal control. Speciﬁc siRNA and transfection cancer but was not speciﬁc to malignancy6,7. The recombination factor Rad51 is highly expressed in pan- creatic adenocarcinoma, and Rad51 autoantibodies have been observed in 7% of patients with pancreatic cancer8. Membrane proteins associated with pancreatic cancer perform many essential cellular functions, and hopefully these proteins become more valuable molecular markers. A previous study identiﬁed autoantibodies of membrane proteins in pancreatic cancer using membrane biology, cellular component proteomic, immuno-proteomic, and membrane proteomic approaches, and prohibitin was noted as a potential immunogenic membrane antigen9. The targeting sequences of a 25-nucleotide siRNA were designed and chemically synthesised (Invitrogen American): 5′-AGCACACGCUCAUCAUAGUCCUCUC-3′ (sense), and 3′-GAGAGGACUAUGAUGAGCGUGUGCU- 5′ (antisense). Negative control siRNA (Invitrogen American) was used as a scrambled siRNA control. A total of 5 × 105 cells were plated in a 6-well plate for 48 h, and siRNA transfection (120 pmol) was performed using the Lipofecta- mine 2000 (6 μl) (Invitrogen) according to the manufacturer’s instructions. Plasmid construction Prohibitin (PHB), including PHB1 (BAP32), PHB2 (BAP37, REA), PHB3 and PHB4, is ubiquitously expressed in multiple cellular compartments, including the mito- chondria, the nucleus and the plasma membrane, and PHB is shuttled between the mitochondria, the cytosol and the nucleus10–12. PHB contains an N-terminal transmembrane domain that is an evolutionarily con- served PHB domain similar to that in lipid raft-associated proteins and a C-terminal coiled-coil domain that is involved in the regulation of protein-protein interac- tions13–15. However, the role of PHB in different cancers is controversial, and the biological functions of PHB in pancreatic cancer is not clear. The current study investi- gated the differential expression of PHB in normal and pancreatic cancer patient sera, tissues and cancer cells; examined the relationship between PHB expression and clinicopathological variables; and explored the biological functions of PHB in cancer cell proliferation, migration, invasion and apoptosis. The pMD18-T-PHB plasmid was digested with BamHI and EcoRI and ligated into the pIRES2-EGFP vector (Novagen, Madison, US). E. coli DH5α cells were trans- formed using the ligation mixture, and colonies were obtained. Presence of the PHB gene was examined using PCR ampliﬁcation, and the products were stored for subsequent analysis. Materials and methods Cell culture Human pancreatic cell lines, including AsPC-1, Capan-1, T3M4, SW1990, SU86.86, Panc-1, Colo-357, BxPC-3, and MiaPaCa-2, were kindly donated by Professor Helmut Freiss from the Technical University Munich of Germany. The MiaPaCa-2 human pancreatic cancer cell line was maintained in Dulbecco’s modiﬁed Eagle’s medium (DMEM), and AsPC-1 was maintained in RPMI 1640 medium. Both media were supplemented with 10% foetal bovine serum (FBS), and the cells were maintained at 37 °C in a humidiﬁed atmosphere of 5% CO2. Introduction 1 (See legend on next page.) Ofﬁcial journal of the American College of Gastroenterology Ofﬁcial journal of the American College of Gastroenterology Page 3 of 12 Wang et al. Clinical and Translational Gastroenterology (2018) 9:178 (see ﬁgure on previous page) Fig. 1 Prohibitin expression in pancreatic cancer cell lines, proliferative, migratory and invasive ability was signiﬁcant reduced after siRNA- Phb transfection in AsPC-1 and MiaPaCa-2 cells. a Prohibitin mRNA and protein expression were generally expressed in 8 pancreatic cancer cell lines with Real-Time PCR and western blot. AsPC-1 and MiaPaCa-2 cells were selected for further functional experiments due to their various PHB expression. β-actin was used as housekeeping. GAPDH was used as a loading control. b AsPC-1 and MiaPaCa-2 cells were transfected with control siRNA or PHB siRNA for 48 h, total proteins were extracted and western blotting analysis was performed, 59.6% and 87.1% PHB proteins were reduced, respectively. c The effects of siRNA-PHB on pancreatic cancer cells AsPC-1 and MiaPaCa-2, the growth after 24 h, 48 h and 72 h were assessed using CCK-8 assay (mean ± SD; p < 0.05). d The migration of AsPC-1 and MiaPaCa-2 cells were assessed after siRNA-PHB transfection and compared with negative control transfected cells (mean ± SD; p < 0.01). e The invasiveness of AsPC-1 and MiaPaCa-2 cells were assessed after siRNA-PHB transfection and compared with negative control transfected cells (mean ± SD; p < 0.01) Immunohistochemical analysis h h ( The migratory abilities of cells were evaluated using Transwell assays. Cell culture inserts with 8-μm micro- porous ﬁlters without extracellular matrix coating (Bec- ton Dickinson Labware, Bedford, MA) were loaded with 200 μl of serum-free DMEM/RPMI 1640 containing 4.0 × 105 pancreatic cancer cells. The bottom chambers were loaded with 500 μl of DMEM/RPMI 1640 contain- ing 10% FBS. The cells on the lower surface of the ﬁlter after a 24-h incubation were ﬁxed and stained. Five random optical ﬁelds (×100 magniﬁcations) from tripli- cate ﬁlters were selected for quantiﬁcation of migrated cells. The invasive abilities of the cells were also evaluated using Transwell assays but with extracellular matrix coating (Sigma). Immunohistochemistry (IHC) was performed to localise PHB expression in 10 normal and 66 pancreatic ductal adenocarcinoma (PDAC) samples. The PHB antibody was diluted 1:3000. Two pathologists who spe- cialised in pancreatic cancer independently rated the staining intensity and percentage of stained cells. Brieﬂy, scores were used to rate the staining intensity of cancer cells (no staining: 0; weak: 1; moderate: 2; and strong: 3) and determine the percentage of stained cells (<5%: (0); 5–25%: (1); >25–50%: (2); >50–75%: (3); and >75%: (4)). The ﬁnal intensity score was equal to the staining intensity score multiplied by the cell percentage score. Staining was stratiﬁed accordingly into low levels of expression (scores <4) or high levels of expression (score ≥4). RNA extraction and quantitative real-time PCR (q-PCR) Total RNA was extracted from the cells and tissues using TRIzol reagent (Invitrogen, CA, USA) according to the manufacturer’s instructions. cDNA was synthesised Ofﬁcial journal of the American College of Gastroenterology Page 4 of 12 Wang et al. Clinical and Translational Gastroenterology (2018) 9:178 g. 2 (See legend on next page.) Fig. 2 (See legend on next page.) Ofﬁcial journal of the American College of Gastroenterology Ofﬁcial journal of the American College of Gastroenterology Page 5 of 12 Wang et al. Clinical and Translational Gastroenterology (2018) 9:178 g p p g Fig. 2 Effects of overexpressed PHB on pancreatic cancer cells AsPC-1 and MiaPaCa-2 proliferation, migration and invasion. a Overexpressed PHB plasmids were transfected into AsPC-1 and MiaPaCa-2 cells for 48 h, total proteins were extracted and Western Blotting analysis was performed, 81.3 and 90.6% PHB prtoteins were overexpressed, respectively. b The effects of PHB overexpressed on pancreatic cancer cells AsPC-1 and MiaPaCa-2, the growth after 24, 48 and 72 h were assessed using CCK-8 assay (mean ± SD; p < 0.05, *p < 0.01). c The migration of AsPC-1 and MiaPaCa-2 cells were assessed after overexpressed PHB plasmid transfection and compared with negative control transfected cells (mean ± SD; p < 0.05). d The invasiveness of AsPC-1 and MiaPaCa-2 cells were assessed after overexpressed PHB transfection and compared with negative control transfected cells (mean ± SD; p < 0.05) instructions 48 h after transfection, washed with cold PBS, and suspended in binding buffer. The cells were incubated for 10 min in the dark at room temperature with Annexin V-FITC and PI in phosphate buffer and analysed using a ﬂow cytometer (FACS CantoII, BD Bioscience, USA) within 1 h of staining. using M-MLV reverse transcriptase (Invitrogen) from 5 μg of total RNA. Quantitative RT-PCR was performed using a Bio-Rad CFX96 real-time PCR system (Bio-Rad, Foster City, CA, USA), KAPA PROBE FAST qPCR kits (Kapa Biosystems, MA, USA) and TaqMan probes (Invitrogen) with the following cycling conditions: 95 °C for 10 min (initial denature); 40 cycles of 95 °C for 15 s; and 60 °C for 60 s. The following sequences were used as PHB primers: 5′-GGGCACAGAGCTGTCATCTT-3′ and 5′-TGACTGGCACATTACGTGT-3′. Caspase-3 activity assay The effect of PHB on caspase-3 activity in AsPC-1 and MiaPaCa-2 cells was determined using a commer- cially available caspase-3 (active) ELISA kit (Applygen Technologies, China). An ELISA for caspase-3 activity was performed according to manufacturer’s instructions. Enzyme-linked immunosorbent assay (ELISA) Sera from 31 pancreatic cancer patients and 31 healthy volunteers were obtained with the consent of patients and donors after approval from the Institutional Human Ethical Committee of the Peking Union Medical College Hospital, China. The human prohibitin ELISA kit (Life Sciences Advanced Technologies, China) was used to determine prohibitin levels according to manufacturer’s instruction. Cell proliferation assay The impact of PHB silencing on the proliferation of pancreatic cancer cells was measured using a Cell Counting Kit-8 (CCK-8) (Dojindo, Japan), according to the manufacturer’s instruction. A total of 1.0 × 103 pan- creatic cancer cells were added to each well of 96-well culture plates 24 h after transfection. Cell proliferation was assessed 0, 24, 48 and 72 h. CCK-8 reagent (10 μl) was added to each well, and the absorbance was measured at 450 nm after a 1.5-h incubation at 37 °C. All experiments were performed in 5 wells per experiment and repeated at least three times. Ofﬁcial journal of the American College of Gastroenterology Statistical analysis The rate of cell apoptosis was quantiﬁed by annexin- V–FITC and propidium iodide double staining using an Annexin-V/FITC kit (Neobiscience, China). Cells were collected according to the manufacturer’s Statistical analysis was performed using SPSS23 software (SPSS, Inc., Chicago, IL). Median values were taken as cutoff limits when two groups were compared. Ofﬁcial journal of the American College of Gastroenterology Wang et al. Clinical and Translational Gastroenterology (2018) 9:178 Page 6 of 12 Fig. 3 siRNA-PHB inhibited apoptosis in pancreatic cancer cells. a After siRNA-PHB transfection 48h, the cells were harvested and double stained with Annexin-V and PI. The early, later and total apoptosis rate of siRNA-PHB cells was obviously lower than the control cells in AsPC-1 and MiaPaCa-2. (p < 0.05, *p < 0.01). b After siRNA-PHB transfection 48h, the protease activity of Caspase-3 notably decreased in AsPC-1 cells and in MiaPaCa-2 cells compared with the control, respectively. (p < 0.05). c Knockdown PHB resulted in a statistically signiﬁcant decrease in the expression of PARP, caspase-3, cleaved caspase-3 and caspase-9 protein in AsPC-1 (p < 0.05) and MiaPaCa-2 (p < 0.05) cells, compared to the control group, respectively. However, the expression of Bcl-2 was signiﬁcantly increased in the AsPC-1 (p < 0.05) and MiaPaCa-2 (p < 0.01) cells after siRNA-PHB transfection 48 h Fig. 3 siRNA-PHB inhibited apoptosis in pancreatic cancer cells. a After siRNA-PHB transfection 48h, the cells were harvested and double stained with Annexin-V and PI. The early, later and total apoptosis rate of siRNA-PHB cells was obviously lower than the control cells in AsPC-1 and MiaPaCa-2. (p < 0.05, p < 0.01). b After siRNA-PHB transfection 48h, the protease activity of Caspase-3 notably decreased in AsPC-1 cells and in MiaPaCa-2 cells compared with the control, respectively. (p < 0.05). c Knockdown PHB resulted in a statistically signiﬁcant decrease in the expression of PARP, caspase-3, cleaved caspase-3 and caspase-9 protein in AsPC-1 (p < 0.05) and MiaPaCa-2 (p < 0.05) cells, compared to the control group, respectively. However, the expression of Bcl-2 was signiﬁcantly increased in the AsPC-1 (p < 0.05) and MiaPaCa-2 (p < 0.01) cells after siRNA-PHB transfection 48 h Fig. 3 siRNA-PHB inhibited apoptosis in pancreatic cancer cells. a After siRNA-PHB transfection 48h, the cells were harvested and double stained with Annexin-V and PI. The early, later and total apoptosis rate of siRNA-PHB cells was obviously lower than the control cells in AsPC-1 and MiaPaCa-2. Prohibitin was expressed in PDAC cell lines, and prohibitin silencing inhibited the proliferation, migration and invasion of AsPC-1 and MiaPaCa-2 cells We investigated PHB expression in 8 pancreatic cancer cell lines, and PHB mRNA and protein were detectable in the cell lines. The AsPC-1 and MiaPaCa-2 cell lines were chosen for further functional experi- ments because of their high and low levels of PHB expression, respectively (Fig. 1a). We performed tran- sient silencing of PHB in AsPC-1 and MiaPaCa-2 cells. Speciﬁc PHB RNA interference (siRNA-PHB) molecules resulted in 71.6 and 87.1% reductions of PHB expression in AsPC-1 and MiaPaCa-2 cells at 48 h, respectively (Fig. 1b). CCK-8 assays were performed to assess the effects of PHB silencing on the proliferation of AsPC-1 and MiaPaCa-2 cells. We observed a sig- niﬁcant decrease in growth rate after effective reduction of endogenous PHB at 24, 48 and 72 h (P < 0.05) com- pared to the control cells (Fig. 1c). Transwell assays were performed to examine the potential role of PHB on cell migration and invasion. Knockdown of PHB expression signiﬁcantly suppressed the migra- tion of AsPC-1 (P < 0.01) and MiaPaCa-2 (P < 0.01) cells (Fig. 1d). The invasive abilities of siRNA-PHB AsPC-1 (P < 0.001) and MiaPaCa-2 (P < 0.001) cells were dramatically decreased compared to the control cells (Fig. 1e). Fig. 4 PHB higher expression in pancreatic cancer patients tissues and sera. a PHB mRNA expression was detected in 8 paired human Fig. 4 PHB higher expression in pancreatic cancer patients tissues and sera. a PHB mRNA expression was detected in 8 paired human pancreatic cancer tissues and normal pancreas with q-PCR analysis, the results were presented as the magnitude of relative expression (means ± SEM). β-actin was used as a housekeeping gene. b PHB protein expression was analysed in 8 paired human pancreatic cancer tissues and normal pancreas. Immunoblot analysis was performed using anti-rabbit antibodies (1:1000), GAPDH was used as a loading control. c The ELISA assay results showed that the level of serum PHB in the 31 PDAC patients was signiﬁcant higher than that of 31 healthy volunteer (5.71 ± 1.85 ng/ml vs. 3.02 ± 0.59 ng/ml, p < 0.01) and sera. a PHB mRNA expression was detected in 8 paired human pancreatic cancer tissues and normal pancreas with q-PCR analysis, the results were presented as the magnitude of relative expression (means ± SEM). β-actin was used as a housekeeping gene. b PHB protein expression was analysed in 8 paired human pancreatic cancer tissues and normal pancreas. Statistical analysis (p < 0.05, p < 0.01). b After siRNA-PHB transfection 48h, the protease activity of Caspase-3 notably decreased in AsPC-1 cells and in MiaPaCa-2 cells compared with the control, respectively. (p < 0.05). c Knockdown PHB resulted in a statistically signiﬁcant decrease in the expression of PARP, caspase-3, cleaved caspase-3 and caspase-9 protein in AsPC-1 (p < 0.05) and MiaPaCa-2 (p < 0.05) cells, compared to the control group, respectively. However, the expression of Bcl-2 was signiﬁcantly increased in the AsPC-1 (p < 0.05) and MiaPaCa-2 (p < 0.01) cells after siRNA-PHB transfection 48 h Ofﬁcial journal of the American College of Gastroenterology Wang et al. Clinical and Translational Gastroenterology (2018) 9:178 Page 7 of 12 Fig. 4 PHB higher expression in pancreatic cancer patients tissues and sera. a PHB mRNA expression was detected in 8 paired human pancreatic cancer tissues and normal pancreas with q-PCR analysis, the results were presented as the magnitude of relative expression (means ± SEM). β-actin was used as a housekeeping gene. b PHB protein expression was analysed in 8 paired human pancreatic cancer tissues and normal pancreas. Immunoblot analysis was performed using anti-rabbit antibodies (1:1000), GAPDH was used as a loading control. c The ELISA assay results showed that the level of serum PHB in the 31 PDAC patients was signiﬁcant higher than that of 31 healthy volunteer (5.71 ± 1.85 ng/ml vs. 3.02 ± 0.59 ng/ml, p < 0.01) 8 Page 7 of 12 Survival analyses were performed using the Kaplan–Meier method log-rank test. Multivariable analysis was per- formed using a Cox proportional hazards model. The median survival and estimations of hazard ratios were reported with 95% conﬁdence intervals. Comparisons of demographic and clinicopathological data between groups were performed using a Χ2 test. The measurement data are presented as means ± SD and compared using Student’s t test or the Mann–Whitney U test. Statistical signiﬁcance was set at a P value less than 0.05. GraphPad Prism 5 (GraphPad, San Diego, CA) software was used to create graphs. Prohibitin was expressed in PDAC cell lines, and prohibitin silencing inhibited the proliferation, migration and invasion of AsPC-1 and MiaPaCa-2 cells Immunoblot analysis was performed using anti-rabbit antibodies (1:1000), GAPDH was used as a loading control. c The ELISA assay results showed that the level of serum PHB in the 31 PDAC patients was signiﬁcant higher than that of 31 healthy volunteer (5.71 ± 1.85 ng/ml vs. 3.02 ± 0.59 ng/ml, p < 0.01) Results Prohibitin was expressed in PDAC cell lines, and prohibitin silencing inhibited the proliferation, migration and invasion of AsPC-1 and MiaPaCa-2 cells Prohibitin was expressed in PDAC cell lines, and prohibitin silencing inhibited the proliferation, migration and invasion of AsPC-1 and MiaPaCa-2 cells Overexpression of the PHB gene in AsPC-1 and MiaPaCa-2 cells increased proliferation, migration and invasion AsPC-1 and MiaPaCa-2 cells were transfected with PHB to conﬁrm the effect of siRNA-PHB on cells pro- liferation, migration and invasion. Speciﬁc pIERS2-EGFP- PHB transfection resulted in 81.3% and 90.6% over- expression of PHB in AsPC-1 and MiaPaCa-2 cells at 48 h, respectively, according to Western blotting analysis (Fig. 2a). CCK-8 assays were performed to assess the effects of PHB overexpression on proliferation. An obvious increase in growth rate was observed after effective PHB transfection at 24, 48 and 72 h in AsPC-1 (P < 0.05) and MiaPaCa-2 (P < 0.05) cells compared to the control cells (Fig. 2b). Transwell assays were performed to examine the impact of PHB overexpression on cell migration and invasion abilities. PHB overexpression Ofﬁcial journal of the American College of Gastroenterology Page 8 of 12 Wang et al. Clinical and Translational Gastroenterology (2018) 9:178 Fig. 5 Location and expression of PHB in normal and cancerous pancreatic tissues. Immunohistochemistry was performed with PHB (1:5000) anti- rabbit antibodies. (magnitude ×200) a Localisation of PHB in normal pancreatic tissues, acinar cells and some ductal cells showed very weak cytoplasmic staining. b Very weak positive staining of PHB in cancer cells. c Weak expression of PHB shown by cytoplasmic staining. d Mild expression of PHB shown by plasma membrane and cytoplasm of cancer cells. e Strong expression of PHB shown by plasma membrane and cytoplasm of cancer cells. f PHB was also strong expressed in pancreatic islet cells Fig. 5 Location and expression of PHB in normal and cancerous pancreatic tissues. Immunohistochemistry was performed with PHB (1:5000) anti- rabbit antibodies. (magnitude ×200) a Localisation of PHB in normal pancreatic tissues, acinar cells and some ductal cells showed very weak cytoplasmic staining. b Very weak positive staining of PHB in cancer cells. c Weak expression of PHB shown by cytoplasmic staining. d Mild expression of PHB shown by plasma membrane and cytoplasm of cancer cells. e Strong expression of PHB shown by plasma membrane and cytoplasm of cancer cells. f PHB was also strong expressed in pancreatic islet cells signiﬁcantly increased the migration ability of AsPC-1 (P < 0.05) and MiaPaCa-2 (P < 0.05) cells (Fig. 2c). PHB overexpression distinctly increased the invasive abilities of AsPC-1 (P < 0.05) and MiaPaCa-2 (P < 0.05) cells compared to controls (Fig. 1d). Ablation of PHB expression reduced apoptosis in PDAC cells Annexin-V and PI staining assays were used with ﬂow cytometry to quantify the effect of siRNA-PHB on apoptosis in human pancreatic cancer cells. Cells were stained with Annexin V-FITC/PI and gated into the lower right (LR) and upper right (UR) quadrants (Fig. 3). Cells in the LR and UR quadrants were considered early apoptotic (Annexin+/PI−−) and late apoptotic (Annexin+/PI+), respectively. Cells in the LL (lower left) and UL (upper left) quadrants were considered alive and necrotic, respectively. The extent of apoptosis is expressed as the sum of the percentages in the LR and UR quadrants. Cells treated with siRNA-PHB exhibited a decrease in apoptosis of 22.8% (siRNA-PHB) to 34.3% (negative control) in AsPC-1 cells (p < 0.01) and from 26.37% (siRNA-PHB) to 29.87% (negative control) in MiaPaCa-2 cells (p < 0.05) (Fig. 3a). The caspase cascade and several closely related mitochon- drial apoptosis proteins were analysed in AsPC-1 and MiaPaCa-2 cells to elucidate the possible pathway of PHB-induced apoptosis. The protease activity of Overexpression of the PHB gene in AsPC-1 and MiaPaCa-2 cells increased proliferation, migration and invasion caspase-3 48 h after PHB RNA interference decreased notably in AsPC-1 cells (p < 0.01) and MiaPaCa-2 cells (p < 0.01) compared to the controls cells (Fig. 3b). PHB knockdown signiﬁcantly decreased the expression of PARP, caspase-3, cleaved caspase-3 and caspase-9 proteins in AsPC-1 (p < 0.05) and MiaPaCa-2 (p < 0.05) cells compared to the control cells. However, Bcl-2 expression increased signiﬁcantly in AsPC-1 (p < 0.05) and MiaPaCa-2 (p < 0.01) cells after siRNA-PHB trans- fection (Fig. 3c). These data further support that the silencing of PHB expression reduces apoptosis in human pancreatic cancer cells via the mitochondrial pathway. Location, expression and clinicopathological relationship analyses of Prohibitin in pancreatic cancer tissues using IHC Table 1 Correlation of PHB expression with clinicopathologic parameters g Immunohistochemistry was performed in 10 normal and 66 PDAC tissues. Acinar cells and some ductal cells in normal tissues exhibited very weak cytoplasmic stain- ing (Fig. 5a). Strong PHB expression was observed in the plasma membrane and cytoplasm of cancer cells and degenerating acinar cells. Various intensities of PHB positivity were observed in cancer cells from the PDAC tissue samples (Fig. 5b–e). PHB was also strongly expressed in pancreatic islet cells (Fig. 5f). IHC revealed that 29 (43.9%) of 66 cases exhibited high PHB expression in the PDAC specimens, and the strong expression rate for para-pancreatic cancer tissues was only 14/66 (21.2%). These results suggest that PHB expression was signiﬁcantly higher in PDAC tissues (P < 0.05). We further divided the PDAC patients into PHB-weak and PHB-strong expression groups based on the differential expression levels of PHB in PDAC tissues. Table 1 shows the demographics and correlations between PHB expression and clinicopathological features. The rate of distant metastasis in follow-up was signiﬁcant higher in the high PHB expression group than the low PHB expression group (79.3 vs. 35.1%) (P < 0.001), which indicates that the overexpression of PHB was associated with the progression of pancreatic cancer. Obviously increased prohibitin expression in pancreatic cancer patient tissues and sera PHB mRNA and protein were detected in 8 paired human pancreatic cancer tissues and normal pancreatic tissues using q-PCR and Western blot (clinical char- acteristics in Supplement Table 1). PHB mRNA and protein expression levels in the pancreatic cancer tissues were signiﬁcantly increased compared to that in the normal pancreas samples (Fig. 4a, b). ELISAs demonstrated that the level of serum PHB in the 31 PDAC patients was signiﬁcantly higher than that in the 31 healthy volunteers (5.71 ± 1.85 ng/ml vs. 3.02 ± 0.59 ng/ml, P < 0.001) (Fig. 4c) (Clinicopathological para- meters in Supplement Table 2). Ofﬁcial journal of the American College of Gastroenterology Page 9 of 12 Wang et al. Clinical and Translational Gastroenterology (2018) 9:178 Table 1 Correlation of PHB expression with clinicopathologic parameters clinicopathologic Characteristics No. of patients Prohibitin expression P value High Low Overall frequency 0.009 PC tissure 66 29 37 Normal tissure 66 14 52 Age 1 <65 43 19 24 ≥65 23 10 13 Gender 0.153 Male 35 12 23 Female 31 17 14 Tumour location 0.314 Head 42 16 26 Body/tail 24 13 11 Histological gradea 0.705 Grade 1/2 37 15 22 Grade 3 29 14 15 Pathological T stage 0.375 T1/T2 11 3 8 T3/T4 55 29 37 Lymph node involvement 0.402 − 30 11 19 + 36 18 18 TNM stage 0.443 I 5 1 4 II 48 23 25 III/IV 13 5 8 Distant metastasis in follow-up <0.001 − 30 6 24 + 36 23 13 P < 0.01 aGrade 1, well differentiated; Grade 2, moderately differentiated; Grade 3, poorly differentiated Survival analysis and univariate/multivariate analyses of the prognostic value of Prohibitin expression in pancreatic cancer Patients with lower levels of PHB expression [n = 37, median survival = 27.4 (21.9–33.1) months] exhibited signiﬁcantly longer survival times compared to patients with higher PHB expression [n = 29, median survival = 11.1 (9.4–12.9) months, p < 0.01] (Fig. 6a). The prognostic value of PHB expression was assessed using a Cox proportional hazards model, and the results revealed that PHB expression (HR = 4.25, 95% CI: 2.09–4.26, P < 0.01) and histological grade (HR = 1.67, 95% CI: 1.07–2.16, P < 0.05) were independent prognostic factors (Fig. 6b). Other known prognostic factors (e.g., TNM stage and lymph node involvement) did not impact the prognosis independently because of the scarcity of early and less aggressive tumours in our cohort of 66 patients (Table 2). P < 0.01 aGrade 1, well differentiated; Grade 2, moderately differentiated; Grade 3, poorly differentiated poor prognosis and promotes cell proliferation and invasion20. Our study detected PHB mRNA and protein expression in 8 paired pancreatic cancer and normal pancreas tissues, and the expression of both entities were obviously higher in pancreatic cancer tissues than normal pancreas. The IHC results demonstrated higher PHB expression in 66 pancreatic cancer tissues compare to para-cancer tissues. We also found Ofﬁcial journal of the American College of Gastroenterology Discussion Recent studies reported that PHB was overexpressed in several tumour types, and it plays crucial roles in cancer development and progression, including hepato- cellular carcinoma, mammary cancer, squamous cell carcinoma of the lung and gastric cancer16–19. PHB is also overexpressed in gallbladder cancer, and it predicts Ofﬁcial journal of the American College of Gastroenterology Wang et al. Clinical and Translational Gastroenterology (2018) 9:178 Page 10 of 12 Fig. 6 Kaplan–Meier and log-rank analyses of prognostic factors assessed in multivariable analysis using a COX proportional hazards model. Median values were taken as cutoff limits when the two groups were compared. Survival analyses were performed using the Kaplan–Meier method for the estimation of event rates, and a log-rank test was used for survival comparisons between patient groups. The P value was 5%. PHB expression level (p < 0.01) and histological G stage (p < 0.05) both were independent prognostic factors, but the others risk factors, although showing the classical tendencies, did not appear to impact on the prognosis independency. (ﬁgures not showed) Fig. 6 Kaplan–Meier and log-rank analyses of prognostic factors assessed in multivariable analysis using a COX proportional hazards model. Median values were taken as cutoff limits when the two groups were compared. Survival analyses were performed using the Kaplan–Meier method for the estimation of event rates, and a log-rank test was used for survival comparisons between patient groups. The P value was 5%. PHB expression level (p < 0.01) and histological G stage (p < 0.05) both were independent prognostic factors, but the others risk factors, although showing the classical tendencies, did not appear to impact on the prognosis independency. (ﬁgures not showed) and invasion via ERK pathway activation in gallbladder cancer20. Zhou Luan et al. reported that blockade of the PHB scaffold-CRAF kinase interaction, which is distinct from direct kinase inhibition, may be a new therapeutic strategy to target oncogenic ERK-driven pancreatic cancers25. Our study veriﬁed that the pro- liferation, migration and invasion abilities were sig- niﬁcantly inhibited in PHB under-expressing pancreatic cancer cells. In contrast, the proliferation, migration and invasion abilities were up-regulated in PHB over- expressing pancreatic cancer cells. that higher PHB expression predicted a shorter overall survival time based on survival analysis. PHB and pathological stage G were independent risk factors in multivariate analyses using a Cox proportional hazards model, which suggests that PHB may be used as a potential factor for survival. Mengwasser et al. Ofﬁcial journal of the American College of Gastroenterology Discussion reported obviously higher PHB expression levels in colorectal cancer patient sera than normal sera21. PHB levels in our study were signiﬁcantly higher in pancreatic cancer patient sera than in healthy volunteer sera according to ELISA. Certainly, in our study, the sample size was small, and we only compared healthy volunteer and pancreatic cancer patients, this result had its own limitations. Next step, we will enlarge sample size and include more category of pancreatic and other digestive system diseases to identify this reselt deeply, but now, which means that PHB may be a new biomarker in pancreatic cancer patients in some extent. PHB is a small conserved protein that is implicated in numerous functions in mitochondria26. PHB appears indispensable for mitochondrial translation, and its abla- tion leads to a decrease in mitochondrial membrane potential27,28. Long Zhang et al. reported increased BAX and Cyt.c expression in PHB over-expressing gastric cancer BGC823 cells and decreased Bcl-2 expression. The activation levels of caspase-3 and caspase-9 were increased, but the activation level of caspase-8 was not changed, which suggests that PHB-induced apoptosis via the mitochondrial pathway29. Consistent with our study results, the expression of PARP, cleaved caspase-3, and caspase-9 was obviously decreased in PHB under- expressing pancreatic cancer cell lines AsPC-1 and MiaPaCa-2, but Bcl-2 was apparently increased. The activation level of caspase-3 was also decreased, which indicates that siRNA-PHB was an inhibitor of apoptosis in pancreatic cancer cells. Bcl-2 family proteins play an important role in regulating mitochondrial function and apoptosis. Bcl-2 family proteins form pore PHB is a hub for many signalling pathways triggered by growth factors, the immune response, steroid hor- mones that regulate metabolism, and mitochondrial biogenesis. PHB is involved in growth, resistance to chemotherapy, and metastasis via several mechanisms, including activation of the Ras-C-Raf-MEK-ERK pathway, modulation of TGF-β signalling, and regulation of tran- scription22. Many studies have demonstrated that PHB played an important role in the activation of the PI3K/Akt and Ras-C-Raf-MEK-ERK signalling pathways, in the modulation of epithelial cell adhesion and migration, and in the promotion of cancer metastasis23,24. Yang Cao et al. reported that PHB promoted cell proliferation Ofﬁcial journal of the American College of Gastroenterology Page 11 of 12 Page 11 of 12 Wang et al. 1. WHAT IS CURRENT KNOWLEDGE More than one-half of pancreatic cancer cases are diagnosed at a distant stage. Novel speciﬁc markers in pancreatic cancer patients are strong demanded. 2. WHAT IS NEW HERE PHB is signiﬁcantly increased in PDAC tissues and patients sera. PHB is an independent prognostic factor in PDAC patients. PHB plays a key role in modulating the malignant phenotype and apoptosis induction. PHB is signiﬁcantly increased in PDAC tissues and patients sera. PHB is an independent prognostic factor in PDAC patients. PHB plays a key role in modulating the malignant phenotype and apoptosis induction. PHB may be a novel prognostic predictor and a candidate for targeted therapy against PDAC. Conﬂict of Interest: Guarantor of the article: Wang Weibin. Speciﬁc author contributions: Z.Y.P. and Z.T.P. conceived and designed the study. W.W.B., X.L. and Y.Y. performed, collected and interpreted the data. W.W. B. and D.L.B. drafted the manuscript. W.W.B., Z.B.B. and LJ reviewed and edited the manuscript. All authors have read and approved the ﬁnal manuscript submitted. Financial support: The study was supported by grants from the 2016 PUMCH Science Found for Junior Faculty (no. pumch-2016-2.6), and National Natural Science Foundation of China (no. 81773215). Potential competing interests: None. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Discussion Clinical and Translational Gastroenterology (2018) 9:178 Table 2 Univariable and multivariable analysis of prognostic factors Univariable analysis Multivariable analysis Comparator N Median survival (95% CI) in months Log-rank p value HR 95% CI p value Median age (years) ≥65.5 vs. <65.5 23/43 18.2 (12.8–23.7) vs. 21.6 (16.6–26.6) 0.291 0.508 1.811 0 0.307–0.791 0.325 Gender M/F 35/31 18.9 (13.7–24.1) vs. 22.1 (16.8–27.4) 0.575 0.239 1.158 0.297–1.463 0.589 Tumour location Head vs. body/tail 42/24 22.5 (17.4–27.5) vs. 17.5 (11.7–23.4) 0.436 0.174 0.976 0.398–1.324 0.435 T-status T1/T2 vs. T3/T4 11/55 29.2 (19.8–38.7) vs. 18.9 (14.8–23.1) 0.194 0.097 1.576 0.12–2.312 0.292 N-status N0 vs. N1 30/36 25.6 (19.5–31.8) vs.16.2 (12.0–20.5) 0.163 0.016 1.18 0.63–2.21 0.61 TNM-stage I/II/III/IV 5/48/6/7 30.1 (22.4–37.6) vs.19.3 (15.3–23.3) vs.26.7 (13.5–39.8) vs.10.4 (2.7–18.2) 0.114 0.025 1.12 0.53–2.36 0.76 Grade G1 vs. G2 vs. G3 9/28/29 37.2 (26.4–48.1) vs. 18.1 (13.4–22.9) vs.17.6 (12.4–22.7) 0.51 0.021 1.67 1.07–2.61 0.026* PHB score ≤4 vs. >4 37/29 27.4 (9.4–12.9) vs. 11.1 (21.9–33.0) 1.446 0.001 4.25 2.09–4.26 0.001** compounds in the mitochondrial inter membrane and release apoptotic factors into the cytoplasm to cause apoptosis30. The exact mechanism of PHB in apoptosis in different cancer cells is not clear. Yingjie Xu et al. reported that PHB1 regulates tumour cell apoptosis via interaction with X-linked inhibitor of apoptosis protein31. Sanchez-Quiles V et al. reported that PHB regulates apoptosis in a mechanism dependent on NFκB signalling in human hepatoma cells32. In summary, we demonstrated that high PHB expres- sion in pancreatic cancer tissues and pancreatic cancer patient sera was associated with poor prognosis in pancreatic cancer patients. Furthermore, down-regulation of PHB expression reduced the proliferation, migra- tion and invasion of pancreatic cancer cells, and the opposite results were obtained in PHB over-expressing cells. Therefore, PHB may be a novel prognosis marker and candidate for targeted therapy against pancreatic cancer. Conﬂict of Interest: Speciﬁc author contributions: Z.Y.P. and Z.T.P. conceived and designed the study. W.W.B., X.L. and Y.Y. performed, collected and interpreted the data. W.W. B. and D.L.B. drafted the manuscript. W.W.B., Z.B.B. and LJ reviewed and edited the manuscript. All authors have read and approved the ﬁnal manuscript submitted. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Page 12 of 12 Page 12 of 12 Page 12 of 12 Wang et al. Clinical and Translational Gastroenterology (2018) 9:178 Wang et al. Clinical and Translational Gastroenterology (2018) 9:178 The online version of this article (https://doi.org/10.1038/s41424-018-0044-1) contains supplementary material, which is available to authorized users. The online version of this article (https://doi.org/10.1038/s41424-018-0044-1) contains supplementary material, which is available to authorized users. 16. Cheng, J. et al. Prohibitin-2 promotes hepatocellular carcinoma malignancy progression in hypoxia based on a label-free quantitative proteomics strategy. Mol. Carcinog. 53, 820–832 (2014). 17. Guo, F. et al. Prohibitin in squamous cell carcinoma of the lung: its expression and possible clinical signiﬁcance. Human. Pathol. 43, 1282–1288 (2012). Received: 25 December 2017 Revised: 4 June 2018 Accepted: 5 June 2018 Received: 25 December 2017 Revised: 4 June 2018 Accepted: 5 June 2018 18. Ko, K. S. et al. Liver-specifc deletion of prohibitin 1 results spontaneous liver injury, ﬁbrosis, hepatocellular carcinoma in mice. Hepatology 52, 2096–2108 (2010). References 19. Kang, X. et al. Prohibitin: a potential biomarker for tissue-based detection of gastric cancer. J. Gastroenterol. 43, 618–625 (2008). 1. Siegel, R. L., Miller, K. D. & Jemal, A. Cancer Statistics, 2017. CA Cancer J. Clin. 67, 7–30 (2017). 20. Cao, Y. et al. Prohibitin overexpression predicts poor prognosis and promotes cell proliferation and invasion through ERK pathway activation in gallbladder cancer. J. Exp. Clin. Cancer Res 35, 68 (2016). 2. Paulson, A. S., Tran Cao, H. S., Tempero, M. A. & Lowy, A. M. Therapeutic advances in pancreatic cancer. Gastroenterology 144, 1316–1326 (2013). 3. Conroy, T. et al. FOLFIRINOX versus gemcitabine for metastatic pancreatic cancer. N Engl. J. Med. 364, 1817–1825 (2011). 21. Mengwasser, J., Piau, A., Schlag, P. & Sleeman, J. P. Differential immunization identiﬁes PHB1/PHB2 as blood-borne tumor antigens. Oncogene 23, 7430–7435 (2004). cancer. N Engl. J. Med. 364, 1817–1825 (2011). 4. Cid-Arregui, A. & Juarez, V. Perspectives in the treatment of pancreatic ade- nocarcinoma. World J. Gastroenterol. 21, 9297–9316 (2015). 22. Thuaud, F., Ribeiro, N., Nebigil, C. G. & Desaubry, L. Prohibitin ligands in cell death and survival: mode of action and therapeutic potential. Chem. Biol. 20, 316–331 (2013). 5. Kotera, Y., Fontenot, J. D., Pecher, G., Metzgar, R. S. & Finn, O. J. Humoral immunity against a tandem repeat epitope of human mucin MUC-1 in sera from breast, pancreatic, and colon cancer patients. Cancer Res. 54, 2856–2860 (1994). 23. Rajalingam, K., & Rudel, T. Ras-Raf signaling eeds prohibitin. Cell Cycle 4, 1503–1505 (2005). 6. Gansauge, S. et al. The role of anti-p53-autoantibodies in pancreatic disorders. Int. J. Pancreatol. 19, 171–178 (1996). 24. Rajalingam, K. et al. Prohibitin is required for Ras-induced Raf-MEK-ERK acti- vation and epithelial cell migration. Nat. Cell Biol. 7, 837–843 (2005). 7. Raedle, J. et al. p53 autoantibodies in patien carcinoma. Pancreas 13, 241–246 (1996). 7. Raedle, J. et al. p53 autoantibodies in patients with pancreatitis and pancreatic carcinoma. Pancreas 13, 241–246 (1996). 25. Luan, Z., He, Y., Alattar, M., Chen, Z. & He, F. Targeting the prohibitin scaffold- CRAF kinase interaction in RAS-ERK-driven pancreatic ductal adenocarcinoma. Mol. Cancer 13, 38 (2014). carcinoma. Pancreas 13, 241–246 (1996). 8. Maacke, H. et al. Autoantibodies in sera of pancreatic cancer patients identify recombination factor Rad51 as a tumour-associated antigen. J. Cancer Res. Clin. Oncol. 128, 219–222 (2002). 8. Maacke, H. et al. Ofﬁcial journal of the American College of Gastroenterology References Autoantibodies in sera of pancreatic cancer patients identify recombination factor Rad51 as a tumour-associated antigen. J. Cancer Res. Clin. Oncol. 128, 219–222 (2002). 26. Tyc, J. et al. Probing for primary functions of prohibitin in Trypanosoma brucei. Int. J. Parasitol. 40, 73–83 (2010). 9. Wang, W. et al. Immunogenic membrane antigen and novel prognostic predictor: voltage-dependent anion channel 1 (VDAC1) in pancreatic cancer. Sci. Rep. 6, 33648 (2016). 27. Ross, J. A., Nagy, Z. S. & Kirken, R. A. The PHB1/2 phosphocomplex is required for mitochondrial homeostasis and survival of human T cells. J. Biol. Chem. 283, 4699–4713 (2008). 10. Nadimpalli, R., Yalpani, N., Johal, G. S. & Simmons, C. R. Prohibitins, stomatins, and plant disease response genes compose a protein superfamily that con- trols cell proliferation, ion channel regulation, and death. J. Biol. Chem. 275, 29579–29586 (2000). 28. Sun, Q. et al. Subproteomic analysis of the mitochondrial proteins in rats 24 h after partial hepatectomy. J. Cell. Biochem. 105, 176–184 (2008). 29. Zhang, L., Ji, Q., Ni, Z.-H. & Sun, J. Prohibitin induces apoptosis in BGC823 gastric cancer cells through the mitochondrial pathway. Asian Pac. J. Cancer Prev. 13, 3803–3807 (2012). 11. Mishra, S., Murphy, L. C. & Murphy, L. J. The Prohibitins: emerging roles in diverse functions. J. Cell. Mol. Med. 10, 353–363 (2006). 30. Korsmeyer, S. J. et al. Pro-apoptotic cascade activates BID, which oligomerizes BAK or BAX into pores that result in the release of cytochrome c. Cell death Differ. 7, 1166–1173 (2000). 12. Chowdhury, I. et al. The emerging roles of prohibitins in folliculogenesis. Front. Biosci. 4, 690–699 (2012). 13. Back, J. W. et al. A structure for the yeast prohibitin complex: Structure pre- diction and evidence from chemical crosslinking and mass spectrometry. Protein Sci. 11, 2471–2478 (2002). 31. Xu, Y., Yang, W., Shi, J. & Zetter, B. R. Prohibitin 1 regulates tumor cell apoptosis via the interaction with X-linked inhibitor of apoptosis protein. J. Mol. Cell Biol. 8, 282–285 (2016). 14. Kuwahara, Y. et al. Unusual thermal disassembly of the SPFH domain oligomer from Pyrococcus horikoshii. Biophys. J. 97, 2034–2043 (2009). 32. Sanchez-Quiles, V. et al. Prohibitin deﬁciency blocks proliferation and induces apoptosis in human hepatoma cells: molecular mechanisms and functional implications. Proteomics 10, 1609–1620 (2010). 15. Winter, A., Kamarainen, O. & Hofmann, A. Molecular modeling of prohibitin domains. Proteins 68, 353–362 (2007). Ofﬁcial journal of the American College of Gastroenterology
https://openalex.org/W3119013845	https://cdm21054.contentdm.oclc.org/digital/api/collection/IR/id/9324/download	English	null	Inhibition of Nonfunctional Ras	Cell chemical biology	2,021	cc-by	13,194	SUMMARY Intuitively, functional states should be targeted; not nonfunctional ones. So why could drugging the inactive K-Ras4BG12C work—but drugging the inactive kinase will likely not? The reason is the distinct oncogenic mechanisms. Kinase driver mutations work by stabilizing the active state and/or destabilizing the inactive state. Either way, oncogenic kinases are mostly in the active state. Ras driver mutations work by quelling its deactivation mechanisms, GTP hydrolysis, and nucleotide exchange. Covalent inhibitors that bind to the inactive GDP-bound K-Ras4BG12C conformation can thus work. By contrast, in kinases, allosteric inhib- itors work by altering the active-site conformation to favor orthosteric drugs. From the translational stand- point this distinction is vital: it expedites effective pharmaceutical development and extends the drug clas- siﬁcation based on the mechanism of action. Collectively, here we postulate that drug action relates to blocking the mechanism of activation, not to whether the protein is in the active or inactive state. Inhibition of Nonfunctional Ras Ruth Nussinov,1,2,* Hyunbum Jang,1 Attila Gursoy,3 Ozlem Keskin,4 and Vadim Gaponenko5,* 1Computational Structural Biology Section, Frederick National Laboratory for Cancer Research in the Laboratory of Cancer Immunometabolism, National Cancer Institute, Frederick, MD 21702, USA 2Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel 3Department of Computer Engineering, Koc University, Istanbul 34450, Turkey 4Department of Chemical and Biological Engineering, Koc University, Istanbul 34450, Turkey 5Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, IL 60607, USA Correspondence: nussinor@mail.nih.gov (R.N.), vadimg@uic.edu (V.G.) https://doi.org/10.1016/j.chembiol.2020.12.012 Ruth Nussinov,1,2, Hyunbum Jang,1 Attila Gursoy,3 Ozlem Keskin,4 and Vadim Gaponenko5,* 1Computational Structural Biology Section, Frederick National Laboratory for Cancer Research in the Laboratory of Cancer Immunometabolism, National Cancer Institute, Frederick, MD 21702, USA 2Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel 3Department of Computer Engineering, Koc University, Istanbul 34450, Turkey 4Department of Chemical and Biological Engineering, Koc University, Istanbul 34450, Turkey 5Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, IL 60607, USA Correspondence: nussinor@mail.nih.gov (R.N.), vadimg@uic.edu (V.G.) https://doi.org/10.1016/j.chembiol.2020.12.012 BACKGROUND However, the location of the drug-binding site matters (Guar- nera and Berezovsky, 2019, 2020), and in kinases the inactive state may also be targeted. For example, Raf’s activation is through homo- or heterodimerization. If a drug binds at the dimer interface to prevent activation via dimerization, it is a successful inhibitor of a nonfunctional kinase conformation. On the other hand, an inhibitor of active guanosine triphosphate (GTP)-bound Ras, binding at the interface of the effector binding site, still pre- vents the signal transduction of active Ras. That is, the drug ac- tion is related to blocking the protein mechanism of activation, not to whether the protein is in the active or inactive state. That is, we suggest drug classiﬁcation based on its mechanism of ac- tion as it would help guide its design. Why can targeting inactive Ras conformations inhibit active ones, but targeting inactive kinase conformations likely will not? There are already examples of inhibition of inactive (nonfunctional) mutant K-Ras4BG12C (Goody et al., 2019; Janes et al., 2018; Ket- tle et al., 2020; Lou et al., 2019; McCarthy et al., 2019; Nagasaka et al., 2020; Nussinov and Tsai, 2015; Ostrem et al., 2013; Zeng et al., 2017), among them some with promising drugs undergoing clinical trials, and ongoing attempts to tame K-Ras dimers (Kess- ler et al., 2020). However, inhibition of nonfunctional kinases was not taken up. This is vastly important to understand, as allosteric inhibitors are increasingly becoming a major drug-discovery route (Gross et al., 2020; Khan et al., 2020b; Kostaras et al., 2020; Lu et al., 2018, 2020; Nussinov and Tsai, 2012, 2013, 2015; Ward et al., 2020). Here, we ﬁrst brieﬂy review Ras biology, highlighting key attributes in Ras activation, some of which have been pursued in Ras pharmacology attempts. We follow with allosteric mutations and inhibitors aimed at nonfunctional Ras states, successes, failures, and considerations. We distinguish these from oncogenic activation of kinases and consequent allo- steric inhibitor strategies. In Ras, activating mutations work by interfering with deactivation; in kinases, they work by increasing the relative stability of the active versus the inactive state, thus vastly increasing the number of molecules in the active state. It is exceedingly challenging to design a high-afﬁnity drug that would bind the inactive state and shift the ensemble back to this state while still at nontoxic dosage. Allosteric drugs that alter the active site appear more tractable. Perspective Inhibition of Nonfunctional Ras Ruth Nussinov,1,2, Hyunbum Jang,1 Attila Gursoy,3 Ozlem Keskin,4 and Vadim Gaponenko5,* 1Computational Structural Biology Section, Frederick National Laboratory for Cancer Research in the Laboratory of Cancer Immunometabolism, National Cancer Institute, Frederick, MD 21702, USA 2Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel 3Department of Computer Engineering, Koc University, Istanbul 34450, Turkey 4Department of Chemical and Biological Engineering, Koc University, Istanbul 34450, Turkey 5Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, IL 60607, USA *Correspondence: nussinor@mail.nih.gov (R.N.), vadimg@uic.edu (V.G.) https://doi.org/10.1016/j.chembiol.2020.12.012 ll OPEN ACCESS ll OPEN ACCESS Cell Chemical Biology 28, February 18, 2021 ª 2021 The Authors. Published by Elsevier Ltd. 121 This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). RAS: AN OVERVIEW It also inﬂuences Ras orientation at the membrane, accessibility, and selective interaction with its effectors (Ahearn et al., 2011; Cao et al., 2019; Chavan et al., 2015; Jang et al., 2016a, 2020; Neale and Garcia, 2020; Nussinov et al., 2018a; Prakash and Gorfe, 2019; Prakash et al., 2020). Membrane attachment can also be regulated by ubiquitination (Baietti et al., 2016; Steklov et al., 2018; Thurman et al., 2020), phosphorylation of the C terminus as well as other post-transla- tional modiﬁcations, such as acetylation, and calmodulin (Jang et al., 2017, 2019; Nussinov et al., 2017b). ) As members of the small GTPase family, Ras proteins function as molecular switches between their two states (Bourne et al., 1991; Cherﬁls and Zeghouf, 2013; Downward, 1990; Geyer and Wittinghofer, 1997; Grand and Owen, 1991; Lamontanara et al., 2014; Lowy et al., 1991; Sprang, 1997; Takai et al., 2001; Vetter and Wittinghofer, 2001; Wittinghofer and Pai, 1991; Wit- tinghofer and Vetter, 2011). Inactivation is mediated by GTPase-activating proteins (GAPs) that hydrolyze the GTP to GDP (Wittinghofer et al., 1997). Strong oncogenic mutations block GAP catalytic activity, retaining the active GTP-bound state, thus promoting downstream signaling and cell prolifera- tion as in the case of G12D K-Ras4B mutant (Adjei, 2001; Schub- bert et al., 2007; Smith and Ikura, 2014; Smith et al., 2013; Vigil et al., 2010). Weaker driver mutations can be associated with promoting a high rate of intrinsic and guanine nucleotide ex- change factor-induced nucleotide exchange, as in the case of A146T K-Ras4B mutant (Poulin et al., 2019). The second major pathway activated by Ras at the membrane is Ras/PI3K/AKT/mTOR (Downward, 2008; Sheridan and Down- ward, 2013). RTK interacts with the nSH2 domain in the p85a regulatory domain of phosphatidylinositol 3-kinase (PI3Ka), re- cruiting the lipid kinase to the membrane and releasing its autoinhibition (Zhang et al., 2019a, 2019b). Subsequent confor- mational changes expose the active site (Zhang et al., 2019a). Ras-GTP assists in the recruitment, acting to enhance the cata- lytically favored PI3K population at the membrane where signaling lipid substrate phosphatidylinositol-3,4-bisphosphate (PIP2) is located. PI3K phosphorylates PIP2 to produce phospha- tidylinositol-3,4,5-bisphosphate (PIP3). PIP3 recruits AKT (also known as protein kinase B [PKB]) and 3-phosphoinositide- dependent protein kinase 1 (PDK1) to the plasma membrane. PDK1 phosphorylates and activates AKT, with the signaling propagating downstream to activate transcription factors whose transcription products also enter the cell cycle at the G1 phase. RAS: AN OVERVIEW Together with the MAPK products, they act in cell growth and di- vision, accelerating proliferation (Gul et al., 2018; Lien et al., 2017; Nussinov et al., 2019a, 2020; Pokrass et al., 2020; Zhang et al., 2020). Countering PIP2 to PIP3 phosphorylation by PI3K is PTEN (phosphatase and tensin homolog), a phosphatase (Hosford et al., 2017; Ijuin, 2019; Yehia et al., 2020). Below, we ﬁrst distinguish between allosteric mutations in ki- nases and in Ras. In Ras, they work by quenching deactivation mechanisms, GTP hydrolysis, and nucleotide exchange; inhibi- tors that bind to the inactive conformation block reactivation. By contrast, in kinases they act by shifting the equilibrium toward the active state; accordingly, their allosteric drugs aim to modu- late the active-site conformation. The distinct mutation scenarios are the key to their inhibition strategy, and these considerations are general. RAS: AN OVERVIEW Wild-type Ras regulates cell growth and division (Bryant et al., 2014; Cox and Der, 2010; Crespo and Leon, 2000; Drosten et al., 2010; Lu et al., 2016b; Pylayeva-Gupta et al., 2011; Wies- muller and Wittinghofer, 1994). An activated receptor tyrosine ki- nase (RTK), such as epidermal growth factor receptor (EGFR) (Moran et al., 1991; Nussinov et al., 2020), recruits the Son of Sevenless (SOS), a Ras-speciﬁc guanine nucleotide exchange factor (GEF). Mediated by adaptor proteins SHC (Src homology 2 domain containing) and Grb2 (growth factor receptor-binding protein 2), RTK activates SOS, which acts to substitute guano- sine diphosphate (GDP) by GTP at the membrane to activate Ras (Bandaru et al., 2019; Huang et al., 2019; Liao et al., 2018, 2020). Membrane-anchored Ras dimers or nanoclusters bind ll OPEN ACCESS Perspective and activate Raf kinase (Nussinov et al., 2019b, 2019c, 2020; Solman et al., 2015; Sutton et al., 2019; Zhou et al., 2018). In the absence of Ras, Raf is autoinhibited. Its Ras-binding domain (RBD) and cysteine-rich domain (CRD) loosely interact and occlude the kinase domain (KD) dimerization surface (Nussinov et al., 2018b). This conformation is stabilized by 14-3-3 (Kondo et al., 2019; Park et al., 2019; Rezaei Adariani et al., 2018). The high-afﬁnity Ras-Raf’s RBD interaction, enhanced by CRD mem- brane attachment (Fang et al., 2020; Li et al., 2018a, 2018b; Okada et al., 1999; Sarkar and Garcia, 2020; Travers et al., 2018), shifts Raf’s equilibrium toward the open state and interac- tion with Ras (Nussinov et al., 2019a). RBDs’ interactions with spatially proximal Ras catalytic domains in Ras dimers or nano- clusters promote Raf’s KD dimerization and activation (Holder- ﬁeld et al., 2014; Lavoie et al., 2013; Lee et al., 2019; Muratcioglu et al., 2015, 2020). Active Raf phosphorylates MEK1/2 (MAPK ki- nase 1/2) (De Luca et al., 2012; Young et al., 2013), with signaling propagating down the mitogen-activated protein kinase (MAPK) pathway to activate extracellular signal-regulated kinase (ERK) and transcription factors, such as Elk-1 (Gao et al., 2019; Nussi- nov et al., 2014), with the resulting expressed proteins entering the G1 phase of the cell cycle, thereby linking the cellular environ- ment to cell-cycle progression. At the G1 phase, cyclin-depen- dent kinases promote passage to the S (synthesis) phase. membrane properties and, thus, segregation into homogeneous nanoclusters and dimers (Fujiwara et al., 2002; Murase et al., 2004; Simons and Vaz, 2004). 122 Cell Chemical Biology 28, February 18, 2021 ALLOSTERIC MUTATIONS IN RAS AND KINASES WORK DIFFERENTLY; THUS, THEIR INHIBITION STRATEGIES SHOULD DIFFER TOO Protein function hinges on the extent to which it populates its active conformation (Lu et al., 2014a, 2014b; Nussinov and Tsai, 2013). Molecular ensembles exist in a conformational equi- librium of active and inactive states (Henley et al., 2020; Nussi- nov, 2016; Nussinov and Wolynes, 2014). Aside from repressors, under physiological conditions most enzymes and receptors are in their inactive states. When stimulated, either by an incoming signal or oncogenic mutations, the ensemble shifts to populate the active state (Tsai and Nussinov, 2014). The relative popula- tions of the states reﬂect their relative stabilities. Allosteric inhib- itors whose shapes and chemistry ﬁt a certain patch of surface of the enzyme or receptor in the inactive (or for repressors, active) state are conformationally selected. The ensemble is equili- brated by a shift toward the stabilized inactive state. Ras anchors in the plasma membrane through its prenylated hypervariable region (HVR) at the C terminus (Barklis et al., 2019; Nussinov et al., 2018a). Whereas the sequences and struc- tures of the catalytic domains of H-Ras and N-Ras, and splice iso- forms K-Ras4A and K-Ras4B, are highly similar, this is not the case for their HVRs (Lu et al., 2016b; Nussinov et al., 2017a; Py- layeva-Gupta et al., 2011) and the distinct combinations of their lipid post-translational modiﬁcations—farnesyl (or geranylger- anyl) and palmitoyl. K-Ras4B is farnesylated, K-Ras4A can be far- nesylated or farnesylated and palmitoylated; N-Ras is also farne- sylated and palmitoylated, and H-Ras is farnesylated and double- palmitoylated. Their distinct chemistry makes them favor different Kinases ﬁt into this description (Figure 1). They switch from the inactive aC-helix-out to the active aC-helix-in conformation 122 Cell Chemical Biology 28, February 18, 2021 ll OPEN ACCESS Perspective Figure 1. The Free Energy Landscape DDG of a Kinase Conformation (here EGFR), the Effect of Mutations, and an Allosteric Inhibitor Oncogenic driver mutations (L858R and T790M) destabilize the inactive state (left plot in the middle panel) and stabilize the active state (center plot), or both (right plot). The outcome is a shift of the ensemble toward the constitutively active EGFR conformation. In the bottom panel, an allosteric drug binding EGFR’s mutant surface stabilizes this conformation, with a population shift toward this conformation. The drug modulates the active-site shape. Perspective the release of the autoinhibition. In the wild type it is triggered by nSH2 domain interacting with RTK’s pYXXM motif in the C ter- minus. ALLOSTERIC MUTATIONS IN RAS AND KINASES WORK DIFFERENTLY; THUS, THEIR INHIBITION STRATEGIES SHOULD DIFFER TOO The outcome is exposure of the active site at the mem- brane. Other mutations, e.g., Glu81, Gly106, Arg108, Lys111, and Gly118, in the adaptor-binding domain, contribute to the exposure as well. These mutations reduce the transition-state barrier (ka). Another common driver, H1047R, enhances the pop- ulation time of the lipid substrate at the active site (km). In TRK tyrosine kinases, xDFG mutations confer resistance to type I in- hibitors but sensitize them to type II by stabilizing the inactive (DFG-out) conformations (Cocco et al., 2020). Not all mutations can, however, be described by the free en- ergy landscape. Ras’s major mutations, e.g., Gly12, Gly13, and Gln61, which block hydrolysis, cannot be described by the free energy landscape (Fernandez-Medarde and Santos, 2011; Nus- sinov et al., 2013; Tsai and Nussinov, 2014). Neither can muta- tions that block nucleotide exchange, such as K-Ras4BA146T. GTP hydrolysis involves proton transfer from an attacking water molecule to another, followed by transfer of a different proton from this water molecule to the GTP. Gln61 can stabilize the tran- sient OH and H3O+ molecules lowering the transition-state bar- rier, which the Leu61 cannot (Martin-Garcia et al., 2012). As to the Gly12 mutants, their longer side chains interfere with GAP’s argi- nine ﬁnger insertion and hydrolysis (Krengel et al., 1990). Despite the different mechanisms, both kinases and Ras GTPases are dynamic molecules, with multiple, not only two discrete, active and inactive states. Both types of proteins can be considered from the mechanistic free energy landscape standpoint, which epitomizes the ‘‘second molecular biological revolution’’ (Nussinov and Wolynes, 2014). The paradigm fo- mented by this revolution postulated that the energy landscape (Frauenfelder et al., 1991) and the laws of quantum mechanics and structural chemistry apply to all biological systems and that biomolecules always interconvert between conformations with varying energies. This view inspired the dynamical statistical description of the molecules (Kumar et al., 2000; Ma et al., 2002; Tsai et al., 1999a, 1999b), whereby the ensembles describe the relative conformational stabilities of the states, their transitions, and their population shifts to retain their equilibrium (Arora and Brooks, 2007; Boehr et al., 2009; Huang et al., 2015; Kar et al., 2010; Mackereth et al., 2011; Motlagh et al., 2014; Nussinov and Ma, 2012; Okazaki and Takada, 2008). GDP-bound Ras can exist in inactive and active conformations. Similarly, Ras- GTP exists in active and inactive states, although the popula- tions vary. ALLOSTERIC MUTATIONS IN RAS AND KINASES WORK DIFFERENTLY; THUS, THEIR INHIBITION STRATEGIES SHOULD DIFFER TOO Indeed, molecular dynamics (MD) simulations and crystallography (Shima et al., 2010) suggest that the inactive state of Ras-GTP may exist in at least three substates. Notably, in reality, there are many inactive protein states and there is also an ensemble at the transition state. Classically, catalysis involves tight binding of the substrate to the transition-state structure to lower the activation energy. However, the transition state is a surface, not a single saddle point on the potential energy surface, and the activated conformations in the transition-state ensemble can catalyze multiple reaction steps (Ma et al., 2000). Nuclear magnetic resonance of GppNHp-bound wild-type H-Ras cata- lytic domain identiﬁed two conformational states that intercon- vert on a millisecond timescale, inactive state 1 and active state 2 (Geyer et al., 1996; Liao et al., 2008). The perturbations induced by the oncogenic mutations promote a redistribution of the Figure 1. The Free Energy Landscape DDG of a Kinase Conformation (here EGFR), the Effect of Mutations, and an Allosteric Inhibitor Oncogenic driver mutations (L858R and T790M) destabilize the inactive state (left plot in the middle panel) and stabilize the active state (center plot), or both (right plot). The outcome is a shift of the ensemble toward the constitutively active EGFR conformation. In the bottom panel, an allosteric drug binding EGFR’s mutant surface stabilizes this conformation, with a population shift toward this conformation. The drug modulates the active-site shape. through a rotation and shift movement, with a salt bridge be- tween the b3-Lys and the aC-Glu, and R-spine formation (Nussi- nov and Tsai, 2013). The triggers for the conformational change vary among the kinase families. Oncogenic mutations stabilize the active aC-helix-in conformation or disrupt interactions that stabilize the aC-helix-out, as in the case of the L858R in EGFR. There, the mutation stabilizes the aC-helix-in conformation by heterodimerization. The mutation is in the middle of a hydropho- bic core. Thus, the change to Arg also disrupts stabilizing hydro- phobic interactions in the aC-helix-out conformation. The T790M mutation in EGFR, T315I in Bcr-Abl, T334I in c-Abl, T341I in Src, T670I in Kit, and T674I in PDGFRa (platelet-derived growth factor receptor A) work by stabilizing the hydrophobic R-spine, pro- moting enzymatically active conformation (Nussinov and Tsai, 2013). ALLOSTERIC MUTATIONS IN RAS AND KINASES WORK DIFFERENTLY; THUS, THEIR INHIBITION STRATEGIES SHOULD DIFFER TOO Early on, a small-molecule compound Kobe0065 and cyclen-metal complexes that bound to the inactive state were developed (Shima et al., 2013). A cova- lent allosteric inhibitor for oncogenic K-RasG12C obtained by a di- sulﬁde fragment-based screening approach (Ostrem et al., 2013) as well as inhibitors that target nonfunctional dimers (Kessler et al., 2019, 2020; Tran et al., 2020) exploit the same rationale. ensemble. Crystal structures reveal allosteric effects on confor- mational states induced by mutations, and mutants display distinct outcomes in effector binding and in oncogenicity, as in the cases of G12V versus G12D. Furthermore, K-Ras populates conformational states differently from its isoform H-Ras and oncogenic mutant K-Ras4BG12D (Parker and Mattos, 2018; Parker et al., 2018). ) The free energy landscape describes the ensemble around the native state as populating the active and inactive states, with the states separated by kinetic barriers. The more stable the confor- mation, the larger its population. Due to functional requirements, the barriers between states are not too high, which allows conformations to switch between them. In kinases, oncogenic mutations work by creating new interactions and/or abolishing existing ones, resulting in higher stability (population) of the active state. In the case of Ras, these free energy considerations do not apply to the mutation actions. GTP binds the active state. With the high-millimolar-range concentration of the guanine nucleotide in the cell and its picomolar afﬁnity, ~75% of K- RasG12C is GTP bound in the steady state. Intrinsic hydrolysis, which is high for this mutant (Hunter et al., 2015), cycles K- Ras4BG12C into its GDP-bound state while allosteric drugs block the nucleotide exchange. Thus, not all mutations can be described by the free energy landscape; only those working via a population shift can (Tsai and Nussinov, 2014). Accordingly, the nature of the drugs should be different. The nonfunctional states of Ras can be targeted; however, for kinases, shifting the ensemble away from the constitutively active state, by for example stabilizing the inactive autoinhibited state, is challenging. Imatinib (STI-571/Gleevec), the ﬁrst synthetic kinase inhibitor to target an oncogene (Bcr-Abl), can serve as a classical example to illustrate our point (Nagar et al., 2003). The physiolog- ical state of Abl is autoinhibited via intradomain interactions be- tween the SH3-linker-SH2 and the KD. The fused Bcr abolishes the autoinhibition to constitutively activate it. ALLOSTERIC MUTATIONS IN RAS AND KINASES WORK DIFFERENTLY; THUS, THEIR INHIBITION STRATEGIES SHOULD DIFFER TOO The landmark crys- tal structure of the KD described by Kuriyan and colleagues with imatinib bound at the ATP-binding site reveals a collapsed acti- vation loop blocking substrate binding, which is open and extended in all active KDs. This binding indicates that imatinib only can bind to the less populated, inactive kinase conformation of Bcr-Abl. The drug-resistant mutations observed by Daley and colleagues (Azam et al., 2003) either directly participate in imati- nib binding or act allosterically to destabilize the autoinhibited Abl kinase conformation to which STI-571 preferentially binds. That is, they reduce drug-binding afﬁnity or increase the popula- tion of the active state by destabilizing the inactive conformation, counteracting the inhibiting action of imatinib. However, as the inhibition of Raf (homo- or hetero-) dimeriza- tion shows, the location of the drug binding matters: if the drug is bound at the dimer interface to prevent activation via dimeriza- tion, it is a successful inhibitor bound to an inactive kinase conformation. ALLOSTERIC MUTATIONS IN RAS AND KINASES WORK DIFFERENTLY; THUS, THEIR INHIBITION STRATEGIES SHOULD DIFFER TOO In another example, in PI3Ka lipid kinase (Zhang et al., 2019a) oncogenic mutations E542K and E545K in the helical domain, with charge reversal (Leontiadou et al., 2018), mimic through a rotation and shift movement, with a salt bridge be- tween the b3-Lys and the aC-Glu, and R-spine formation (Nussi- nov and Tsai, 2013). The triggers for the conformational change vary among the kinase families. Oncogenic mutations stabilize the active aC-helix-in conformation or disrupt interactions that stabilize the aC-helix-out, as in the case of the L858R in EGFR. There, the mutation stabilizes the aC-helix-in conformation by heterodimerization. The mutation is in the middle of a hydropho- bic core. Thus, the change to Arg also disrupts stabilizing hydro- phobic interactions in the aC-helix-out conformation. The T790M mutation in EGFR, T315I in Bcr-Abl, T334I in c-Abl, T341I in Src, T670I in Kit, and T674I in PDGFRa (platelet-derived growth factor receptor A) work by stabilizing the hydrophobic R-spine, pro- moting enzymatically active conformation (Nussinov and Tsai, 2013). In another example, in PI3Ka lipid kinase (Zhang et al., 2019a) oncogenic mutations E542K and E545K in the helical domain, with charge reversal (Leontiadou et al., 2018), mimic Cell Chemical Biology 28, February 18, 2021 123 ll OPEN ACCESS Perspective massive community efforts to identify effective inhibitors have proved to be challenging (Cox et al., 2014; Ledford, 2015; Lu et al., 2016c; Milroy and Ottmann, 2014; Samatar and Poulika- kos, 2014; Shao et al., 2014; Spiegel et al., 2014; Stites and Rav- ichandran, 2009; Wang et al., 2013; Zimmermann et al., 2013). There are many reasons for the difﬁculty, including Ras’s picomolar afﬁnity for guanine nucleotide whose cellular concen- tration is in the millimolar range, making binding of nucleotide an- alogs disfavored (Khan et al., 2020a; Waters and Der, 2018). In addition, despite searches, deep pockets for small-molecule binding have not been identiﬁed (Spiegel et al., 2014). ALLOSTERIC MUTATIONS IN RAS AND KINASES WORK DIFFERENTLY; THUS, THEIR INHIBITION STRATEGIES SHOULD DIFFER TOO The diverse strategies of potential Ras inhibitors have been reviewed extensively in the literature (Chatani and Yang, 2020; Cox et al., 2014, 2015; Dang et al., 2017; Gentile et al., 2017; Gorfe and Cho, 2019; Gupta et al., 2019; Khan et al., 2020a; Liu et al., 2010, 2019; Lu et al., 2016a, 2016c; Mattingly, 2013; McCor- mick, 2018; Mullard, 2019; O’Bryan, 2019; Papke and Der, 2017; Papke et al., 2016; Patricelli et al., 2016; Sakamoto et al., 2017; Sheridan, 2020; Singh et al., 2015; Spencer-Smith and O’Bryan, 2019; Spiegel et al., 2014; Zhang and Shokat, 2019; Zimmermann et al., 2013). Among those that have been explored are agents blocking Ras dimerization (e.g., NS1 and K13) (Spencer-Smith et al., 2017) and inhibition of farnesyl trans- ferase (FTase), thus blocking cysteine farnesylation at the C ter- minus, translocation from the ER to the plasma membrane, and anchorage (e.g., tipifarnib, deltasonamide) (Baranyi et al., 2020; Cheng et al., 2020; Martin-Gago et al., 2017; Reid and Beese, 2004). Blocking Ras-GEF interaction (e.g., DCAI, HBS3) (Maurer et al., 2012; Patgiri et al., 2011), and Ras-Raf (e.g., Abd-7, com- pound 3344) (Bery et al., 2018; Quevedo et al., 2018) (Figure 2) have also been explored. In another strategy, an unbiased approach using synthetic single-domain monobodies (Spencer-Smith et al., 2017) was employed. An NS1 monobody for Ras isoforms selectively inhibited oncogenic H-Ras and K- Ras, but not N-Ras, proliferation (Khan et al., 2020a). Additional strategies for preventing Ras-plasma membrane anchorage include enhancing K-Ras phosphorylation, inhibiting K-Ras interaction with its chaperone protein, and more (Cheng et al., 2020; Gorfe and Cho, 2019). With the aim of identifying function- ally productive states with the effector interacting surface exposed, Ras orientation at the membrane has also been considered (Cao et al., 2019; Chavan et al., 2015; Jang et al., 2016a, 2020; Neale and Garcia, 2020; Nussinov et al., 2018a; Prakash and Gorfe, 2019; Prakash et al., 2020). These ap- proaches are hindered, however, by membrane dynamics and ﬂuidity, toxicity, speciﬁcity, and thus altogether effectiveness. SML-8-73-1 (Hunter et al., 2014; Lim et al., 2014; Xiong et al., 2017) and SML-10-70-1 (Lim et al., 2014) are covalent small- molecule inhibitors binding at the active site of oncogenic G12C K-Ras mutant. Direct approaches of targeting Ras appear more promising but still face hurdles. 124 Cell Chemical Biology 28, February 18, 2021 ALLOSTERIC DRUGS TARGETING NONFUNCTIONAL RAS The pocket it binds differs from that of covalent K-RasG12C inhibitors, which bind to the SII-P pocket in the Switch II region and is present in both the active (GTP- bound) and inactive (GDP-bound) forms of Ras proteins. It mod- ulates MAPK signaling (pERK phosphorylation) and PI3K/AKT (pAKT) and inhibits KRAS mutant cell proliferation, suggesting that this pocket can be druggable. Tran et al. (2020) proposed an alternative interpretation to the observation by Kessler et al. (2019) of BI-2852 inhibition at nano- molar afﬁnity. Their analysis of the crystal structure of K-Ras4B with BI-2852, which included the neighboring unit cell, observed that BI-2852 induces a K-Ras4B dimer consisting of four mole- cules (two of BI-2852 and two of K-Ras4B) with rotational sym- metry (Figure 3, right panel). This was conﬁrmed experimentally by size-exclusion column chromatography at a concentration 1,000 times higher (3 mM) than those in which the cellular effects of BI-2852 were observed (Kessler et al., 2020), mass spectrom- etry, measurement of binding afﬁnity (isothermal titration calo- rimetry), and surface plasmon resonance. Analysis of the crystal structure (PDB: 6GJ8) indicated that the pocket is formed by two K-Ras4B molecules where one copy of BI-2852 binds, suggest- ing an asymmetry, with one K-Ras4B presenting a larger pocket (Switch I/II pocket) and the other a smaller one. The second BI- 2852 indicates symmetric formation. The complex is stabilized by four salt bridges altogether, pointing to the discovery by Kess- ler et al. (2019) of formation of a nonfunctional dimer stabilized by two molecules of BI-2852. The covalent attachment ﬁrst designed by Shokat and col- leagues involved disulﬁde tethering to the Cys, exploiting the Cys thiol reactivity (Ostrem et al., 2013). Exploiting this method, they obtained compounds that bound in an unknown pocket in the Switch II region near the nucleotide binding pocket (SW2 binding pocket). The pocket exists in the GDP-bound but not in the GTP-bound conformation; thus, these compounds bound only the GDP-bound state. Their binding resulted in conforma- tional changes in the Switch I and Switch II regions, inhibiting in- teractions with Ras effectors and activators. The impact of this work resulted from its signiﬁcant and novel components: it was the ﬁrst to exploit covalent allosteric drug on Ras and it exploited its inactive state. It provided hope that this concept can ﬁnally produce the highly coveted drug against a mutant Ras. ALLOSTERIC DRUGS TARGETING NONFUNCTIONAL RAS Using a fragment-based screening approach, Shokat and co- workers pioneered the ﬁrst compounds that covalently link to the cysteine in GDP-bound K-RasG12C, blocking SOS-catalyzed nucleotide exchange (Ostrem et al., 2013). Their initial leads had only millimolar afﬁnity, although subsequent work by several groups led to other high-afﬁnity drugs with some undergoing clinical trials (Sheridan, 2020). Among the more potent com- pounds are AMG510 (Sotorasib) by Amgen (phase I trial), MRTX849 (phase I/II) (Canon et al., 2019; Hallin et al., 2020; Mir- ati Therapeutics, 2020), JNJ-74699157 (formerly ARS-3248) (phase I, Johnson & Johnson, Wellspring Biosciences; earlier ARS-1620 [Janes et al., 2018]), and LY3499446 (phase I/II clinical trials, Eli Lilly). Illustrating the hardships in developing a drug against K-Ras, Eli Lilly exited the K-Ras race due to toxicity in its phase I effort, and Johnson & Johnson stopped recruiting (Adams, 2020). Initial results in patients with K-RasG12C mutation indicated that MRTX849 is a promising clinical candidate (Fell et al., 2020; Gabizon and London, 2020). Both AMG510 and MRTX seem to be moving at equal pace in the clinic. Phase I data for AMG510 in advanced colorectal cancer suggested that monotherapy provided prolonged disease control. Anti- tumor activity was also observed across multiple solid tumors (Hong et al., 2020). A phase II monotherapy study in advanced colorectal cancer is getting under way. A targeted degradation strategy for oncogenic K-RasG12C by construction of a library of C12-directed covalent degrader molecules (PROTACs) was also explored; however, initially the lead degrader was unable to effectively polyubiquitinate cellular K-RasG12C (Zeng et al., 2020). More recently, LC-2, a PROTAC capable of degrading endogenous K-RasG12C via E3 ligase VHL, was developed. It too covalently binds K-RasG12C with an MRTX849 warhead, re- sulting in MAPK suppression (Bond et al., 2020). These allosteric compounds bind to nonfunctional K-RasG12C. Kessler et al. (2019) have targeted a pocket in K-Ras4B previ- ously considered ‘‘undruggable’’ due to its comparative shallow- ness and polarity, rather than hydrophobic, between Switch I and Switch II (SI/II pocket) (Cruz-Migoni et al., 2019; Maurer et al., 2012; Quevedo et al., 2018; Sun et al., 2012). Starting from very weakly binding fragments and structure-based drug design, they discovered BI-2852 (compound 1) (Figure 3, upper left panel), a nanomolar inhibitor to this pocket distinct from co- valent K-RasG12C inhibitors. APPROACHES TO RAS INHIBITION Driver mutations in K-Ras are common in cancers (Chen et al., 2015; Eser et al., 2014; Holderﬁeld et al., 2014; Krens et al., 2010; Prior et al., 2012; Stephen et al., 2014); however, the Allosteric compounds that target inactive monomers and di- mers may hold the key for future successes. Nonetheless, 124 Cell Chemical Biology 28, February 18, 2021 ll OPEN ACCESS Perspective targeting dimers may also face additional hurdles: spatially prox- imal Ras nanoclusters can play a major role in MAPK activation; however, PI3K activation does not require Ras dimers, so its activation can still go through (Nussinov et al., 2018a, 2019b). Raf and, consequently, MAPK signaling. It resulted in several lead compounds by pharmaceutical companies with subse- quent clinical trials as described above. Covalent drugs, especially allosteric ones, are powerful. How- ever, to date their applicability has been limited to few cancers, particularly small cell lung cancer where the G12C mutation is abundant (Gorfe and Cho, 2019; Stephen et al., 2014). The ac- count and promise shown by the impressive optimization of MRTX849 (Figure 2) that targets K-RasG12C emphasizes, however, the challenges in the development of covalent drugs in structure-based design to improve drug potency and pharma- cological properties, reaching nanomolar potency and selectivity in vivo (Fell et al., 2020; Gabizon and London, 2020). ALLOSTERIC DRUGS TARGETING NONFUNCTIONAL RAS ALLOSTERIC DRUGS TARGETING NONFUNCTIONAL RAS Further- more, it showed that a drug that interacts with the inactive Ras state can work (Khan et al., 2020a). Continued efforts led to ARS-1620 (Janes et al., 2018) (Figure 2), with higher reaction ki- netics and more potent drug action, inhibiting interaction with To further explore stabilization of nonfunctional K-Ras4B di- mers for KRAS-driven cancers, Kessler et al. (2020) synthesized other possible dimeric Switch I/II-pocket binders. They discov- ered compound 2, which dimerizes K-Ras4B with a KD of 3.8 mM. Soaking active K-Ras4BG12D (Bergner et al., 2019), they obtained K-Ras4B crystal structures of dimers with this compound at 1.9-A˚ resolution. Subsequent co-crystallization with compound 2 obtained a dimeric structure (at 1.57-A˚ resolution), a similar b-sandwich interface as with BI-2852, that resembles the one previously postulated by modeling and MD simulations of K-Ras4B molecules (Jang et al., 2016b). The Cell Chemical Biology 28, February 18, 2021 125 ll OPEN ACCESS ll OPEN ACCESS Perspective Figure 2. Examples of Ras Inhibitors The ﬁrst row shows crystal structures of the GDP-bound H-Ras in complex with NS1 monobody (PDB: 5E95), farnesyltransferase (FTase) containing the a and b subunits in complex with farnesyl diphosphate (FPP) and the inhibitor tipifarnib (R115777) (PDB: 1SA4), and phosphodiesterase d subunit (PDEd) inhibited by deltasonamide (PDB: 5ML3). Covalent drugs to the G12C mutation are shown in the second row; crystal structures of the GDP-bound K-Ras4BG12C with MRTX849 (PDB: 6UT0), the GDP-bound K-Ras4BG12C with ARS-1620 (PDB: 5V9U), and K-Ras4BG12C with the covalent GTP-competitive inhibitor SML-8-73-1 (PDB: 5KYK). The third row shows crystal structures of active Ras inhibited by noncovalent, small molecular drugs for the GCP-bound K-Ras4BG12D with DCAI (PDB: 4DST), the GNP-bound K-Ras4BQ61H with Abd-7 (PDB: 6FA4), and the GNP-bound K-Ras4BQ61H with 3344 (Abd-8) (PDB: 6F76). Figure 2. Examples of Ras Inhibitors The ﬁrst row shows crystal structures of the GDP-bound H-Ras in complex with NS1 monobody (PDB: 5E95), farnesyltransferase (FTase) containing the a and b subunits in complex with farnesyl diphosphate (FPP) and the inhibitor tipifarnib (R115777) (PDB: 1SA4), and phosphodiesterase d subunit (PDEd) inhibited by deltasonamide (PDB: 5ML3). Covalent drugs to the G12C mutation are shown in the second row; crystal structures of the GDP-bound K-Ras4BG12C with MRTX849 (PDB: 6UT0), the GDP-bound K-Ras4BG12C with ARS-1620 (PDB: 5V9U), and K-Ras4BG12C with the covalent GTP-competitive inhibitor SML-8-73-1 (PDB: 5KYK). 126 Cell Chemical Biology 28, February 18, 2021 ALLOSTERIC DRUGS TARGETING NONFUNCTIONAL RAS Ras Inhibitor with Nanomolar Afﬁnity Figure 3. Ras Inhibitor with Nanomolar Afﬁnity Crystal structure of the GCP-bound K-Ras4BG12D with BI-2852 (PDB: 6GJ8) (upper left panel). The compound 1 drug, BI-2852, exhibits nanomolar afﬁnity to a pocket between Switch I and Switch II. The BI-2852-induced Ras dimerization (right panel). Symmetric rotation of the K-Ras4B crystal structure reveals a b-sandwich dimer aligning its b1, b2, and b3 strands by two BI-2852 molecules. The ﬁrst BI-2852 (ligand-1) binds the Switch I and II pocket, the deep pocket formed by Lys5, Val7, Asp54, and Leu56 on the ﬁrst Ras (Ras-1). The second BI-2852 (ligand-2), a copy of the ﬁrst, binds the shallow pocket formed by Glu3, Leu52, and Asp54 on Ras-1. The complex is stabilized by several salt bridges. The molecular dynamics (MD) simulations of Ras molecules at the anionic membrane (Jang et al., 2016b) previously reported the nonfunctional b-sandwich dimer for K-Ras4B-GTP and H-Ras-GTP (lower left panel). Small molecules have also been developed to inhibit Ral GTPases, which are Ras GTPases identical in structure to K- Ras. The innovative strategy employed there similarly proposed to target GDP- rather than GTP-bound Ral, exploiting a new pocket (Yan et al., 2014). This pocket, which was not targeted before, harbors the K-RasG12C mutation. This work also led to a follow-up discovery in Meroueh’s lab of a covalent inhibitor that forms a bond with a tyrosine on Ral and Ras GTPases (Bum-Erdene et al., 2020), raising the possibility that covalent tyrosine linkage can open another venue in targeting of the Ras superfamily. Allosteric drugs are highly speciﬁc with fewer side effects and can tune the inhibition. Nonetheless, they still need to stably bind the protein, requiring some pockets. Covalent drugs also require a sufﬁciently reactive warhead, though not too reactive so as to avoid toxicity. The pocket should permit speciﬁc interactions that allow such reduction in warhead strength. Finally, K-RasG12C inhibitors target a somatic cysteine muta- tion. Since all cells are targeted, concerns about their likely toxicity persist (Sheridan, 2020). Selectivity against the wild type (therapeutic index) also depends upon the oncogene spec- iﬁcity in addition to the selectivity. Drugging small GTPase Rheb is similarly challenging. Rheb activates the mechanistic target of rapamycin complex 1 (mTORC1), a master regulator of cellular growth and metabolism in the Ras/PI3K/AKT/mTOR pathway. ALLOSTERIC DRUGS TARGETING NONFUNCTIONAL RAS Small molecule NR1 binds Rheb in the Switch II domain and selectively blocks mTORC1 signaling, but not mTORC2 (Mahoney et al., 2018). ALLOSTERIC DRUGS TARGETING NONFUNCTIONAL RAS The third row shows crystal structures of active Ras inhibited by noncovalent, small molecular drugs for the GCP-bound K-Ras4BG12D with DCAI (PDB: 4DST), the GNP-bound K-Ras4BQ61H with Abd-7 (PDB: 6FA4), and the GNP-bound K-Ras4BQ61H with 3344 (Abd-8) (PDB: 6F76). modeled structure dimerizes through the effector lobe b-sand- wich interface involving side-chain interactions of b1, b2, and b3 strands (Jang et al., 2016b) (Figure 3, lower left panel). The b-sandwich interface emerged from the exact b-sheet alignment due to hydrogen bond (H-bond) mismatch be- tween the b2 strands. Especially noteworthy is that this dimeric interface was a minor dimeric form in the simulations, that is, with a smaller population, suggesting that compound 2 selects a minor species and stabilizes it. state. Interestingly, the inactive K-Ras4B dimer that dimerizes through the effector lobe interface favors a shifted b-sheet extension through intermolecular H-bonds between the b2 strands formed by Glu37, Ser39, and Arg41 (Jang et al., 2016b). However, Raf easily competes with Ras molecules assembled through the effector lobe dimer interface due to the high-afﬁnity interaction of RBD with the Ras effector binding site. The active K-Ras4B dimer, where the monomers interact through their allosteric lobes, favors an interaction through the a3-a4 helices, while H-Ras allosteric lobe dimerization favors the a4-a5 helical interface (Jang et al., 2016b, 2020). Thus, whether monomers or dimers, allosteric interaction with a nonfunctional state will shift the ensemble away from the active 126 Cell Chemical Biology 28, February 18, 2021 ll OPEN ACCESS Perspective Perspective Figure 3. Ras Inhibitor with Nanomolar Afﬁnity Crystal structure of the GCP-bound K-Ras4BG12D with BI-2852 (PDB: 6GJ8) (upper left panel). The compound 1 drug, BI-2852, exhibits nanomolar afﬁnity to a pocket between Switch I and Switch II. The BI-2852-induced Ras dimerization (right panel). Symmetric rotation of the K-Ras4B crystal structure reveals a b-sandwich dimer aligning its b1, b2, and b3 strands by two BI-2852 molecules. The ﬁrst BI-2852 (ligand-1) binds the Switch I and II pocket, the deep pocket formed by Lys5, Val7, Asp54, and Leu56 on the ﬁrst Ras (Ras-1). The second BI-2852 (ligand-2), a copy of the ﬁrst, binds the shallow pocket formed by Glu3, Leu52, and Asp54 on Ras-1. The complex is stabilized by several salt bridges. The molecular dynamics (MD) simulations of Ras molecules at the anionic membrane (Jang et al., 2016b) previously reported the nonfunctional b-sandwich dimer for K-Ras4B-GTP and H-Ras-GTP (lower left panel). Perspective Figure 3. Cell Chemical Biology 28, February 18, 2021 127 SIGNIFICANCE The mechanism of action of many drugs is known. Prior knowledge in the development stage of the mechanism of action—at the molecular level—of effective drugs for the Cell Chemical Biology 28, February 18, 2021 127 ll OPEN ACCESS Perspective targeted protein is enormously advantageous and reduces late-stage failures. Here we interrogate distinct mecha- nisms embodied by allosteric drugs for two classes of major drug targets: Ras superfamily and kinases. Among the emerging allosteric strategies for Ras is targeting its nonfunctional state—which differs from the allosteric drug strategies for kinases. In kinases, allosteric drugs mostly target the active, functional state. We ask why drugging the inactive K-Ras4BG12C can work but drugging the inactive kinase will likely not work. We propose that the reason is their distinct oncogenic mechanisms. In kinases, the mech- anism of oncogenic kinase mutations involves increasing the population of the active conformations as compared with the inactive conformations. By contrast, Ras oncogenic mutations work by quenching the deactivation mechanisms, GTP hydrolysis, and nucleotide exchange. Thus, covalent allosteric inhibitors that bind to the inactive GDP-bound K- Ras4BG12C conformation can work—which is not the case in kinases, whose allosteric inhibitors work by altering the active-site conformation. Arora, K., and Brooks, C.L., 3rd. (2007). Large-scale allosteric conformational transitions of adenylate kinase appear to involve a population-shift mecha- nism. Proc. Natl. Acad. Sci. U S A 104, 18496–18501. Arora, K., and Brooks, C.L., 3rd. (2007). Large-scale allosteric conformational transitions of adenylate kinase appear to involve a population-shift mecha- nism. Proc. Natl. Acad. Sci. U S A 104, 18496–18501. Azam, M., Latek, R.R., and Daley, G.Q. (2003). Mechanisms of autoinhibition and STI-571/imatinib resistance revealed by mutagenesis of BCR-ABL. Cell 112, 831–843. Baietti, M.F., Simicek, M., Abbasi Asbagh, L., Radaelli, E., Lievens, S., Crowther, J., Steklov, M., Aushev, V.N., Martinez Garcia, D., Tavernier, J., et al. (2016). OTUB1 triggers lung cancer development by inhibiting RAS monoubiquitination. EMBO Mol. Med. 8, 288–303. Bandaru, P., Kondo, Y., and Kuriyan, J. (2019). The interdependent activation of son-of-sevenless and ras. Cold Spring Harb. Perspect. Med. 9, a031534. Baranyi, M., Buday, L., and Hegedus, B. (2020). K-Ras prenylation as a poten- tial anticancer target. Cancer Metastasis Rev. 39, 1127–1141. Barklis, E., Stephen, A.G., Staubus, A.O., Barklis, R.L., and Alfadhli, A. (2019). Organization of farnesylated, carboxymethylated KRAS4B on membranes. J. Mol. Biol. 431, 3706–3717. SIGNIFICANCE Bergner, A., Cockcroft, X., Fischer, G., Gollner, A., Hela, W., Kousek, R., Man- toulidis, A., Martin, L.J., Mayer, M., Mullauer, B., et al. (2019). KRAS binders hidden in nature. Chemistry 25, 12037–12041. Bery, N., Cruz-Migoni, A., Bataille, C.J., Quevedo, C.E., Tulmin, H., Miller, A., Russell, A., Phillips, S.E., Carr, S.B., and Rabbitts, T.H. (2018). BRET-based RAS biosensors that show a novel small molecule is an inhibitor of RAS- effector protein-protein interactions. eLife 7, e37122. Drug discovery-wise the signiﬁcance of our premise here is 3-fold: (1) it postulates that the mechanism of drug action relates to the mechanism of the oncogenic driver mutations; not to whether the protein is in the active or inactive state. (2) However, if drug binding allosterically blocks the target functional site, as in the case of Raf dimerization, it will work also for the inactive kinase states, and the GTP-bound Ras. That is, the location of the drug-binding site matters. (3) From the translational standpoint, this mechanism of action classiﬁcation accelerates pharmaceutical development. It postulates that drug action is related to blocking the mech- anism of activation—not to the active or inactive state, ex- tending drug classiﬁcation charts. Boehr, D.D., Nussinov, R., and Wright, P.E. (2009). The role of dynamic confor- mational ensembles in biomolecular recognition. Nat. Chem. Biol. 5, 789–796. Bond, M.J., Chu, L., Nalawansha, D.A., Li, K., and Crews, C.M. (2020). Tar- geted degradation of oncogenic KRAS(G12C) by VHL-recruiting PROTACs. ACS Cent. Sci. 6, 1367–1375. Bourne, H.R., Sanders, D.A., and McCormick, F. (1991). The GTPase super- family: conserved structure and molecular mechanism. Nature 349, 117–127. Bryant, K.L., Mancias, J.D., Kimmelman, A.C., and Der, C.J. (2014). KRAS: feeding pancreatic cancer proliferation. Trends Biochem. Sci. 39, 91–100. Bum-Erdene, K., Liu, D., Gonzalez-Gutierrez, G., Ghozayel, M.K., Xu, D., and Meroueh, S.O. (2020). Small-molecule covalent bond formation at tyrosine creates a binding site and inhibits activation of Ral GTPases. Proc. Natl. Acad. Sci. U S A 117, 7131–7139. ACKNOWLEDGMENTS This project has been funded in whole or in part with federal funds from the Na- tional Cancer Institute, National Institutes of Health, under contract HHSN261200800001E. The content of this publication does not necessarily reﬂect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products or organizations imply endorsement by the US Government. This Research was supported (in part) by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research, and the Intramural Research Program of the NIH Clinical Center. Canon, J., Rex, K., Saiki, A.Y., Mohr, C., Cooke, K., Bagal, D., Gaida, K., Holt, T., Knutson, C.G., Koppada, N., et al. (2019). The clinical KRAS(G12C) inhibitor AMG 510 drives anti-tumour immunity. Nature 575, 217–223. Cao, S., Chung, S., Kim, S., Li, Z., Manor, D., and Buck, M. (2019). K-Ras G- domain binding with signaling lipid phosphatidylinositol (4,5)-phosphate (PIP2): membrane association, protein orientation, and function. J. Biol. Chem. 294, 7068–7084. Chatani, P.D., and Yang, J.C. (2020). Mutated RAS: targeting the "untarget- able" with T cells. Clin. Cancer Res. 26, 537–544. AUTHOR CONTRIBUTIONS Chavan, T.S., Muratcioglu, S., Marszalek, R., Jang, H., Keskin, O., Gursoy, A., Nussinov, R., and Gaponenko, V. (2015). Plasma membrane regulates Ras signaling networks. Cell. Logist. 5, e1136374. R.N. and H.J. designed research. R.N. performed research and prepared the initial draft. H.J. generated the ﬁgures. R.N. and H.J. wrote and edited the pa- per. R.N., H.J., A.G., O.K., and V.G. have given approval to the ﬁnal version of the manuscript. Chen, L., Tao, Y., and Jiang, Y. (2015). Apelin activates the expression of in- ﬂammatory cytokines in microglial BV2 cells via PI-3K/Akt and MEK/Erk path- ways. Sci. China Life Sci. 58, 531–540. Perspective (1990). The ras superfamily of small GTP-binding proteins. Trends Biochem. Sci. 15, 469–472. Hallin, J., Engstrom, L.D., Hargis, L., Calinisan, A., Aranda, R., Briere, D.M., Sudhakar, N., Bowcut, V., Baer, B.R., Ballard, J.A., et al. (2020). The KRAS(G12C) inhibitor MRTX849 provides insight toward therapeutic suscep- tibility of KRAS-mutant cancers in mouse models and patients. Cancer Discov. 10, 54–71. Downward, J. (2008). Targeting RAS and PI3K in lung cancer. Nat. Med. 14, 1315–1316. Drosten, M., Dhawahir, A., Sum, E.Y., Urosevic, J., Lechuga, C.G., Esteban, L.M., Castellano, E., Guerra, C., Santos, E., and Barbacid, M. (2010). Genetic analysis of Ras signalling pathways in cell proliferation, migration and survival. EMBO J. 29, 1091–1104. Henley, M.J., Linhares, B.M., Morgan, B.S., Cierpicki, T., Fierke, C.A., and Mapp, A.K. (2020). Unexpected speciﬁcity within dynamic transcriptional pro- tein-protein complexes. Proc. Natl. Acad. Sci. U S A 117, 27346–27353. Eser, S., Schnieke, A., Schneider, G., and Saur, D. (2014). Oncogenic KRAS signalling in pancreatic cancer. Br. J. Cancer 111, 817–822. Holderﬁeld, M., Deuker, M.M., McCormick, F., and McMahon, M. (2014). Tar- geting RAF kinases for cancer therapy: BRAF-mutated melanoma and beyond. Nat. Rev. Cancer 14, 455–467. Fang, Z., Lee, K.Y., Huo, K.G., Gasmi-Seabrook, G., Zheng, L., Moghal, N., Tsao, M.S., Ikura, M., and Marshall, C.B. (2020). Multivalent assembly of KRAS with the RAS-binding and cysteine-rich domains of CRAF on the mem- brane. Proc. Natl. Acad. Sci. U S A 117, 12101–12108. Hong, D.S., Fakih, M.G., Strickler, J.H., Desai, J., Durm, G.A., Shapiro, G.I., Falchook, G.S., Price, T.J., Sacher, A., Denlinger, C.S., et al. (2020). KRAS(G12C) inhibition with sotorasib in advanced solid tumors. N. Engl. J. Med. 383, 1207–1217. Fell, J.B., Fischer, J.P., Baer, B.R., Blake, J.F., Bouhana, K., Briere, D.M., Brown, K.D., Burgess, L.E., Burns, A.C., Burkard, M.R., et al. (2020). Identiﬁca- tion of the clinical development candidate MRTX849, a covalent KRAS(G12C) inhibitor for the treatment of cancer. J. Med. Chem. 63, 6679–6693. Hosford, S.R., Dillon, L.M., Bouley, S.J., Rosati, R., Yang, W., Chen, V.S., Demidenko, E., Morra, R.P., Jr., and Miller, T.W. (2017). Combined inhibition of both p110alpha and p110beta isoforms of phosphatidylinositol 3-kinase is required for sustained therapeutic effect in PTEN-deﬁcient, ER(+) breast can- cer. Clin. Cancer Res. 23, 2795–2805. Fernandez-Medarde, A., and Santos, E. (2011). Ras in cancer and develop- mental diseases. Genes Cancer 2, 344–358. Frauenfelder, H., Sligar, S.G., and Wolynes, P.G. (1991). The energy land- scapes and motions of proteins. Science 254, 1598–1603. Perspective Perspective Goody, R.S., Muller, M.P., and Rauh, D. (2019). Mutant-speciﬁc targeting of ras G12C activity by covalently reacting small molecules. Cell Chem. Biol. 26, 1338–1348. Cox, A.D., and Der, C.J. (2010). Ras history: the saga continues. Small GTPases 1, 2–27. Cox, A.D., Der, C.J., and Philips, M.R. (2015). Targeting RAS membrane asso- ciation: back to the future for anti-RAS drug discovery? Clin. Cancer Res. 21, 1819–1827. Gorfe, A.A., and Cho, K.J. (2019). Approaches to inhibiting oncogenic K-Ras. Small GTPases, 1–10. Grand, R.J., and Owen, D. (1991). The biochemistry of ras p21. Biochem. J. 279, 609–631. Cox, A.D., Fesik, S.W., Kimmelman, A.C., Luo, J., and Der, C.J. (2014). Drug- ging the undruggable RAS: mission possible? Nat. Rev. Drug Discov. 13, 828–851. Gross, L.Z.F., Sacerdoti, M., Piiper, A., Zeuzem, S., Leroux, A.E., and Biondi, R.M. (2020). ACE2, the receptor that enables infection by SARS-CoV-2: biochemistry, structure, allostery and evaluation of the potential development of ACE2 modulators. ChemMedChem 15, 1682–1690. Crespo, P., and Leon, J. (2000). Ras proteins in the control of the cell cycle and cell differentiation. Cell Mol Life Sci 57, 1613–1636. Cruz-Migoni, A., Canning, P., Quevedo, C.E., Bataille, C.J.R., Bery, N., Miller, A., Russell, A.J., Phillips, S.E.V., Carr, S.B., and Rabbitts, T.H. (2019). Struc- ture-based development of new RAS-effector inhibitors from a combination of active and inactive RAS-binding compounds. Proc. Natl. Acad. Sci. U S A 116, 2545–2550. Guarnera, E., and Berezovsky, I.N. (2019). Toward comprehensive allosteric control over protein activity. Structure 27, 866–878 e861. Guarnera, E., and Berezovsky, I.N. (2020). Allosteric drugs and mutations: chances, challenges, and necessity. Curr. Opin. Struct. Biol. 62, 149–157. Dang, C.V., Reddy, E.P., Shokat, K.M., and Soucek, L. (2017). Drugging the ‘undruggable’ cancer targets. Nat. Rev. Cancer 17, 502–508. Gul, A., Leyland-Jones, B., Dey, N., and De, P. (2018). A combination of the PI3K pathway inhibitor plus cell cycle pathway inhibitor to combat endocrine resistance in hormone receptor-positive breast cancer: a genomic algo- rithm-based treatment approach. Am. J. Cancer Res. 8, 2359–2376. De Luca, A., Maiello, M.R., D’Alessio, A., Pergameno, M., and Normanno, N. (2012). The RAS/RAF/MEK/ERK and the PI3K/AKT signalling pathways: role in cancer pathogenesis and implications for therapeutic approaches. Expert Opin. Ther. Targets 16, S17–S27. Gupta, A.K., Wang, X., Pagba, C.V., Prakash, P., Sarkar-Banerjee, S., Putkey, J., and Gorfe, A.A. (2019). Multi-target, ensemble-based virtual screening yields novel allosteric KRAS inhibitors at high success rate. Chem. Biol. Drug Des. 94, 1441–1456. Downward, J. REFERENCES Cheng, J., Li, Y., Wang, X., Dong, G., and Sheng, C. (2020). Discovery of novel PDEd degraders for the treatment of KRAS mutant colorectal cancer. J. Med. Chem. 63, 7892–7905. Adams, B. (2020). Eli Lilly crashes out of KRAS race as toxicity sees it ditch phase 1 effort. https://www.ﬁercebiotech.com/biotech/eli-lilly-crashes-out- kras-race-as-it-ditches-phase-1-effort. Adams, B. (2020). Eli Lilly crashes out of KRAS race as toxicity sees it ditch phase 1 effort. https://www.ﬁercebiotech.com/biotech/eli-lilly-crashes-out- kras-race-as-it-ditches-phase-1-effort. Cherﬁls, J., and Zeghouf, M. (2013). Regulation of small GTPases by GEFs, GAPs, and GDIs. Physiol. Rev. 93, 269–309. Adjei, A.A. (2001). Blocking oncogenic Ras signaling for cancer therapy. J. Natl. Cancer Inst. 93, 1062–1074. Adjei, A.A. (2001). Blocking oncogenic Ras signaling for cancer therapy. J. Natl. Cancer Inst. 93, 1062–1074. Cocco, E., Lee, J.E., Kannan, S., Schram, A.M., Won, H.H., Shifman, S., Kulick, A., Baldino, L., Toska, E., Arruabarrena-Aristorena, A., et al. (2020). TRK xDFG mutations trigger a sensitivity switch from type I to II kinase inhibitors. Cancer Discov. https://doi.org/10.1158/2159-8290.CD-1120-0571. Ahearn, I.M., Haigis, K., Bar-Sagi, D., and Philips, M.R. (2011). Regulating the regulator: post-translational modiﬁcation of RAS. Nat. Rev. Mol. Cell. Biol. 13, 39–51. Ahearn, I.M., Haigis, K., Bar-Sagi, D., and Philips, M.R. (2011). Regulating the regulator: post-translational modiﬁcation of RAS. Nat. Rev. Mol. Cell. Biol. 13, 39–51. 128 Cell Chemical Biology 28, February 18, 2021 ll OPEN ACCESS Perspective Huang, W., Wang, G., Shen, Q., Liu, X., Lu, S., Geng, L., Huang, Z., and Zhang, J. (2015). ASBench: benchmarking sets for allosteric discovery. Bioinformatics 31, 2598–2600. Fujiwara, T., Ritchie, K., Murakoshi, H., Jacobson, K., and Kusumi, A. (2002). Phospholipids undergo hop diffusion in compartmentalized cell membrane. J. Cell Biol. 157, 1071–1081. Huang, W.Y.C., Alvarez, S., Kondo, Y., Lee, Y.K., Chung, J.K., Lam, H.Y.M., Biswas, K.H., Kuriyan, J., and Groves, J.T. (2019). A molecular assembly phase transition and kinetic proofreading modulate Ras activation by SOS. Science 363, 1098–1103. Gabizon, R., and London, N. (2020). Hitting KRAS when it’s down. J. Med. Chem. 63, 6677–6678. Hunter, J.C., Gurbani, D., Ficarro, S.B., Carrasco, M.A., Lim, S.M., Choi, H.G., Xie, T., Marto, J.A., Chen, Z., Gray, N.S., et al. (2014). In situ selectivity proﬁling and crystal structure of SML-8-73-1, an active site inhibitor of oncogenic K- Ras G12C. Proc. Natl. Acad. Sci. U S A 111, 8895–8900. Gao, Y., Zhou, J., Xie, Z., Wang, J., Ho, C.K., Zhang, Y., and Li, Q. (2019). Me- chanical strain promotes skin ﬁbrosis through LRG-1 induction mediated by ELK1 and ERK signalling. Commun. Biol. 2, 359. Gentile, D.R., Rathinaswamy, M.K., Jenkins, M.L., Moss, S.M., Siempelkamp, B.D., Renslo, A.R., Burke, J.E., and Shokat, K.M. (2017). Ras binder induces a modiﬁed switch-II pocket in GTP and GDP states. Cell Chem. Biol. 24, 1455– 1466 e1414. Hunter, J.C., Manandhar, A., Carrasco, M.A., Gurbani, D., Gondi, S., and Westover, K.D. (2015). Biochemical and structural analysis of common can- cer-associated KRAS mutations. Mol. Cancer Res. 13, 1325–1335. Ijuin, T. (2019). Phosphoinositide phosphatases in cancer cell dynamics- Beyond PI3K and PTEN. Semin. Cancer Biol. 59, 50–65. Geyer, M., Schweins, T., Herrmann, C., Prisner, T., Wittinghofer, A., and Kal- bitzer, H.R. (1996). Conformational transitions in p21ras and in its complexes with the effector protein Raf-RBD and the GTPase activating protein GAP. Biochemistry 35, 10308–10320. Janes, M.R., Zhang, J., Li, L.S., Hansen, R., Peters, U., Guo, X., Chen, Y., Bab- bar, A., Firdaus, S.J., Darjania, L., et al. (2018). Targeting KRAS mutant cancers with a covalent G12C-speciﬁc inhibitor. Cell 172, 578–589 e517. Geyer, M., and Wittinghofer, A. (1997). GEFs, GAPs, GDIs and effectors: taking a closer (3D) look at the regulation of Ras-related GTP-binding proteins. Curr. Opin. Struct. Biol. 7, 786–792. Jang, H., Banerjee, A., Chavan, T., Gaponenko, V., and Nussinov, R. (2017). yield a high-afﬁnity interaction enabling K-Ras4B membrane extraction. J. Biol. Chem. 292, 12544–12559. Leontiadou, H., Galdadas, I., Athanasiou, C., and Cournia, Z. (2018). Insights into the mechanism of the PIK3CA E545K activating mutation using MD simu- lations. Sci. Rep. 8, 15544. Jang, H., Banerjee, A., Chavan, T.S., Lu, S., Zhang, J., Gaponenko, V., and Nussinov, R. (2016a). The higher level of complexity of K-Ras4B activation at the membrane. FASEB J. 30, 1643–1655. Li, S., Jang, H., Zhang, J., and Nussinov, R. (2018a). Raf-1 cysteine-rich domain increases the afﬁnity of K-Ras/Raf at the membrane, promoting MAPK signaling. Structure 26, 513–525 e512. Jang, H., Banerjee, A., Marcus, K., Makowski, L., Mattos, C., Gaponenko, V., and Nussinov, R. (2019). The structural basis of the farnesylated and methyl- ated KRas4B interaction with calmodulin. Structure 27, 1647–1659 e1644. Li, Z.L., Prakash, P., and Buck, M. (2018b). A "tug of war" maintains a dynamic protein-membrane complex: molecular dynamics simulations of C-Raf RBD- CRD bound to K-Ras4B at an anionic membrane. ACS Cent. Sci. 4, 298–305. Jang, H., Muratcioglu, S., Gursoy, A., Keskin, O., and Nussinov, R. (2016b). Membrane-associated Ras dimers are isoform-speciﬁc: K-Ras dimers differ from H-Ras dimers. Biochem. J. 473, 1719–1732. Liao, J., Shima, F., Araki, M., Ye, M., Muraoka, S., Sugimoto, T., Kawamura, M., Yamamoto, N., Tamura, A., and Kataoka, T. (2008). Two conformational states of Ras GTPase exhibit differential GTP-binding kinetics. Biochem. Bio- phys. Res. Commun. 369, 327–332. Jang, H., Zhang, M., and Nussinov, R. (2020). The quaternary assembly of KRas4B with Raf-1 at the membrane. Comput. Struct. Biotechnol. J. 18, 737–748. Liao, T.J., Jang, H., Fushman, D., and Nussinov, R. (2018). Allosteric KRas4B can modulate SOS1 fast and slow ras activation cycles. Biophys. J. 115, 629–641. Kar, G., Keskin, O., Gursoy, A., and Nussinov, R. (2010). Allostery and popula- tion shift in drug discovery. Curr. Opin. Pharmacol. 10, 715–722. Liao, T.J., Jang, H., Nussinov, R., and Fushman, D. (2020). High-afﬁnity inter- actions of the nSH3/cSH3 domains of Grb2 with the C-terminal proline-rich domain of SOS1. J. Am. Chem. Soc. 142, 3401–3411. Kessler, D., Gmachl, M., Mantoulidis, A., Martin, L.J., Zoephel, A., Mayer, M., Gollner, A., Covini, D., Fischer, S., Gerstberger, T., et al. (2019). Drugging an undruggable pocket on KRAS. Proc. Natl. Acad. Sci. U S A 116, 15823–15829. Lien, E.C., Dibble, C.C., and Toker, A. (2017). PI3K signaling in cancer: beyond AKT. Curr. Opin. Cell Biol. 45, 62–71. Lu, S., Huang, W., and Zhang, J. (2014a). Recent computational advances in the identiﬁcation of allosteric sites in proteins. Drug Discov. Today 19, 1595–1600. Lu, S., Jang, H., Gu, S., Zhang, J., and Nussinov, R. (2016a). Drugging Ras GTPase: a comprehensive mechanistic and signaling structural view. Chem. Soc. Rev. 45, 4929–4952. Krengel, U., Schlichting, I., Scherer, A., Schumann, R., Frech, M., John, J., Kabsch, W., Pai, E.F., and Wittinghofer, A. (1990). Three-dimensional struc- tures of H-ras p21 mutants: molecular basis for their inability to function as signal switch molecules. Cell 62, 539–548. Lu, S., Jang, H., Muratcioglu, S., Gursoy, A., Keskin, O., Nussinov, R., and Zhang, J. (2016b). Ras conformational ensembles, allostery, and signaling. Chem. Rev. 116, 6607–6665. Krens, L.L., Baas, J.M., Gelderblom, H., and Guchelaar, H.J. (2010). Therapeu- tic modulation of k-ras signaling in colorectal cancer. Drug Discov. Today 15, 502–516. Lu, S., Jang, H., Zhang, J., and Nussinov, R. (2016c). Inhibitors of ras-SOS in- teractions. ChemMedChem 11, 814–821. Kumar, S., Ma, B., Tsai, C.J., Sinha, N., and Nussinov, R. (2000). Folding and binding cascades: dynamic landscapes and population shifts. Protein Sci. 9, 10–19. Lu, S., Ji, M., Ni, D., and Zhang, J. (2018). Discovery of hidden allosteric sites as novel targets for allosteric drug design. Drug Discov. Today 23, 359–365. Lamontanara, A.J., Georgeon, S., Tria, G., Svergun, D.I., and Hantschel, O. (2014). The SH2 domain of Abl kinases regulates kinase autophosphorylation by controlling activation loop accessibility. Nat. Commun. 5, 5470. Lu, S., Li, S., and Zhang, J. (2014b). Harnessing allostery: a novel approach to drug discovery. Med. Res. Rev. 34, 1242–1285. Lu, S., Qiu, Y., Ni, D., He, X., Pu, J., and Zhang, J. (2020). Emergence of allo- steric drug-resistance mutations: new challenges for allosteric drug discovery. Drug Discov. Today 25, 177–184. Lavoie, H., Thevakumaran, N., Gavory, G., Li, J.J., Padeganeh, A., Guiral, S., Duchaine, J., Mao, D.Y., Bouvier, M., Sicheri, F., et al. (2013). Inhibitors that stabilize a closed RAF kinase domain conformation induce dimerization. Nat. Chem. Biol. 9, 428–436. Ma, B., Kumar, S., Tsai, C.J., Hu, Z., and Nussinov, R. (2000). Transition-state ensemble in enzyme catalysis: possibility, reality, or necessity? J. Theor. Biol. 203, 383–397. Ledford, H. (2015). Cancer: the ras renaissance. Nature 520, 278–280. Lee, Y., Phelps, C., Huang, T., Mostoﬁan, B., Wu, L., Zhang, Y., Tao, K., Chang, Y.H., Stork, P.J., Gray, J.W., et al. (2019). Kessler, D., Gollner, A., Gmachl, M., Mantoulidis, A., Martin, L.J., Zoephel, A., Mayer, M., Covini, D., Fischer, S., Gerstberger, T., et al. (2020). Reply to Tran et al.: dimeric KRAS protein-protein interaction stabilizers. Proc. Natl. Acad. Sci. U S A 117, 3365–3367. Lim, S.M., Westover, K.D., Ficarro, S.B., Harrison, R.A., Choi, H.G., Pacold, M.E., Carrasco, M., Hunter, J., Kim, N.D., Xie, T., et al. (2014). Therapeutic tar- geting of oncogenic K-Ras by a covalent catalytic site inhibitor. Angew. Chem. Int. Ed. Engl. 53, 199–204. Kettle, J.G., Bagal, S.K., Bickerton, S., Bodnarchuk, M.S., Breed, J., Carbajo, R.J., Cassar, D.J., Chakraborty, A., Cosulich, S., Cumming, I., et al. (2020). Structure-based design and pharmacokinetic optimization of covalent allo- steric inhibitors of the mutant GTPase KRAS(G12C). J. Med. Chem. 63, 4468–4483. Liu, M., Sjogren, A.K., Karlsson, C., Ibrahim, M.X., Andersson, K.M., Olofsson, F.J., Wahlstrom, A.M., Dalin, M., Yu, H., Chen, Z., et al. (2010). Targeting the protein prenyltransferases efﬁciently reduces tumor development in mice with K-RAS-induced lung cancer. Proc. Natl. Acad. Sci. U S A 107, 6471–6476. Khan, I., Rhett, J.M., and O’Bryan, J.P. (2020a). Therapeutic targeting of RAS: new hope for drugging the "undruggable. Biochim. Biophys. Acta Mol. Cell Res. 1867, 118570. Liu, P., Wang, Y., and Li, X. (2019). Targeting the untargetable KRAS in cancer therapy. Acta Pharm. Sin B 9, 871–879. Khan, Z.M., Real, A.M., Marsiglia, W.M., Chow, A., Duffy, M.E., Yerabolu, J.R., Scopton, A.P., and Dar, A.C. (2020b). Structural basis for the action of the drug trametinib at KSR-bound MEK. Nature 588, 509–514. Lou, K., Steri, V., Ge, A.Y., Hwang, Y.C., Yogodzinski, C.H., Shkedi, A.R., Choi, A.L.M., Mitchell, D.C., Swaney, D.L., Hann, B., et al. (2019). KRAS(G12C) inhi- bition produces a driver-limited state revealing collateral dependencies. Sci. Signal. 12, eaaw9450. Kondo, Y., Ognjenovic, J., Banerjee, S., Karandur, D., Merk, A., Kulhanek, K., Wong, K., Roose, J.P., Subramaniam, S., and Kuriyan, J. (2019). Cryo-EM structure of a dimeric B-Raf:14-3-3 complex reveals asymmetry in the active sites of B-Raf kinases. Science 366, 109–115. Lowy, D.R., Zhang, K., DeClue, J.E., and Willumsen, B.M. (1991). Regulation of p21ras activity. Trends Genet. 7, 346–351. Kostaras, E., Kaserer, T., Lazaro, G., Heuss, S.F., Hussain, A., Casado, P., Hayes, A., Yandim, C., Palaskas, N., Yu, Y., et al. (2020). A systematic molec- ular and pharmacologic evaluation of AKT inhibitors reveals new insight into their biological activity. Br. J. Cancer 123, 542–555. Perspective Flexible-body motions of calmodulin and the farnesylated hypervariable region Cell Chemical Biology 28, February 18, 2021 129 Cell Chemical Biology 28, February 18, 2021 129 ll OPEN ACCESS Perspective Nussinov, R., Jang, H., Tsai, C.J., Liao, T.J., Li, S., Fushman, D., and Zhang, J. (2017a). Intrinsic protein disorder in oncogenic KRAS signaling. Cell. Mol. Life Sci. 74, 3245–3261. Mackereth, C.D., Madl, T., Bonnal, S., Simon, B., Zanier, K., Gasch, A., Rybin, V., Valcarcel, J., and Sattler, M. (2011). Multi-domain conformational selection underlies pre-mRNA splicing regulation by U2AF. Nature 475, 408–411. Nussinov, R., and Ma, B. (2012). Protein dynamics and conformational selec- tion in bidirectional signal transduction. BMC Biol. 10, 2. Mahoney, S.J., Narayan, S., Molz, L., Berstler, L.A., Kang, S.A., Vlasuk, G.P., and Saiah, E. (2018). A small molecule inhibitor of Rheb selectively targets mTORC1 signaling. Nat. Commun. 9, 548. Nussinov, R., and Tsai, C.J. (2012). The different ways through which speci- ﬁcity works in orthosteric and allosteric drugs. Curr. Pharm. Des. 18, 1311–1316. Martin-Gago, P., Fansa, E.K., Klein, C.H., Murarka, S., Janning, P., Schur- mann, M., Metz, M., Ismail, S., Schultz-Fademrecht, C., Baumann, M., et al. (2017). A PDE6delta-KRas inhibitor chemotype with up to seven H-bonds and picomolar afﬁnity that prevents efﬁcient inhibitor release by Arl2. Angew. Chem. Int. Ed. Engl. 56, 2423–2428. Nussinov, R., and Tsai, C.J. (2013). Allostery in disease and in drug discovery. Cell 153, 293–305. Martin-Garcia, F., Mendieta-Moreno, J.I., Lopez-Vinas, E., Gomez-Puertas, P., and Mendieta, J. (2012). The Role of Gln61 in HRas GTP hydrolysis: a quan- tum mechanics/molecular mechanics study. Biophys. J. 102, 152–157. Nussinov, R., and Tsai, C.J. (2015). The design of covalent allosteric drugs. Annu. Rev. Pharmacol. Toxicol. 55, 249–267. Nussinov, R., Tsai, C.J., and Jang, H. (2018a). Oncogenic ras isoforms signaling speciﬁcity at the membrane. Cancer Res. 78, 593–602. Mattingly, R.R. (2013). Activated ras as a therapeutic target: constraints on directly targeting ras isoforms and wild-type versus mutated proteins. ISRN Oncol. 2013, 536529. Nussinov, R., Tsai, C.J., and Jang, H. (2019a). Does ras activate Raf and PI3K allosterically? Front. Oncol. 9, 1231. Maurer, T., Garrenton, L.S., Oh, A., Pitts, K., Anderson, D.J., Skelton, N.J., Fauber, B.P., Pan, B., Malek, S., Stokoe, D., et al. (2012). Small-molecule li- gands bind to a distinct pocket in Ras and inhibit SOS-mediated nucleotide ex- change activity. Proc. Natl. Acad. Sci. U S A 109, 5299–5304. Nussinov, R., Tsai, C.J., and Jang, H. (2019b). Is nanoclustering essential for all oncogenic KRas pathways? Can it explain why wild-type KRas can inhibit its oncogenic variant? Semin. Cancer Biol. 54, 114–120. Perspective McCarthy, M.J., Pagba, C.V., Prakash, P., Naji, A.K., van der Hoeven, D., Liang, H., Gupta, A.K., Zhou, Y., Cho, K.J., Hancock, J.F., et al. (2019). Discov- ery of high-afﬁnity noncovalent allosteric KRAS inhibitors that disrupt effector binding. ACS Omega 4, 2921–2930. Nussinov, R., Tsai, C.J., and Jang, H. (2019c). Oncogenic KRas mobility in the membrane and signaling response. Semin. Cancer Biol. 54, 109–113. Nussinov, R., Tsai, C.J., and Jang, H. (2020). Ras assemblies and signaling at the membrane. Curr. Opin. Struct. Biol. 62, 140–148. McCormick, F. (2018). Targeting KRAS directly. In Annual Review of Cancer Biology, T. Jacks and C.L. Sawyers, eds. (Annual Reviews), pp. 81–90. Nussinov, R., Tsai, C.J., and Mattos, C. (2013). Pathway drug cocktail’: target- ing Ras signaling based on structural pathways. Trends Mol. Med. 19, 695–704. Milroy, L.G., and Ottmann, C. (2014). The renaissance of Ras. ACS Chem. Biol. 9, 2447–2458. Nussinov, R., Wang, G., Tsai, C.J., Jang, H., Lu, S., Banerjee, A., Zhang, J., and Gaponenko, V. (2017b). Calmodulin and PI3K signaling in KRAS cancers. Trends Cancer 3, 214–224. Mirati Therapeutics (2020). KRAS G12C inhibitor. https://www.mirati.com/ pipeline/kras-g12c/. Moran, M.F., Polakis, P., McCormick, F., Pawson, T., and Ellis, C. (1991). Pro- tein-tyrosine kinases regulate the phosphorylation, protein interactions, sub- cellular distribution, and activity of p21ras GTPase-activating protein. Mol. Cell Biol. 11, 1804–1812. Nussinov, R., and Wolynes, P.G. (2014). A second molecular biology revolu- tion? The energy landscapes of biomolecular function. Phys. Chem. Chem. Phys. 16, 6321–6322. Nussinov, R., Zhang, M., Tsai, C.J., Liao, T.J., Fushman, D., and Jang, H. (2018b). Autoinhibition in Ras effectors Raf, PI3Kalpha, and RASSF5: a comprehensive review underscoring the challenges in pharmacological inter- vention. Biophys. Rev. 10, 1263–1282. Motlagh, H.N., Wrabl, J.O., Li, J., and Hilser, V.J. (2014). The ensemble nature of allostery. Nature 508, 331–339. Mullard, A. (2019). Cracking KRAS. Nat. Rev. Drug Discov. 18, 887–891. O’Bryan, J.P. (2019). Pharmacological targeting of RAS: recent success with direct inhibitors. Pharmacol. Res. 139, 503–511. Murase, K., Fujiwara, T., Umemura, Y., Suzuki, K., Iino, R., Yamashita, H., Saito, M., Murakoshi, H., Ritchie, K., and Kusumi, A. (2004). Ultraﬁne mem- brane compartments for molecular diffusion as revealed by single molecule techniques. Biophys. J. 86, 4075–4093. Okada, T., Hu, C.D., Jin, T.G., Kariya, K., Yamawaki-Kataoka, Y., and Kataoka, T. (1999). The strength of interaction at the Raf cysteine-rich domain is a critical determinant of response of Raf to Ras family small GTPases. Mol. Cell Biol. High-throughput, single-particle tracking reveals nested membrane domains that dictate KRas(G12D) diffusion and trafﬁcking. eLife 8, e46393. Ma, B., Shatsky, M., Wolfson, H.J., and Nussinov, R. (2002). Multiple diverse ligands binding at a single protein site: a matter of pre-existing populations. Protein Sci. 11, 184–197. 130 Cell Chemical Biology 28, February 18, 2021 130 Cell Chemical Biology 28, February 18, 2021 ll OPEN ACCESS Perspective Perspective Structure 26, 810–820.e4. cule Ras inhibitors that display antitumor activity by blocking the Ras-effector interaction. Proc. Natl. Acad. Sci. U S A 110, 8182–8187. Simons, K., and Vaz, W.L. (2004). Model systems, lipid rafts, and cell mem- branes. Annu. Rev. Biophys. Biomol. Struct. 33, 269–295. Patgiri, A., Yadav, K.K., Arora, P.S., and Bar-Sagi, D. (2011). An orthosteric in- hibitor of the Ras-Sos interaction. Nat. Chem. Biol. 7, 585–587. Singh, H., Longo, D.L., and Chabner, B.A. (2015). Improving prospects for tar- geting RAS. J. Clin. Oncol. 33, 3650–3659. Patricelli, M.P., Janes, M.R., Li, L.S., Hansen, R., Peters, U., Kessler, L.V., Chen, Y., Kucharski, J.M., Feng, J., Ely, T., et al. (2016). Selective inhibition of oncogenic KRAS output with small molecules targeting the inactive state. Cancer Discov. 6, 316–329. Smith, M.J., and Ikura, M. (2014). Integrated RAS signaling deﬁned by parallel NMR detection of effectors and regulators. Nat. Chem. Biol. 10, 223–230. Smith, M.J., Neel, B.G., and Ikura, M. (2013). NMR-based functional proﬁling of RASopathies and oncogenic RAS mutations. Proc. Natl. Acad. Sci. U S A 110, 4574–4579. Pokrass, M.J., Ryan, K.A., Xin, T., Pielstick, B., Timp, W., Greco, V., and Regot, S. (2020). Cell-cycle-dependent ERK signaling dynamics direct fate speciﬁca- tion in the mammalian preimplantation embryo. Dev. Cell 55, 328–340.e5. Solman, M., Ligabue, A., Blazevits, O., Jaiswal, A., Zhou, Y., Liang, H., Lectez, B., Kopra, K., Guzman, C., Harma, H., et al. (2015). Speciﬁc cancer-associated mutations in the switch III region of Ras increase tumorigenicity by nanocluster augmentation. eLife 4, e08905. Poulin, E.J., Bera, A.K., Lu, J., Lin, Y.J., Strasser, S.D., Paulo, J.A., Huang, T.Q., Morales, C., Yan, W., Cook, J., et al. (2019). Tissue-speciﬁc oncogenic activity of KRAS(A146T). Cancer Discov. 9, 738–755. Prakash, P., and Gorfe, A.A. (2019). Probing the conformational and energy landscapes of KRAS membrane orientation. J. Phys. Chem. B 123, 8644–8652. Spencer-Smith, R., Koide, A., Zhou, Y., Eguchi, R.R., Sha, F., Gajwani, P., San- tana, D., Gupta, A., Jacobs, M., Herrero-Garcia, E., et al. (2017). Inhibition of RAS function through targeting an allosteric regulatory site. Nat. Chem. Biol. 13, 62–68. Prakash, P., Litwin, D., Liang, H., Sarkar-Banerjee, S., Dolino, D., Zhou, Y., Hancock, J.F., Jayaraman, V., and Gorfe, A.A. (2020). Dynamics of mem- brane-bound G12V-KRAS from simulations and single-molecule FRET in native nanodiscs. Biophys. J. 118, 532. Spencer-Smith, R., and O’Bryan, J.P. (2019). Direct inhibition of RAS: quest for the holy grail? Semin. Cancer Biol. 54, 138–148. Perspective 19, 6057–6064. Muratcioglu, S., Aydin, C., Odabasi, E., Ozdemir, E.S., Firat-Karalar, E.N., Jang, H., Tsai, C.J., Nussinov, R., Kavakli, I.H., Gursoy, A., et al. (2020). Onco- genic K-Ras4B dimerization enhances downstream mitogen-activated protein kinase signaling. J. Mol. Biol. 432, 1199–1215. Okazaki, K., and Takada, S. (2008). Dynamic energy landscape view of coupled binding and protein conformational change: induced-ﬁt versus popu- lation-shift mechanisms. Proc. Natl. Acad. Sci. U S A 105, 11182–11187. Muratcioglu, S., Chavan, T.S., Freed, B.C., Jang, H., Khavrutskii, L., Freed, R.N., Dyba, M.A., Stefanisko, K., Tarasov, S.G., Gursoy, A., et al. (2015). GTP-dependent K-ras dimerization. Structure 23, 1325–1335. Ostrem, J.M., Peters, U., Sos, M.L., Wells, J.A., and Shokat, K.M. (2013). K- Ras(G12C) inhibitors allosterically control GTP afﬁnity and effector interac- tions. Nature 503, 548–551. Nagar, B., Hantschel, O., Young, M.A., Scheffzek, K., Veach, D., Bornmann, W., Clarkson, B., Superti-Furga, G., and Kuriyan, J. (2003). Structural basis for the autoinhibition of c-Abl tyrosine kinase. Cell 112, 859–871. Papke, B., and Der, C.J. (2017). Drugging RAS: know the enemy. Science 355, 1158–1163. Nagasaka, M., Li, Y., Sukari, A., Ou, S.I., Al-Hallak, M.N., and Azmi, A.S. (2020). KRAS G12C Game of Thrones, which direct KRAS inhibitor will claim the iron throne? Cancer Treat. Rev. 84, 101974. Papke, B., Murarka, S., Vogel, H.A., Martin-Gago, P., Kovacevic, M., Truxius, D.C., Fansa, E.K., Ismail, S., Zimmermann, G., Heinelt, K., et al. (2016). Iden- tiﬁcation of pyrazolopyridazinones as PDEdelta inhibitors. Nat. Commun. 7, 11360. Neale, C., and Garcia, A.E. (2020). The plasma membrane as a competitive in- hibitor and positive allosteric modulator of KRas4B signaling. Biophys. J. 118, 1129–1141. Park, E., Rawson, S., Li, K., Kim, B.W., Ficarro, S.B., Pino, G.G., Sharif, H., Marto, J.A., Jeon, H., and Eck, M.J. (2019). Architecture of autoinhibited and active BRAF-MEK1-14-3-3 complexes. Nature 575, 545–550. Nussinov, R. (2016). Introduction to protein ensembles and allostery. Chem. Rev. 116, 6263–6266. Parker, J.A., and Mattos, C. (2018). The K-ras, N-ras, and H-ras isoforms: unique conformational preferences and implications for targeting oncogenic mutants. Cold Spring Harb Perspect. Med. 8, a031427. Nussinov, R., Jang, H., and Tsai, C.J. (2014). The structural basis for cancer treatment decisions. Oncotarget 5, 7285–7302. Cell Chemical Biology 28, February 18, 2021 131 Cell Chemical Biology 28, February 18, 2021 131 ll OPEN ACCESS Parker, J.A., Volmar, A.Y., Pavlopoulos, S., and Mattos, C. (2018). K-ras pop- ulates conformational states differently from its isoform H-ras and oncogenic mutant K-RasG12D. Perspective Spiegel, J., Cromm, P.M., Zimmermann, G., Grossmann, T.N., and Waldmann, H. (2014). Small-molecule modulation of Ras signaling. Nat. Chem. Biol. 10, 613–622. Prior, I.A., Lewis, P.D., and Mattos, C. (2012). A comprehensive survey of Ras mutations in cancer. Cancer Res. 72, 2457–2467. Pylayeva-Gupta, Y., Grabocka, E., and Bar-Sagi, D. (2011). RAS oncogenes: weaving a tumorigenic web. Nat. Rev. Cancer 11, 761–774. Sprang, S.R. (1997). G proteins, effectors and GAPs: structure and mecha- nism. Curr. Opin. Struct. Biol. 7, 849–856. Quevedo, C.E., Cruz-Migoni, A., Bery, N., Miller, A., Tanaka, T., Petch, D., Ba- taille, C.J.R., Lee, L.Y.W., Fallon, P.S., Tulmin, H., et al. (2018). Small molecule inhibitors of RAS-effector protein interactions derived using an intracellular antibody fragment. Nat. Commun. 9, 3169. Steklov, M., Pandolﬁ, S., Baietti, M.F., Batiuk, A., Carai, P., Najm, P., Zhang, M., Jang, H., Renzi, F., Cai, Y., et al. (2018). Mutations in LZTR1 drive human disease by dysregulating RAS ubiquitination. Science 362, 1177–1182. Stephen, A.G., Esposito, D., Bagni, R.K., and McCormick, F. (2014). Dragging ras back in the ring. Cancer Cell 25, 272–281. Reid, T.S., and Beese, L.S. (2004). Crystal structures of the anticancer clinical candidates R115777 (Tipifarnib) and BMS-214662 complexed with protein far- nesyltransferase suggest a mechanism of FTI selectivity. Biochemistry 43, 6877–6884. Stites, E.C., and Ravichandran, K.S. (2009). A systems perspective of ras signaling in cancer. Clin. Cancer Res. 15, 1510–1513. Rezaei Adariani, S., Buchholzer, M., Akbarzadeh, M., Nakhaei-Rad, S., Dvor- sky, R., and Ahmadian, M.R. (2018). Structural snapshots of RAF kinase inter- actions. Biochem. Soc. Trans. 46, 1393–1406. Sun, Q., Burke, J.P., Phan, J., Burns, M.C., Olejniczak, E.T., Waterson, A.G., Lee, T., Rossanese, O.W., and Fesik, S.W. (2012). Discovery of small mole- cules that bind to K-Ras and inhibit Sos-mediated activation. Angew. Chem. Int. Ed. Engl. 51, 6140–6143. Sakamoto, K., Kamada, Y., Sameshima, T., Yaguchi, M., Niida, A., Sasaki, S., Miwa, M., Ohkubo, S., Sakamoto, J.I., Kamaura, M., et al. (2017). K- Ras(G12D)-selective inhibitory peptides generated by random peptide T7 phage display technology. Biochem. Biophys. Res. Commun. 484, 605–611. Sutton, M.N., Lu, Z., Li, Y.C., Zhou, Y., Huang, T., Reger, A.S., Hurwitz, A.M., Palzkill, T., Logsdon, C., Liang, X., et al. (2019). DIRAS3 (ARHI) blocks RAS/ MAPK signaling by binding directly to RAS and disrupting RAS clusters. Cell Rep. 29, 3448–3459.e6. Samatar, A.A., and Poulikakos, P.I. (2014). Targeting RAS-ERK signalling in cancer: promises and challenges. Nat. Rev. Drug Discov. 13, 928–942. Perspective Perspective Vigil, D., Cherﬁls, J., Rossman, K.L., and Der, C.J. (2010). Ras superfamily GEFs and GAPs: validated and tractable targets for cancer therapy? Nat. Rev. Cancer 10, 842–857. Young, A., Lou, D., and McCormick, F. (2013). Oncogenic and wild-type Ras play divergent roles in the regulation of mitogen-activated protein kinase signaling. Cancer Discov. 3, 112–123. Wang, Y., Kaiser, C.E., Frett, B., and Li, H.Y. (2013). Targeting mutant KRAS for anticancer therapeutics: a review of novel small molecule modulators. J. Med. Chem. 56, 5219–5230. Zeng, M., Lu, J., Li, L., Feru, F., Quan, C., Gero, T.W., Ficarro, S.B., Xiong, Y., Ambrogio, C., Paranal, R.M., et al. (2017). Potent and selective covalent quina- zoline inhibitors of KRAS G12C. Cell Chem. Biol. 24, 1005–1016.e3. Ward, R.A., Fawell, S., Floc’h, N., Flemington, V., McKerrecher, D., and Smith, P.D. (2020). Challenges and opportunities in cancer drug resistance. Chem. Rev. https://doi.org/10.1021/acs.chemrev.1020c00383. Zeng, M., Xiong, Y., Safaee, N., Nowak, R.P., Donovan, K.A., Yuan, C.J., Na- bet, B., Gero, T.W., Feru, F., Li, L., et al. (2020). Exploring targeted degradation strategy for oncogenic KRAS(G12C). Cell Chem. Biol. 27, 19–31.e6. Waters, A.M., and Der, C.J. (2018). KRAS: the critical driver and therapeutic target for pancreatic cancer. Cold Spring Harb. Perspect. Med. 8, a031435. Zhang, M., Jang, H., and Nussinov, R. (2019a). The mechanism of PI3Kalpha activation at the atomic level. Chem. Sci. 10, 3671–3680. Wiesmuller, L., and Wittinghofer, F. (1994). Signal transduction pathways involving Ras. Mini review. Cell. Signal. 6, 247–267. Zhang, M., Jang, H., and Nussinov, R. (2019b). The structural basis for Ras activation of PI3Kalpha lipid kinase. Phys. Chem. Chem. Phys. 21, 12021–12028. Wittinghofer, A., and Pai, E.F. (1991). The structure of Ras protein: a model for a universal molecular switch. Trends Biochem. Sci. 16, 382–387. Zhang, M., Jang, H., and Nussinov, R. (2020). PI3K inhibitors: review and new strategies. Chem. Sci. 11, 5855–5865. Wittinghofer, A., Scheffzek, K., and Ahmadian, M.R. (1997). The interaction of Ras with GTPase-activating proteins. FEBS Lett. 410, 63–67. Wittinghofer, A., and Vetter, I.R. (2011). Structure-function relationships of the G domain, a canonical switch motif. Annu. Rev. Biochem. 80, 943–971. Zhang, Z., and Shokat, K.M. (2019). Bifunctional small-molecule ligands of K- ras induce its association with immunophilin proteins. Angew. Chem. Int. Ed. Engl. 58, 16314–16319. Xiong, Y., Lu, J., Hunter, J., Li, L., Scott, D., Choi, H.G., Lim, S.M., Manandhar, A., Gondi, S., Sim, T., et al. (2017). Zimmermann, G., Papke, B., Ismail, S., Vartak, N., Chandra, A., Hoffmann, M., Hahn, S.A., Triola, G., Wittinghofer, A., Bastiaens, P.I., et al. (2013). Small molecule inhibition of the KRAS-PDEdelta interaction impairs oncogenic KRAS signalling. Nature 497, 638–642. Cell Chemical Biology 28, February 18, 2021 133 Perspective Takai, Y., Sasaki, T., and Matozaki, T. (2001). Small GTP-binding proteins. Physiol. Rev. 81, 153–208. Sarkar, S., and Garcia, A.E. (2020). Presence or absence of ras dimerization shows distinct kinetic signature in ras-raf interaction. Biophys. J. 118, 1799–1810. Thurman, R., Siraliev-Perez, E., and Campbell, S.L. (2020). RAS ubiquitylation modulates effector interactions. Small GTPases 11, 180–185. Schubbert, S., Shannon, K., and Bollag, G. (2007). Hyperactive Ras in devel- opmental disorders and cancer. Nat. Rev. Cancer 7, 295–308. Tran, T.H., Alexander, P., Dharmaiah, S., Agamasu, C., Nissley, D.V., McCor- mick, F., Esposito, D., Simanshu, D.K., Stephen, A.G., and Balius, T.E. (2020). The small molecule BI-2852 induces a nonfunctional dimer of KRAS. Proc. Natl. Acad. Sci. U S A 117, 3363–3364. Shao, T., Zheng, Y., Zhao, B., Li, T., Cheng, K., and Cai, W. (2014). Recombi- nant expression of different mutant K-ras gene in pancreatic cancer Bxpc-3 cells and its effects on chemotherapy sensitivity. Sci. China Life Sci. 57, 1011–1017. Travers, T., Lopez, C.A., Van, Q.N., Neale, C., Tonelli, M., Stephen, A.G., and Gnanakaran, S. (2018). Molecular recognition of RAS/RAF complex at the membrane: role of RAF cysteine-rich domain. Sci. Rep. 8, 8461. Sheridan, C. (2020). Grail of RAS cancer drugs within reach. Nat. Biotechnol. 38, 6–8. Tsai, C.J., Kumar, S., Ma, B., and Nussinov, R. (1999a). Folding funnels, bind- ing funnels, and protein function. Protein Sci. 8, 1181–1190. Sheridan, C., and Downward, J. (2013). Inhibiting the RAS-PI3K pathway in cancer therapy. Enzymes 34 Pt. B, 107–136. Tsai, C.J., Ma, B., and Nussinov, R. (1999b). Folding and binding cascades: shifts in energy landscapes. Proc. Natl. Acad. Sci. U S A 96, 9970–9972. Shima, F., Ijiri, Y., Muraoka, S., Liao, J., Ye, M., Araki, M., Matsumoto, K., Ya- mamoto, N., Sugimoto, T., Yoshikawa, Y., et al. (2010). Structural basis for conformational dynamics of GTP-bound Ras protein. J. Biol. Chem. 285, 22696–22705. Tsai, C.J., and Nussinov, R. (2014). The free energy landscape in translational science: how can somatic mutations result in constitutive oncogenic activa- tion? Phys. Chem. Chem. Phys. 16, 6332–6341. Shima, F., Yoshikawa, Y., Ye, M., Araki, M., Matsumoto, S., Liao, J., Hu, L., Su- gimoto, T., Ijiri, Y., Takeda, A., et al. (2013). In silico discovery of small-mole- Vetter, I.R., and Wittinghofer, A. (2001). The guanine nucleotide-binding switch in three dimensions. Science 294, 1299–1304. 132 Cell Chemical Biology 28, February 18, 2021 ll OPEN ACCESS ll OPEN ACCESS Perspective Covalent guanosine mimetic inhibitors of G12C KRAS. ACS Med. Chem. Lett. 8, 61–66. Zhou, Y., Prakash, P., Gorfe, A.A., and Hancock, J.F. (2018). Ras and the plasma membrane: a complicated relationship. Cold Spring Harb. Perspect. Med. 8, a031831. Yan, C., Liu, D., Li, L., Wempe, M.F., Guin, S., Khanna, M., Meier, J., Hoffman, B., Owens, C., Wysoczynski, C.L., et al. (2014). Discovery and characterization of small molecules that target the GTPase Ral. Nature 515, 443–447. Zimmermann, G., Papke, B., Ismail, S., Vartak, N., Chandra, A., Hoffmann, M., Hahn, S.A., Triola, G., Wittinghofer, A., Bastiaens, P.I., et al. (2013). Small molecule inhibition of the KRAS-PDEdelta interaction impairs oncogenic KRAS signalling. Nature 497, 638–642. Yehia, L., Keel, E., and Eng, C. (2020). The clinical spectrum of PTEN muta- tions. Annu. Rev. Med. 71, 103–116.
https://openalex.org/W2950156458	https://zenodo.org/records/3639596/files/THE%20ANALYSIS%20OF%20EXISTING%20EXPERIENCE%20FOR%20THE%20ETHNOBOTANICAL%20INFORMATION%20SYSTEM.pdf	English	null	THE ANALYSIS OF EXISTING EXPERIENCE FOR THE ETHNOBOTANICAL INFORMATION SYSTEM	Eureka, Life Sciences/Eureka, Life Sciences.	2,019	cc-by	5,950	Reports on research projects Reports on research projects Reports on research projects (2019), «EUREKA: Life Sciences» Number 3 Abstract Keywords: ethnobotanical biodiversity, herbal medicine, medicinal plants, ethnobotanical database, key components, struc- ture, main tables of the ethnobotanical database. THE ANALYSIS OF EXISTING EXPERIENCE FOR THE ETHNOBOTANICAL INFORMATION SYSTEM Laman Faig Hamidova Department of System analysis, control and information processing Institute of Information Technology of ANAS 9A B.Vahabzade str., Baku, Azerbaijan, AZ1141 leman.hemidova92@gmail.com DOI: 10.21303/2504-5695.2019.00897 © Laman Faig Hamidova Abstract Ethnobotanical researches reflect the conventional learning of a region. Over the previous decade, medical plants which used for healing indigenous people has become a significant notion among the people and impacted improvement of scientific and ethnobotanical knowledge and investigations of eliminating health problems. A public database has been based on data assembled from various verifiable sources, including journals, travel records, and treatises on therapeutic plants, composed by explorers, botanists, doctors, researchers who went to the nations during the most recent three centuries. In addition, ethnobotanical data depicted in chronicled natural accumulations and in Ancient and Medieval writings from the inquired district have been incorpo- rated into the database. The databases have to be sufficiently adaptable to illustrate a valuable tool for analysts who need to store and analyze present and past ethnobotanical data from the researched location. The ethnobotanical researches are improved in Azerbaijan day by day. The database is used for informing people about some national plants which are growing in the different region of Azerbaijan. The ethnobotanical databases from different countries are analyzed in this article. There are used some special methods for comparing the differences among these databases as data mining and text min- ing. As a first step the suitable databases are gathered for our investigation, then are defined the best information systems that are used in many countries’ biologists and scientists and the end is observed advantages and disadvantages of all existing ethno- botanical databases which we researched. The features of information systems are evaluated. The results demonstrated each of databases has its very own quality, but none has turned a standard form for universal research. The reason is very basic: none of these databases enable specialists to include their own information. There is also illustrated sample structure, main tables and key components of the ethnobotanical database. The obtained results, while a few ethnobotanical databases existing, none are satisfactory answers for worldwide work, and none enable analysts to include their very own information. There is a need brought together all essential properties of exist- ing databases, and creating a free database that encourages ethnobotanical research. Due to the rise and quick improvement in the field of data advances, it has now turned out to be conceivable to digitize, oversee and make ethnobotanical information accessible to a more extensive gathering of people. 1. Introduction Besides, alongside the loss of biodiversity, social assorted variety related with important therapeutic learning is additionally vanishing at a disturbing rate, because of cultural assimilation and urbanization. Therefore, scientists perceive that the answer of the protection of biodiversity lies in a multi-disciplinary and coordinated methodology alongside the fundamental contribution of indigenous. Furthermore, there is additionally a worldwide accord that simple and productive access to data on medical plants information and practices are basic for proficient pro- tection methodologies and the economical utilization of biodiversity [6]. Associations including the United Nations (UN) are scanning for approaches to protect customary learning and find a use for it to help both indigenous and non-indigenous individuals.i ganization (WHO), 80 % of the populace from various nations relies upon customarily used thera- peutic plants for their essential medicinal services [3]. It is evaluated that there are 300,000 plants on the planet of which 75,000 plant species are used as customary medication [4]. As indicated by the writing information, the ﬂora of Azerbaijan comprises of about 5000 plant species, having a place with 1142 genera of 186 families, including 200 endemics of Azerbaijan and 950 endemics of the Caucasus. The types of the ﬂora of Azerbaijan are circulated in 20 phytogeographical lo- cales. Among them, the hilly piece of Nakhchivan emerges as far as species decent variety, with about 60 % of all plant types of the country occurring there, trailed by Greater Caucasus and the Lankaran-Mughan plain [5]. Be that as it may, proceeding natural surroundings annihilation and human-incited ecological changes have prompted the loss of vital therapeutic plants used custom- arily by indigenous. Besides, alongside the loss of biodiversity, social assorted variety related with important therapeutic learning is additionally vanishing at a disturbing rate, because of cultural assimilation and urbanization. Therefore, scientists perceive that the answer of the protection of biodiversity lies in a multi-disciplinary and coordinated methodology alongside the fundamental contribution of indigenous. Furthermore, there is additionally a worldwide accord that simple and productive access to data on medical plants information and practices are basic for proficient pro- tection methodologies and the economical utilization of biodiversity [6]. Associations including the United Nations (UN) are scanning for approaches to protect customary learning and find a use for it to help both indigenous and non-indigenous individuals. 1. Introduction Over a time of four billion years, development has brought about overwhelming biodiversity on earth and has remained the essential power for the advancement of assorted life shapes over the globe [1]. It is assessed that of the aggregate number of life forms going from 7–20 million, just 1.8 million species are logically named. Overwhelmingly, the world’s biodiversity alluded to as “biodiversity storage facilities” with remarkable taxon and species assorted variety [1]. Environ- mental change and human impacts have brought about a phenomenal loss of biodiversity, with an expected 100–1000 overlap speeding up in the annihilation rate of botanical and faunal species contrasted with their normal rate. Shockingly, moderates are looked with a scarcity of information as just 1 % of every single recognized species has been considered past essential classification [2]. Truly, people have depended on biodiversity as an essential wellspring of sustenance, pro- tect and for profound and mental prosperity. Medical plants involve a noteworthy piece of the biodiversity and shape an inseparable connection between natural and social variety. Their co-ad- vancement has delivered neighborhood conventional restorative plant learning and practices. This wide knowledgebase is passed on from age to age, moves as orally like stories, melodies and frames for the manageable misuse and preservation of biodiversity. As indicated by the World Health Or- 15 Agricultural and Biological Scienсes (2019), «EUREKA: Life Sciences» (2019), «EUREKA: Life Sciences» Number 3 Reports on research projects Number 3 ganization (WHO), 80 % of the populace from various nations relies upon customarily used thera- peutic plants for their essential medicinal services [3]. It is evaluated that there are 300,000 plants on the planet of which 75,000 plant species are used as customary medication [4]. As indicated by the writing information, the ﬂora of Azerbaijan comprises of about 5000 plant species, having a place with 1142 genera of 186 families, including 200 endemics of Azerbaijan and 950 endemics of the Caucasus. The types of the ﬂora of Azerbaijan are circulated in 20 phytogeographical lo- cales. Among them, the hilly piece of Nakhchivan emerges as far as species decent variety, with about 60 % of all plant types of the country occurring there, trailed by Greater Caucasus and the Lankaran-Mughan plain [5]. Be that as it may, proceeding natural surroundings annihilation and human-incited ecological changes have prompted the loss of vital therapeutic plants used custom- arily by indigenous. Agricultural and Biological Scienсes 2. 1. Using Innovative Systems for Ethnobotanical Information Ethnobotany has advanced to address complex human-plant interrelation compare to ba- sic classifying of customarily used plants by people. Ethnobotanical information is urgent for an assortment of logical investigations, for example, pharmacology, veterinary science, biology, and customary learning protection. The obstructions for achieving ethnobotanical data restrict the ca- pacity of analysts and decision-makers producers to address issues in the regions of the investiga- tion, protection, manageable use, and basic leadership. The different idea of accessible ethnobotan- ical information has presented difficulties for information access, administration, and joining the different ethnobotanical resources. At present, the best way is to use informatics tools to digitize both unpublished and distributed ethnobotanical information (recorded and contemporary) held in different configurations for straightforward entry. A second way is encouraging promotion and consistent mix of information from various areas, for example, scientific classification, environ- ment, phytochemistry, pharmacology, microbiology, and atomic science. Such a coordinated data framework can conceivably corresponding ethnobotanical information with datasets from differ- ent controls, along these lines encouraging better comprehension for taking educated choices. For instance, incorporating ethnobotanical data with ordered datasets, for example, the International Plant Name Index will resolve the logical name and related issues concerning ethnobotanical plant types of essentialness [7]. In the ongoing past, there has been an expansion in the accessibility of ethnobotanical in- formation over the web. In spite of an expansion in the number of activities giving information in advanced configurations, for example, public databases and HTML site pages, the aggregate ac- cessible online information are divided and just a little part of what is known. In addition, these endeavors do not equivalent to the measure of gathered ethnobotanical information throughout the hundreds of years and the rate at which it is produced in electronic systems. Further, be- cause of the absence of informatics foundation encouraging simple access and confinements in information administration programming, huge measures of information could not be coordi- nated, prompting excess and wasteful use. For instance, a database, for example, NAPRALERT, with overall inclusion, gives ethnobotanical data on 20,000 medical plants including some In- dian species. In correlation, the Encyclopedia of Indian Medicinal Plants gives ethnobotanical data on 6,198 restorative Indian plant species, yet not these are incorporated into NAPRALERT. Since these datasets are not connected there is no simple method for checking the cover between them. 2. 1. Using Innovative Systems for Ethnobotanical Information Hence, similarly as with entries, for example, the Global Biodiversity Information Facility (GBIF) information entryway, Species 2000 and ITIS Catalog of Life, the Consortium for the Barcode of Life (CBOL), Australia’s Virtual Herbarium (AVH) and the Universal Biological In- dexer and Organizer (uBio), a bound together or combined information gateway is required for getting to ethnobotanical data all around [8]. (2019), «EUREKA: Life Sciences» Number 3 (2019), «EUREKA: Life Sciences» Number 3 other common items got from nature, for example, nourishment, plants used in ceremonies, fiber plants, harms, building materials for houses, and so on. other common items got from nature, for example, nourishment, plants used in ceremonies, fiber plants, harms, building materials for houses, and so on. 1. Introduction Ethnobotany, as the extensive number of educational and medical fields, sites, courses, workshops scholarly projects and media consideration propose, has encountered an unparalleled time of development in the previous twenty years. To be sure, inside this period, “ethnobotany” has been moved out of them to some degree elusive edges of science into the open standard. This re- newal of ethnobotany is apparent in post-modern, industrializing and non-industrialized countries, recommending it connect to more extensive auxiliary procedures. The improvement and make use of databases incorporated with conventional learning is ex- tending. While there’s an expected 40 years history of using databases in ethnoscientific subfields (e. g. ethnobotany, ethnobotanics, ethnozoology, ethnopharmacology and so on.) we know nearly nothing of their potential hugeness to indigenous people group. People need to keep up control of a database will probably require self-ruling advancement or community accomplices focused on strategies that stick to social conventions and the insurance of customary learning. While thinking about the significance and potential advancement of a database it is basic to survey its incentive as a computerized innovation. Ethnobotany is recently improved field of the request which is getting more consideration from the academic network in Azerbaijan as of late. There are some distributed works in this field (Fig. 1). Fig. 1. Fields of related to ethnobotany Ethnobotany •Chemistry •Genetics •Microbiology •Pharmacology •Anthroplogy •Ethnobiography •Indegenous law •Economics •Linguistics •Geography •Folklore •Ecology Fig. 1. Fields of related to ethnobotany The order of ethnobotany has extensive control what is keen on all investigations about the connection among people and plants. Ethnobotany bases around medicinal plants as well as on 16 Reports on research projects 2. 2. The Analysis of some of the Existing Ethnobotanical Databases There are a few well-known ethnobotanical databases in our time. While each of these stores has its very own quality, but none has turned a standard form for universal research. The reason is very basic: none of these databases enable specialists to include their own information. Let’s use two methods for comparing ethnobotanical databases. Text mining or content mining is the way toward investigating and examining a lot of un- structured content information supported by programming that can distinguish ideas, designs, subjects, and different traits in the data. Text mining has turned out to be increasingly viable for information researchers and different users because of the improvement of huge information stag- es [9]. One of the initial phases in the content mining process is composing and structuring the information in some material so it very well may be exposed to both subjective and quantitative analysis. The forthright work incorporates ordering, grouping, condensing informational collec- 17 Agricultural and Biological Scienсes (2019), «EUREKA: Life Sciences» (2019), «EUREKA: Life Sciences» Number 3 Reports on research projects tions, making scientific classifications, and extricating data about things like word frequencies and connections between information substances (Fig. 2). tions, making scientific classifications, and extricating data about things like word frequencies and connections between information substances (Fig. 2). Fig. 2. Text mining steps Fig. 2. Text mining steps Data mining is the way toward dealing with expansive informational collections to dis- tinguish designs and set up connections to take care of issues through information examination. Information mining devices enable institutes or any companies to anticipate future patterns. In information mining, affiliation rules are made by breaking down information for successive on the off chance that designs, utilizing the help and certainty criteria to find the most essential connec- tions inside the information. Support is the means the information show up in the database now and after amending process, while certainty is the occasions if proclamations are accurate. Other data mining parameters incorporate Sequence or Path Analysis, Classification, Clustering and Fore- casting. Grouping or Path Analysis parameters search for examples where one occasion prompts another later occasion. A Sequence is an arranged rundown of sets of things, and it is a typical sort of information structure found in numerous databases. A Classification parameter searches new examples, and as a result information is sorted out. Grouping calculations anticipate factors depen- dent on different factors inside the database [10], (Fig. Agricultural and Biological Scienсes 2. 2. The Analysis of some of the Existing Ethnobotanical Databases Additionally, it was noted the using qualities of the plants: disease or indication treated, recurrence of use, sort of drug (human or veterinary). There were illustrated 251 different species in Africa [12]. ethnobotanical data, vernacular names of the plant. Additionally, it was noted the using qualities of the plants: disease or indication treated, recurrence of use, sort of drug (human or veterinary). There were illustrated 251 different species in Africa [12]. FRLHT Encyclopedia of Indian Medicinal Plants, a database that was prepared on In- dian Medicinal Plants is currently accessible for analysts, academicians, students, ethnobotanists, preservationists, nature friends. Thus, FRLHT has been creating in recent years a thorough data- base on Indian Medicinal Plants. It covers different subject connected to normal assets used by the Indian System of Medicine, for example, natural and common names relationship, geo-dissemina- tion information, maps, exchange data and so on. This is an all-around referenced unique database. This restrictive and creative pursuit based database stores 7,637 herbal names (6,198 restorative plants species) with 119183 vernacular names from 12 dialects crosswise over India. Almost 2688 plant pictures are additionally accessible in the database [13]. TradiMed database, in view of different Chinese and Korean therapeutic plants, is the con- solidating aftereffect of the conventional drug and the advanced science, which advances compre- hension of customary therapeutic medicines from the perspective of the cutting-edge science, and supplements for new improvements of items. Herbal Taxonomy and Miscellaneous: Aggregate of 3,199 natural data with images, symp- toms, adequacy, target organs, property, and well-being measure can be sought by names. What’s more, there are 2,944 orders on creatures and herbs. Formulae: Aggregate of 11,810 medications from 17 works with points of interest, for ex- ample, target organs, symptoms, adequacy, readiness, use, alert, reference, and measurement can be recovered. Constituents (chemical data with range investigation information): Aggregate of around 20,012 constituents from crude materials can be seen with synthetic data, investigative spectrum (UV, NMR, IR, Mass), and conceivable cross-reference. Manifestation/Disease (cross-reference between the Eastern and the Western medications): Aggregate of 4,080 customary infections are referenced into the cutting edge malady arrangement with titles and side effects. Preparing: It is the customary technique to plan crude herbs previously use. The aggregate of 95 general processing and 502 herbs are recorded [14]. Dr. Duke’s Phytochemical and Ethnobotanical databases’ parameters look into accu- mulated by Dr. 2. 2. The Analysis of some of the Existing Ethnobotanical Databases 3). Fig. 3. Data mining steps There are additionally a few different shortcomings and qualities of every one of these ap- plications. There are additionally a few different shortcomings and qualities of every one of these ap- plications. There are additionally a few different shortcomings and qualities of every one of these ap- plications. International Ethnobotany Database is completely multilingual and underpins interpreta- tion, information section, and survey for any dialect. In particular, the database is available to any scientist to include new information, datasets, and dialects. The ebDB is overseen by the Founda- tion for Open EthnoBotany Research (FOER), an association devoted exclusively to dealing with the database and giving a worldwide store to ethnobotanical information. Therefore, it is protected from institutional political strife and business interests and gives a reliable environment for differ- ent information. The biologists have been worked more than four years, and look into the fields of Peru, the ebDB contains an expansive list of capabilities and is structured particularly for ethno- botanical explore [11]. It is completely multilingual, has a glossary, in excess of 20 classes of infor- mation, effective searching process, and information send out highlights. Right now, the database preserves information from Ecuador, Peru, Kenya, and Hawai’i. It is the expectation of this venture the ebDB turns into a worldwide standard for putting away ethnobotanical information [11].i Prelude Medicinal Plants Database, this specialized database is identified with the uses of a few plants in customary veterinary and human prescription in Africa. It is helpful for analysts, specialists, veterinary specialists to have documentation available to them that gives access to con- solidated data from an assortment of sources in regards to the make use of customary therapeutic plants in various areas of Africa. The point of this database is to address this issue. The data put away in the database is gathered from articles, books, papers given to congresses or straightfor- wardly to the Prelude Sub-Network on “Well-being, creature preparations, condition”. The information has relegated a data for each plant under the accompanying headings in this database: name of the plant, name of a sort, bibliographical reference, people who were gathered 18 (2019), «EUREKA: Life Sciences» (2019), «EUREKA: Life Sciences» Number 3 Reports on research projects Number 3 ethnobotanical data, vernacular names of the plant. Agricultural and Biological Scienсes 2. 2. The Analysis of some of the Existing Ethnobotanical Databases Jim Duke of the Green Pharmacy Garden and the United States Department of Agriculture (USDA). The USDA’s Agricultural Research Service offers help for the database. Dr. Duke’s database has a few key qualities: countless, last information on most database sections, and to reveal concoction data on an assortment of plants. There are 49,788 entities such as phytochemi- cal (biological activity, chemicals, plant, syndrome) and ethnobotanical (ethnobotanical plants and ethnobotanical uses). It is possible to see 13,010 different ethnobotanical plants on this database and their growing place, for what using, common names of some plants. Researchers can also be downloaded data which is necessary for them as pdf or spreadsheet form. Ethnobotanists can find some useful information for pharmaceutical, nutritional, and biomedical research, as well as alter- native therapies and herbal products. It additionally has a few basic shortcomings, most strikingly the absence of open information, and restricted help for an assortment of ethnobotany information. The “EthnobotDB” itself just has fields for natural portrayal and use. While this makes the data- base a successful tool for spreading Dr. Duke’s work, it does nothing to help different specialists to viably detail the products of their exploration [15]. A Modern HerbalDatabase was composed in England with an atmosphere like the Pacific Northwest in America, and keeps a top priority. For Medicinal Use – a primary concern of this database was composed that indigenous people used a plant with true way of thinking of the mid-1900s. This ought to be considered as a portion of the data may now be viewed as erroneous, or not as per the current drug [16]. Plant and Herb Index: More than 800 assortments of herbs and plants. Record of Recipes: A posting of 29 plants that incorporate formulas. Record of Poisons: A posting of 44 plants that are recorded as harmful. 19 Agricultural and Biological Scienсes Agricultural and Biological Scienсes (2019), «EUREKA: Life Sciences» (2019), «EUREKA: Life Sciences» Number 3 Reports on research projects Number 3 NAPRALERT contains data on a wide assortment of plants and their uses, but its use to sci- entists start and closure there. In contrast to alternate databases, NAPRALERT is compensation for every framework, with an expense at composing of 8.90 USD per search. While the University of Illinois at Chicago is qualified for reserve its examination, obviously this is no summed up a storage facility for ethnobotanical information. 2. 2. The Analysis of some of the Existing Ethnobotanical Databases It’s essential to likewise take note of that NAPRALERT centers around the phytochemical data for the plants in its database. While phytochemistry is an essential science, with the worries of bio-prospecting it is more dependable to keep phytochemical information separate from unadulterated ethnobotanical information. At last, similar to the previ- ously mentioned two databases, NAPRALERT is monolingual framework [17]. Native American Ethnobotany, a database of plants used medications, sustenance, colors by local Peoples of North America. The online database was a great extent finished in the late 1990s. The database currently contains 44,691 things. This form included nourishments, drugs, colors, strands and different employments of plants (an aggregate of more than 44,000 things). This information was assembled by 291 Native American gatherings of 4,029 species from 243 distinc- tive plant families. A portion of them is therapeutic. This development of the database was upheld by allowing from the National Science Foundation [18]. The second form of the database was distributed in 1986 by the Museum of Anthropology of the University of Michigan with the title Medicinal Plants of Native America. That database – which contained 17,634 things illustrating to the therapeutic employment of 2,147 species from 760 genera and 142 families by 123 diverse local American gatherings – was worked overtime of around 10 years with help from the National Endowment for the Humanities, the National Science Foundation, and the University of Michigan-Dearborn. The information was entered utilizing an intricate plan checking for information uprightness which electronically settled numerous organic equivalent words (which happen when the “same” plant gets diverse logical names, something that frequently occurs over significant lots of time) [18]. A few databases for plant optional metabolic pathways, organic exercises, synthetic struc- tures, ethnobotanical uses, content in nourishments, and pharmacology are open source. A couple of valuable databases are recorded beneath and may keep on filling in as refreshed bases of data. Table 1 The ethnobotanical databases spreading throughout the world 20 Table 1 The ethnobotanical databases spreading throughout the world Ethnobotanical Databases Links Languages 1 2 3 Dictionary of Natural Compounds http://dnp.chemnetbase.com English PubChem http://pubchem.ncbi.nlm.nih.gov English NAPRALERT http://www.napralert.org/about.aspx English Dr. Agricultural and Biological Scienсes Reports on research projects 2. 2. The Analysis of some of the Existing Ethnobotanical Databases Dukes’s Phytochemical and Ethnobotanical DB https://phytochem.nal.usda.gov/phytochem/search English Plant Metabolic Network http://www.plantcyc.org USDA databases http://fnic.nal.usda.gov/food-composition /phytonutrients English MetaCYC http://metacyc.org/ English Phenol-Explorer http://phenol-explorer.eu English Prelude Medicinal Plants Database www.metafro.be/prelude English, French FRLHT Encyclopedia of Indian Medicinal Plants http://www.frlht.org/newenvis/ indian-medicinalplants-database.php English Modern Herbal Database http://www.botanical.com/botanical/mgmh/ mgmh.html English International Ethnobotany Database https://olorien.org/ebDB English Native American Ethnobotany http://naeb.brit.org English Agricultural and Biological Scienсes 5. 1. The Determination of Structure and Schematic Illustration of the Ethnobotanical Database The database model has been executed as a local database. Distinctive execution arrange- ments were tried, as indicated by the prerequisites and find a use for cases. Present and old ethnobo- tanical information was composed in a similar table structure, permitting a few expenses of excess for the homogenization of information and inquiries. Since adaptability was a noteworthy issue for our file, the questions were situated center of ideas which were actualized, for example, boundless various leveled structures, especially for classified tables and scientific species categorization, and legacy ideas, being a reference to data sources, a person, a natural or an archeological remain. Those procedures enabled individuals to build up an insignificant information structure fitting diverse sort of data and to perform questions at various levels of detail (Fig. 5). Fig. 5. A schematic illustration of the key components of the ethnobotanical database Fig. 5. A schematic illustration of the key components of the ethnobotanical database The document presently permits the capacity and administration of ethnobotanical data from sources, recorded herbarium examples and archives, as indicated by a various leveled inven- tory of plant uses and arrangements (therapeutic, nutritious, stylized, veterinary, local, medical, food). It is additionally conceivable to incorporate ethnobotanical data from archeological remains, in spite of the fact that the connections to the relative databases have not yet been executed. Plants’ names data: plant prevalent names (local, botanical, ancient), and their relationship with species scientific classification. i Ethnobotanical information was composed in a social structure: an identified table can gath- er data on various plants, which can be used for various degrees. Conventional learning can’t be effectively ordered and remedial utilizations are regularly intermixed with formal and representa- tive ones. Be that as it may, to permit a more compelling hunt inside the database, convictions, and works on managing people employments of an animal varieties were assembled being used clas- sifications, going over restorative (counting warmth and method of organization of the plant), for- mal (representative, custom, religious including any conviction concerning the plant), wholesome, local, veterinary employment. These classifications have been sorted out in a various leveled struc- ture and might be relegated by the detail level of the data accumulated. A dependability level of those utilization assignments is given, especially for information from the antiquated treatises and from archeological remains. Agricultural and Biological Scienсes 5. Results 5. 1. The Determination of Structure and Schematic Illustration of the Ethnobotanical Database 5. Results 5. 1. The Determination of Structure and Schematic Illustration of the Ethnobotanical Database Table 1 20 Reports on research projects (2019), «EUREKA: Life Sciences» Number 3 Source: https://www.wipo.int/edocs/mdocs/tk/en/wipo_grtkf _ic_3/wipo_grtkf _ic_3_6-main1.pdf It i ill t t d th di th t i t f th t f th b t i l d t b i t 1 2 3 TradiMed http://www.tradimed.com English AGRICOLA http://www.nal.usda.gov/ag98 English The Arctic Science and Technology Information System database http://www.aina.ucalgary.ca/astis English, French Alaska Traditional Knowledge and Native Foods Database http://www.nativeknowledge.org/start.htm English American Folk Medicine http://www.folkmed.ucla.edu/index.html English Ayurveda http://www.pharm.uu.nl/home/smit English BIOSIS http://www.biosis.org/products_services/ba.html English CAB HEALTH Database http://www.cabi-publishing.org/Products/ Database/Health/Index.asp English CHIMERA http://www.rosenthal.hs.columbia.edu/databases/ TradMed_CMMRC.html English Crane Herb Company http://www.craneherb.com English Economic Botany Bibliographic Database http://www.kew.org/data/ecbot-biblio.html English GRIN http://www.ars-grin.gov/npgs/tax/index.html English, Portuguese, Spanish Gernot Katzer’s Spice Pages http://www-ang.kfunigraz.ac.at/ ~katzer/engl/index.html English, German HerbMed http://www.herbmed.org English Herbal Medicine Databases http://www.holisticmed.com/www/herbdb.html English MAROWINA Database http://www.tropilab.com/medsupp.html English Medicinal Herbs On-line http://www.egregore.com/misc/herbindx.htm English Medicinal Plants Catalogue http://www.ayuherbal.com/ medplantcatalogue.htm English, French, Ger- man, Spanish Rainforest Tropical Plant Database http://www.rain-tree.com/plants.htm English, French, Ger- man, Italian, Norwegian, Portuguese, Spanish TRAMIL Medicinal Plant Database http://www.funredes.org/tramil English, French, Spanish (2019), «EUREKA: Life Sciences» Number 3 It is illustrated the diagram that consist of the percentage of ethnobotanical databases exist- ing in the different countries according to their using more by experts and users (Fig. 4). 21 Fig. 4. The percentages of using ehnobotanical databases according to countries Source: https://www.wipo.int/edocs/mdocs/tk/en/wipo_grtkf _ic_3/wipo_grtkf _ic_3_6-main1.pdf 33 % 29 % 10 % 10 % 6 % 6 % 6 % USA China UK India Canada Australia France 21 Fig. 4. The percentages of using ehnobotanical databases according to countries Source: https://www.wipo.int/edocs/mdocs/tk/en/wipo_grtkf _ic_3/wipo_grtkf _ic_3_6-main1.pdf Fig. 4. The percentages of using ehnobotanical databases according to countries Source: https://www.wipo.int/edocs/mdocs/tk/en/wipo_grtkf _ic_3/wipo_grtkf _ic_3_6-main1.pdf 21 Agricultural and Biological Scienсes (2019), «EUREKA: Life Sciences» Number 3 Reports on research projects Reports on research projects 5. 1. The Determination of Structure and Schematic Illustration of the Ethnobotanical Database The guideline of putting away data with various levels of exactness has been executed additionally for the arrangement steps and for the plant parts included (Fig. 6). Data sources can be numerous sorts, in particular, bibliographic references, gatherers, herb- als or archeological remains. All or subsets of these sorts can be referenced, as indicated by the tables where a source data is required. 22 22 (2019), «EUREKA: Life Scien Number 3 Fig. 6. The structure and main tables of the ethnobotanical database rts on research ects (2019), «EUREKA: Life Sciences» Number 3 Fig. 6. The structure and main tables of the ethnobotanical database Reports on research projects Fig. 6. The structure and main tables of the ethnobotanical database The database of medicinal plants for plant biodiversity conservation was prepared for Azer- baijan nature. The database consists of 178 families, 740 genera, 1547 species which grow in Azer- baijan. It was observed when analyzing this database that 34.3 % plants are medicinal plant among 4500 plant species. Each species has 24 parameters which reflect the whole taxonomic characteris- tics, ecology, the territory of distribution, contents of the biologically active compounds, applica- tion forms, therapeutic properties, the status of plant species as endemics, relicts, rarity, references, their photographs in natural ecosystems. Among these medicinal plants, 77 herbal plants species are Caucasian endemics, 5 species are national endemics, 44 species are relicts, 112 medicinal spe- cies are rare and endangered. The collected information on different herbs gives valid data on its high assorted variety and endemism, and also information on scarce and endangered plant species in the certain districts of the nation. It permits to effectively make an oversee framework to charac- terize the conveyance and centralization of biodiversity spots of herbs and to apportion the regions, particularly critical for conservation [19]. It is planned to create a new information system for Azerbaijani ethnobotanical plants which used OLAP (Online Analytical Processing) technology for illustrating ethnobotanical data about plants. As a result, it will be possible to find plant information according to years, medical and chemical properties, also to evaluate various components of multidimensional information. (2019), «EUREKA: Life Sciences» Number 3 (2019), «EUREKA: Life Sciences» Number 3 Reports on research projects Acknowledgements g I would like to thank my advisor, DScTech Rasim Mahammad Alguliyev, PhD Farhad Firu- din Yusifov and Phd Naiba Pirverdi Mehtiyeva for lots of great inspiration, ideas, comments, and feedbacks. I certainly could not have completed this article without their expertise and advice. Agricultural and Biological Scienсes 6. Conclusions Massive ethnobotanical information having social hugeness and financial esteem is con- veyed among various indigenous peoples and research associations. Licensed innovation rights are vital for tending to worldwide issues, for example, decreasing biodiversity, sustenance security, lessening conventional learning and the requirement for new prescriptions to battle expanding drug obstruction. While a few ethnobotanical databases exist, none are satisfactory answers for worldwide work, and none enable analysts to include their very own information. There is a need brought together all essential properties of existing databases, and creating a free database that encourages ethnobotanical research. Due to the rise and quick improvement in the field of data advances, it has now turned out to be conceivable to digitize, oversee and make ethnobotanical information acces- sible to a more extensive gathering of people. By grasping the intensity of innovation data, ethnobotanists and decision-makers can con- nect the substantial gap between the created databases. The way to accomplishing these objectives would be the inception of communitarian associations and familiarity with the centrality of IT among ethnobotanists, indigenous peoples and decision-makers from different nations. It is vital to guarantee protection and reasonable find a use for this data for human prosperity. 23 Agricultural and Biological Scienсes (2019), «EUREKA: Life Sciences» Number 3 References [1] Ahmed, H. M. (2016). Ethnopharmacobotanical study on the medicinal plants used by herbalists in Sulaymaniyah Province, Kurdistan, Iraq. Journal of Ethnobiology and Ethnomedicine, 12 (1). doi: https:// doi.org/10.1186/s13002-016-0081-3 [1] Ahmed, H. M. (2016). Ethnopharmacobotanical study on the medicinal plants used by herbalists in Sulaymaniyah Province, Kurdistan, Iraq. Journal of Ethnobiology and Ethnomedicine, 12 (1). doi: https:// doi.org/10.1186/s13002-016-0081-3 [2] Baydoun, S., Chalak, L., Dalleh, H., Arnold, N. (2015). Ethnopharmacological survey of medic- inal plants used in traditional medicine by the communities of Mount Hermon, Lebanon. Journal of Ethno- pharmacology, 173, 139–156. doi: https://doi.org/10.1016/j.jep.2015.06.052 [2] Baydoun, S., Chalak, L., Dalleh, H., Arnold, N. (2015). Ethnopharmacological survey of medic- inal plants used in traditional medicine by the communities of Mount Hermon, Lebanon. Journal of Ethno- pharmacology, 173, 139–156. doi: https://doi.org/10.1016/j.jep.2015.06.052 [3] WHO Traditional Medicine Strategy: 2014–2023 (2013). Geneva: World Health Organiza- tion, 76. [4] Sarkar, I. N. (2007). Biodiversity informatics: organizing and linking information across the spectrum of life. Briefings in Bioinformatics, 8 (5), 347–357. doi: https://doi.org/10.1093/bib/bbm037 [5] Mehdiyeva, N. P., Guliyev, I. S., Alizade, V. M., Alirzayeva, E. H., Bussmann, R. W. (2017). Eth- nobotany of the Caucasus – Azerbaijan. Ethnobotany of the Caucasus, 37–46. doi: https://doi.org/10.1007/978- 3-319-49412-8_16 [6] Maryo, M., Nemomissa. S., Bekele, T. (2015). An ethnobotanical study of medicinal plants of the Kembatta ethnic group in Enset-based agricultural landscape of Kembatta Tembaro (KT) Zone, Southern Ethiopia. Asian Journal of Plant Science and Research, 5 (7), 42–61. [7] Buenz, E., Schnepple, D., Bauer, B., Elkin, P., Riddle, J., Motley, T. (2004). Techniques: Bio- prospecting historical herbal texts by hunting for new leads in old tomes. Trends in Pharmacological Scienc- es, 25 (9), 494–498. doi: https://doi.org/10.1016/j.tips.2004.07.003 [8] Buneman, P. (2005). What the Web Has Done for Scientific Data – and What It Hasn’t. Lecture Notes in Computer Science, 1–7. doi: https://doi.org/10.1007/11563952_1 [9] Shinde, P., Govilkar, S. (2015). A Systematic study of Text Mining Techniques. International Journal on Natural Language Computing, 4 (4), 54–62. doi: https://doi.org/10.5121/ijnlc.2015.4405 [10] Haripriya, P., Porkodi, R. (2016). A Survey Paper on Data mining Techniques and Challenges in Distributed DICOM. International Journal of Advanced Research in Computer and Communication Engi- neering, 5 (3), 741–747. [11] International Ethnobotany Database. Available at: https://olorien.org/ebDB [12] Prelude Medicinal Plants Database. Available at: https://www.africamuseum.be/research/col- lections_libraries/biology/prelude [13] FRLHT Encyclopedia of Indian Medicinal Plants. Available at: http://www.frlht.org/newenvis/ indian-medicinalplants-database.php [14] TradiMed. References Available at: http://www.tradimed.com/ [15] Dr Duke’s Phytochemical and Ethnobotanical Databases Available at: https://phytochem nal 14] TradiMed. Available at: http://www.tradimed.com/ [14] TradiMed. Available at: http://www.tradimed.com/ [15] Dr. Duke’s Phytochemical and Ethnobotanical Databases. Available at: https://phytochem.nal. usda.gov/phytochem/search [15] Dr. Duke’s Phytochemical and Ethnobotanical Databases. Available at: https://phytochem.nal. usda.gov/phytochem/search [16] A Modern Herbal Database. Available at: http://www.botanical.com/botanical/mgmh/mgmh.html [17] NAPRALERT. Available at: https://napralert.org/ [17] NAPRALERT. Available at: https://napralert.org/ [18] Native American Ethnobotany. Available at: http://naeb.brit.org/ [18] Native American Ethnobotany. Available at: http://naeb.brit.org/ [19] Mehtiyeva, N. (2013). Importance database of medicinal plants for plant biodiversity conserva- tion in Azerbaijan. International conference, Environmental changes conservation of plant diversity. 24 24
https://openalex.org/W3108253228	https://www.emerald.com/insight/content/doi/10.1108/IJOPM-09-2020-832/full/pdf?title=operations-adding-value-to-society	English	null	Operations adding value to society	International journal of operations & production management	2,020	cc-by	5,141	Operations adding value to society Operations adding value to society Markku Kuula Department of Information and Service Management, School of Business, Aalto University, Espoo, Finland Stefan Seuring Department of Supply Chain Management, University of Kassel, Kassel, German, and Constantin Blome Revised 4 November 2020 23 November 2020 Accepted 23 November 2020 Stefan Seuring Department of Supply Chain Management, University of Kassel, Kassel, German, and Constantin Blome Revised 4 November 2020 23 November 2020 Accepted 23 November 2020 School of Business, Management and Economics, University of Sussex, Falmer, UK Operations adding value to society Gy€ongyi Kovacs Humanitarian Logistics and Supply Chain Research Institute (HUMLOG Institute), Hanken School of Economics, Helsinki, Finland Markku Kuula Department of Information and Service Management, School of Business, Aalto University, Espoo, Finland Stefan Seuring Department of Supply Chain Management, University of Kassel, Kassel, German, and Constantin Blome School of Business Management and Economics University of Sussex Falmer UK Operations adding value to society 1293 Received 29 October 2020 Revised 4 November 2020 23 November 2020 Accepted 23 November 2020 The current issue and full text archive of this journal is available on Emerald Insight at: https://www.emerald.com/insight/0144-3577.htm Operations adding value to society Gy€ongyi Kovacs Humanitarian Logistics and Supply Chain Research Institute (HUMLOG Institute), Hanken School of Economics, Helsinki, Finland Markku Kuula Department of Information and Service Management, School of Business, Aalto University, Espoo, Finland Stefan Seuring Department of Supply Chain Management, University of Kassel, Kassel, German, and Constantin Blome School of Business Management and Economics University of Sussex Falmer UK Operations adding value to society 1293 Received 29 October 2020 Revised 4 November 2020 23 November 2020 Accepted 23 November 2020 The current issue and full text archive of this journal is available on Emerald Insight at: https://www.emerald.com/insight/0144-3577.htm The current issue and full text archive of this journal is available on Emerald Insight at: https://www.emerald.com/insight/0144-3577.htm © Gy€ongyi Kovacs, Markku Kuula, Stefan Seuring and Constantin Blome. Published by Emerald Publishing Limited. This article is published under the Creative Commons Attribution (CC BY 4.0) licence. Anyone may reproduce, distribute, translate and create derivative works of this article (for both commercial and non-commercial purposes), subject to full attribution to the original publication and authors. The full terms of this licence may be seen at http://creativecommons.org/licences/by/4.0/legalcode Publishers note: The publisher would like to inform readers that the editorial article, ‘Kovacs, G., Kuula, M., Seuring, S. and Blome, C. (2020), “Guest editorial”, International Journal of Operations & Production Management, Vol. 40 No. 9, pp. 1293-1299. https://doi.org/10.1108/IJOPM-09-2020-832’ was mistakenly published as non-article content. This error was introduced as part of the production process, and the publisher sincerely apologizes for this error.The article should be cited as Kovacs, G., Kuula, M., Seuring,S. and Blome, C. (2020), “Operations Adding Value to Society”, International Journal of Operations & Production Management, Vol. 40 No. 9, pp. 1293-1300. https://doi.org/10.1108/IJOPM-09-2020-832. Abstract Purpose – The purpose of this article is to discuss the role of operations management in society. The article detects trends, raises critical questions to operations management research and articulates a research agenda to increase the value of such research in addressing societal problems. Design/methodology/approach – This paper evaluates the papers presented at the EurOMA 2019 conference to detect trends and discuss the contributions of operations management research to society. It further goes to identify gaps in the research agenda. Findings – The article finds several important streams of research in operations management: sustainable operations and supply chains, health care and humanitarian operations, innovation, digitalisation and 4.0, risk and resilience. It highlights new trends such as circular economy research and problematises when to stop implementing innovation and how to address and report their potential failure. Importantly, it shows how it is not just a question of offshoring vs reshoring but of constant change in manufacturing that operations management addresses. Originality/value – The article highlights not just novel research areas but also gaps in the research agenda where operations management seeks to add value to society. Paper type Research paper Paper type Research paper “Operations Adding Value to Society” was the theme of the 26th conference of the European Operations Management Association (EurOMA). The idea behind the theme was to extend previous conference themes beyond manufacturing and services to wider societal and community implications and impact. The conference theme highlighted a move beyond goods manufacturing to considerations of the shared economy, the circular economy, and also, to critical infrastructure and supply chains. EurOMA has had a tradition of tracks in © Gy€ongyi Kovacs, Markku Kuula, Stefan Seuring and Constantin Blome. Published by Emerald Publishing Limited. This article is published under the Creative Commons Attribution (CC BY 4.0) licence. Anyone may reproduce, distribute, translate and create derivative works of this article (for both commercial and non-commercial purposes), subject to full attribution to the original publication and authors. The full terms of this licence may be seen at http://creativecommons.org/licences/by/4.0/legalcode Publishers note: The publisher would like to inform readers that the editorial article, ‘Kovacs, G., Kuula, M., Seuring, S. and Blome, C. (2020), “Guest editorial”, International Journal of Operations & Production Management, Vol. 40 No. 9, pp. 1293-1299. https://doi.org/10.1108/IJOPM-09-2020-832’ was mistakenly published as non-article content. International Journal of Operations & Production Management Vol. 40 No. 9, 2020 pp. 1293-1300 Emerald Publishing Limited 0144-3577 DOI 10.1108/IJOPM-09-2020-832 Abstract This error was introduced as part of the production process, and the publisher sincerely apologizes for this error.The article should be cited as Kovacs, G., Kuula, M., Seuring,S. and Blome, C. (2020), “Operations Adding Value to Society”, International Journal of Operations & Production Management, Vol. 40 No. 9, pp. 1293-1300. https://doi.org/10.1108/IJOPM-09-2020-832. International Journal of Operations & Production Management Vol. 40 No. 9, 2020 pp. 1293-1300 Emerald Publishing Limited 0144-3577 DOI 10.1108/IJOPM-09-2020-832 various industries, and this theme specifically welcomed also health care operations, humanitarian operations and public sector management. Notwithstanding the more outspoken humanitarian operations topic, who would have thought this to be one of the last face-to-face events of the association for such a while? While it was wonderful to see the full auditorium that even had to be streamed to another room for people to follow, our congratulations go to the conference organisers in 2020 who managed such a lively online conference during the current pandemic! 1294 The 26th conference of the EurOMA was jointly hosted by the Aalto University Business School, Hanken School of Economics and the HUMLOG Institute, in Helsinki, Finland, 15–20 June 2019. “Operations adding value to society” strongly relates to the research at our universities, but also to the mandate of universities, and of research, to impact on society. This was the more emphasised by the rector of the Hanken School of Economics, Karen Spens, herself a professor in supply chain management, who gave the opening keynote at the conference on how to use operations management principles when leading a university. After all, service operations, but also total quality management, come together in the continuous improvement efforts of universities as well. The dissemination of knowledge at conferences, the outreach to society and the interaction with society are key to operations management. As for us organisers, we considered organising EurOMA as a way to pay back our debts to the EurOMA community and taking a more active role. We are still incredibly proud of all the reviewers of the conference who supported us in getting the review process done ahead of time! Operations management seeks to have an impact on business and is crucial to the economic bottom line of companies but also plays an important role in making operations sustainable, in relating to the environment and to society. The very impact of research is something the next keynote, Prof. Informing the operations management research agenda The conference theme of “Operations Adding Value to Society” was well represented in the tracks with most submissions, which were the sustainability track (50 papers), SCM (46 papers), health care operations (33), innovation, product and service development (21), and risk and resilience (19). Interestingly, sustainability papers even outnumbered the traditionally strong supply chain management track. A somewhat different picture emerges if analysing paper titles for keywords: according to this, 25 papers focussed on digitalisation, with another 16 on industry 4.0, compared to 14 papers on various food-related operations, 12 on blockchain, 8 on big data as well as 8 on the circular economy. Digitalisation and technology were also embraced by the teaching operations track where one could for example visit a French warehouse virtually from the conference in Finland. This is arguably even more important now in the age of distance education. Let us analyse the track as well as prominent cross-cutting keywords for some highlights for the current and future operations management research agenda. Abstract Janet Godsell, underscored when talking about engaged scholarship and the “time for a rethink” in operations and supply chain management through storytelling, videos, art and social media. Societal impact was even at the heart of the reception at the Helsinki city, where the deputy mayor emphasises the importance of operations to the city’s mantra of making Helsinki the “most functional” city. Yet another angle to it came through the practitioner keynote, with Kalle L€o€ovi, the head of international operations of the Finnish Red Cross, sharing his stories and perspective on humanitarian operations, according to which, occasionally, Finnish midsummer can cause supply chain disruptions in Yemen. But EurOMA 2019 was not just about talking about societal impact but about making one. Minimising the ecological footprint, there were no prints nor conference bags but an app, no buses but walking around the city, no bottled water but vegetarian and vegan meals with flowers on dessert. New rules were applied to all panels and special sessions, which required people to come together from various countries, and have representatives of both genders. Some of the new sessions truly embraced the conference theme, with there being one on increasing the relevance and reach of O&SCM research; and another hands-on workshop on the bottom-up nurturing of an inclusive operations management community. It is high time for our discipline to embrace scholars of diverse backgrounds, in the veins of Black Lives Matter. Being in Europe, EurOMA itself has made quite an effort to include scholars from the “Danube” section, building bridges rather than walls. Where else is that better placed than in Helsinki, the neutral city where East meets West? The story does not end there. The programme finished with industry visits to the Science Centre Heureka, and the new cargo terminal of Finnair Cargo. At the conference, the outgoing EurOMA president Giovanni Perrone also passed the baton the incoming president Taco van der Vaart. Besides embracing Finnish oddities from bird karaoke to playing air guitar at the conference dinner, let us have a more serious look at the implications of the 26th EurOMA conference for the operations management research agenda. Operations adding value to society Operations adding value to society implications of the 26th EurOMA conference for the operations management research agenda. Sustainable operations and supply chains Two streams of sustainable operations research were particularly prominently represented at the conference: (1) the sustainability of agri-food supply chains and (2) circular economy research. The interest in agri-food supply chains spans both a variety of types of food (especially cocoa, coffee, but also whitefish) but also the entire supply chain. Interestingly, it was not only the sustainable performance of these supply chains and especially of food retail that was in the limelight, but more in detail, questions of fairness in retailer-led greening, or incentive alignment for collaborative carbon reduction. Research has also moved on to more concrete questions such as shelf-life-based inventory management, household food waste flows and how to enhance the last mile in grocery e-commerce. Overall, it can be stated that sustainability questions in this industry have gone beyond mere mission statements and have started to be addressed more seriously also empirically, and in detail. This is no surprise, as food and food supply chains affect every single person on the planet. The aspect of value to society starts to emerge by including performance measures that also capture environmental and social impacts. This gives hope for the potential impact of this research as well, where the impacts such supply chain have in emerging economies and at the base-of-the-pyramid still offers many research opportunities y pp The same applies to circular economy research. Conceptual papers notwithstanding, the circular economy has evolved from being a buzzword to research on its implementation. Conference papers reported on specific applications, from manufacturing in Norway, to the offshore wind energy sector. Beyond the use of the circular economy concept as yet another way to describe reverse logistics and closed loop supply chains, by now, research has moved on to embracing its novel aspects of the sharing economy (from truck sharing and bike sharing to new product-service systems), upcycling and product development overall, and the new business models the concept gives rise to. Even more to the point were the specifics of loop flows and networks in the circular economy, the purchasing of remanufactured materials, and the mitigation of intertemporal tensions in the circular economy. Relating again to the topic of “Operations adding Value to Society” the circular supply chains have to take all aspects of value into account, thereby moving beyond narrowly defined operational measures. Sustainable operations and supply chains Health care and humanitarian operations Following a long tradition, lean health care and health supply chain integration were rather prominent at the conference. Health care operations are often characterised by their complexity, and by multiple organisations being involved in the care of a single patient. Thus, there is a general strong focus on patient flows, care pathways, as well as process improvements across these myriads of organisations, and the modules of care they provide. Digitalisation in health, and the operational adoption and scalability of medical technology have also become rather classic topics by now. This was interspersed with a few new topics, from applying sharing economy principles to health care, to mapping the hospital supply chain of medical consumables, and even calculating the CO2 emissions of mobile health care units. Given the current global pandemic, such research is particularly relevant and impactful. Not understanding the operation and supply chain logic puts many lives at risk. Health care and humanitarian operations 1296 The humanitarian operations section of the conference on the other hand is still less established; which shows in the abundance of conceptual rather than empirical papers. The focus drifts from the big picture looking at hunger, gender (in)equality, GDP and the environment; to climate science in humanitarian supply chains. The section has started to embrace more theoretical approaches, such as the social capital lens on humanitarian supply chains. These topics certainly set the agenda for research to come; but the devil is in the detail and the data. Few papers at the conference stand out with empirical studies, though the relevance and impact of research also in this sector will come with that. Hence, collecting data in such settings, often taking place in emerging and developing countries are encouraged. Innovation, digitalisation and Industry 4.0 As innovation literature goes, we cannot be but fascinated with novel fashion styles and trends, and novel concepts, of course. It was about time to investigate the value of social media to the supply chain, or how to split team rewards in supply chain innovation. One would expect the innovation section of the conference to be the most innovative, of course. That may be the case conceptually, but the overwhelming majority of systematic literature reviews rather highlight the still outstanding gaps and rarely go beyond mission statements. Blockchain as well as drones are still searching for their potential application. As one paper title amply puts it, if blockchain is the answer, what is the question? It is not much a surprise, that again a lack of empirical data can be observed. Here, we hope that companies implementingsuch solutionswouldbenefitfromtheresearch athand, butalsobe willing toallow us insights into their operations, so to learn and abstract from single cases. Given the huge potential and impact of digital technologies on many people’s lives, this could be of great societal impact. Digitalisation is perhaps too broad a term for the variety of operations management research that it covers. Papers referring to digitalisation investigated the traceability of pharmaceuticals; the performance of manufacturing networks, the use of 3D models in the workplace or the supply chain of satellite imagery. What stays quite open seems to be the link to some of the already mentioned tracks, such as the impact digitalisation has on sustainability, on health care operations or on humanitarian supply chains, but also into risk and resilience issue addressed in the next section. This seems to be a field warranting future research. Other earlier buzzwords have come a long way. Additive manufacturing has now been studied in its application. Industry 4.0, often combined with leanness, has been evaluated in various countries and industries. There are case studies from Central and Eastern Europe, Operations adding value to society impact measurements on buyer–supplier relationships and even board games to teach Industry 4.0 to enable the next industrial revolution. We cannot wait to see these results published in good journals. impact measurements on buyer–supplier relationships and even board games to teach Industry 4.0 to enable the next industrial revolution. We cannot wait to see these results published in good journals. Operations adding value to society 1297 1297 Risk and resilience Risk and resilience Studies in risk and resilience tackle operational glitches to larger risks. From an operational perspective, studies were presented on the risk and resilience in export, contingencies in emerging economies, risks in food and vegetable transportation, food loss and waste, to the procedural integration of spare parts in material flows. Different approaches are also highlighted in managing these, from predictive analytics, to buffering vs bridging, to the use of relational capital in the supply chain. p pp y Some larger risks were either predictable or have already occurred, with studies addressing their aftermath. These include the imminence of Brexit, or the evolution of the Egyptian tourism industry after the Arab spring. The bigger picture further extends to resilience with regards to natural disasters, environmental risk management, food safety risks and more novel to this year, the focus on modern slavery in the supply chain. Others question the tensions between sustainability and resilience, or address the very risks of Industry 4.0 applications. An interesting common theme could be observed in the focus on changes in operations, whether form public to private, or from own to outsourced. An increasing number of papers were focussing on reshoring rather than offshoring from a risk management perspective. This is to stress that reshoring has been on the (research) agenda for a while and is not in fact just a consequence of the current pandemic. Perhaps most interesting is the approach from one of the papers focussing on how to manage the continuous migration of manufacturing locations in the supply chain. The actual change of out- vs insourcing, off- vs reshoring may be different over time, what is constant is change itself and that is worth focussing on in operations management research. Innovation, digitalisation and Industry 4.0 With the maturing of innovation, one of the more interesting questions becomes when to stop the implementation of an innovation, and how to evaluate supplier development initiatives that have failed. This is a research area that would deserve more interest. Finally, the selected few – the papers in this special issue Finally, the selected few the papers in this special issue A total of 561 presenters came from 44 countries to the conference, with presentations of 409 papers taking place in 20 parallel tracks. It is always difficult to summarise a conference in a few words or to select the creme de la creme of papers for a special issue. Luckily there are the award nominations that guide the selection, apart from journal editors attending the conference and spotting interesting papers and results. In 2019, 231 papers wanted to be considered for the Chris Voss Award, 79 of which had received the highest ratings by both reviewers. A further 170 papers wanted to be considered for the Harry Boer Award, with 61 receiving highest rates and 15 for the Nigel Slack Award, with five receiving highest ratings. The tremendous work of the award committees in their selections of further shortlists and winners was then combined with further readings of the proceedings to shortlist 35 papers, of which 15 were finally selected for further review for the special issue. It is of course based on this review that we can now present the final few that have also survived the rigour of the review process. At the end, they now give quite an interesting snapshot of both established and novel topics at the pulse of time. Silvestre et al.’s (2020) article on “Supply chain sustainability trajectories: learning through sustainability initiatives” studies five supply chains from quite different industries, and the evolution of their sustainability considerations over time. Based on contingency theory, they develop a framework that categorises these trajectories in terms of path dependence between sustainability orientation and the implementation of sustainability initiatives and the capabilities of supply chain members to embrace and learn from these. The framework helps in singling out the triggers that have led these supply chains to embark on their sustainability trajectory, as well as the supply chain partnerships that were required for their implementation. Rather than merely categorising the supply chains in the end, the framework is also used to illustrate different elements of these trajectories within these supply chains and contrast differing viewpoints in the supply chain that may distort the outcomes of sustainability initiatives. 1298 Fracarolli Nunes, Lee Park and Paiva (2020) ask the important question of “Can we have it all?” when examining “Sustainability trade-offs and cross-insurance mechanisms in supply chains”. Finally, the selected few – the papers in this special issue The focus on insurance and cross-insurance mechanisms in corporate social responsibility is certainly novel and adds to the estimation of corporate credibility and reputation. Through their study in the food industry, they show how sustainability trade-offs impact on different supply chain members differently. This further leads to hierarchies amongst sustainability objectives in the supply chain as a move away from the triple bottom line. It seems we cannot have our cake and eat it, too. Liu et al. (2020) posit a different question in “When do 3PLs initiate low-carbon supply chain integration?”. They discover a paradox of the social network in this regard, where 3PLs that were more embedded in their network, and more integrated with their customers, had less room to lead and implement their own decarbonisation initiatives. On the other hand, low carbon supply chain integration has a positive effect on the 3PL’s firm performance, but also, 3PLs have a critical role to play as the orchestrators of such integration regardless of their degree of embeddedness in the network. Peters et al. (2020) present the findings from the Chris Voss award-winning paper when “Elaborating on modular interfaces in multi-provider contexts” in health care operations. They look at the various closed vs open interfaces between health care providers and customers and across health care providers and investigate cases where the delivery of integrated patient care was jeopardised. Service modularity is used to understand the types of interfaces in multi-provider contexts. Stefanini et al. (2020) embraced Finnish silence in their article “Silence is golden: The role of team coordination in health operations”. They report the findings from a study of routine surgeries where health care professionals wear devices monitoring every of their moves The study focusses on implicit, non-verbal, vs explicit, verbal communication and finds that the lower the use of explicit communication, the higher a surgical team performs. That said, implicit communication occurs where team members can anticipate the behaviour of one another. Thus, team cohesion and soft skills decrease the risk for glitches. The study by Schiffling et al. (2020) on the “Coopetition in temporary contexts: examining swift trust and swift distrust in humanitarian operations” was one of the few empirical studies in humanitarian operations at the conference. Operations adding value to society Cotta, D. and Salvador, F. (2020), “Exploring the antecedents of organizational resilience practices – a transactive memory systems approach”, International Journal of Operations and Production Management, Vol. 40 No. 9, pp. 1531-1559. Famous last words Combined, the conference papers and their topics are a good reflection of the current research interests of the operations management community. Albeit conference abstract reviews are not quite the same as is then the peer review process of a journal – also the one for this special issue – a conference still provides a quick glimpse of what is current, topical and which insights are to come, quite a long time ahead of the articles being published. Attending scientific conferences such as EurOMA will continue to be of importance, whether we need to continue doing so virtually or can do so in person. At the end, when it comes to the final papers selected for this special issue, they embrace International Journal of Operations & Production Management’s new mantra of being bold or italic in operations management research – but to stand out in quality, rigour and relevance. The latter is the more important for leaving our mark in society. Operations management may sound mechanic and technical at times, but at the end of the day, it is what makes our world work, albeit from the backstage. Let us end with a quote from Kalle L€o€ovi’s keynote at EurOMA 2019: “Supply chains are solidarity chains”. Now more than ever. Fabbe-Costes, N., Lechaptois, L. and Spring, M. (2020), “The map is not the territory’: a boundary objects perspective on supply chain mapping”, International Journal of Operations and Production Management, Vol. 40 No. 9, pp. 1475-1497. 1299 Cotta and Salvador (2020) on the other hand, focus on more operational aspects of organisational resilience in their article “Exploring the antecedents of organizational resilience practices – a transactive memory systems approach”. Apart from investigating the antecedents of resilience practices, they further combine these with the individual characteristics of managers (heads of manufacturing) to understand their decisions when faced with operational disruptions. In the end, transactive memory systems are a combination of individual and firm capabilities. p Sousa and da Silveira’s (2020) “Advanced services and differentiation advantage: An empirical investigation” investigate a rather classical operations management topic. The servitisation of manufacturing industries supposedly brings great potential for market differentiation; which is corroborated here as well. Interestingly, servitisation itself is influenced by market complexity, however; indicating that it is more of a strategic move rather than a reaction to the market. On a further important note, the article also addresses the shortcomings of the IMSS data it uses as a basis of the study, which are important to bear in mind when using it. Finally, the selected few – the papers in this special issue In this study, they expand on a previous study on swift trust by including also swift distrust in their scope and find evidence for the development of both in situations with a limited set of co-operation partners. The same operations are characterised by high uncertainty and interdependence, which is why it is the more interesting that swift distrust can in fact aid in managing uncertainty, enable co-opetitive relationships and contribute to risk management. Fabbe-Costes et al. (2020) put supply chain mapping back on the agenda in their article on “‘The map is not the territory’: a boundary objects perspective on supply chain mapping”. There are so many different approaches to supply chain mapping that even in the same company, no two maps look alike. Boundary objects are used to overcome differences and align the understanding of a map and its purpose, in order to be able to increase its usefulness. Operations adding value to society The study by Roscoe et al. (2020) puts the spotlight on the imminent event that was most discussed in the corridors of the conference: Brexit. “Managing supply chain uncertainty arising from geopolitical disruptions: Evidence from the pharmaceutical industry and Brexit” follows a particular industry in its journey to manage the already foreseeable supply chain disruptions. The study contrasts the approaches and possibilities of MNEs and SMEs in this regard, finding, perhaps less surprisingly, that approaches vary across company size, with larger companies having more resources at their availability to cope with a disruption. Apart from the use of tangible resources such as additional inventory, capacity and assets; the study highlights the importance of intangible ones such as the investment of management time and knowledge acquisition to reduce uncertainties related to the disruption. 1299 References Cotta, D. and Salvador, F. (2020), “Exploring the antecedents of organizational resilience practices – a transactive memory systems approach”, International Journal of Operations and Production Management, Vol. 40 No. 9, pp. 1531-1559. Fabbe-Costes, N., Lechaptois, L. and Spring, M. (2020), “The map is not the territory’: a boundary objects perspective on supply chain mapping”, International Journal of Operations and Production Management, Vol. 40 No. 9, pp. 1475-1497. Fracarolli Nunes, M., Lee Park, C. and Paiva, E.L. (2020), “Can we have it all? Sustainability trade-offs and cross-insurance mechanisms in supply chains”, International Journal of Operations and Production Management, Vol. 40 No. 9, pp. 1339-1366. Liu, X., Xian, C. and Wang, S. (2020), “When do 3PLs initiate low-carbon supply chain integration?”, International Journal of Operations and Production Management, Vol. 40 No. 9, pp. 1367-1395. Peters, V., V€ah€atalo, M., Meijboom, B., Barendregt, A., Bok, L. and de Vries, E. (2020), “Elaborating on modular interfaces in multi-provider contexts”, International Journal of Operations and Production Management, Vol. 40 No. 9, pp. 1397-1419. 1300 Roscoe, S., Skipworth, H., Aktas, E. and Habib, F. (2020), “Managing supply chain uncertainty arising from geopolitical disruptions: evidence from the pharmaceutical industry and Brexit”, International Journal of Operations and Production Management, Vol. 40 No. 9, pp. 1499-1529. Schiffling, S., Hannibal, C., Fan, Y. and Tickle, M. (2020), “Coopetition in temporary contexts: examining swift trust and swift distrust in humanitarian operations”, International Journal of Operations and Production Management, Vol. 40 No. 9, pp. 1449-1473. Silvestre, B., Silva, M.E., Cormack, A. and Tavares Thome, A.M. (2020), “Supply chain sustainability trajectories: learning through sustainability initiatives”, International Journal of Operations and Production Management, Vol. 40 No. 9, pp. 1301-1337. Sousa, R. and da Silveira, G. (2020), “Advanced services and differentiation advantage: an empirical investigation”, International Journal of Operations and Production Management, Vol. 40 No. 9, pp. 1561-1587. Stefanini, A., Aloini, D. and Gloor, P. (2020), “Silence is golden: the role of team coordination in health operations”, International Journal of Operations and Production Management, Vol. 40 No. 9, pp. 1421-1447. Corresponding author G i b Gy€ongyi Kovacs can be contacted at: kovacs@hanken.fi For instructions on how to order reprints of this article, please visit our w www.emeraldgrouppublishing.com/licensing/reprints.htm Or contact us for further details: permissions@emeraldinsight.com
https://openalex.org/W3131547510	https://publications.goettingen-research-online.de/bitstream/2/84914/1/1-s2.0-S1535947621000347-main.pdf	English	null	Protein Phosphorylation in Depolarized Synaptosomes: Dissecting Primary Effects of Calcium from Synaptic Vesicle Cycling	Molecular & cellular proteomics	2,021	cc-by	21,567	In Brief Analysis of protein phosphorylation in isolated nerve terminals (synaptosomes) treated with C. botulinum neurotoxins (BoNT) to inhibit synaptic vesicle (SV) cycling reveals phosphorylation events that are primarily dependent on depolarization-induced Ca2+ inﬂux and those that also require active SV-cycling machinery. In particular, SV-cycling-dependent phosphorylation sites on synaptobrevin (Vamp2), syntaxin-1 (Stx1a), and cannabinoid receptor-1 (Cnr1) are capable of changing the rate of exo- and endocytosis in cultured hippocampal neurons. Depolarized Synaptosomes SV-cycling-dependent Phosphorylation Sites Primary Ca2+-dependent Phosphorylation Sites LC-MS/MS Protein Phosphorylation in Depolarized Synaptosomes: Dissecting Primary Effects of Calcium from Synaptic Vesicle Cycling Authors Ivan Silbern, Kuan-Ting Pan, Maksims Fiosins, Stefan Bonn, Silvio O. Rizzoli, Eugenio F. Fornasiero, Henning Urlaub, and Reinhard Jahn Graphical Abstract Correspondence efornas@gwdg.de; henning. urlaub@mpibpc.mpg.de; rjahn@gwdg.de Ca2+ K+ Ca2+ K+ BoNT TiO2 P P P PP SV-cycling-dependent Phosphorylation Sites Primary Ca2+-dependent Phosphorylation Sites LC-MS/MS Depolarized Synaptosomes rjahn@gwdg.de In Brief Analysis of protein phosphorylation in isolated nerve terminals (synaptosomes) treated with C. botulinum neurotoxins (BoNT) to inhibit synaptic vesicle (SV) cycling reveals phosphorylation events that are primarily dependent on depolarization-induced Ca2+ inﬂux and those that also require active SV-cycling machinery. In particular, SV-cycling-dependent phosphorylation sites on synaptobrevin (Vamp2), syntaxin-1 (Stx1a), and cannabinoid receptor-1 (Cnr1) are capable of changing the rate of exo- and endocytosis in cultured hippocampal neurons. Ca2+ K+ Ca2+ K+ BoNT TiO2 P P P PP SV-cycling-dependent Phosphorylation Sites Primary Ca2+-dependent Phosphorylation Sites LC-MS/MS Depolarized Synaptosomes Ca2+ K+ Ca2+ K+ BoNT TiO2 P P P PP SV-cycling-dependent Phosphorylation Sites Primary Ca2+-dependent Phosphorylation Sites LC-MS/MS Depolarized Synaptosomes RESEARCH RESEARCH 2021, Mol Cell Proteomics 20, 100061 © 2021 THE AUTHORS. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology. This is an open access article under the CC BY license (http://creativecommons.org/ licenses/by/4.0/). https://doi.org/10.1016/j.mcpro.2021.100061 Ivan Silbern1,2 , Kuan-Ting Pan2 , Maksims Fiosins3,4, Stefan Bonn3,4 , Silvio O. Rizzoli5,6 , Eugenio F. Fornasiero5,* , Henning Urlaub1,2,* , and Reinhard Jahn7,* Ivan Silbern1,2 , Kuan-Ting Pan2 , Maksims Fiosins3,4, Stefan Bonn Silvio O. Rizzoli5,6 , Eugenio F. Fornasiero5,* , Henning Urlaub1,2,* , a Synaptic transmission is mediated by the regulated exocytosis of synaptic vesicles. When the presynaptic membrane is depolarized by an incoming action potential, voltage-gated calcium channels open, resulting in the inﬂux of calcium ions that triggers the fusion of synaptic vesicles (SVs) with the plasma membrane. SVs are recy- cled by endocytosis. Phosphorylation of synaptic proteins plays a major role in these processes, and several studies have shown that the synaptic phosphoproteome changes rapidly in response to depolarization. However, it is un- clear which of these changes are directly linked to SV cycling and which might regulate other presynaptic func- tions that are also controlled by calcium-dependent ki- nases and phosphatases. To address this question, we analyzed changes in the phosphoproteome using rat synaptosomes in which exocytosis was blocked with botulinum neurotoxins (BoNTs) while depolarization- induced calcium inﬂux remained unchanged. BoNT- treatment signiﬁcantly alters the response of the synap- tic phoshoproteome to depolarization and results in reduced phosphorylation levels when compared with stimulation of synaptosomes by depolarization with KCl alone. We dissect the primary Ca2+-dependent phos- phorylation from SV-cycling-dependent phosphorylation and conﬁrm an effect of such SV-cycling-dependent phosphorylation events on syntaxin-1a-T21/T23, synapto- brevin-S75, and cannabinoid receptor-1-S314/T322 on exo- and endocytosis in cultured hippocampal neurons. the presynaptic compartment (1). When an action potential arrives in the nerve terminal, the presynaptic membrane de- polarizes, which elicits a rapid inﬂux of calcium ions through voltage-dependent calcium channels clustered around the active zone, an electron-dense protein complex connecting the presynaptic release site with the neurotransmitter re- ceptors in the postsynaptic membrane (2). Major protein constituents of the active zone include RIM proteins, RIM- binding protein, and Munc13, as well as bassoon, piccolo, and liprins. Among other functions, these proteins are required for the clustering of calcium channels and govern the priming and docking of SVs. The calcium ions entering the presynapse are recognized by the calcium-sensor synaptotagmin 1 that triggers exocytosis by lowering the energy barrier for fusion. Exocytotic fusion itself is accomplished by soluble N-ethyl- maleimide-sensitive factor-attachment protein receptors (SNARE) (3–6). This process requires additional cytoplasmic proteins, such as Munc18 and complexins, which modulate the efﬁciency of SNARE zippering (7, 8). Highlights • KCl-depolarization induces phosphoproteome changes in isolated nerve terminals (synaptosomes). • KCl-depolarization induces phosphoproteome changes in isolated nerve term • KCl-depolarization induces phosphoproteome changes in isolated nerve terminals (synaptosomes). • Botulinum neurotoxin affects protein phosphorylation in depolarized synaptosomes. • Botulinum neurotoxin affects protein phosphorylation in depolarized synaptosomes. ulinum neurotoxin affects protein phosphorylation in depolarized synaptosomes. • BoNT treatment reveals phosphorylation sites that depend on SV-cycling activity. • BoNT treatment reveals phosphorylation sites that depend on SV-cycling • BoNT treatment reveals phosphorylation sites that depend on SV-cycling activity. • SV-cycling-dependent sites on Vamp2, Stx1a, and Cnr1 affect exo- and endocytosis. • SV-cycling-dependent sites on Vamp2, Stx1a, and Cnr1 affect exo- and endocytosis. • SV-cycling-dependent sites on Vamp2, Stx1a, and Cnr1 affect exo- and endocytosis. RESEARCH From the 1Institute of Clinical Chemistry, University Medical Center Goettingen, Goettingen, Germany; 2Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, Goettingen, Germany; 3German Center for Neurodegenerative Diseases, Tübingen, Germany; 4Institute for Medical Systems Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; 5Department of Neuro- and Sensory Physiology, University Medical Center Göttingen, Göttingen, Germany; 6Cluster of Excellence “Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells” (MBExC), University of Goettingen, Göttingen, Germany; and 7Laboratory of Neurobiology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany For correspondence: Eugenio F Fornasiero efornas@gwdg de; Henning Urlaub henning urlaub@mpibpc mpg de; Reinhard Jahn Ivan Silbern1,2 , Kuan-Ting Pan2 , Maksims Fiosins3,4, Stefan Bonn3,4 , Silvio O. Rizzoli5,6 , Eugenio F. Fornasiero5, , Henning Urlaub1,2,* , and Reinhard Jahn7,* In order to sustain continuous neurotransmitter release and to maintain the structure and composition of the plasma membrane, fused SVs are retrieved by endocytosis, followed by rapid regeneration of SV precursors that are ﬁlled with neurotransmitters and prepared for the next round of neuro- transmitter release. It is still debated which mechanisms are primarily responsible for endocytosis and recycling, with several occurring in parallel (9–16). The classical pathway of clathrin-mediated endocytosis requires coordinated actions of a protein machinery responsible for coat formation, invagina- tion, and formation of a coated pit, followed by scission, removal of the vesicle from the plasma membrane, and In the nervous system, synapses represent specialized cellular junctions that are able to transmit signals from the presynaptic to the postsynaptic neuron. Signaling is mediated by the release of neurotransmitter molecules that are stored in j g g Present address for Kuan-Ting Pan: Frankfurt Cancer Institute, Goethe University, D-60596, Frankfurt am Main, Germany; Hematology/ Oncology, Department of Medicine II, Johann Wolfgang Goethe University, D-60590 Frankfurt am Main, Germany. Mol Cell Proteomics (2021) 20 100061 1 © 2021 THE AUTHORS. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1016/j.mcpro.2021.100061 From the 1Institute of Clinical Chemistry, University Medical Center Goettingen, Goettingen, Germany; 2Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, Goettingen, Germany; 3German Center for Neurodegenerative Diseases, Tübingen, Germany; 4Institute for Medical Systems Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; 5Department of Neuro- and Sensory Physiology, University Medical Center Göttingen, Göttingen, Germany; 6Cluster of Excellence “Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells” (MBExC), University of Goettingen, Göttingen, Germany; and 7Laboratory of Neurobiology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany For correspondence: Eugenio F. Fornasiero, efornas@gwdg.de; Henning Urlaub, henning.urlaub@mpibpc.mpg.de; Reinhard Jahn, rjahn@gwdg.de. Present address for Kuan-Ting Pan: Frankfurt Cancer Institute, Goethe University, D-60596, Frankfurt am Main, Germany; Hematology/ Oncology, Department of Medicine II, Johann Wolfgang Goethe University, D-60590 Frankfurt am Main, Germany. j g g Present address for Kuan-Ting Pan: Frankfurt Cancer Institute, Goethe University, D-60596, Frankfurt am Main, Germany; Hematology/ Oncology, Department of Medicine II, Johann Wolfgang Goethe University, D-60590 Frankfurt am Main, Germany. We stimulated mock- and BoNT-treated synapto- somes with potassium chloride, which is known to elicit dose- dependent Ca2+ inﬂux and neurotransmitter release (56, 57). By quantitatively monitoring changes of phosphorylation sites of synaptosomal proteins, we found that BoNT treatment af- fects almost 1500 phosphorylation sites implying that they are directly dependent on SV cycling. Strikingly, most of the sites show reduced phosphorylation intensity in depolarized syn- aptosomes following BoNT treatment. We further identiﬁed SV-cycling-dependent phosphorylation sites on syntaxin-1a (Stx1a-T21, T23), synaptobrevin (Vamp2-S75), and cannabi- noid receptor-1 (Cnr1-T314, T322). Finally, we demonstrated that phosphorylation of these sites elicits a pronounced effect on exo- and endocytosis in cultured hippocampal neurons by expressing phosphomimetic and nonphosphorylatable vari- ants of these proteins. An increasing body of data suggests that the basic exo– endocytotic machinery is ﬁne-tuned by regulatory control mechanisms that mainly operate by phosphorylations and dephosphorylations of proteins involved in the SV cycle. In particular, calcium plays an important role not only as a trigger of exocytosis, but also as a modulator of kinase and phosphatase activity in the presynapse (19). Calcium/ calmodulin-dependent kinase 2 (CaMKII) residing in synap- ses is one of the major targets activated by calcium inﬂux (20–23). Its ﬁrst known substrate was the SV resident protein synapsin-1, and it is now established that its phosphoryla- tion inﬂuences the available pool of SVs for exocytosis (24–26). Other kinases such as protein kinase C (PKC) and cyclin-dependent kinase 5 (CDK5) have been shown to be important for SV endocytosis and control, together with the calcium-dependent protein phosphatase calcineurin, the phosphorylation status of many endocytosis-associated proteins such as dynamin-1, amphiphysin, and AP180 (27–36). Mitogen-activated protein kinases (MAPK) appear to operate on parallel pathways as these can regulate syn- aptic plasticity either by directly modifying synaptic proteins (37, 38) or by affecting gene expression (39, 40), thereby contributing to both rapid and long-lasting modulatory effects. Earlier phosphoproteomics studies have shown system- atic changes in phosphorylation of synaptic proteins caused by depolarization-evoked calcium inﬂux, and they have pointed to involvement of CaMKII and MAP kinases in this process (41, 42). However, it remains elusive which of the observed phosphorylation changes caused by calcium inﬂux have a direct functional impact on SV cycling. Indeed, following the initial discovery of synapsin-1 phosphorylation, many studies have demonstrated that phosphorylation of speciﬁc synaptic proteins at speciﬁc sites does have an impact on exo- or endocytosis. © 2021 THE AUTHORS. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Protein Phosphorylation in Depolarized Synaptosomes exocytosis (43–48). Munc18 phosphorylation can further be a result of the MAP kinase activation due to cannabinoid receptor-1 signaling (37); the latter was also shown to bear a phosphorylation site (Cnr1-S317) that is controlled by PKC (49). disassembly of the clathrin coat. Key proteins involved in these steps include, among others, clathrin adaptor proteins (AP2, AP180), clathrin itself, the GTPase dynamin-1, the phosphatase synaptojanin-1, endophilins, and related pro- teins, as well as the clathrin uncoating ATPase Hsc70 and its cofactor auxilin (17). Finally, re-endocytosed SVs are ﬁlled with the respective neurotransmitter by speciﬁc vesicular neuro- transmitter transporters that are fueled by the activity of a vacuolar proton pump (18). In this article, we refer to SV cycling as a multistep process, which includes SV fusion with the plasma membrane mediated by the exocytotic machinery (exocytosis) and SV recycling through available endocytotic mechanisms (endocytosis), followed by the steps in which SVs become ready for the next round of SV cycle (i.e., reﬁlling of the neurotransmitter, SV priming, and docking). However, it remains to be established which of the vast amount of phosphosite changes observed in recent phos- phoproteomic studies are dependent on the membrane traf- ﬁcking steps of the SV cycle and which are, while being targeted by the activated network of kinases and phospha- tases, not directly related to exo–endocytotic cycling. Changes in the protein conformation that accompany as- sembly and disassembly of multimolecular protein complexes during SV cycling may alter the accessibility of phosphoryla- tion sites as well as the availability of docking sites for kinases and phosphatases. To address this issue, we conducted a quantitative phosphoproteome analysis using synaptosomes as a functional model of the synapse. Synaptosomes contain the complete SV-cycling machinery, can maintain the mem- brane potential and ATP at physiological levels, and respond to depolarization by Ca2+-dependent neurotransmitter release (50–53). To differentiate between calcium-induced changes and phosphorylation events that are directly connected to SV cycling, we utilized botulinum neurotoxins (BoNT). These bacterial protein toxins represent a group of endoproteinases that selectively cleave individual SNARE proteins and thus block exocytosis and, consequently, SV recycling without compromising Ca2+ inﬂux or synaptosomal ATP levels (54, 55). For instance, phosphoryla- tion of SNARE proteins (Snap25-S187, Stx1a-S14, S188, Vamp2-T35, S75), as well as of the SNARE-interacting pro- tein Munc18 (Stxbp1-S313), can indeed interfere with the formation of the SNARE complex, which results in altered EXPERIMENTAL PROCEDURES Ethical Statement All experiments involving animals complied with the regulations as designated in the section 4 of the Animal Welfare Law of the Federal Republic of Germany (section 4 Tierschutzgesetz der Bundesrepublik Deutschland, TierSchG). All experiments were conducted in the animal facility at the Max-Planck-Institute for Biophysical Chemistry, Göttin- gen, Germany, and registered accordingly to the section 11 Abs. 1 2 Mol Cell Proteomics (2021) 20 100061 Protein Phosphorylation in Depolarized Synaptosomes 1 mg of estimated synaptosomal protein (the ﬁnal BoNT concentration of 100–200 nM was estimated on the basis of the total protein content of the semipuriﬁed BoNT, which amounted to approximately 10 μg/μl). The following combinations of C. botulinum cell culture supernatants were used: (i) C. botulinum A and D or (ii) C. botulinum C1 and B. Alternatively, synaptosomes were treated with the same amount of respective cell culture supernatants inactivated by heating at 95 ◦C for 1 h (mock control). BoNT- and mock-treated synaptosomes were incubated at 37 ◦C for 90 min and then directly subjected to the glutamate release assay. In brief, synaptosomal suspension was transferred into a quartz glass cuvette (Hellma, Müllheim, Germany) and kept stirred at 37 ◦C during the measurements. In total, 1 mM NADP and 50 U per 1 mg of synaptosomal protein of GluDH were stepwise added to the suspension. Synaptosomes were depolarized by adding KCl to a ﬁnal concentration of 50 mM. NADPH-ﬂuorescence at 440 nm was monitored at each step using Fluorolog-3 ﬂuorometer (Horiba Jobin Yvon, Bensheim, Germany). After 2 min of stimulation, the reaction was quenched with an equal volume of lysis buffer (6 M Guanidine hydrochloride, 200 mM HEPES, 20 mM TCEP, 80 mM CAA, 1 × Halt Protease, and phosphatase inhibitor cocktail). The synapto- somal sample was incubated for 5 min at 95 ◦C, chilled on ice, and then sonicated for 10 min using 30 s on/30 s off cycles at the maximum output of Bioruptor ultrasonication device (Diagenode, Seraing, Belgium). The sample was repeatedly incubated for 5 min at 95 ◦C, and proteins were precipitated following methanol/chloroform protein precipitation method (60). TierSchG as documented by 39 20 00_2a Si/rö, dated Dec 11, 2013 (“Erlaubnis, Wirbeltiere zu Versuchszwecken zu züchten und zu hal- ten”) by the Veterinär-und Verbraucherschutzamt für den Landkreis und die Stadt Göttingen and examined regularly by the supervisory veterinary authority of the Landkreis Göttingen. EXPERIMENTAL PROCEDURES Ethical Statement All procedures were supervised by the animal welfare ofﬁcer and the animal welfare committee of the Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany, established in accordance with the TierSchG and the regulation of animal experiments, dated Aug 31, 2015 (Tier- schutz-Versuchstier-Verordung, TierSchVersV). Phosphopeptide Enrichment and TMT Labeling Precipitated synaptosomal proteins (1–2 mg per condition) were redissolved in a digestion buffer (100 mM TEAB, 10% TFE, 0.1% Rapigest) and sonicated in the Bioruptor for 10 min at maximum in- tensity using 30 s on/30 s off cycles. Trypsin was added at 1:40 trypsin-to-protein ratio (wt/wt), and proteins were digested overnight at 37 ◦C. Next day, the sample was treated with 2.5 U of PNGase F per 1 mg of the initial protein amount for 1 h. Afterward, ACN was added to a ﬁnal concentration of 45% (v/v), and the sample was incubated with a second portion of trypsin (1:100 trypsin-to-protein ratio, wt/wt) for 1 h. The sample was centrifuged for 15 min at 17,000×g in a table- top centrifuge, and the cleared supernatant was subjected to phos- phopeptide enrichment as previously described with modiﬁcations (61). Brieﬂy, equal input peptide amounts were assured by measuring peptide concentration in Nanodrop-1000 (Thermo Fisher Scientiﬁc, Waltham, USA) using an A280 method. KCl, KH2PO4, and TFA were added to the peptide sample to reach ﬁnal concentrations of 228 mM, 3.9 mM, and 38% (v/v), respectively. Peptides were incubated with TiO2 beads (GL Sciences, Tokyo, Japan) at 1:10 protein-to-beads ratio for 20 min at 40 ◦C. Unbound peptide fraction (nonphosphorylated peptides) was collected, and TiO2 beads were subjected to four washing steps using washing buffer (60% (v/v) ACN, 1% (v/v) TFA in water). Bound phosphopeptides were eluted using 3.75% (v/v) NH4OH 40% (v/v) ACN in water, snap-frozen in liquid nitrogen, and dried in a centrifugal Savant SpeedVac vacuum concentrator (Thermo Fisher Scientiﬁc, Waltham, USA). Dried phosphopeptides and pep- tides in the unbound fraction were subjected to desalting using pre- packed C18 spin columns (Harvard Apparatus, Holliston, USA). Desalted and dried peptides were redissolved in 50 mM TEAB 50% (v/ v) ACN in water, and TMT6-labeling reaction was conducted ac- cording to manufacturer’s instructions. After the labeling, peptide samples were accordingly combined, cleaned using the prepacked C18 spin columns, and concentrated in a SpeedVac. Isolated Nerve Terminals Isolated nerve terminals were prepared from brains of 5–6 weeks old Wistar rats as described previously (58). Brieﬂy, brains were quickly homogenized in ice-cold homogenization buffer (320 mM sucrose, 5 mM HEPES is water) using a Teﬂon/glass homogenizer. The homogenate was cleared by centrifugation for 2 min at 2988×g in an SS-34 ﬁxed angle rotor (Thermo Fisher Scientiﬁc, Waltham, USA). The supernatant was collected and recentrifuged for 12 min at 14,462×g. The synaptosomal pellet was resuspended in the homogenization buffer and loaded onto discontinuous Ficoll gradient (6%/9%/13% (wt/v) Ficoll in the homoge- nization buffer) and centrifuged for 35 min at 86,575×g in an SW-41 swinging bucket rotor (Beckman Coulter, Krefeld, Germany). The syn- aptosomal band at the interface between 9% and 13% Ficoll was collected and washed with the homogenization buffer. Synaptosomal protein concentration was estimated using Pierce 660 nm protein assay according to manufacturer’s instructions. The viability of synaptosomes was conﬁrmed by glutamate release assay (59). Chemicals LC/MS-grade water, methanol, and acetonitrile (ACN) were used in this study if not otherwise stated and were purchased together with chloroform, 25% (v/v) NH4OH, CaCl2, KCl, KH2PO4, MgCl2, NaCl, NaHCO3, NaHPO4, sucrose, and glucose from Merck, Darmstadt, Germany. Triﬂuoroethanol (TFE), triethylammonium bicarbonate buffer (TEAB), formic acid (FA), EGTA, PM400-Ficoll (Ficoll), NADP, L-Glu- tamic dehydrogenase from bovine liver (GluDH), guanidine hydro- chloride, tris(2-carboxyethyl)phosphine (TCEP), chloroacetamide (CAA), Triton-X100, and MS-grade trypsin were obtained from Sigma- Aldrich, Taufkirchen, Germany. HEPES was purchased from VWR Chemicals, Darmstadt, Germany. SDS was purchased from Serva Electrophoresis GmbH, Heidelberg, Germany. TFA was obtained from Roth, Karlsruhe, Germany. Peptide-N(4)-(N-acetyl-beta-glucosaminyl) asparagine amidase (PNGase F) was obtained from Roche, Man- nheim, Germany. Rapigest was obtained from Waters, Milford, USA. Pierce 660 nm protein assay, Halt Protease and phosphatase inhibitor cocktail, and TMTsixplex isobaric labeling reagents (TMT6) were purchased from Thermo Fisher Scientiﬁc, Bleiswijk, Netherlands. C. botulinum strain A–D cell culture supernatants (BoNT A, B, C1, D) were obtained from Miprolab, Göttingen, Germany. BoNT Treatment and Glutamate Release Assay In total, 1–1.5 ml of synaptosomal suspension was centrifuged at 6900×g for 3 min in a table-top centrifuge, and synaptosomal pellet was resuspended in the sodium-containing buffer (10 mM glucose, 5 mM KCl, 140 mM NaCl, 5 mM NaHCO3, 1 mM MgCl2, 1.2 mM NaHPO4, 20 mM HEPES in water) to achieve a ﬁnal concentration of 1 mg/ml of synaptosomal proteins. CaCl2 was added to a ﬁnal con- centration of 1.3 mM, and synaptosomes were incubated for 5 min at 37 ◦C before adding BoNT. For Ca versus EGTA experiments, syn- aptosomes were preincubated with 1.3 mM CaCl2 or 0.5 mM EGTA for 15 min at 37 ◦C before stimulation with KCl (50 mM). To block SNARE- assisted neurotransmitter release, 20 μl of each BoNT was added per Basic Reversed-Phase (bRP) Chromatography Basic Reversed-Phase (bRP) Chromatography TMT6-labeled peptides were fractionated using Agilent 1100 series HPLC system (Agilent, Santa Clara, USA) equipped with a Mol Cell Proteomics (2021) 20 100061 3 Protein Phosphorylation in Depolarized Synaptosomes (February 2019, 29,951 entries). Reference proteome of C. botulinum (strain Hall/ATCC 3502/NCTC 13319/Type A) containing canonical protein sequences (Uniprot, January 2020, 3590 entries) was sup- plemented with sequences of BoNT B-D (strain Okra/Type B1, C. botulinum C phage, C. botulinum D phage, respectively). Peptide- spectrum matches (PSM) and protein FDR threshold were kept at 0.01. Following steps in data analysis were conducted in R statistical programming language using custom scripts. The scripts are available at GitHub (https://github.com/IvanSilbern/2021_Silbern_etal_Synapt_ BoNT_KCl). For each phosphorylation site, a leading protein was selected based on the list of potential candidate proteins for this site reported by MaxQuant. Proteins were ranked based on (listed from the most to the least signiﬁcant) (i) the number of the unique phosphor- ylation sites assigned to the protein in the data set; (ii) the number of all phosphorylation sites per protein in the data set; (iii) “reviewed” status and Uniprot-annotation score of the protein sequence; (iv) isoform status of the protein sequence. The ofﬁcial gene name and Uniprot accession of the leading protein were used in the subsequent analyses. Impurity-corrected reporter ion intensities for each phos- phorylation site identiﬁed at one of the three multiplicity levels were extracted from MaxQuant “Phospho(S, T, Y).txt” output table. Po- tential contaminants, reversed sequences, and phosphorylation sites identiﬁed with localization probability <0.75 as determined by Max- Quant (63, 64) were excluded from further analysis. Phosphorylation sites quantiﬁed at different multiplicity levels (“1” means that the phosphorylation site was quantiﬁed on singly, “2” on doubly, and “3” on multiply phosphorylated peptides) were considered as separate entities or phosphorylation events. If a phosphorylation event con- tained <3 nonzero intensity values for a given labeling experiment, the event was considered as not quantiﬁed in this labeling experiment. Otherwise, missing values were imputed for each TMT channel/ experiment pair individually by random sampling from a Gauss dis- tribution with a mean at the 5% quantile and a double-standard de- viation of the log-transformed intensities. Only a minor proportion of phosphorylation events required imputation (<1% per channel). Log2- transformed reporter ion intensities were then normalized using Tukey median polishing individually for each labeling experiment and sub- jected to statistical testing using limma package (66). Basic Reversed-Phase (bRP) Chromatography C18-X-Bridge column (3.5 μm particles, 1.0 mm inner diameter, 150 mm length; Waters, Milford, USA). The HPLC was operated at the ﬂow rate of 60 μl/min under basic pH (buffer A: 10 mM NH4OH in water, pH ~10; buffer B: 10 mM NH4OH and 80% (v/v) ACN in water, pH ~10). The column was equilibrated with the 95% buffer A and 5% buffer B mixture. Peptides were resolved using a linear gradient ranging from 10% to 35% buffer B for 35 min followed by a linear increase to 60% over 5 min and a washing step at 90% buffer B for 5 min. One-minute fractions were collected and concatenated into 24 (for Mock/BoNT phosphorylated peptides) or 12 (for Mock/BoNT nonphosphorylated peptides and Ca/EGTA phosphorylated peptides) ﬁnal fractions as suggested elsewhere (62). BRP fractions were snap- frozen in liquid nitrogen and dried in the SpeedVac. Experimental Design and Statistical Rationale In total, 216 .raw ﬁles were analyzed in order to quantify changes in phosphorylation site intensities in BoNT- or mock-treated synapto- somes or synaptosomes stimulated in the presence of calcium or EGTA. Out of the 216 .raw ﬁles, 144 .raw ﬁles were obtained from two TMT6-labeling sets of mock- versus BoNT-treated synaptosomes. These ﬁles correspond to 24 concatenated BRP-fractions and three injection replicates per fraction. Remaining 72 .raw ﬁles were obtained from another two TMT6-labeling experiments of synaptosomes stim- ulated in the presence of Ca2+ or EGTA. The ﬁles correspond to 12 concatenated bRP-fractions and three injection replicates per fraction. Each set of TMT6-labeled samples contained three independent replicates for each of the two treatment conditions, resulting in six independent replicates for each treatment regime (Mock, BoNT, Ca, or EGTA). The choice for this number of replicates was dictated by the constraints of the TMT6 labeling and the previous study (41). The phosphorylation site intensities resulting from the three technical replicates were summed in order to obtain a single value reported by MaxQuant (64). We used log2-transformed and normalized reporter ion intensities to assess changes in the phosphorylation site intensities. Speciﬁcally, we used the limma package (66) to test the difference in phosphorylation site intensities between mock- and BoNT-treated synaptosomes and synaptosomes stimulated in the presence of cal- cium or EGTA. Linear models were speciﬁed to account for treatment differences (Mock, BoNT, Ca, EGTA) and batch effects between TMT6 LC-MS/MS Dried peptides were redissolved in 2% (v/v) ACN 0.1% (v/v) TFA in water. Each concatenated BRP fraction was analyzed in triplicates (phosphorylated peptides) or as a single injection (nonphosphorylated peptides). Dissolved peptides were injected onto a C18 PepMap100- trapping column (0.3 x 5 mm, 5 μm, Thermo Fisher Scientiﬁc, Wal- tham, USA) connected to an in-house packed C18 analytical column (75 μm x 300 mm; Reprosil-Pur 120C18-AQ, 1.9 μm, Dr Maisch GmbH, Ammerbuch, Germany). The columns were pre-equilibrated using a mixture of 98% buffer A (0.1% (v/v) FA in water), 2% buffer B (80% (v/v) ACN, 0.1% (v/v) FA in water), or 95% buffer A, 5% buffer B (nonphosphorylated peptides). Liquid chromatography was oper- ated on an UltiMate-3000 RSLC nanosystem (Thermo Fisher Scien- tiﬁc, Waltham, USA). Phosphopeptides were eluted using a 60 min- linear gradient ranging from 10% to 25% buffer B (80% (v/v) ACN, 0.1% (v/v) FA in water) followed by a linear increase to 45% buffer B over 10 min and a washing step at 90% of buffer B for 5 min. For nonphosphorylated peptides, a steeper gradient was applied ranging from 10% to 45% buffer B followed by an increase to 60% buffer B and a washing step at 90% buffer B. Eluting peptides were sprayed into a QExactive HF-X or Orbitrap Exploris 480 (Thermo Fisher Sci- entiﬁc, Bremen, Germany). For the analysis of phosphorylated pep- tides, MS1 scans (350–1600 m/z) were acquired with a resolution of 120,000 at 200 m/z, 3e6 automatic gain control (AGC) target, and 50 ms maximum injection time. Precursor ions were isolated using a 0.8 m/z isolation window, and a normalized collision energy of 33% was applied to obtain fragment spectra. Only charge states from 2+ to 6+ were considered, and the dynamic exclusion was set to 25 s. The fragment spectra were acquired with a resolution of 30,000, 1e5 AGC target, and 130 ms maximum injection time. For the analysis of non- phosphorylated peptides, following parameters were set differently: MS1 scans were acquired with a resolution of 60,000 at 200 m/z, 300% normalized AGC target. Fragment spectra were acquired with the resolution of 15,000 at 200 m/z, 50% of the normalized AGC target and 54 ms maximum injection time. Data Analysis The kinase prediction with the highest NetworKIN score was selected for each phosphorylation site. Predicted kinases were orga- nized in the kinase groups introduced by NetPhorest classiﬁcation (73). The second-level and top-level kinase groups in the kinase hi- erarchy were used for enrichment analyses and visualizations. The overrepresentation analysis of predicted kinase–substrate interactions for phosphorylation sites signiﬁcantly regulated in Ca/EGTA or Mock/ BoNT experiments was conducted in comparision with NetworKIN kinase–substrate predictions for human proteome. To assess the disbalance in the number of up/downregulated phosphorylation events, the number of up/downregulated phosphorylation events controlled by each kinase group was compared with the total number of up/down phosphorylation events among signiﬁcantly regulated phosphorylated sites. All tests of differential term or kinase enrichment between two groups were done using Fisher’s exact test. Benjamini– Hochberg adjusted p-values are reported throughout the study if not speciﬁed otherwise. The anticipated phosphatase–substrate in- teractions rely upon described protein phosphatase-1(PP1) and Cal- cineurin docking motifs (76). To demonstrate possible phosphatase– substrate connections, interaction maps were extracted from protein–protein interactions annotated for R. norvegicus (STRING (75) database version 10.5). Shortest paths from phosphatase to target proteins (phosphoproteins involved in endo/exocytosis and containing PP1 or Calcineurin docking motif) were computed using shortest_path function from igraph package (77) (version 1.4.6). Only proteins pre- viously identiﬁed in synaptosomes and interactions with experimental evidence or annotated in curated databases were included in the analysis. Phosphorylation site occupancy was calculated using a 3D model presented by Hoegrebe et al (78). In brief, a linear model of the form Ph = m1∙Prot – m2∙NPh, where Ph is the normalized (by median polishing) intensity of the phosphorylated peptide; Prot is the normalized protein intensity, and NPh is the normalized intensity of nonphosphorylated peptide. The model was ﬁt by using intensity data from Mock/BoNT experiments. Phosphorylation occupancy was then calculated using the following formula: Occupancy = a/(1+a), where a = m2∙Ph/NPh. “Illegal” stoichiometry values, i.e., values outside the interval [0;1] were excluded from the analysis. Only m2 values with a p value below 0.1 were considered. Deﬁning Primary Ca2+-Dependent and SV-Cycling-Dependent Phosphorylation Sites Deﬁning Primary Ca2+-Dependent and SV-Cycling-Dependent Phosphorylation Sites Phophorylation events were deﬁned as “primary Ca2+-dependent” if they show absolute log2 (Ca/EGTA) > 0.263 and absolute log2 (Mock/ BoNT) < 0.263 or were not quantiﬁed in the Mock/BoNT experiment. Data Analysis Raw ﬁles were processed using MaxQuant version 1.6.10.2 (63, 64) under default settings with some exceptions. Speciﬁcally, cysteine carbamidomethylation was selected as a ﬁxed modiﬁcation, whereas methionine oxidation, acetylation of protein N termini, and phos- phorylation of serine, threonine, and tyrosine (only for phosphorylated peptides) were set as variable modiﬁcations. Up to ﬁve variable modiﬁcations were allowed per peptide. Speciﬁc trypsin digestion was selected with up to two missed cleavage sites allowed per peptide. Maximal peptide mass was increased to 6000. Quantiﬁcation using reporter ions in MS2 (TMT6plex) and minimal parent ion fraction of 0.75 were selected. MS1 and MS2 mass tolerances were kept at 4.5 ppm and 20 ppm, respectively. Canonical amino acid sequences of Rattus norvegicus proteins were retrieved from Uniprot (65) 4 Mol Cell Proteomics (2021) 20 100061 Protein Phosphorylation in Depolarized Synaptosomes sets. Differences in the peptide intensities between mock- and BoNT- treated synaptosomes and synaptosomes stimulated in the presence/ absence of calcium were tested. Empirical-Bayes moderated p-values (66) were subjected to multiple testing correction using q-value approach (67). Phosphorylation events (phosphorylation site at a given multiplicity) were considered as signiﬁcantly regulated when showing at least 1.2 intensity fold change (FC, corresponds to absolute log2 FC > 0.263) and q-value < 0.01 (corresponds to false discovery rate (FDR) < 1%). sets. Differences in the peptide intensities between mock- and BoNT- treated synaptosomes and synaptosomes stimulated in the presence/ absence of calcium were tested. Empirical-Bayes moderated p-values (66) were subjected to multiple testing correction using q-value approach (67). Phosphorylation events (phosphorylation site at a given multiplicity) were considered as signiﬁcantly regulated when showing at least 1.2 intensity fold change (FC, corresponds to absolute log2 FC > 0.263) and q-value < 0.01 (corresponds to false discovery rate (FDR) < 1%). Phosphorylation sites with no assignment were subjected to Net- worKIN kinase–substrate prediction tool (73, 74). Speciﬁcally, rat protein sequences were aligned against the reference set of human protein sequences (STRING (75) database version 9.05) by using BLAST+ (additional command line parameters: -max_target_seqs 40 -word_size 6 -gapopen 12 -gapextend 1). The best alignment for each rat protein sequence was selected based on the rat protein sequence coverage, number of amino acid positions considered identical, alignment bitscore, number of open sequence gaps (listed with decreasing importance). Positions of phosphorylation sites in the best matching human protein sequences were subjected to NerworKIN tool. Data Analysis Conversely, events showing absolute log2 (Mock/BoNT) > 0.263 were termed as “SV-cycling-dependent.” Phosphorylation events that exhibited only small changes in both experiments (absolute log2 (Ca/ EGTA) < 0.263 and absolute log2 (Mock/BoNT) < 0.263) were considered as being “not-affected.” In the following analyses, only phosphorylation events that surpassed an FDR-threshold of <1% were considered. Speciﬁcally, “primary Ca2+-dependent” phosphory- lation events had to satisfy a q-value threshold of <0.01 in the Ca/ EGTA experiment, whereas “SV-cycling-dependent” sites had to meet a q-value threshold of <0.01 in Mock/BoNT experiment or a q-value- threshold of <0.01 and an absolute log2 FC threshold of >0.263 in Ca/ EGTA experiment. Phosphorylation events that did not match these criteria were considered as being “not-affected” as well. If a phos- phorylation site was quantiﬁed at different multiplicities (phosphory- lation events), whereby one of which was classiﬁed as being “SV- cycling-dependent,” the site was considered as being “SV-cycling- dependent.” To deﬁne regulation groups at protein level, number of signiﬁcantly regulated phosphorylation sites were counted per protein (gene). Proteins were distributed in the following groups (i) “mostly primary Ca2+-dependent” (>60% of sites being categorized as “pri- mary Ca2+-dependent”); (ii) “mostly SV-cycling-dependent” (>60% of sites being categorized as “SV-cycling-dependent”); (iii) “mixed” (none of the groups was dominating). Bioinformatic Analysis Gene enrichment analyses were conducted using gene names of proteins carrying signiﬁcantly regulated phosphorylation sites in Ca/ EGTA or Mock/BoNT experiments. Gene ontology enrichment anal- ysis was performed using David web interface and whole R. norvegicus proteome as a background (68). Direct GO term cate- gories surpassing Benjamini–Hochberg-adjusted p-value threshold of 0.001 were considered for further analysis. For reactome pathway analysis, only enriched (Benjamini–Hochberg-adjusted p-value < 0.1) “neuronal system” categories were selected (69). Synapse-speciﬁc SynGO database was used to retrieve signiﬁcantly enriched (GSEA “gene cluster” FDR corrected p-value < 0.001) GO-biological function terms (70). Experimentally validated kinase–substrate interactions were derived from PhosphoSitePlus database (“Kinase_ Substrate_Dataset.txt”, version of 10.04.2018) (71). In order to ac- count for possible differences in phosphorylation site annotation, sequence windows of the phosphorylation sites in the data set were aligned against sequence windows reported in the PhosphoSitePlus database using BLAST+ software (version: 2.7.1, additional command line parameters: -max_target_seqs 20) (72). Only exact alignments of the phosphorylation sites showing the minimum bitscore of 20 were considered. The best match was selected from ﬁltered alignments ranked based on the alignment bitscore, number of amino acid po- sitions considered identical (nident), number of open sequence gaps, kinase, and substrate species (rat > mouse > human > rabbit). C. botulinum Cell Culture Supernatants Proﬁling For protein proﬁling, supernatants of C. botulinum cell culture were digested overnight using trypsin. Digested peptides were desalted using prepacked C18 spin columns. Samples were analyzed on QExactive HF-X and liquid chromatography setup as described above. To test for unspeciﬁc phosphatase activity, C. botulinum cell culture supernatants were incubated with the nuclear extract of HeLa cells. The nuclear extract was prepared as previously described (79) in Roeder D buffer (20 mM Hepes-KOH pH 7.9, 100 mM KCl, 1.5 mM MgCl2, 10% (v/v) glycerol, 0.5 mM DTT, 0.5 mM PMSF, 0.2 mM EDTA) and was a generous gift of Prof. Dr Lührmann laboratory. The extract was supplemented with 1.3 mM CaCl2 and 20 μl of each C. botulinum cell culture supernatant (A and D or C1 and B), heat-inactivated cell Mol Cell Proteomics (2021) 20 100061 5 Protein Phosphorylation in Depolarized Synaptosomes (previously described, (83)) allowing neuron-speciﬁc expression under the human synapsin-1 promoter. For all the following subcloning steps, we relied on commercial chemically competent SURE bacteria from Agilent, to avoid the loss of ITR sequences. Positive clones were conﬁrmed by sequencing, and before transfection in the packaging HEK293 T cells, the presence of ITRs and the correct length of all plasmids were checked by restriction enzyme analysis. Syntaxin-1a (Stx1a; P32851) and cannabinoid receptor 1 (Cnr1; P20272) and their mutants (respectively T21E-T23 E pseudo-phosphorylated Stx1a, T21A-T23 A nonphosphorylatable Stx1a, T314E-T322 E pseudo- phosphorylated Cnr1 and T314A-T322 A nonphosphorylatable Cnr1) were inserted in the WT Vamp2-pHluorin viral vector by cutting with the enzymes SgsI (AscI) and SbfI (SdaI). Their expression was driven by mean of a second human synapsin-1 promoter from the same viral plasmid to allow consistent coexpression. To distinguish these pro- teins from the endogenous, an ALFA-tag was added (84). Also in this case, positive clones were conﬁrmed by sequencing, and before transfection in the packaging HEK293 T cells, the presence of ITRs and the correct length of all plasmids were checked by restriction enzyme analysis. All synthetic plasmids and their respective se- quences are available from the authors upon request. culture supernatants, or 40 μl of the sodium buffer per 1 mg of nuclear proteins. Each treatment condition was performed in triplicate, resulting in a total of nine samples. The samples were incubated for 1.5 h at 37 ◦C. Synaptic Vesicle Exo-/Endocytosis Measurements SV exo/endocytosis assays were performed analogously to what previously described (81, 85). Brieﬂy, neurons were ﬁeld-stimulated with platinum electrodes in custom-made chambers housing 18 mm coverslips (8 mm distance between electrodes). We used a Stimulus Isolator combined with an A310 Accupulser Stimulator (both from World Precision Instruments, Sarasota, USA) with a nominal output of 100 mA for stimulation. In the case of Stx1a and Cnr1 coexpression was conﬁrmed by imaging. Live experiments were performed in Tyrode’s solution (124 mM NaCl, 5 mM KCl, 30 mM glucose, 25 mM HEPES, 2 mM CaCl2, 1 mM MgCl2, pH 7.4) supplemented with 10 μM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; Tocris Bioscience, Cambridge, UK) and 50 μM 2-amino-5-phosphonopentanoic acid (D- AP5; Tocris Bioscience, Cambridge, UK) to avoid spontaneous network activation. At the end of the imaging, 50 mM NH4Cl was Adeno-Associated Virus Preparation Recombinant AAV particles were prepared as previously described (83). Brieﬂy, vectors were packaged in HEK293 T cells using the pDP6 helper plasmid (avoiding adenoviral contamination). Packaging cells were lysed in Tyrode’s solution (124 mM NaCl, 5 mM KCl, 30 mM glucose, 25 mM HEPES, 2 mM CaCl2, 1 mM MgCl2, pH 7.4), passed through 0.22 μm syringe ﬁlters, and viral titer was adjusted by serial dilution on primary hippocampal neurons monitoring pHluorin levels (always expressed under the neurospeciﬁc human synapsin-1 promoter). C. botulinum Cell Culture Supernatants Proﬁling Afterward, urea in 100 mM HEPES pH 8 was added to the ﬁnal concentration of 1 M and proteins were digested overnight at 30 ◦C using MS-grade trypsin at a trypsin-to-protein ration (wt/wt) of 1:50. Next day, peptides were subjected to phosphopeptide enrich- ment as described above excluding the deglycosylation and second endoproteinase digestion steps. Enriched phosphopeptides were cleaned using the prepacked C18 spin columns, redissolved in 2% (v/ v) ACN, 0.1% (v/v) TFA in water, and analyzed on UltiMate 3000 RSLC nanosystem connected to Orbitrap Exploris 480 mass spectrometer. Raw phosphorylation site intensities as reported by MaxQuant were analyzed as described before using R scripts and the limma package for data normalization, visualization, and statistical testing. Calcium Inﬂux in Synaptosomes Following BoNT Treatment and KCl Stimulation Calcium Inﬂux in Synaptosomes Following BoNT Treatment and KCl Stimulation Synaptosome suspensions containing 1 mg of synaptosomal pro- teins were prepared as described for glutamate release assay. Sus- pensions were supplemented with 1.3 mM CaCl2 and additional 40 μl of the sodium-containing buffer or 1.3 mM CaCl2 and 20 μl of each C. botulinum cell culture supernatants in two combinations (A and D or C1 and B). Synaptosomes were then incubated for 1 h at 37 ◦C under mild agitation. Afterward, synaptosome suspensions were supple- mented with 5 μM of Fura-2AM dye (Molecular probes, Eugene, USA) and incubated for additional 30 min at 37 ◦C. Following steps were conducted as previously described (80). Brieﬂy, synaptosomes were resuspended in 1 ml of fresh sodium-containing buffer and loaded into the quartz glass cuvette. The synaptosomal suspension was stirred and kept at 37 ◦C during the measurements. The ﬂuorescence was continuously measured using Fluorolog-3 at excitation wavelengths of 340 and 380 nm and emission at 505 nm, for 2 s at each wavelength. In total, 1.3 mM CaCl2, 50 mM KCl, 0.4% (wt/v) SDS, 1 mM EGTA were added stepwise and the signal was acquired for 3 min at each step. Fluorescence intensity at 340 nm excitation was divided by the intensity at 380 nm excitation and then by the maximum ﬂuorescence ratio following SDS lysis. Median ﬂuorescence ratio before stimulation with KCl was subtracted to obtain proportional changes in ﬂuores- cence intensity relatively to its maximum. Immuno Staining with NbALFA The expression of Stx1a and Cnr1 in all viruses and mutants was conﬁrmed by staining with the NbALFA (84). Brieﬂy, neurons were ﬁxed with 4% paraformaldehyde (PFA; w/v) for 30 min at room tem- perature (RT). Cells were quenched in 100 mM NH4Cl and per- meabilized in PBS containing 4% bovine serum albumin (BSA; wt/v) and 0.1% (v/v) Triton-X 100 for 15 min at RT. Fluorescently labeled NbALFA (FluoTag-X2 anti-ALFA AbberiorStar635P, NanoTag Bio- technologies #N1502-Ab635P-L, Germany) was diluted 1:200 in blocking solution for 1 h at RT and subsequently washed three times for 5 min with PBS. Coverslips were mounted on glass slides using Mowiol solution, dried, and imaged. Note that superecliptic GFP (pHluorin) keeps its ﬂuorescence in Mowiol solution since its pH is ~7.5 and can thus be imaged with no additional staining. Cell Cultures and Transfections Primary hippocampal neuronal cultures were prepared from P2 rats as previously described (81). HEK293 T cells were obtained by a commercial supplier (Sirion Biotech GmbH; Germany). HEK293 T cells were grown in DMEM supplemented with 10% FBS, 4 mM L-glutamine, and 600 U/ml penicillin-streptomycin (Lonza). For transfection Lipofectamine 2000 transfection reagent was used following the guidelines of the manufacturer (Thermo Fisher Scientiﬁc, Invitrogen, USA). All plasmids for transfection were produced in bacteria and puriﬁed with the NucleoBond Xtra Midi endotoxin-free plasmid DNA kit (Macherey-Nagel, Germany). RESULTS applied to evaluate total SV content. Live imaging was performed with an inverted Nikon Ti epiﬂuorescence microscope (Nikon, Tokyo, Japan) equipped with a Plan Apochromat 60x 1.4 NA oil-immersion objective, an HBO-100W Lamp, an IXON X3897 Andor camera (Northern Ireland, UK), and an OKOLab cage incubator system (OKOLab, Ottaviano, Italy) to maintain a constant temperature of 37 ◦C. ND2 Images were imported using the Bio-Formats (OME) plug-in. For quantiﬁcations, the ﬂuorescence intensity was measured with custom-made Matlab (USA) code that can be provided on an indi- vidual basis if requested. Brieﬂy, multiple regions containing synaptic boutons were manually selected, in 5–8 independent experiments. Between 140 and 345 regions were selected for each condition, over all independent experiments. The Vamp2-pHluorin signal was then measured in the respective regions, and the background intensity (obtained in identically sized regions from the neuron-free adjoining regions) was subtracted. The Vamp2-pHluorin signal curves were then normalized to the maximum intensity obtained during the addi- tion of NH4Cl. To compare easily different conditions and mutant variants, all curves were furthermore normalized to their initial (pres- timulus) baselines. Statistical differences were tested in Matlab, relying on Kruskal–Wallis tests, followed by Tukey–Kramer post-hoc tests. applied to evaluate total SV content. Live imaging was performed with an inverted Nikon Ti epiﬂuorescence microscope (Nikon, Tokyo, Japan) equipped with a Plan Apochromat 60x 1.4 NA oil-immersion objective, an HBO-100W Lamp, an IXON X3897 Andor camera (Northern Ireland, UK), and an OKOLab cage incubator system (OKOLab, Ottaviano, Italy) to maintain a constant temperature of 37 ◦C. ND2 Images were imported using the Bio-Formats (OME) plug-in. For quantiﬁcations, the ﬂuorescence intensity was measured with custom-made Matlab (USA) code that can be provided on an indi- vidual basis if requested. Brieﬂy, multiple regions containing synaptic boutons were manually selected, in 5–8 independent experiments. Between 140 and 345 regions were selected for each condition, over all independent experiments. The Vamp2-pHluorin signal was then measured in the respective regions, and the background intensity (obtained in identically sized regions from the neuron-free adjoining regions) was subtracted. The Vamp2-pHluorin signal curves were then normalized to the maximum intensity obtained during the addi- tion of NH4Cl. To compare easily different conditions and mutant variants, all curves were furthermore normalized to their initial (pres- timulus) baselines. Statistical differences were tested in Matlab, relying on Kruskal–Wallis tests, followed by Tukey–Kramer post-hoc tests. DNA Cloning of Wild-Type Proteins and Respective Phosphomimetic Mutants Replicates within one experiment were labeled with a set of TMT6 isobaric labeling reagents. A pooled peptide sample was fractionated by reversed-phased chromatography at basic pH and measured in a high-resolution quadrupole-orbitrap mass spectrometer. Reporter ion intensities were utilized to infer relative abundances of phosphorylated peptides. FIG. 1. Workﬂow for the phosphoproteome analyses of synaptosomes under different stimuli. Brains of 5–6 week-old Wistar rats were homogenized and the synaptosomal fraction was enriched by differential centrifugation in discontinuous Ficoll gradient. Two sets of experiments were performed that compared a) stimulation of synaptosomes in the presence of Ca2+ (1.3 mM) or Ca2+-chelator (EGTA, 0.5 mM), or b) stimulation of synaptosomes pretreated with inactivated toxins (Mock), or a combination of two botulinum toxins (BoNT, A + D or C1+B). Synaptosomes were depolarized by 50 mM KCl for 2 min. Each experiment included three independent control stimulations (EGTA or Mock- treated) and three independent test stimulations (Ca or BoNT). Samples were processed in a conventional bottom-up proteomics workﬂow and phosphorylated peptides were enriched by metal-oxide chromatography (TiO2). Replicates within one experiment were labeled with a set of TMT6 isobaric labeling reagents. A pooled peptide sample was fractionated by reversed-phased chromatography at basic pH and measured in a high-resolution quadrupole-orbitrap mass spectrometer. Reporter ion intensities were utilized to infer relative abundances of phosphorylated peptides. DNA Cloning of Wild-Type Proteins and Respective Phosphomimetic Mutants All the sequences (including mutants) were designed in silico based on rat genes with the appropriate restriction enzymes for viral cloning and ordered at GenScript (USA). The synaptobrevin (Vamp2) mutants were based on rat Vamp2 (Uniprot: P63045) fused to the optimized version of superecliptic GFP (pHluorin) (82) by mean of a short peptide linker. We used AgeI and SdaI (SbfI) restriction sites for inserting Vamp2-pHluorin and the respective mutants (S75D pseudo- phosphorylated and S75A non-phosphorylatable) in the viral vector 6 Mol Cell Proteomics (2021) 20 100061 Protein Phosphorylation in Depolarized Synaptosomes FIG. 1. Workﬂow for the phosphoproteome analyses of synaptosomes under different stimuli. Brains of 5–6 week-old Wistar rats were homogenized and the synaptosomal fraction was enriched by differential centrifugation in discontinuous Ficoll gradient. Two sets of experiments were performed that compared a) stimulation of synaptosomes in the presence of Ca2+ (1.3 mM) or Ca2+-chelator (EGTA, 0.5 mM), or b) stimulation of synaptosomes pretreated with inactivated toxins (Mock), or a combination of two botulinum toxins (BoNT, A + D or C1+B). Synaptosomes were depolarized by 50 mM KCl for 2 min. Each experiment included three independent control stimulations (EGTA or Mock- treated) and three independent test stimulations (Ca or BoNT). Samples were processed in a conventional bottom-up proteomics workﬂow and phosphorylated peptides were enriched by metal-oxide chromatography (TiO2). Replicates within one experiment were labeled with a set of TMT6 isobaric labeling reagents. A pooled peptide sample was fractionated by reversed-phased chromatography at basic pH and measured in a high-resolution quadrupole-orbitrap mass spectrometer. Reporter ion intensities were utilized to infer relative abundances of phosphorylated peptides. FIG. 1. Workﬂow for the phosphoproteome analyses of synaptosomes under different stimuli. Brains of 5–6 week-old Wistar rats were homogenized and the synaptosomal fraction was enriched by differential centrifugation in discontinuous Ficoll gradient. Two sets of experiments were performed that compared a) stimulation of synaptosomes in the presence of Ca2+ (1.3 mM) or Ca2+-chelator (EGTA, 0.5 mM), or b) stimulation of synaptosomes pretreated with inactivated toxins (Mock), or a combination of two botulinum toxins (BoNT, A + D or C1+B). Synaptosomes were depolarized by 50 mM KCl for 2 min. Each experiment included three independent control stimulations (EGTA or Mock- treated) and three independent test stimulations (Ca or BoNT). Samples were processed in a conventional bottom-up proteomics workﬂow and phosphorylated peptides were enriched by metal-oxide chromatography (TiO2). Synaptosome Depolarization by KCl in the Presence of Ca2+ Results in Substantial Changes in Protein Phosphorylation A, Number of quantiﬁed phosphorylation sites in Ca versus EGTA and Mock versus BoNT experiments (panel 1), Ca versus EGTA experiments only (panel 2) and recently published data set (Engholm-Keller (42), panel 3). B and C, volcano plots depicting -log10 (q-value) versus log2 (intensity fold change) of phosphorylation sites quantiﬁed within Ca versus EGTA and Mock versus BoNT experiments, respectively. D, Comparison of log2 fold changes between our and Engholm-Keller et al (42) data sets. Selected phosphorylation sites are marked as ofﬁcial gene symbol with phosphorylated amino acid and position. E and F, The stacked bar graphs show the total number of signiﬁcantly regulated phosphorylation events in Ca versus EGTA (E) or Mock versus BoNT (F) experiments that can be a result of known (PhosphositePlus database (71)) or predicted (NetworKIN (73, 74)) kinase–substrate interactions. Orange bars depict the number of phosphorylation events that show signiﬁcant intensity increase in Ca2+ or Mock-treated synaptosomes versus respective control (EGTA or BoNT). Blue bars correspond to downregulated events, respectively. The kinase classiﬁcation (NetPhorest Groups) follows one introduced by NetPhorest (73) and uses its second-level group anno- tation. Colored vertical bars delineate the highest-level group in the kinase classiﬁcation. Asterisks mark kinase groups that are predicted to control signiﬁcantly more phosphorylation sites than expected as based on predicted kinase–substrate interactions in human proteome (p < 0.01). AGC, protein kinase A, G, C kinase group; CaMKII, calcium-calmodulin kinase 2; CDK, cyclin-dependent kinase; CK, casein kinase; CK1, casein kinase 1; CK2, casein kinase 2; CLK, SRPK1 and Clk/Sty protein kinase; CMGC, CDK, MAP, GSK, CDKL kinase group; DAPK, death- associated protein kinase; GSK3, glycogen synthase kinase 3; MAPK, mitogen-activated protein kinase; PAK, p21-activated kinase; PKC, protein kinase C; STE, “sterile” serine/threonine protein kinases. FIG. 2. Substantial changes in synaptic phosphoproteome following depolarization and responsible kinase groups. A, Number of quantiﬁed phosphorylation sites in Ca versus EGTA and Mock versus BoNT experiments (panel 1), Ca versus EGTA experiments only (panel 2) and recently published data set (Engholm-Keller (42), panel 3). B and C, volcano plots depicting -log10 (q-value) versus log2 (intensity fold change) of phosphorylation sites quantiﬁed within Ca versus EGTA and Mock versus BoNT experiments, respectively. D, Comparison of log2 fold changes between our and Engholm-Keller et al (42) data sets. Selected phosphorylation sites are marked as ofﬁcial gene symbol with phosphorylated amino acid and position. Synaptosome Depolarization by KCl in the Presence of Ca2+ Results in Substantial Changes in Protein Phosphorylation Our analysis shows that phosphorylation is a prominent modiﬁcation of synaptosomal proteins. Speciﬁcally, we iden- tiﬁed and quantiﬁed phosphorylation under stimulated and nonstimulated conditions on 12,021 sites belonging to more When analyzing synaptosomes depolarized in the pres- ence of calcium ions in comparison with those depolarized in FIG. 2. Substantial changes in synaptic phosphoproteome following depolarization and responsible kinase groups. A, Number of quantiﬁed phosphorylation sites in Ca versus EGTA and Mock versus BoNT experiments (panel 1), Ca versus EGTA experiments only (panel 2) and recently published data set (Engholm-Keller (42), panel 3). B and C, volcano plots depicting -log10 (q-value) versus log2 (intensity fold change) of phosphorylation sites quantiﬁed within Ca versus EGTA and Mock versus BoNT experiments, respectively. D, Comparison of log2 fold changes between our and Engholm-Keller et al (42) data sets. Selected phosphorylation sites are marked as ofﬁcial gene symbol with phosphorylated amino acid and position. E and F, The stacked bar graphs show the total number of signiﬁcantly regulated phosphorylation events in Ca versus EGTA (E) or Mock versus BoNT (F) experiments that can be a result of known (PhosphositePlus database (71)) or predicted (NetworKIN (73, 74)) kinase–substrate interactions. Orange bars depict the number of phosphorylation events that show signiﬁcant intensity increase in Ca2+ or Mock-treated synaptosomes versus respective control (EGTA or BoNT). Blue bars correspond to downregulated events, respectively. The kinase classiﬁcation (NetPhorest Groups) follows one introduced by NetPhorest (73) and uses its second-level group anno- tation. Colored vertical bars delineate the highest-level group in the kinase classiﬁcation. Asterisks mark kinase groups that are predicted to control signiﬁcantly more phosphorylation sites than expected as based on predicted kinase–substrate interactions in human proteome (p < 0.01). AGC, protein kinase A, G, C kinase group; CaMKII, calcium-calmodulin kinase 2; CDK, cyclin-dependent kinase; CK, casein kinase; CK1, casein kinase 1; CK2, casein kinase 2; CLK, SRPK1 and Clk/Sty protein kinase; CMGC, CDK, MAP, GSK, CDKL kinase group; DAPK, death- associated protein kinase; GSK3, glycogen synthase kinase 3; MAPK, mitogen-activated protein kinase; PAK, p21-activated kinase; PKC, protein kinase C; STE, “sterile” serine/threonine protein kinases. FIG. 2. Substantial changes in synaptic phosphoproteome following depolarization and responsible FIG. 2. Substantial changes in synaptic phosphoproteome following depolarization and responsible kinase groups. RESULTS We performed a quantitative in-depth analysis of changes in protein phosphorylation events in active synaptosomes. We ﬁrst quantitatively compared the phosphorylation status of proteins in chemically stimulated synaptosomes under Ca2+- deprived and free Ca2+ conditions; for the latter, depolariza- tion of the plasma membrane of synaptosomes was induced by KCl in buffer containing EGTA or CaCl2 (Fig. 1). The viability of the synaptosomes was conﬁrmed by monitoring release of the neurotransmitter (glutamate) (59) (supplemental Fig. S1A). We applied depolarization for 2 min; this duration was chosen in order (i) to achieve comparability with our previous studies (41) and (ii) to allow the capture of short- and medium-term changes in phosphorylation. Proteins from stimulated and nonstimulated synaptosomes were extracted, digested, and enriched for phosphopeptides in parallel; thereafter, the (phospho)peptides were labeled with unique isotopically labeled TMT6 reagents. Peptides were then pooled, Mol Cell Proteomics (2021) 20 100061 7 Protein Phosphorylation in Depolarized Synaptosomes than 2600 proteins (unique gene names) with a localization probability of >75% as determined by MaxQuant (63, 64). This coverage greatly exceeds our earlier one (41) and is compa- rable to a recent study from another laboratory (42). When the phosphosites quantiﬁed from BoNT-treated synaptosomes are included, the number of sites quantiﬁed in this study in- creases to 18,981 sites belonging to 3840 proteins (Fig. 2A, left panel). prefractionated by basic reverse-phase chromatography, and ﬁnally analyzed by LC-MS/MS (Fig. 1). In the second approach, we inhibited SV cycling using BoNT and monitored quantitatively the changes in the phosphoproteome after K+- mediated depolarization, comparing synaptosomes that were treated with active or, respectively, with inactive BoNT (Fig. 1). Phosphopeptides were enriched and analyzed as described above. This second approach should differentiate phosphor- ylation events that are dependent on SV exo–endocytotic cycling. Synaptosome Depolarization by KCl in the Presence of Ca2+ Results in Substantial Changes in Protein Phosphorylation Synaptosome Depolarization by KCl in the Presence of Ca2+ Results in Substantial Changes in Protein Phosphorylation E and F, The stacked bar graphs show the total number of signiﬁcantly regulated phosphorylation events in Ca versus EGTA (E) or Mock versus BoNT (F) experiments that can be a result of known (PhosphositePlus database (71)) or predicted (NetworKIN (73, 74)) kinase–substrate interactions. Orange bars depict the number of phosphorylation events that show signiﬁcant intensity increase in Ca2+ or Mock-treated synaptosomes versus respective control (EGTA or BoNT). Blue bars correspond to downregulated events, respectively. The kinase classiﬁcation (NetPhorest Groups) follows one introduced by NetPhorest (73) and uses its second-level group anno- tation. Colored vertical bars delineate the highest-level group in the kinase classiﬁcation. Asterisks mark kinase groups that are predicted to control signiﬁcantly more phosphorylation sites than expected as based on predicted kinase–substrate interactions in human proteome (p < 0.01). AGC, protein kinase A, G, C kinase group; CaMKII, calcium-calmodulin kinase 2; CDK, cyclin-dependent kinase; CK, casein kinase; CK1, casein kinase 1; CK2, casein kinase 2; CLK, SRPK1 and Clk/Sty protein kinase; CMGC, CDK, MAP, GSK, CDKL kinase group; DAPK, death- associated protein kinase; GSK3, glycogen synthase kinase 3; MAPK, mitogen-activated protein kinase; PAK, p21-activated kinase; PKC, protein kinase C; STE, “sterile” serine/threonine protein kinases. 8 Mol Cell Proteomics (2021) 20 100061 Protein Phosphorylation in Depolarized Synaptosomes the absence of calcium (EGTA control), we detected sub- stantial changes in protein phosphorylation. Speciﬁcally, there are 1363 phosphorylated sites mapped to approxi- mately 470 proteins that show intensity changes of at least 20% at an FDR of 1% (Fig. 2B). Using the PhosphositePlus database (71) to annotate known kinase–substrate relations and the NetworKIN tool (73, 74) to predict possible kinase– substrate pairs, we observed that certain kinase families were overrepresented including the CaMKII, MAPK, and GSK3 kinase groups (supplemental Table S1). Importantly, the distribution of up- or downregulated sites between ki- nase groups is not random: CaMKII and PKC kinase groups are responsible for phosphorylation sites that appear upre- gulated, while the majority of sites controlled by MAPK, CDK, and CK1 show reduced intensity in the presence of Ca2+ as compared with EGTA controls (Fig. 2E and supplemental Fig. S2). The latter observation is in line with previous studies that demonstrated upregulation of putative CaMKII substrates and downregulation of predicted MAPK substrates following potassium-triggered depolarization of synaptosomes (41, 42). Synaptosome Depolarization by KCl in the Presence of Ca2+ Results in Substantial Changes in Protein Phosphorylation We also observed changes of phosphorylated sites in response to KCl treatment that were similar to changes re- ported in earlier studies (42) for bassoon (Bsn), piccolo (Pclo), synapsin-1 (Syn1), and dynamin-1 (Dnm1), as well as the microtubule-associated protein Tau (Mapt) and the dihydropyrimidinase-related proteins 2 and 5 (Dpysl2 and Dpysl5), and others (Fig. 2D). Overall, the overlap of identiﬁed and quantiﬁed sites with the results of previous studies is approximately 60% as determined by the overlap in protein sequences ﬂanking phosphorylation sites (sequence windows). Enrichment analysis of the biological function of proteins carrying regulated phosphorylation sites (DAVID (68)) revealed gene ontology (GO) terms tightly connected with synaptic functioning, cytoskeleton organization, regulation of synaptic functions, and neuronal projections development (Fig. 3A). A similar picture (Fig. 3B) was obtained when using the synapse- speciﬁc SynGo database in which a large set of synaptic proteins was annotated by experts (70). It shows that the signiﬁcantly enriched terms describe biological functions connected with SV cycling, regulation of neurotransmitter FIG. 3. Functional annotation of proteins carrying regulated phosphorylation sites based on Ca versus EGTA experiments. A, Asso- ciation between signiﬁcantly enriched GO terms and kinase groups. The enriched biological function-GO terms (background: Rattus norvegicus, p < 0.001) were derived using DAVID web service (68) for proteins carrying regulated phosphorylation sites. Overrepresentation of putative kinase–substrates relationships was tested for proteins annotated by each GO term. Frequencies of putative kinase–substrate relationships across all regulated sites for Ca versus EGTA experiments were taken as a background. The color encodes raw p-values (Fisher’s exact test). B, Sunburst diagram showing enriched biological function terms based on the synapse-speciﬁc SynGo database annotation (70). The color en- codes the signiﬁcance of the enrichment (-log10 (q-value)). Full list of enriched biological functions can be found in supplemental Table S2. FIG. 3. Functional annotation of proteins carrying regulated phosphorylation sites based on Ca versus EGTA experiments. A, Asso- ciation between signiﬁcantly enriched GO terms and kinase groups. The enriched biological function-GO terms (background: Rattus norvegicus, p < 0.001) were derived using DAVID web service (68) for proteins carrying regulated phosphorylation sites. Overrepresentation of putative kinase–substrates relationships was tested for proteins annotated by each GO term. Frequencies of putative kinase–substrate relationships across all regulated sites for Ca versus EGTA experiments were taken as a background. The color encodes raw p-values (Fisher’s exact test). Synaptosome Depolarization by KCl in the Presence of Ca2+ Results in Substantial Changes in Protein Phosphorylation B, Sunburst diagram showing enriched biological function terms based on the synapse-speciﬁc SynGo database annotation (70). The color en- codes the signiﬁcance of the enrichment (-log10 (q-value)). Full list of enriched biological functions can be found in supplemental Table S2. FIG. 3. Functional annotation of proteins carrying regulated phosphorylation sites based on Ca versus EGTA experiments. A, Asso- ciation between signiﬁcantly enriched GO terms and kinase groups. The enriched biological function-GO terms (background: Rattus norvegicus, p < 0.001) were derived using DAVID web service (68) for proteins carrying regulated phosphorylation sites. Overrepresentation of putative kinase–substrates relationships was tested for proteins annotated by each GO term. Frequencies of putative kinase–substrate relationships across all regulated sites for Ca versus EGTA experiments were taken as a background. The color encodes raw p-values (Fisher’s exact test). B, Sunburst diagram showing enriched biological function terms based on the synapse-speciﬁc SynGo database annotation (70). The color en- codes the signiﬁcance of the enrichment (-log10 (q-value)). Full list of enriched biological functions can be found in supplemental Table S2. Mol Cell Proteomics (2021) 20 100061 9 Protein Phosphorylation in Depolarized Synaptosomes MAPK, PKC, and GSK3 kinase groups (supplemental Table S3). When comparing the quantitative Ca versus EGTA phosphorylation data, signiﬁcantly fewer (P ≅1.0 × 10-5) regulated phosphorylation sites could be ascribed to being CaMKII substrates, whereas putative sites for MAPK (1), CLK (2), and PAK (3) were slightly over- (1, 2) or under- (3) repre- sented, respectively (Fig. 2F, supplemental Table S4). There- fore, the crucial point in this analysis is the distribution of up- or downregulated phosphorylation sites for the different ki- nase groups. While depolarization of synaptosomes by KCl in the presence of Ca2+ leads to signiﬁcantly reduced intensities of phosphorylation sites presumably regulated by MAPK and CDK kinases, the intensities of the putative MAPK, CDK, and GSK3 substrates appear higher when comparing mock- treated with BoNT-treated synaptosomes (compare Fig. 2, E and F). The balance of up- and downregulated phosphoryla- tion sites was also signiﬁcantly altered (p < 0.01) for CLK, CK1, and PAK kinases. release, and synapse organization (for a complete list of enriched terms, see supplemental Table S2). Interestingly, regulation of some functions might be linked to the activity of certain kinase groups (Fig. 3A). Synaptosome Depolarization by KCl in the Presence of Ca2+ Results in Substantial Changes in Protein Phosphorylation For example, the number of sites predicted to be substrates for GSK3 and CDK is pro- portionally higher among proteins involved into the GO term “axonogenesis” as compared with other GO terms. Similarly, many phosphorylated sites on proteins that are involved in the “calcium-dependent regulation of synaptic functions” were also predicted as CaMKII substrates. The proportion of pu- tative MAPK substrates is higher for proteins associated with “synapse assembly” and “cytoskeleton organization” as compared with other GO terms. This analysis reﬂects, in accordance with recent studies (41, 42), the overall changes of phosphorylation in isolated nerve terminals upon stimulation—including the phosphosites that are important for exocytosis and endocytosis, i.e., for SV cycling. However, the observed effects can be a result of both Ca2+ inﬂux and active SV cycling. Dissecting Primary Ca2+-Driven Phosphorylation Events from Events Linked to SV Cycling Block of SV Cycling with Botulinum Neurotoxins Induces Changes in Protein Phosphorylation Assuming that the comparison of mock-treated synapto- somes with the BoNT-treated ones reveals the phosphoryla- tion changes caused by Ca2+-induced SV cycling, we were able to identify phosphorylation events that are primarily dependent on Ca2+ inﬂux and are not affected if SV cycling is inhibited (Figs. 4A and S3, orange dots). In the following, we refer to these sites as “primary Ca2+-dependent” sites. They are presumably directly (de)phosphorylated by Ca2+-depen- dent protein kinases and phosphatases upon Ca2+ inﬂux. Conversely, a large group of phosphorylation events has shown changes when comparing mock- and BoNT-treated synaptosomes. Hence, we termed these sites “SV-cycling- dependent.” We noted with interest that the majority of “SV- cycling-dependent” sites did not show signiﬁcant intensity changes in the Ca/EGTA experiment. We also noted that many proteins tend to carry regulated phosphorylation sites of one of the two groups introduced above, e.g., mostly “primary Ca2+-dependent” or “SV-cycling-dependent” sites, while only a few contain sites of various categories (Fig. 4B). Interest- ingly, the number of proteins that carry mostly “primarily Ca2+- dependent” sites is lower than the number of proteins that carry mostly “SV-cycling-dependent” sites (Fig. 4B). It in- dicates the tendency that “primarily Ca2+-dependent” phos- phorylation sites appear clustered on a few proteins, whereas “SV-cycling-dependent” sites are often found as the only signiﬁcantly regulated phosphorylation site in a single protein. The site occupancy assessment (based on the model of Hoegrebe et al. Synaptosome Depolarization by KCl in the Presence of Ca2+ Results in Substantial Changes in Protein Phosphorylation A, Categorization of phosphorylation events based on the magnitude of the log2 fold changes in Mock versus BoNT experiment (x-axis) and log2 fold changes in Ca versus EGTA experiment (y-axis). Only events quantiﬁed in both experiments and satisfying a q-value threshold of <0.01 are shown. The events are classiﬁed into two regulation groups: (i) “primary Ca2+-dependent” (sites with no signiﬁcant or not-quantiﬁed SV cycling effect, i.e., absolute log2 (Mock/BoNT) < 0.263 and absolute log2 (Ca/EGTA) > 0.263), shown as orange dots; and (ii) “SV-cycling-dependent” (absolute log2 (Mock/BoNT) > 0.263). B, Proportion of proteins that can be organized in one of the regulation groups based on the categorization of phosphorylation sites they carry. Only sites that surpassed the FDR-threshold of <1% were considered. If > 60% of the signiﬁcantly regulated sites on a protein belong to one of the two regulation groups (“primary Ca2+-dependent” or “SV-cycling-dependent”), the protein is categorized in “mostly primary Ca2+-dependent” or “mostly SV-cycling-dependent,” respectively. C, Enrichment of predicted kinase–substrate interactions for “primary Ca2+-dependent” and “SV- cycling-dependent” regulation groups. The left panel shows the percentage and the absolute number of sites in each category (x-axis) that can be regulated by a particular kinase group (y-axis). The number under the black line provides the total number of sites with predicted kinase– substrate relationship in each category. The right panel shows Benjamini–Hochberg adjusted p-values after a Fisher’s exact test. An equal distribution of kinase–substrates relationships between the “primary Ca2+-dependent” and “SV-cycling-dependent” categories was set as a null hypothesis. D, Reactome pathway terms of neuronal origin (69). Reactome pathway terms of neuronal origin were selected based on their enrichment (Benjamini–Hochberg adjusted p-value < 0.1) for all proteins carrying signiﬁcantly regulated phosphorylation sites. Proportion of “primary Ca2+-dependent” and “SV-cycling-dependent” sites of all “primary Ca2+-dependent” or “SV-cycling-dependent” sites per functional term is shown as color code (magenta). Fisher’s exact test was applied to test equal distribution of “primary Ca2+-dependent” and “SV-cycling- dependent” sites for each term. Benjamini–Hochberg adjusted p-values are represented as a color code (green). Abbreviations: AMPAR stays for α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor; CREB1 ~ CAMP-responsive element binding protein 1; GABA ~ γ-ammino- butyric acid; NMDAR ~ N-methyl-d-aspartate receptor; RAS ~ rat sarcoma gene. FIG. 4. Dependence of regulated phosphorylation sites on calcium inﬂux and SV cycling. Synaptosome Depolarization by KCl in the Presence of Ca2+ Results in Substantial Changes in Protein Phosphorylation A, Categorization of phosphorylation events based on the magnitude of the log2 fold changes in Mock versus BoNT experiment (x-axis) and log2 fold changes in Ca versus EGTA experiment (y-axis). Only events quantiﬁed in both experiments and satisfying a q-value threshold of <0.01 are shown. The events are classiﬁed into two regulation groups: (i) “primary Ca2+-dependent” (sites with no signiﬁcant or not-quantiﬁed SV cycling effect, i.e., absolute log2 (Mock/BoNT) < 0.263 and absolute log2 (Ca/EGTA) > 0.263), shown as orange dots; and (ii) “SV-cycling-dependent” (absolute log2 (Mock/BoNT) > 0.263). B, Proportion of proteins that can be organized in one of the regulation groups based on the categorization of phosphorylation sites they carry. Only sites that surpassed the FDR-threshold of <1% were considered. If > 60% of the signiﬁcantly regulated sites on a protein belong to one of the two regulation groups (“primary Ca2+-dependent” or “SV-cycling-dependent”), the protein is categorized in “mostly primary Ca2+-dependent” or “mostly SV-cycling-dependent,” respectively. C, Enrichment of predicted kinase–substrate interactions for “primary Ca2+-dependent” and “SV- cycling-dependent” regulation groups. The left panel shows the percentage and the absolute number of sites in each category (x-axis) that can be regulated by a particular kinase group (y-axis). The number under the black line provides the total number of sites with predicted kinase– substrate relationship in each category. The right panel shows Benjamini–Hochberg adjusted p-values after a Fisher’s exact test. An equal distribution of kinase–substrates relationships between the “primary Ca2+-dependent” and “SV-cycling-dependent” categories was set as a null hypothesis. D, Reactome pathway terms of neuronal origin (69). Reactome pathway terms of neuronal origin were selected based on their enrichment (Benjamini–Hochberg adjusted p-value < 0.1) for all proteins carrying signiﬁcantly regulated phosphorylation sites. Proportion of “primary Ca2+-dependent” and “SV-cycling-dependent” sites of all “primary Ca2+-dependent” or “SV-cycling-dependent” sites per functional term is shown as color code (magenta). Fisher’s exact test was applied to test equal distribution of “primary Ca2+-dependent” and “SV-cycling- dependent” sites for each term. Benjamini–Hochberg adjusted p-values are represented as a color code (green). Abbreviations: AMPAR stays for α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor; CREB1 ~ CAMP-responsive element binding protein 1; GABA ~ γ-ammino- butyric acid; NMDAR ~ N-methyl-d-aspartate receptor; RAS ~ rat sarcoma gene. FIG. 4. Dependence of regulated phosphorylation sites on calcium inﬂux and SV cycling. Synaptosome Depolarization by KCl in the Presence of Ca2+ Results in Substantial Changes in Protein Phosphorylation (78)) suggests high phosphorylation levels (>60%) for a number of phosphoproteins, including Bsn, Pclo, Dnm1, Camk2a, Mapt, and Dplysl5, underlining the impor- tance of the observed phosphorylation events (supplemental Fig. S4). It should be noted that the estimated phosphoryla- tion levels represent only a rough estimate, as the ratio As indicated above, our ﬁrst quantitative analysis does not allow for differentiating phosphorylation changes that are directly dependent on active membrane trafﬁcking in the synapse rather than being caused by the activation of calcium-dependent kinases and phosphatases such as CaMKII (23), calcineurin (86), etc. Of course, these processes are intertwined since these enzymes are known to regulate the phosphorylation status of proteins closely associated with the process of SV cycling (87, 88). We suppressed SV cycling by incubating synaptosomes with BoNT that block exocytosis by cleaving SNARE proteins. Toxin-treated synaptosomes and (as controls) synaptosomes incubated with inactivated toxins were stimulated with the high-potassium buffer in the presence of Ca2+ ions (Fig. 1). The toxin treatment greatly inhibited neurotransmitter release (supplemental Fig. S1B) but did not compromise Ca2+ inﬂux (supplemental Fig. S1C). It led to signiﬁcant changes in the phosphoproteome of the synaptosomes: 1497 sites showed signiﬁcant difference with a minimum of 20% intensity changes at 1% FDR that can be mapped to approximately 700 proteins. Strikingly, BoNT treatment reduced the intensity of many phosphorylation sites (Fig. 2C). The phosphorylation status of these sites and their corresponding proteins is apparently dependent on the Ca2+-induced SV cycling activity in the presynapse. The enrichment analyses for GO terms did not show sub- stantial differences when compared with proteins carrying regulated sites in Ca versus EGTA experiments (not shown). The impact of BoNT treatment became apparent when we investigated possible substrate–kinase relationships. The enrichment analysis showed signiﬁcant overrepresentation (p < 0.01) of phosphorylation sites that can be attributed to 10 Mol Cell Proteomics (2021) 20 100061 Protein Phosphorylation in Depolarized Synaptosomes FIG. 4. Dependence of regulated phosphorylation sites on calcium inﬂux and SV cycling. A, Categorization of phosphorylation events based on the magnitude of the log2 fold changes in Mock versus BoNT experiment (x-axis) and log2 fold changes in Ca versus EGTA experiment (y-axis). Only events quantiﬁed in both experiments and satisfying a q-value threshold of <0.01 are shown. The events are classiﬁed into two FIG. 4. Dependence of regulated phosphorylation sites on calcium inﬂux and SV cycling. Synaptosome Depolarization by KCl in the Presence of Ca2+ Results in Substantial Changes in Protein Phosphorylation In contrast, terms associated with “Neurotransmitter release cycle”, e.g., glutamate, acetylcholine, etc., release, are barely enriched in phosphorylated proteins that carry “SV- cycling-dependent” sites (Fig. 4D, top). Reactome pathway annotation aligned well with the manual annotation of key- words describing protein function or location according to in- formation available in the literature (supplemental Fig. S8). On the basis of our annotation, we noted that 234 out of 301 phosphorylation sites on proteins directly involved in the pro- cesses of exo- and endocytosis belong to the “primary Ca2+- dependent” group. For example, phosphorylation of RIM, RIM- binding protein, synapsin-1, and “dephosphins” such as dynamin-1 (Dnm1), amphiphysin (Amph), and AP180 (Snap91) showed a clear Ca2+-dependent character (supplemental Figs. S9 and S10). Importantly, phosphorylation sites on synapsin-1 followed the previously described pattern of phosphorylation changes when comparing Ca with the EGTA condition: increased phosphorylation of S9 and S603 and dephosphorylation of S62, S67, S549, and S551 (90–95) (supplemental Fig. S11). In contrast, the “SV-cycling-depen- dent” phosphorylation sites tended to be present on proteins that are described as being associated with cytoskeleton ele- ments and channels, for example, spectrin beta (Sptb, Sptan1, Sptbn1, Sptbn2, Sptbn4) and spectrin-binding proteins addu- cin alpha (Add1) and beta (Add2), as well as potassium channel subunits that are known constituents or modulators of Kv1 (Kcna2, Kcna4, Kcnab2), Kv2 (Kcnb1, Kcnb2), Kv4.3 (Kcnd2), Kv7.2 (Kcnq2), K2p10.1 (Kcnk10), Hcn (Hcn1, Hcn2, Kctd3) potassium channels (96) (supplemental Figs. S12–S15). As mentioned above, the observed SV cycling effect on several potassium channels seems to be very speciﬁc for the particular proteins and results in increased phosphorylation of the ma- jority of those proteins except Add1 and Kcnma2 (supplemental Fig. S16). Conversely, the Ca2+-activated po- tassium channel subunit alpha-1 (Kcnma1) and several other potassium channel subunits and modulators (Kcnc3, Kcnh1, Kcnip2, Kcnq5) have shown the “primary Ca2+-dependent” character of phosphorylation (supplemental Fig. S17). We summarize changes in phosphorylation site intensities for protein sequences that contain phosphorylation sites signiﬁ- cantly regulated in one of the experiments and provide them as graphics in supplemental Data 5 and 6 (log2(Ca/EGTA) and log2(Mock/BoNT), respectively). These can be also visualized using a shiny app accessible at https://s1608-shiny.mpibpc. mpg.de. Figure 5 summarizes our results according to the schematic representation of proteins at the presynapse (97). compression, which is a known issue for the TMT-based quantiﬁcation, might severely affect the occupancy calcula- tion. In terms of probable kinase regulation (Fig. Synaptosome Depolarization by KCl in the Presence of Ca2+ Results in Substantial Changes in Protein Phosphorylation As mentioned above, the observed SV cycling effect on several potassium channels seems to be very speciﬁc for the particular proteins and results in increased phosphorylation of the ma- jority of those proteins except Add1 and Kcnma2 (supplemental Fig. S16). Conversely, the Ca2+-activated po- tassium channel subunit alpha-1 (Kcnma1) and several other potassium channel subunits and modulators (Kcnc3, Kcnh1, Kcnip2, Kcnq5) have shown the “primary Ca2+-dependent” character of phosphorylation (supplemental Fig. S17). We summarize changes in phosphorylation site intensities for protein sequences that contain phosphorylation sites signiﬁ- cantly regulated in one of the experiments and provide them as graphics in supplemental Data 5 and 6 (log2(Ca/EGTA) and log2(Mock/BoNT), respectively). These can be also visualized using a shiny app accessible at https://s1608-shiny.mpibpc. mpg.de. Figure 5 summarizes our results according to the schematic representation of proteins at the presynapse (97). Importantly, the proteins of the core exocytotic machinery including synaptobrevin, SNAP-25, syntaxin-1a, and Munc18 exhibit SV-cycling-dependent sites, while Munc13 reveals one SV-cycling-dependent site, which is close to the signiﬁcance cutoff. y ( pp g ) We noticed that numerous phosphorylation events on phosphatases and phosphatase regulators were signiﬁcantly regulated in our study (supplemental Fig. S7A). Particularly, Neurabin-1 (Ppp1r9a), a PP1 regulator, showed increased phosphorylation in the Mock/BoNT experiment, suggesting the involvement of PP1 in regulating SV cycling. Although phosphatase–substrate interactions are more difﬁcult to analyze due to the degenerated nature of phosphatase docking motifs, previous studies led to the proposal of docking motifs of PP1 and the Ca2+-calmodulin-dependent phosphatase calcineurin (76). We therefore examined possible interactions between exo- and endocytosis-related proteins containing PP1 or Calcineurin docking motifs and the respective phosphatase (supplemental Fig. S7, B and C). Available interactome data suggest a partially overlapping set of proteins that can be regulated by the phosphatases (e.g., Rims2, Bsn, Bin1, Stxbp1), which does not allow conﬁdent assignment of a phosphatase to a particular phosphorylation event. When considering all potential targets, our results show a minor increase in the percentage of “SV-cycling-dependent” sites over “primary Ca2+-dependent” sites on proteins that can be regulated by PP1 (Fisher’s exact test, p = 0.075, supplemental Fig. S7D). Because of the tendency of proteins to carry preferentially one of the two groups of signiﬁcantly regulated phosphoryla- tion sites, we set out to investigate the putative biological functions of these proteins in order to delineate processes that can be controlled by “primary Ca2+-dependent” or “SV-cycling dependent” phosphorylations. Synaptosome Depolarization by KCl in the Presence of Ca2+ Results in Substantial Changes in Protein Phosphorylation A, Categorization of phosphorylation events based on the magnitude of the log2 fold changes in Mock versus BoNT experiment (x-axis) and log2 fold changes in Ca versus EGTA experiment (y-axis). Only events quantiﬁed in both experiments and satisfying a q-value threshold of <0.01 are shown. The events are classiﬁed into two regulation groups: (i) “primary Ca2+-dependent” (sites with no signiﬁcant or not-quantiﬁed SV cycling effect, i.e., absolute log2 (Mock/BoNT) < 0.263 and absolute log2 (Ca/EGTA) > 0.263), shown as orange dots; and (ii) “SV-cycling-dependent” (absolute log2 (Mock/BoNT) > 0.263). B, Proportion of proteins that can be organized in one of the regulation groups based on the categorization of phosphorylation sites they carry. Only sites that surpassed the FDR-threshold of <1% were considered. If > 60% of the signiﬁcantly regulated sites on a protein belong to one of the two regulation groups (“primary Ca2+-dependent” or “SV-cycling-dependent”), the protein is categorized in “mostly primary Ca2+-dependent” or “mostly SV-cycling-dependent,” respectively. C, Enrichment of predicted kinase–substrate interactions for “primary Ca2+-dependent” and “SV- cycling-dependent” regulation groups. The left panel shows the percentage and the absolute number of sites in each category (x-axis) that can be regulated by a particular kinase group (y-axis). The number under the black line provides the total number of sites with predicted kinase– substrate relationship in each category. The right panel shows Benjamini–Hochberg adjusted p-values after a Fisher’s exact test. An equal distribution of kinase–substrates relationships between the “primary Ca2+-dependent” and “SV-cycling-dependent” categories was set as a null hypothesis. D, Reactome pathway terms of neuronal origin (69). Reactome pathway terms of neuronal origin were selected based on their enrichment (Benjamini–Hochberg adjusted p-value < 0.1) for all proteins carrying signiﬁcantly regulated phosphorylation sites. Proportion of “primary Ca2+-dependent” and “SV-cycling-dependent” sites of all “primary Ca2+-dependent” or “SV-cycling-dependent” sites per functional term is shown as color code (magenta). Fisher’s exact test was applied to test equal distribution of “primary Ca2+-dependent” and “SV-cycling- dependent” sites for each term. Benjamini–Hochberg adjusted p-values are represented as a color code (green). Abbreviations: AMPAR stays for α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor; CREB1 ~ CAMP-responsive element binding protein 1; GABA ~ γ-ammino- butyric acid; NMDAR ~ N-methyl-d-aspartate receptor; RAS ~ rat sarcoma gene. Mol Cell Proteomics (2021) 20 100061 11 Protein Phosphorylation in Depolarized Synaptosomes Another striking difference is that phosphorylated proteins termed with “Synaptic adhesion-like molecules” and “Neurex- ins and neuroligins” are enriched in SV-cycling-dependent group. Synaptosome Depolarization by KCl in the Presence of Ca2+ Results in Substantial Changes in Protein Phosphorylation 4C), sites predicted to be CaMKII substrates include signiﬁcantly more “primary Ca2+-dependent” sites than sites classiﬁed as “SV- cycling dependent.” This harmonizes well with the strict Ca2+ dependence of CaMKII activity (89). Conversely, the MAPK group controls signiﬁcantly more “SV-cycling dependent” sites than “primary Ca2+-dependent” sites. Similarly, predicted PAK substrates are overrepresented among “primary Ca2+- dependent” sites, whereas predicted PKC, CDK, and CLK- substrates appear more often among “SV-cycling-depen- dent” sites (Fig. 4C). The majority of phosphorylation sites on CAMKII, MAPK1, MAPK3, and PRKCB themselves show dependence on calcium ions. Increased phosphorylation of the regulatory sites of CaMKII, MAPK1, MAPK3, and PRKCB (Camk2a-T286, Camk2b-T287, Camk2d-T287, Mapk1-T183, Y185, Mapk3-T203, Y205, Prkcb-T640) points to the activa- tion of those kinases following depolarization of synaptosmes by KCl (supplemental Figs. S5 and S6). Another striking difference is that phosphorylated proteins termed with “Synaptic adhesion-like molecules” and “Neurex- ins and neuroligins” are enriched in SV-cycling-dependent group. In contrast, terms associated with “Neurotransmitter release cycle”, e.g., glutamate, acetylcholine, etc., release, are barely enriched in phosphorylated proteins that carry “SV- cycling-dependent” sites (Fig. 4D, top). Reactome pathway annotation aligned well with the manual annotation of key- words describing protein function or location according to in- formation available in the literature (supplemental Fig. S8). On the basis of our annotation, we noted that 234 out of 301 phosphorylation sites on proteins directly involved in the pro- cesses of exo- and endocytosis belong to the “primary Ca2+- dependent” group. For example, phosphorylation of RIM, RIM- binding protein, synapsin-1, and “dephosphins” such as dynamin-1 (Dnm1), amphiphysin (Amph), and AP180 (Snap91) showed a clear Ca2+-dependent character (supplemental Figs. S9 and S10). Importantly, phosphorylation sites on synapsin-1 followed the previously described pattern of phosphorylation changes when comparing Ca with the EGTA condition: increased phosphorylation of S9 and S603 and dephosphorylation of S62, S67, S549, and S551 (90–95) (supplemental Fig. S11). In contrast, the “SV-cycling-depen- dent” phosphorylation sites tended to be present on proteins that are described as being associated with cytoskeleton ele- ments and channels, for example, spectrin beta (Sptb, Sptan1, Sptbn1, Sptbn2, Sptbn4) and spectrin-binding proteins addu- cin alpha (Add1) and beta (Add2), as well as potassium channel subunits that are known constituents or modulators of Kv1 (Kcna2, Kcna4, Kcnab2), Kv2 (Kcnb1, Kcnb2), Kv4.3 (Kcnd2), Kv7.2 (Kcnq2), K2p10.1 (Kcnk10), Hcn (Hcn1, Hcn2, Kctd3) potassium channels (96) (supplemental Figs. S12–S15). Synaptosome Depolarization by KCl in the Presence of Ca2+ Results in Substantial Changes in Protein Phosphorylation In most cases, an ofﬁcial gene symbol was used to name proteins, except: Bassoon stays for Bsn; Ca2+-ATPase ~ Atp2b1, Atp2b2, Atp2b3; Camsap ~ Camsap1, Camsap3; CatininA ~ Cttna2; CatininD ~ Ctnnd1, Ctnnd2; Dystrobrevin ~ Dtnb; Dystrophin-related ~ Drp2; Liprin ~ Ppﬁa1, Ppﬁa2, Ppﬁa3, Ppﬁa4; Map ~ Map1a, Map1b, Map1s, Map2, Map4, Map6, Map7d1, Map7d2; Mapk ~ Mapk1, Mapk3; Na+/K+ ATPase ~ Atp1a1, Atp1a2, Atp1a3; Na+-Channel ~ Scn1a, Scn1b, Scn2b, Scn3a; Neurexins ~ Nrxn1; Piccolo ~ Pcl; RIM-BPs ~ Rimbp1, Rimbp2; RIMs ~ Rim1, Rim2; Spectrin ~ Sptan1, Sptb, Sptbn1, Sptbn2, Sptbn4; Stathmin ~ Stmn1; Synaptotagmin ~ Syt1, Syt6, Syt9, Syt10, Syt17, Styl5, Esyt2; Syntaxin ~ Stx1a, Stx1b, Stx12; Syntrophin ~ Sntb2; Tomosyn ~ Stxbp5; VAMP ~ Vamp2; V-ATPase ~ Atp6v1d; VELI ~ Lin7a, Lin7b, Lin7c. Asterisks mark sites that do not meet signiﬁcance criteria of q-value < 0.01: Unc13a- pS389 (q-value = 0.013), Rab3a-pS188 (q-value = 0.012). Synaptosome Depolarization by KCl in the Presence of Ca2+ Results in Substantial Changes in Protein Phosphorylation Reactome pathway analysis (69) sheds light on the putative different function of the involved phosphorylated proteins (Fig. 4D). One obvious difference regarding the two groups is that proteins are connected to potassium channels (“Voltage-gated potassium channels”), which are more enriched in the “SV-cycling-dependent” group. 12 Mol Cell Proteomics (2021) 20 100061 Protein Phosphorylation in Depolarized Synaptosomes FIG. 5. Schematic representation of presynaptic compartment with a single docked synaptic vesicle, plasma membrane, and cyto- skeleton elements, based on Chua et al (97). The represented proteins are categorized in “mostly primary Ca2+-dependent” (yellow), “mostly SV-cycling-dependent” (blue), “Mixed” (brown) based on the regulation type of the phosphorylation sites they carry. Proteins with no signiﬁ- cantly regulated phosphorylation sites are represented in gray; proteins for which no ﬁrst-class phosphorylation sites (MaxQuant localization probability <0.75) were identiﬁed are represented in white. In most cases, an ofﬁcial gene symbol was used to name proteins, except: Bassoon stays for Bsn; Ca2+-ATPase ~ Atp2b1, Atp2b2, Atp2b3; Camsap ~ Camsap1, Camsap3; CatininA ~ Cttna2; CatininD ~ Ctnnd1, Ctnnd2; Dystrobrevin ~ Dtnb; Dystrophin-related ~ Drp2; Liprin ~ Ppﬁa1, Ppﬁa2, Ppﬁa3, Ppﬁa4; Map ~ Map1a, Map1b, Map1s, Map2, Map4, Map6, Map7d1, Map7d2; Mapk ~ Mapk1, Mapk3; Na+/K+ ATPase ~ Atp1a1, Atp1a2, Atp1a3; Na+-Channel ~ Scn1a, Scn1b, Scn2b, Scn3a; Neurexins ~ Nrxn1; Piccolo ~ Pcl; RIM-BPs ~ Rimbp1, Rimbp2; RIMs ~ Rim1, Rim2; Spectrin ~ Sptan1, Sptb, Sptbn1, Sptbn2, Sptbn4; Stathmin ~ Stmn1; Synaptotagmin ~ Syt1, Syt6, Syt9, Syt10, Syt17, Styl5, Esyt2; Syntaxin ~ Stx1a, Stx1b, Stx12; Syntrophin ~ Sntb2; Tomosyn ~ Stxbp5; VAMP ~ Vamp2; V-ATPase ~ Atp6v1d; VELI ~ Lin7a, Lin7b, Lin7c. Asterisks mark sites that do not meet signiﬁcance criteria of q-value < 0.01: Unc13a- pS389 (q-value = 0.013), Rab3a-pS188 (q-value = 0.012). FIG. 5. Schematic representation of presynaptic compartment with a single docked synaptic vesicle, plasma membrane, and cyto- skeleton elements, based on Chua et al (97). The represented proteins are categorized in “mostly primary Ca2+-dependent” (yellow), “mostly SV-cycling-dependent” (blue), “Mixed” (brown) based on the regulation type of the phosphorylation sites they carry. Proteins with no signiﬁ- cantly regulated phosphorylation sites are represented in gray; proteins for which no ﬁrst-class phosphorylation sites (MaxQuant localization probability <0.75) were identiﬁed are represented in white. Phosphomimetic Sites on SNARE Proteins and Cannabinoid Receptor-1 Modulate Neurotransmitter Release Surprisingly, we found that inhibition of exocytosis by BoNT had a profound effect on the phosphoproteome despite unchanged calcium inﬂux. These ﬁndings reveal complex signaling cascades during which regulatory changes in protein phosphorylation are not only dependent on the activation of kinase/phospha- tase networks but also on the progression of vesicular trafﬁcking. By using isobaric tandem-mass tags for labeling of phosphorylated peptides, we were able to substantially in- crease the phosphoproteome coverage and to achieve reli- able quantiﬁcation for thousands of phosphorylation sites. Despite differences in the synaptosome preparation (Ficoll- puriﬁed synaptosomes versus crude synaptosomal pellet) and stimulation (50 mM KCl for 2 min versus 75 mM KCl for 10 s), many of the regulated phosphorylation sites observed by us are in excellent agreement with earlier results from another laboratory (Fig. 2D) (42). Our data show higher magnitudes of changes, which are probably a consequence of the prolonged stimulation time used in our study. The most drastic changes were observed for pacsin (Pacs1- S517, log2 fold change of 3.98), thymosin beta (Tmsb4x-T23, 2.63), ADP-ribosylation factor GTPase activating protein 1 (Arfgap1-S247 2.57), calcium/calmodulin-dependent protein kinase type II subunit delta (Camk2d-S315/S319, 2.49), protein bassoon (Bsn-S1354, –2.06), septin 5 (Sept5-S17, –1.93), and RIM-binding protein 2 (Rimbp2-S554, –1.86). We also conﬁrm phosphorylation changes in response to stimulation that were reported earlier for synapsin-1, dyna- min-1, Tau protein, as well as the multisite phosphorylations of the giant active zone proteins bassoon and piccolo (26, 31, 36, 101). The cannabinoid receptor-1 is known for modulation of the neurotransmitter release (49, 99, 100), and its activation can also be linked, through the activation of MAP kinases, to the phosphorylation of Munc-18 (37). Our experiments show that the pseudo-phosphorylated variant Cnr1-T314E/T322E reduced exocytosis (most evident during short stimulation), while the nonphosphorylatable variant Cnr1-T314A/T322A increased exocytosis in response to long or short stimulation. The effect on endocytosis was observed only for the pseudo- phosphorylatable Cnr1 variant after long stimulation (Fig. 6, H and I). These experiments tested only three of the synaptic phos- phorylation sites that we found differentially phosphorylated in this work. Nevertheless, even if these experiments are based on overexpression in a background containing normal levels of WT proteins, all tested candidates resulted in signiﬁcant modulations of exo- or endocytosis. Phosphomimetic Sites on SNARE Proteins and Cannabinoid Receptor-1 Modulate Neurotransmitter Release expressed the respective wild-type (WT) proteins, their phos- phomimetic mutants, and phosphorylation-null mutants in cultured hippocampal neurons (Fig. 6A). With this strategy, we addressed Vamp2-S75, Stx1-S21/ S23, Cnr1-T314/T322, and their respective mutants (Vamp2- S75D; Vamp2-S75A; Stx1-S21E/S23E; Stx1-S21A/S23A; Cnr1-T314E/T322E, and Cnr1-T314A/T322A). For these pro- teins we created adeno-associated viruses (AAVs) that allowed us to mimic the effects of phosphorylation, and we expressed them in neurons together with Vamp2-pHluorin, as a sensor for exo-/endocytosis (Fig. 6, B and C). Since protein phosphorylation can be an epiphenomenon of intracellular signaling, we selected a few of the changing phosphosites for determining directly whether they indeed have a modulatory effect on synaptic transmission. We noted with interest that certain phosphorylation sites on SNARE proteins, including synaptobrevin (Vamp2) and syntaxin-1a (Stx1a), have a phenotype, which belongs to the “SV-cycling-dependent” group (supplemental Fig. S18). More intriguing, some of those sites (such as Vamp2-S75) were previously described as being important for the interaction with Munc18-1, a protein that can exhibit a stimulatory effect on trans-SNARE zippering (46). Similarly, we noted that two sites on the cannabinoid receptor-1 (Cnr1), T314 and T322, belong to the “SV-cycling-dependent” group as well. In order to test the effects of phosphorylation at these sites, we Our stimulation experiments demonstrated that both phosphomimetic and nonphosphorylatable amino-acid sub- stitutions, in both proteins, inﬂuence exo- and endocytosis in the cultured neurons. Speciﬁcally, we stimulated neurons with either 60 action potentials (AP) at 20 Hz (short stimulation) targeting the readily releasable pool of SVs and thus reﬂecting the immediate exocytotic response, or using 600 AP at 20 Hz Mol Cell Proteomics (2021) 20 100061 13 Protein Phosphorylation in Depolarized Synaptosomes (long stimulation) to mobilize the more inert recycling SV pools, reaching a state in which the entire vesicle cycle is fully activated (98). The expression of phosphomimetic Vamp2- S75D reduced exocytosis following short and long stimula- tion; it also reduced endocytosis in response to long stimulation. Expression of nonphosphorylatable Vamp2-S75A reduced exo- and endocytosis following both long and short stimulations (Fig. 6, D and E). Conversely, pseudo- phosphorylated Stx1a-T21E/T23E enhances exocytosis efﬁciency during short stimulation, while the non- phosphorylatable variant does the opposite (Fig. 6F). The effects on exocytosis become negligible during long stimula- tion, but both mutant variants result in an enhancement of endocytosis under these conditions (Fig. 6G). require active endo–exocytotic SV cycling. Phosphomimetic Sites on SNARE Proteins and Cannabinoid Receptor-1 Modulate Neurotransmitter Release Although in the case of Vamp2 both mutants led to a substantial reduction in exocy- tosis rates, its pseudo-phosphorylated form (Vamp2-S75D) showed a greater effect on exocytosis than the non- phosphorylatable form (Vamp2-S75A). Presumably, the absence of a charged residue did not fully compensate the steric effect caused by the amino acid substitution at position 75 of Vamp2, which has apparently a stronger impact on protein function than the double mutant at positions 21/23 of Stx1a. The S75 phosphorylation site is located within the R-SNARE motif required for forming a coiled-coil complex with Stx1a and Snap25 during exocytosis. In contrast, the T21/T23 positions of Stx1a are located in the regulatory N-terminal helical bundle, explaining why mutations show different effects on exocytosis. Overall, these ﬁndings support the concept that protein phosphorylation is a key regulatory mechanism in neurotransmitter release that modulates different steps in the SV cycle. It seems that several proteins known to function at the postsynapse respond to potassium chloride stimulation and BoNT treatment in synaptosomes (e.g., Shank1, 2, 3; com- ponents of NMDAR, Grin1, Grin2a, Grin2b). It would be of interest to investigate whether these proteins are also present at measurable levels in the presynapse and whether they are affected by synaptic activity. Although synaptosomal prepa- rations are known to contain fragments of the postsynaptic membrane (102), they usually do not retain metabolic activity, in contrast to functionally active membrane-enclosed nerve terminals that can maintain the ATP levels needed for phos- phorylation (50–53). Clearly, the primary trigger for the phosphorylation changes is calcium that enters synaptosomes following potassium- induced depolarization. Calcium directly regulates the activ- ity of kinases and phosphatases, including CaMKII, PKC, calcineurin, PP1, etc. (23, 89, 103, 104). Accordingly, we observe increased phosphorylation of speciﬁc sites on CaM- KII and MAP kinases that are known to result in activation of the kinases as well as enrichment of possible substrates for CaMKII, MAPK, and GSK3 kinases among regulated phos- phorylation sites. 14 Mol Cell Proteomics (2021) 20 100061 Protein Phosphorylation in Depolarized Synaptosomes dephosphorylation of many substrates, the observed effect can be explained either by activation of phosphatase or by suppression of kinase activities that are not caused directly by calcium but by changes in proteins that are themselves regulated by the activity of the SV cycle and in turn control kinases or phosphatases. Moreover, it is conceivable that the availability of phosphorylation sites on substrate proteins is coupled to its “activity cycle.” Trafﬁcking steps such as SV docking, priming, fusion, coat formation, ﬁssion, and SV movement are associated with numerous changes in protein conformation and the dynamic assembly and disassembly of multimolecular complexes. Thus, it is to be expected that phosphorylation site accessibility is dependent on these dy- namic changes, many of which are likely to be arrested following inhibition of the SV cycle. However, the fact that selective inhibition of calcium- activated SV cycling results in major changes of the phos- phoproteome documents that the activation status of the ki- nase/phosphatase networks is directly dependent on the progression of the exo–endocytotic cycle. In most cases, BoNT treatment inhibited phosphorylation, and these results are in line with a very early study that used BoNT-treated synaptosomes from the electric organ of Torpedo marmor- ata and radioactive phosphate for protein labeling (105). Interestingly, phosphorylation events known to be directly regulated by some of the major calcium-dependent kinases and phosphatases such as those on synapsin-1 and dynamin- 1, as well as on many of the predicted CaMKII- phosphorylation sites, were almost unaffected by the BoNT treatment. Conversely, predicted sites of MAPK, PKC, and CDK were affected more strongly by the BoNT treatment. These observations support the view that the activity of MAPK is dependent on active SV cycling, resulting in the phos- phorylation of delta catenin, metabotropic glutamate receptor, and other proteins in the synapse (106–109). Indeed, we observe SV-cycling-dependent phosphorylation changes not only in catenin-delta (Cttnd2) but also in other proteins that might take part in the Wnt pathway such as Apc, Amotl1, and Tnik. Given the connection between the Wnt pathway and the regulation of cell–cell contacts and cytoskeletal elements, it is not surprising that many cytoskeleton- and cell-adhesion- related proteins contain BoNT-responsive phosphorylation sites (e.g., spectrin beta, adducin alpha and beta, neurofascin, neuronal cell adhesion molecule). neurons and exo-/endocytosis was measured 4 days post infection (d.p.i.). In order to evaluate exo-/endocytosis, neurons were ﬁeld-stimulated and changes in ﬂuorescence were measured (82). B, Immunoﬂuorescence of infected neurons. The high efﬁciency of infection and the coex- pression of the POIs (Stx1a; Cnr1) is conﬁrmed by immunoﬂuorescence using a nanobody directed against the ALFA tag (NbALFA; (84)). Scale bar 10 μm (C) The basal ﬂuorescence of Vamp2-pHluorin (left image) allows to identify single synaptic boutons (inset on the right). Following a deﬁned number of action potentials (AP), SVs fuse with the plasma membrane (middle insets on the right). Upon fusion, the intravesicular pH increases, resulting in higher pHluorin ﬂuorescence intensity. Ammonium chloride (NH4Cl) is used at the end of each experiment to reveal the localization of SVs. Scale bars: 10 μm (left image) and 5 μm (right insets). D and E, Both the phosphomimetic and the phosphorylation-null mutants of Vamp2 showed decreased exocytosis, for both short and long train of stimuli, respectively 60 and 600 action potentials (APs). The maximum peak intensity was signiﬁcantly lower in both stimuli (p < 0.001). The fraction of endocytosed Vamp2 (measured at 30 s after the stimulation, as % of the exocytosed Vamp2) was also decreased, especially for the longer stimulus. F and G, The phosphomimetic mutant of Stx1a (T21-T23) enhanced exocytosis for short stimuli, while the phosphorylation-null mutant left exocytosis unaffected, while enhancing endocytosis. Under long stimuli the enhancement of exocytosis was no longer signiﬁcant, but the increase in endocytosis was, for both phosphomimetic and phosphorylation-null mutants. H and I, The phosphomimetic mutants of Cnr1 (T314E-T322 E) reduced exocytosis during short stimuli, while the nonphosphorylatable variant increased exocytosis. The latter effect was also observed during long stimuli. Neither of the Cnr1 variants had a substantial effect on endocytosis. In all panels statistical differences were tested relying on Kruskal–Wallis tests, followed by Tukey–Kramer post-hoc tests ( p < 0.05, p < 0.01 and * p < 0.001). DISCUSSION In this study, we have used isolated nerve terminals as a model to differentiate phosphorylation changes that are induced by calcium inﬂux alone from those that in addition 14 Mol Cell Proteomics (2021) 20 100061 Protein Phosphorylation in Depolarized Synaptosomes FIG. 6. Phosphomimetic modulation of phosphorylation sites found differentially regulated in synaptobrevin (Vamp2), syntaxin-1, and cannabinoid receptor-1 affects SV recycling properties. A, Experimental scheme. Two versions of the adeno-associated viral (AAV) con- structs were prepared (V1 and V2). V1 contained Vamp2 wild-type or the respective Vamp2 mutants (Vamp2-S75D and Vamp2-S75A) fused to the superecliptic (pH-dependent) green ﬂuorescence protein (pHluorin; (82)). The expression of recombinant proteins is driven by the neuron- speciﬁc synapsin-1 promoter. In these experiments the Vamp2-pHluorin is used both as the test protein and as a sensor for exo- and endo- cytosis. V2 contained both WT Vamp2-pHluorin (used as an exo-/endocytosis sensor) and the proteins of interest (POI; respectively Stx1a-WT, Stx1a-T21E-T23E; Stx1a-T21A-T23A; Cnr1-WT; Cnr1-T314E-T322E, and Cnr1-T314A-T322A); see methods for details.To distinguish these proteins from the endogenous versions, an ALFA-tag sequence has been added (84). Mutants were expressed in rat primary hippocampal Protein Phosphorylation in Depolarized Synaptosomes FIG. 6. Phosphomimetic modulation of phosphorylation sites found differentially regulated in synaptobrevin (Vamp2), syntaxin-1, and cannabinoid receptor-1 affects SV recycling properties. A, Experimental scheme. Two versions of the adeno-associated viral (AAV) con- structs were prepared (V1 and V2). V1 contained Vamp2 wild-type or the respective Vamp2 mutants (Vamp2-S75D and Vamp2-S75A) fused to the superecliptic (pH-dependent) green ﬂuorescence protein (pHluorin; (82)). The expression of recombinant proteins is driven by the neuron- speciﬁc synapsin-1 promoter. In these experiments the Vamp2-pHluorin is used both as the test protein and as a sensor for exo- and endo- cytosis. V2 contained both WT Vamp2-pHluorin (used as an exo-/endocytosis sensor) and the proteins of interest (POI; respectively Stx1a-WT, Stx1a-T21E-T23E; Stx1a-T21A-T23A; Cnr1-WT; Cnr1-T314E-T322E, and Cnr1-T314A-T322A); see methods for details.To distinguish these proteins from the endogenous versions, an ALFA-tag sequence has been added (84). Mutants were expressed in rat primary hippocampal Mol Cell Proteomics (2021) 20 100061 15 DATA AVAILABILITY under stimulation. One possible mechanism is the impaired interaction with Munc18-1, which can facilitate SNARE zip- pering and thereby promote SV fusion with the plasma membrane (46). The changes of endocytosis are possibly a consequence of altered exocytosis. In contrast to many other BoNT-responsive phosphorylation sites, the phosphorylation sites on Vamp2 and Stx1a that we examined showed increased phosphorylation following toxin treatment, sug- gesting that the phosphorylation is due to adaptive changes in the synapse rather than a result of SNARE cleavage by BoNT. under stimulation. One possible mechanism is the impaired interaction with Munc18-1, which can facilitate SNARE zip- pering and thereby promote SV fusion with the plasma membrane (46). The changes of endocytosis are possibly a consequence of altered exocytosis. In contrast to many other BoNT-responsive phosphorylation sites, the phosphorylation sites on Vamp2 and Stx1a that we examined showed increased phosphorylation following toxin treatment, sug- gesting that the phosphorylation is due to adaptive changes in the synapse rather than a result of SNARE cleavage by BoNT. In contrast to the phosphomimetic and non- phosphorylatable variants of the SNARE proteins, the corre- sponding variants of cannabinoid recepor-1 (Cnr1) had an effect only on exocytosis in cultured hippocampal neurons. The effect can be also linked through the activation of MAPK to Munc-18 phosphorylation and regulation of SNARE as- sembly (99). Similarly, the intensity of phosphorylation of T314/T321 sites was increased following toxin treatment. Although Vamp2-S75, Stx1a-S21/S23, Cnr1-T314/T321 phosphorylation sites were identiﬁed in previous studies (46, 47, 117, 118), it is the ﬁrst time, to the best of our knowledge, that phosphorylation of these sites has been shown to have an impact on the exo- and endocytosis in the context of stimulation experiments. The mass spectrometry data have been deposited to the ProteomeXchange Consortium via the PRIDE (121) partner repository with the data set identiﬁer PXD020564. R scripts used for data analysis are available at GitHub: https://github.com/IvanSilbern/2021_Silbern_etal_Synapt_ BoNT_KCl. We also provide a visualization tool in a shiny-app (https:// cran.r-project.org/web/packages/shiny/index.html) format. The source data and code are available at GitHub: https:// github.com/IvanSilbern/2021_Silbern_etal_ShinyApp. The app is deployed and accessible at: https://s1608-shiny. mpibpc.mpg.de. Supplemental data —This article contains supplemental data (59, 65, 66, 70, 71, 73–76, 78, 122, 123). DATA AVAILABILITY Acknowledgments —We sincerely thank Thomas Gundlach and Sascha Krause for assistance in animal experiments; Soﬁa Ainatzi and Dr Momchil Ninov for help in synaptosome preparations; Monika Raabe, Uwe Plessmann, Dr Sabine König, and Dr Ralf Pﬂanz for technical support; Heiko Ludwig for launching of the shiny-server. Interestingly, we also found phosphorylation sites on Munc18 that are regulated in SV-cycling-dependent manner, namely Stxbp1-S313 and Stxbp1-S506. Both sites were signiﬁcantly downregulated in the BoNT-treated samples. Although not much known about S506, S313 site was previ- ously described as a possible target of PKC and that its phosphorylation can negatively affect interaction of Munc18 with syntaxin-1 (48, 119). Funding and additional information —S. B., S. O. R., H. U., and R. J. are funded by the Deutsche For- schungsgemeinschaft through collaborative research center SFB1286, projects A08, A03, Z02, and Z03. Funding and additional information —S. B., S. O. R., H. U., and R. J. are funded by the Deutsche For- schungsgemeinschaft through collaborative research center SFB1286, projects A08, A03, Z02, and Z03. Author contributions —R. J., H. U., and I. S. conceptualized the study; E. F. conceptualized and performed phosphomi- metic studies in neurons; I. S. performed synaptosomal experiments under the supervision of K. T. P., H. U., and R. J.; I. S. analyzed the data; M. F. and S. B. contributed to the bioinformatics analysis; initial draft was written by I. S. and H. U., reviewed and edited by H. U, R. J., E. F., S. O. R., and K. T. P. Author contributions —R. J., H. U., and I. S. conceptualized the study; E. F. conceptualized and performed phosphomi- metic studies in neurons; I. S. performed synaptosomal experiments under the supervision of K. T. P., H. U., and R. J.; I. S. analyzed the data; M. F. and S. B. contributed to the bioinformatics analysis; initial draft was written by I. S. and H. U., reviewed and edited by H. U, R. J., E. F., S. O. R., and K. T. P. A remarkable aspect of the synapse phosphoproteome regulation has recently been addressed by F. Brüning, et al. (120). Their analysis shows that phosphorylation sites on many synaptic proteins undergo oscillations during the course of the day. Protein Phosphorylation in Depolarized Synaptosomes It is tempting to speculate that these phosphorylation changes represent the activation of pathways directed at increasing synaptic strength (most importantly LTP) by mechanisms such as size increase of the synaptic contact zone, enlargement of the presynaptic bou- ton, and adaptations of the presynaptic cytoskeleton to in- crease the efﬁciency of SV cycling (110–113). An interesting example is given by the PP1 whose post- synaptic activation appears to be dependent on synaptic ac- tivity (114). Neurabin-1 (Ppp1r9a) can play a crucial role in this, by recruiting PP1 to the synapse by regulating its binding to actin ﬁlaments, which impacts PP1 activity (115, 116). Our observations suggest that PP1 undergoes similar activity- dependent regulation also in the presynapse as we observe “SV-cycling-dependent” sites within the actin-binding region of Neurabin-1. It is tempting to speculate whether SV-cycling- dependent activity of PP1 or other phosphatases can be opposed by Ca2+-activated kinases such as CaMKII, MAPK1, MAPK3. This would explain reduced intensities of predicted MAPK substrates in Ca/EGTA experiments despite apparent activation of MAPK following K+-induced depolarization and Ca2+ inﬂux. Furthermore, it would explain why many phos- phosites appear signiﬁcantly regulated when comparing mock-with BoNT-treated synaptosomes, but do not show signiﬁcant changes in Ca/EGTA experiments, since compari- son of Ca2+ with EGTA conditions reveals a composite effect of Ca2+ inﬂux and SV cycling on protein phosphorylation. Experiments involving BoNT also led us to the discovery of phosphorylation sites on the core SNARE proteins, sites that otherwise did not appear signiﬁcant. We showed that these sites can affect exo- and endocytosis in hippocampal neurons How can the wide-ranging neurotoxin effects be explained when considering the extraordinary speciﬁcity of the SNARE proteases, which should have no direct effect on kinases or phosphatases? Given that toxin treatment causes 16 Mol Cell Proteomics (2021) 20 100061 Protein Phosphorylation in Depolarized Synaptosomes Protein Phosphorylation in Depolarized Synaptosomes REFERENCES 26. De Camilli, P., Benfenati, F., Valtorta, F., and Greengard, P. (1990) The synapsins. Annu. Rev. Cel. Biol. 6, 433–460 1. Del Castillo, J., and Katz, B. (1956) Biophysical aspects of neuromuscular transmission. Prog. Biophys. Biophys. Chem. 6, 121–170 1. Del Castillo, J., and Katz, B. (1956) Biophysical aspects of neuromuscular transmission. Prog. Biophys. Biophys. Chem. 6, 121–170 27. Robinson, P. J. (1992) Differential stimulation of protein kinase C activity by phorbol ester or calcium/phosphatidylserine in vitro and in intact synaptosomes. J. Biol. Chem. 267, 21637–21644 2. Schneggenburger, R., and Neher, E. (2005) Presynaptic calcium and control of vesicle fusion. Curr. Opin. Neurobiol. 15, 266–274 28. Robinson, P. J., Sontag, J.-M., Liu, J.-P., Fykse, E. M., Slaughter, C., McMahontt, H., and Südhof, T. C. (1993) Dynamin GTPase regulated by protein kinase C phosphorylation in nerve terminals. Nature 365, 163– 166 3. Geppert, M., Goda, Y., Hammer, R. E., Li, C., Rosahl, T. W., Stevens, C. F., and Südhof, T. C. (1994) Synaptotagmin I: A major Ca2+ sensor for transmitter release at a central synapse. Cell 79, 717–727 2 4. Chapman, E. R. (2002) Synaptotagmin: A Ca2+ sensor that triggers exocytosis? Nat. Rev. Mol. Cell Biol. 3, 498–508 29. Tan, T. C., Valova, V. A., Malladi, C. S., Graham, M. E., Berven, L. A., Jupp, O. J., Hansra, G., McClure, S. J., Sarcevic, B., and Boadle, R. A. (2003) Cdk5 is essential for synaptic vesicle endocytosis. Nat. Cel. Biol. 5, 701–710 5. Söllner, T., Whiteheart, S. W., Brunner, M., Erdjument-Bromage, H., Ger- omanos, S., Tempst, P., and Rothman, J. E. (1993) SNAP receptors implicated in vesicle targeting and fusion. Nature 362, 318–324 30. Floyd, S. R., Porro, E. B., Slepnev, V. I., Ochoa, G.-C., Tsai, L.-H., and De Camilli, P. (2001) Amphiphysin 1 binds the cyclin-dependent kinase (cdk) 5 regulatory subunit p35 and is phosphorylated by cdk5 and cdc2. J. Biol. Chem. 276, 8104–8110 2 6. Chapman, E. R., An, S., Barton, N., and Jahn, R. (1994) SNAP-25, a t- SNARE which binds to both syntaxin and synaptobrevin via domains that may form coiled coils. J. Biol. Chem. 269, 27427–27432 31. Sihra, T., Bogonez, E., and Nicholls, D. (1992) Localized Ca2+ entry pref- erentially effects protein dephosphorylation, phosphorylation, and glutamate release. J. Biol. Chem. 267, 1983–1989 7. Toonen, R. F., and Verhage, M. (2007) Munc18-1 in secretion: Lonely Munc joins SNARE team and takes control. Trends Neurosci. 30, 564– 572 32. DATA AVAILABILITY Interestingly, a few of these sites are shown to be regulated upon depolarization by potassium chloride and treatment with botulinum neurotoxins, including phosphory- lation sites on proteins previously discussed: Cnr1, Vamp2, Stx1a, Cttnd2, Add1, Add2, Sptbn1, Sptbn4, Kcnb1, Kcnd2, Hcn2, Kcnma1, Tnik, Apc, Camk2a, etc. This also suggests that, despite the caveat that potassium-induced depolariza- tion is nonphysiological (42), it allows for identifying regulated phosphorylation sites that are of high functional relevance for normal synapse physiology. Conﬂict of interest—The authors declare that they have no conﬂicts of interest with the contents of this article. Abbreviations —The abbreviations used are: AAV, adeno- associated virus; ACN, acetonitrile; AGC, protein kinase A, G, C kinase group; AGC, automatic gain control; AMPAR, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid re- ceptor; AP, action potential; BoNT, botulinum neurotoxin; bRP, basic reversed-phase chromatography; CAA, chlor- oacetamide; CaMKII, calcium-calmodulin kinase 2; CDK, cyclin-dependent kinase; CK1, casein kinase 1; CK2, casein kinase 2; CLK, SRPK1 and Clk/Sty protein kinase; CMGC, CDK, MAP, GSK, CDKL kinase group; CREB1, CAMP- responsive element binding protein 1; DAPK, death-associ- ated protein kinase; FA, formic acid; GABA, γ-aminobutyric acid; GluDH, glutamate dehydrogenase; GO, gene ontology; GRK, G-protein-coupled receptor kinase; GSK3, glycogen Despite major progress, many aspects of synapse regula- tion remain to be uncovered. Our study has added another layer of complexity by showing how intricately the phosphorylation–dephosphorylation cascade in the synapse is linked to SV cycling. We believe that the results presented here provide a ﬁrm basis for future investigations about how synaptic activity and synaptic plasticity are regulated at the molecular level. Mol Cell Proteomics (2021) 20 100061 17 Protein Phosphorylation in Depolarized Synaptosomes synthase kinase 3; ITR, inverted terminal repeat (sequence); MAPK, mitogen-activated protein kinase; NMDAR, N-methyl- d-aspartate receptor; PAK, p21-activated kinase; PKC, protein kinase C; PNGase F, peptide-N(4)-(N-acetyl-beta- glucosaminyl)asparagine amidase; POI, protein of interest; PP1, protein phosphatase 1; RAS, rat sarcoma gene; RT, room temperature; SNAP-25, synaptosomal-associated pro- tein, 25kDa; SNARE, N-ethylmaleimide-sensitive factor- attachment protein receptors; STE, “sterile” serine/threonine protein kinases; SV, synaptic vesicle; TCEP, tris(2- carboxyethyl)phosphine; TEAB, triethylammonium bicarbon- ate buffer; TFE, triﬂuoroethanol. 20. Gorelick, F., Wang, J., Lai, Y., Nairn, A., and Greengard, P. (1988) Auto- phosphorylation and activation of Ca2+/calmodulin-dependent protein kinase II in intact nerve terminals. J. Biol. Chem. 263, 17209–17212 21. Walaas, S. I., Gorelick, F. S., and Greengard, P. (1989) Presence of cal- cium/calmodulin-dependent protein Kinase II in Nerve terminals of rat brain. Synapse 3, 356–362 22. REFERENCES Liu, J.-P., Sim, A., and Robinson, P. J. (1994) Calcineurin inhibition of dynamin I GTPase activity coupled to nerve terminal depolarization. Science 265, 970–973 8. Brose, N. (2008) Altered complexin expression in psychiatric and neuro- logical disorders: Cause or consequence? Mol. Cell. 25, 7–19 9. Ceccarelli, B., Hurlbut, W., and Mauro, A. (1973) Turnover of transmitter and synaptic vesicles at the frog neuromuscular junction. J. Cel. Biol. 57, 499–524 33. Nichols, R. A., Suplick, G. R., and Brown, J. M. (1994) Calcineurin-medi- ated protein dephosphorylation in brain nerve terminals regulates the release of glutamate. J. Biol. Chem. 269, 23817–23823 10. Fesce, R., Grohovaz, F., Valtorta, F., and Meldolesi, J. (1994) Neuro- transmitter release: Fusion or ‘kiss-and-run’? Trends Cell Biol. 4, 1–4 34. Bauerfeind, R., Takei, K., and De Camilli, P. (1997) Amphiphysin I is associated with coated endocytic intermediates and undergoes stimulation-dependent dephosphorylation in nerve terminals. J. Biol. Chem. 272, 30984–30992 11. Heuser, J., and Reese, T. (1973) Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction. J. Cel. Biol. 57, 315–344 35. Marks, B., and McMahon, H. T. (1998) Calcium triggers calcineurin- dependent synaptic vesicle recycling in mammalian nerve terminals. Curr. Biol. 8, 740–749 12. Von Gersdorff, H., and Mathews, G. (1994) Dynamics of synaptic vesicle fusion and membrane retrieval in synaptic terminals. Nature 367, 735– 739 36. Cousin, M. A., Tan, T. C., and Robinson, P. J. (2001) Protein phosphory- lation is required for endocytosis in nerve terminals: Potential role for the dephosphins dynamin I and synaptojanin, but not AP180 or amphi- physin. J. Neurochem. 76, 105–116 13. Boucrot, E., Ferreira, A. P., Almeida-Souza, L., Debard, S., Vallis, Y., Howard, G., Bertot, L., Sauvonnet, N., and McMahon, H. T. (2015) Endophilin marks and controls a clathrin-independent endocytic pathway. Nature 517, 460–465 37. Schmitz, S. K., King, C., Kortleven, C., Huson, V., Kroon, T., Kevenaar, J. T., Schut, D., Saarloos, I., Hoetjes, J. P., and de Wit, H. (2016) Pre- synaptic inhibition upon CB 1 or mG lu2/3 receptor activation requires ERK/MAPK phosphorylation of Munc18-1. EMBO J. 35, 1236–1250 14. Clayton, E. L., Evans, G. J., and Cousin, M. A. (2008) Bulk synaptic vesicle endocytosis is rapidly triggered during strong stimulation. J. Neurosci. 28, 6627–6632 15. Watanabe, S., Rost, B. R., Camacho-P ´erez, M., Davis, M. W., Söhl- Kielczynski, B., Rosenmund, C., and Jorgensen, E. M. (2013) Ultrafast endocytosis at mouse hippocampal synapses. DATA AVAILABILITY Ninan, I., and Arancio, O. (2004) Presynaptic CaMKII is necessary for synaptic plasticity in cultured hippocampal neurons. Neuron 42, 129– 141 23. Lisman, J., Schulman, H., and Cline, H. (2002) The molecular basis of CaMKII function in synaptic and behavioural memory. Nat. Rev. Neu- rosci. 3, 175–190 24. Greengard, P., Benfenati, F., and Valtorta, F. (1994) Synapsin I, an actin- binding protein regulating synaptic vesicle trafﬁc in the nerve terminal. Adv. second messenger phosphoprotein Res. 29, 31–45 25. Ceccaldi, P.-E., Grohovaz, F., Benfenati, F., Chieregatti, E., Greengard, P., and Valtorta, F. (1995) Dephosphorylated synapsin I anchors synaptic vesicles to actin cytoskeleton: An analysis by videomicroscopy. J. Cell Biol. 128, 905–912 Received January 15, 2021 Published, MCPRO Papers in Press, February 12, 2021, https://doi.org/10.1016/j.mcpro.2021.100061 Received January 15, 2021 Published, MCPRO Papers in Press, February 12, 2021, https://doi.org/10.1016/j.mcpro.2021.100061 Protein Phosphorylation in Depolarized Synaptosomes Jassal, B., Matthews, L., Viteri, G., Gong, C., Lorente, P., Fabregat, A., Sidiropoulos, K., Cook, J., Gillespie, M., and Haw, R. (2020) The reac- tome pathway knowledgebase. Nucleic Acids Res. 48, D498–D503 0 K F Ni P A d Al M B A Cij T 47. Li, J., Han, Y. R., Plummer, M. R., and Herrup, K. (2009) Cytoplasmic ATM in neurons modulates synaptic function. Curr. Biol. 19, 2091–2096 48 C i T J E G J d M A (2003) Ph i l i l l i f 70. Koopmans, F., van Nierop, P., Andres-Alonso, M., Byrnes, A., Cijsouw, T., Coba, M. P., Cornelisse, L. N., Farrell, R. J., Goldschmidt, H. L., and Howrigan, D. P. (2019) SynGO: An evidence-based, expert-curated knowledge base for the synapse. Neuron 103, 217–234.e214 48. Craig, T. J., Evans, G. J., and Morgan, A. (2003) Physiological regulation of Munc18/nSec1 phosphorylation on serine-313. J. Neurochem. 86, 1450–1457 71. Hornbeck, P. V., Zhang, B., Murray, B., Kornhauser, J. M., Latham, V., and Skrzypek, E. (2015) PhosphoSitePlus, 2014: Mutations, PTMs and recalibrations. Nucleic Acids Res. 43, D512–D520 49. Garcia, D., Brown, S., Hille, B., and Mackie, K. (1998) Protein kinase C disrupts cannabinoid actions by phosphorylation of the CB1 cannabi- noid receptor. J. Neurosci. 18, 2834–2841 72. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) Basic local alignment search tool. J. Mol. Biol. 215, 403–410 50. Scott, I., and Nicholls, D. (1980) Energy transduction in intact synapto- somes. Inﬂuence of plasma-membrane depolarization on the respiration and membrane potential of internal mitochondria determined in situ. Biochem. J. 186, 21–33 73. Miller, M. L., Jensen, L. J., Diella, F., Jørgensen, C., Tinti, M., Li, L., Hsiung, M., Parker, S. A., Bordeaux, J., and Sicheritz-Ponten, T. (2008) Linear motif atlas for phosphorylation-dependent signaling. Sci. signaling 1, ra2 51. Kauppinen, R. A., and Nicholls, D. G. (1986) Synaptosomal bioenergetics. Eur. J. Biochem. 158, 159–165 74. Horn, H., Schoof, E. M., Kim, J., Robin, X., Miller, M. L., Diella, F., Palma, A., Cesareni, G., Jensen, L. J., and Linding, R. (2014) KinomeXplorer: An integrated platform for kinome biology studies. Nat. Methods 11, 603– 604 52. Nicholls, D. G. (2003) Bioenergetics and transmitter release in the isolated nerve terminal. Neurochem. Res. 28, 1433–1441 75. Szklarczyk, D., Gable, A. L., Lyon, D., Junge, A., Wyder, S., Huerta-Cepas, J., Simonovic, M., Doncheva, N. T., Morris, J. Protein Phosphorylation in Depolarized Synaptosomes phase chromatography with multiple fraction concatenation strategy for proteome proﬁling of human MCF10A cells. Proteomics 11, 2019–2026 phase chromatography with multiple fraction concatenation strategy for proteome proﬁling of human MCF10A cells. Proteomics 11, 2019–2026 41. Kohansal-Nodehi, M., Chua, J. J., Urlaub, H., Jahn, R., and Czernik, D. (2016) Analysis of protein phosphorylation in nerve terminal reveals extensive changes in active zone proteins upon exocytosis. Elife 5, e14530 63. Cox, J., Neuhauser, N., Michalski, A., Scheltema, R. A., Olsen, J. V., and Mann, M. (2011) Andromeda: A peptide search engine integrated into the MaxQuant environment. J. proteome Res. 10, 1794–1805 42. Engholm-Keller, K., Waardenberg, A. J., Müller, J. A., Wark, J. R., Fer- nando, R. N., Arthur, J. W., Robinson, P. J., Dietrich, D., Schoch, S., and Graham, M. E. (2019) The temporal proﬁle of activity-dependent pre- synaptic phospho-signalling reveals long-lasting patterns of post- stimulus regulation. PLoS Biol. 17, e3000170 64. Tyanova, S., Temu, T., and Cox, J. (2016) The MaxQuant computational platform for mass spectrometry-based shotgun proteomics. Nat. Pro- toc. 11, 2301 65. Consortium, U. (2019) UniProt: A worldwide hub of protein knowledge. Nucleic Acids Res. 47, D506–D515 43. Gao, J., Hirata, M., Mizokami, A., Zhao, J., Takahashi, I., Takeuchi, H., and Hirata, M. (2016) Differential role of SNAP-25 phosphorylation by protein kinases A and C in the regulation of SNARE complex formation and exocytosis in PC12 cells. Cell Signal. 28, 425–437 66. Smyth, G. K. (2005) Limma: Linear models for microarray data. Bioinfor- matics Computational Biology Solutions Using R. Bioconductor, 397– 420. Springer 44. Tian, J.-H., Das, S., and Sheng, Z.-H. (2003) Ca2+-dependent phosphor- ylation of syntaxin-1A by the death-associated protein (DAP) kinase regulates its interaction with Munc18. J. Biol. Chem. 278, 26265–26274 67. Storey, J. D., and Tibshirani, R. (2003) Statistical signiﬁcance for genomewide studies. Proc. Natl. Acad. Sci. 100, 9440–9445 45. Rickman, C., and Duncan, R. R. (2010) Munc18/Syntaxin interaction ki- netics control secretory vesicle dynamics. J. Biol. Chem. 285, 3965– 3972 68. Jiao, X., Sherman, B. T., Huang, D. W., Stephens, R., Baseler, M. W., Lane, H. C., and Lempicki, R. A. (2012) DAVID-WS: A stateful web service to facilitate gene/protein list analysis. Bioinformatics 28, 1805– 1806 46. Shen, C., Rathore, S. S., Yu, H., Gulbranson, D. R., Hua, R., Zhang, C., Schoppa, N. E., and Shen, J. (2015) The trans-SNARE-regulating function of Munc18-1 is essential to synaptic exocytosis. Nat. Com- mun. 6, 1–10 69. Protein Phosphorylation in Depolarized Synaptosomes H., and Bork, P. (2019) STRING v11: Protein–protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets. Nucleic Acids Res. 47, D607–D613 53. Choi, S. W., Gerencser, A. A., Lee, D. W., Rajagopalan, S., Nicholls, D. G., Andersen, J. K., and Brand, M. D. (2011) Intrinsic bioenergetic properties and stress sensitivity of dopaminergic synaptosomes. J. Neurosci. 31, 4524–4534 54. Sanchez-Prieto, J., Sihra, T. S., Evans, D., Ashton, A., Dolly, J. O., and Nicholls, D. G. (1987) Botulinum toxin A blocks glutamate exocytosis from Guinea-pig cerebral cortical synaptosomes. Eur. J. Biochem. 165, 675–681 76. Roy, J., and Cyert, M. S. (2009) Cracking the phosphatase code: Docking interactions determine substrate speciﬁcity. Sci. Signaling 2, re9 77. Csardi, G., and Nepusz, T. (2006) The igraph software package for com- plex network research. Inter J Complex Syst. 1695, 1–9 55. McMahon, H., Foran, P., Dolly, J., Verhage, M., Wiegant, V. M., and Nicholls, D. (1992) Tetanus toxin and botulinum toxins type A and B inhibit glutamate, gamma-aminobutyric acid, aspartate, and met- enkephalin release from synaptosomes. Clues to the locus of action. J. Biol. Chem. 267, 21338–21343 78. Hogrebe, A., von Stechow, L., Bekker-Jensen, D. B., Weinert, B. T., Kelstrup, C. D., and Olsen, J. V. (2018) Benchmarking common quan- tiﬁcation strategies for large-scale phosphoproteomics. Nat. Commun. 9, 1–13 79. Dignam, J. D., Lebovitz, R. M., and Roeder, R. G. (1983) Accurate tran- scription initiation by RNA polymerase II in a soluble extract from iso- lated mammalian nuclei. Nucleic Acids Res. 11, 1475–1489 56. Blaustein, M. (1975) Effects of potassium, veratridine, and scorpion venom on calcium accumulation and transmitter release by nerve terminals in vitro. J. Physiol. 247, 617–655 57. McMahon, H. T., and Nicholls, D. G. (1991) Transmitter glutamate release from isolated nerve terminals: Evidence for biphasic release and trig- gering by localized Ca2+. J. Neurochem. 56, 86–94 80. Komulainen, H., and Bondy, S. (1987) The estimation of free calcium within synaptosomes and mitochondria with fura-2; comparison to quin-2. Neurochem. Int. 10, 55–64 g g y 58. von Mollard, G. F., Südhof, T. C., and Jahn, R. (1991) A small GTP-binding protein dissociates from synaptic vesicles during exocytosis. Nature 349, 79–81 81. Truckenbrodt, S., Viplav, A., Jähne, S., Vogts, A., Denker, A., Wildhagen, H., Fornasiero, E. F., and Rizzoli, S. O. (2018) Newly produced synaptic vesicle proteins are preferentially used in synaptic transmission. EMBO J. 37, e98044 59. Nicholls, D. REFERENCES Nature 504, 242–247 38. Jovanovic, J. N., Benfenati, F., Siow, Y., Sihra, T. S., Sanghera, J. S., Pelech, S. L., Greengard, P., and Czernik, A. J. (1996) Neurotrophins stimulate phosphorylation of synapsin I by MAP kinase and regulate synapsin I-actin interactions. Proc. Natl. Acad. Sci. 93, 3679–3683 16. Gan, Q., and Watanabe, S. (2018) Synaptic vesicle endocytosis in different model systems. Front. Cell. Neurosci. 12, 171 39. Impey, S., Obrietan, K., Wong, S. T., Poser, S., Yano, S., Wayman, G., Deloulme, J. C., Chan, G., and Storm, D. R. (1998) Cross talk between ERK and PKA is required for Ca2+ stimulation of CREB-dependent transcription and ERK nuclear translocation. Neuron 21, 869–883 17. Chanaday, N. L., Cousin, M. A., Milosevic, I., Watanabe, S., and Morgan, J. R. (2019) The synaptic vesicle cycle revisited: New insights into the modes and mechanisms. J. Neurosci. 39, 8209–8216 18. Edwards, R. H. (2007) The neurotransmitter cycle and quantal size. Neuron 55, 835–858 40. Levenson, J. M., O'Riordan, K. J., Brown, K. D., Trinh, M. A., Molfese, D. L., and Sweatt, J. D. (2004) Regulation of histone acetylation during memory formation in the hippocampus. J. Biol. Chem. 279, 40545– 40559 19. Sihra, T. S. (1997) Protein phosphorylation and dephosphorylation in iso- lated nerve terminals (synaptosomes). Regul. Protein Modiﬁcation, 67– 119. Springer 18 Mol Cell Proteomics (2021) 20 100061 Protein Phosphorylation in Depolarized Synaptosomes Protein Phosphorylation in Depolarized Synaptosomes Protein Phosphorylation in Depolarized Synaptosomes Opazo, F. (2019) The ALFA-tag is a highly versatile tool for nanobody- based bioscience applications. Nat. Commun. 10, 1–12 Opazo, F. (2019) The ALFA-tag is a highly versatile tool for nanobody- based bioscience applications. Nat. Commun. 10, 1–12 105. Guitart, X., Egea, G., Solsona, C., and Marsal, J. (1987) Botulinum neurotoxin inhibits depolarization-stimulated protein phosphorylation in pure cholinergic synaptosomes. FEBS Lett. 219, 219–223 85. Fornasiero, E. F., Raimondi, A., Guarnieri, F. C., Orlando, M., Fesce, R., Benfenati, F., and Valtorta, F. (2012) Synapsins contribute to the dy- namic spatial organization of synaptic vesicles in an activity-dependent manner. J. Neurosci. 32, 12214–12227 106. Boggio, E. M., Putignano, E., Sassoe-Pognetto, M., Pizzorusso, T., and Giustetto, M. (2007) Visual stimulation activates ERK in synaptic and somatic compartments of rat cortical neurons with parallel kinetics. PLoS One 2, e604 86. Klee, C., Crouch, T., and Krinks, M. (1979) Calcineurin: A calcium-and calmodulin-binding protein of the nervous system. Proc. Natl. Acad. Sci. 76, 6270–6273 107. Edbauer, D., Cheng, D., Batterton, M. N., Wang, C.-F., Duong, D. M., Yaffe, M. B., Peng, J., and Sheng, M. (2009) Identiﬁcation and charac- terization of neuronal mitogen-activated protein kinase substrates using a speciﬁc phosphomotif antibody. Mol. Cell Proteomics 8, 681–695 87. Wang, Z.-W. (2008) Regulation of synaptic transmission by presynaptic CaMKII and BK channels. Mol. Neurobiol. 38, 153–166 88. Cousin, M. A., and Robinson, P. J. (2001) The dephosphins: Dephos- phorylation by calcineurin triggers synaptic vesicle endocytosis. Trends Neurosci. 24, 659–665 108. Park, J. M., Hu, J.-H., Milshteyn, A., Zhang, P.-W., Moore, C. G., Park, S., Datko, M. C., Domingo, R. D., Reyes, C. M., and Wang, X. J. (2013) A prolyl-isomerase mediates dopamine-dependent plasticity and cocaine motor sensitization. Cell 154, 637–650 89. Rosenberg, O. S., Deindl, S., Sung, R.-J., Nairn, A. C., and Kuriyan, J. (2005) Structure of the autoinhibited kinase domain of CaMKII and SAXS analysis of the holoenzyme. Cell 123, 849–860 109. Mao, L.-M., and Wang, J. Q. (2016) Synaptically localized mitogen- activated protein kinases: Local substrates and regulation. Mol. Neu- robiol. 53, 6309–6315 90. Huttner, W., DeGennaro, L., and Greengard, P. (1981) Differential phos- phorylation of multiple sites in puriﬁed protein I by cyclic AMP- dependent and calcium-dependent protein kinases. J. Biol. Chem. 256, 1482–1488 110. Ataman, B., Ashley, J., Gorczyca, M., Ramachandran, P., Fouquet, W., Sigrist, S. J., and Budnik, V. Protein Phosphorylation in Depolarized Synaptosomes Mol. Cell. Biol. 22, 4690–4701 96. Gonz ´alez, C., Baez-Nieto, D., Valencia, I., Oyarzún, I., Rojas, P., Naranjo, D., and Latorre, R. (2012) K+ channels: Function-structural overview. Compr. Physiol. 2, 2087–2149 97. Chua, J. J., Kindler, S., Boyken, J., and Jahn, R. (2010) The architecture of an excitatory synapse. J. Cel. Sci. 123, 819–823 117. Dubois, T., Kerai, P., Learmonth, M., Cronshaw, A., and Aitken, A. (2002) Identiﬁcation of syntaxin-1A sites of phosphorylation by casein kinase I and casein kinase II. Eur. J. Biochem. 269, 909–914 98. Rizzoli, S. O., and Betz, W. J. (2005) Synaptic vesicle pools. Nat. Rev. Neurosci. 6, 57–69 118. Trinidad, J. C., Barkan, D. T., Gulledge, B. F., Thalhammer, A., Sali, A., Schoepfer, R., and Burlingame, A. L. (2012) Global identiﬁcation and characterization of both O-GlcNAcylation and phosphorylation at the murine synapse. Mol. Cell Proteomics 11, 215–229 99. Marsicano, G., Goodenough, S., Monory, K., Hermann, H., Eder, M., Cannich, A., Azad, S. C., Cascio, M. G., Guti ´errez, S. O., and Van der Stelt, M. (2003) CB1 cannabinoid receptors and on-demand defense against excitotoxicity. Science 302, 84–88 119. Barclay, J. W., Craig, T. J., Fisher, R. J., Ciufo, L. F., Evans, G. J., Morgan, A., and Burgoyne, R. D. (2003) Phosphorylation of Munc18 by protein kinase C regulates the kinetics of exocytosis. J. Biol. Chem. 278, 10538– 10545 100. Kellogg, R., Mackie, K., and Straiker, A. (2009) Cannabinoid CB1 receptor- dependent long-term depression in autaptic excitatory neurons. J. Neurophysiol. 102, 1160–1171 101. Hsieh-Wilson, L. C., Benfenati, F., Snyder, G. L., Allen, P. B., Nairn, A. C., and Greengard, P. (2003) Phosphorylation of spinophilin modulates its interaction with actin ﬁlaments. J. Biol. Chem. 278, 1186–1194 120. Brüning, F., Noya, S. B., Bange, T., Koutsouli, S., Rudolph, J. D., Tya- garajan, S. K., Cox, J., Mann, M., Brown, S. A., and Robles, M. S. (2019) Sleep-wake cycles drive daily dynamics of synaptic phosphorylation. Science 366, eaav3617 102. Dunkley, P. R., Heath, J. W., Harrison, S. M., Jarvie, P. E., Glenﬁeld, P. J., and Rostas, J. A. (1988) A rapid Percoll gradient procedure for isolation of synaptosomes directly from an S1 fraction: Homogeneity and morphology of subcellular fractions. Brain Res. 441, 59–71 121. Perez-Riverol, Y.,Csordas,A.,Bai, J.,Bernal-Llinares,M.,Hewapathirana, S., Kundu, D. J., Inuganti, A., Griss, J., Mayer, G., and Eisenacher, M. (2019) The PRIDE database and related tools and resources in 2019: Improving support for quantiﬁcation data. Nucleic Acids Res. Protein Phosphorylation in Depolarized Synaptosomes (2008) Rapid activity-dependent modiﬁca- tions in synaptic structure and function require bidirectional Wnt signaling. Neuron 57, 705–718 91. Czernik, A. J., Pang, D. T., and Greengard, P. (1987) Amino acid se- quences surrounding the cAMP-dependent and calcium/calmodulin- dependent phosphorylation sites in rat and bovine synapsin I. Proc. Natl. Acad. Sci. 84, 7518–7522 111. Cerpa, W., Godoy, J. A., Alfaro, I., Farías, G. G., Metcalfe, M. J., Fuen- tealba, R., Bonansco, C., and Inestrosa, N. C. (2008) Wnt-7a modulates the synaptic vesicle cycle and synaptic transmission in hippocampal neurons. J. Biol. Chem. 283, 5918–5927 92. Wang, J., Walaas, S., and Greengard, P. (1988) Protein phosphorylation in nerve terminals: Comparison of calcium/calmodulin-dependent and calcium/diacylglycerol-dependent systems. J. Neurosci. 8, 281–288 112. Budnik, V., and Salinas, P. C. (2011) Wnt signaling during synaptic development and plasticity. Curr. Opin. Neurobiol. 21, 151–159 93. Matsubara, M., Kusubata, M., Ishiguro, K., Uchida, T., Titani, K., and Taniguchi, H. (1996) Site-speciﬁc phosphorylation of synapsin I by mitogen-activated protein kinase and Cdk5 and its effects on physio- logical functions. J. Biol. Chem. 271, 21108–21113 113. Ciani, L., Marzo, A., Boyle, K., Stamatakou, E., Lopes, D. M., Anane, D., McLeod, F., Rosso, S. B., Gibb, A., and Salinas, P. C. (2015) Wnt sig- nalling tunes neurotransmitter release by directly targeting Synapto- tagmin-1. Nat. Commun. 6, 1–13 114. Woolfrey, K. M., and Dell'Acqua, M. L. (2015) Coordination of protein phosphorylation and dephosphorylation in synaptic plasticity. J. Biol. Chem. 290, 28604–28612 94. Menegon, A., Dunlap, D. D., Castano, F., Benfenati, F., Czernik, A. J., Greengard, P., and Valtorta, F. (2000) Use of phosphosynapsin I-speciﬁc antibodies for image analysis of signal transduction in single nerve terminals. J. Cel. Sci. 113, 3573–3582 115. Allen, P. B., Greenﬁeld, A. T., Svenningsson, P., Haspeslagh, D. C., and Greengard, P. (2004) Phactrs 1–4: A family of protein phosphatase 1 and actin regulatory proteins. Proc. Natl. Acad. Sci. 101, 7187–7192 95. Cesca, F., Baldelli, P., Valtorta, F., and Benfenati, F. (2010) The synapsins: Key actors of synapse function and plasticity. Prog. Neurobiol. 91, 313– 348 actin regulatory proteins. Proc. Natl. Acad. Sci. 101, 718 16 Oli C J T L R T Elli E Bl W 116. Oliver, C. J., Terry-Lorenzo, R. T., Elliott, E., Bloomer, W. A. C., Li, S., Brautigan, D. L., Colbran, R. J., and Shenolikar, S. (2002) Targeting protein phosphatase 1 (PP1) to the actin cytoskeleton: The neurabin I/ PP1 complex regulates cell morphology. Protein Phosphorylation in Depolarized Synaptosomes G., Sihra, T. S., and Sanchez-Prieto, J. (1987) Calcium- dependent and-independent release of glutamate from synaptosomes monitored by continuous ﬂuorometry. J. Neurochem. 49, 50–57 82. Sankaranarayanan, S., De Angelis, D., Rothman, J. E., and Ryan, T. A. (2000) The use of pHluorins for optical measurements of presynaptic activity. Biophysical J. 79, 2199–2208 60. Wessel, D.m., and Flügge, U. (1984) A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal. Biochem. 138, 141–143 83. Keihani, S., Kluever, V., Mandad, S., Bansal, V., Rahman, R., Fritsch, E., Gomes, L. C., Gärtner, A., Kügler, S., and Urlaub, H. (2019) The long noncoding RNA neuroLNC regulates presynaptic activity by interacting with the neurodegeneration-associated protein TDP-43. Sci. Adv. 5, eaay2670 61. Humphrey, S. J., Azimifar, S. B., and Mann, M. (2015) High-throughput phosphoproteomics reveals in vivo insulin signaling dynamics. Nat. Biotechnol. 33, 990–995 62. Wang, Y., Yang, F., Gritsenko, M. A., Wang, Y., Clauss, T., Liu, T., Shen, Y., Monroe, M. E., Lopez-Ferrer, D., and Reno, T. (2011) Reversed- 84. Götzke, H., Kilisch, M., Martínez-Carranza, M., Sograte-Idrissi, S., Rajavel, A., Schlichthaerle, T., Engels, N., Jungmann, R., Stenmark, P., and Mol Cell Proteomics (2021) 20 100061 19 19 Protein Phosphorylation in Depolarized Synaptosomes 47, D442–D450 103. Grossman, S. D., Futter, M., Snyder, G. L., Allen, P. B., Nairn, A. C., Greengard, P., and Hsieh-Wilson, L. C. (2004) Spinophilin is phos- phorylated by Ca2+/calmodulin-dependent protein kinase II resulting in regulation of its binding to F-actin. J. Neurochem. 90, 317–324 122. Popoff, M. R., and Poulain, B. (2010) Bacterial toxins and the nervous system: Neurotoxins and multipotential toxins interacting with neuronal cells. Toxins 2, 683–737 123. Schwanhäusser, B., Busse, D., Li, N., Dittmar, G., Schuchhardt, J., Wolf, J., Chen, W., and Selbach, M. (2011) Global quantiﬁcation of mammalian gene expression control. Nature 473, 337–342 104. Turner, K. M., Burgoyne, R. D., and Morgan, A. (1999) Protein phosphor- ylation and the regulation of synaptic membrane trafﬁc. Trends Neuro- sciences 22, 459–464 20 Mol Cell Proteomics (2021) 20 100061
https://openalex.org/W1605817811	https://europepmc.org/articles/pmc2614187?pdf=render	English	null	Electrostatic and Functional Analysis of the Seven-Bladed WD β-Propellers	Evolutionary bioinformatics online	2,008	cc-by	8,839	Introduction WD40 repeat-like proteins form a structurally conserved superfamily and are part of the β-propeller fold. The WD repeat was ﬁ rst characterized by Van der Voorn and Ploegh (van der Voorn and Ploegh, 1992). It is a motif containing about 40–60 amino acids ending in a tryptophan and aspartic acid (WD). Multiple X-ray structures of this repeat revealed that it represents a blade formed from a four-stranded antiparallel β sheet. These blades are arranged radially forming a β-propeller structure and this propeller is structurally conserved despite high divergence in sequence amongst β-propeller proteins (Paoli, 2001) (Fig. 1). Perhaps surprisingly, in general a single WD repeat forms parts of two blades within a propeller. p g y g g p p p p The inner strand α of each blade is situated in the centre of the propeller and strand d (Fig. 1A) forms the “outer” side of the propeller. Superimposition and analysis of the structures by various authors sug- gested that strands a–c are generally conserved and most variability is observed in strand d and in the connecting segments between blades (Murzin, 1992). The innermost strands form a channel which has a conical shape. In most of the propeller proteins the joining of the N- and C-termini to enclose the propeller circle is achieved by the pairing of strand d from the N-terminus with three strands (a–c) from the C-terminus in the same blade (Fig. 1A). g Murzin (Murzin, 1992) developed a model for the propeller assembly of β-sheets and carried out an analysis on the multi-sheet packing of the blades into propeller structures. His calculations showed that four is the only possible number of strands in a blade and that any additional strands would generate unacceptably close packing of the strands. This geometrical model also predicted that an ideal seven- bladed β-propeller satisﬁ es average β-geometry best. He found that the seven-fold symmetry has no strong sequence constraints and is preferable to six- or eight-fold blades for assembly. g q p g y The seven-bladed β-propellers in SCOP (Murzin et al. 1995) are classiﬁ ed into several superfamilies, the one of interest to this work is the WD40-repeat like superfamily. This superfamily is further split into two families: the WD40-repeat, the family we shall study further, and the cell cycle arrest protein BUB3. Correspondence: Najl V. Valeyev. Tel: +44 (0) 7817 068953; Email: nv28@le.ac.uk Copyright in this article, its metadata, and any supplementary data is held by its author or authors. It is published under the Creative Commons Attribution By licence. For further information go to: http://creativecommons.org/licenses/by/3.0/. ORIGINAL RESEARCH ORIGINAL RESEARCH Electrostatic and Functional Analysis of the Seven-Bladed WD β-Propellers Najl V. Valeyev1,4, A. Kristina Downing1, John Sondek2 and Charlott 1Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, U.K. 2Department of Pharmacology, University of North Carolina, 27599-7365, U.S.A. 3Department of Statistics, University of Oxford, 1 South Parks Road, Oxford, OX1 3TG, U.K. 4Current address: Systems Biology Lab, Department of Engineering, University of Leicester, University Road, Leicester, LE1 7RH, U.K. Abstract: β-propeller domains composed of WD repeats are highly ubiquitous and typically used as multi-site docking platforms to coordinate and integrate the activities of groups of proteins. Here, we have used extensive homology modelling of the WD40-repeat family of seven-bladed β-propellers coupled with subsequent structural classiﬁ cation and clustering of these models to deﬁ ne subfamilies of β-propellers with common structural, and probable, functional characteristics. We show that it is possible to assign seven-bladed WD β-propeller proteins into functionally different groups based on the information gained from homology modelling. We examine general structural diversity within the WD40-repeat family of seven-bladed β-propellers and demonstrate that seven-bladed β-propellers composed of WD-repeats are structurally distinct from other seven-bladed β-propellers. We further provide some insights into the multifunctional diversity of the seven-bladed WD β-propeller surfaces. This report once again reinforces the importance of structural data and the usefulness of homology models in functional classiﬁ cation. Keywords: β-propeller, electrostatics, evolutionary trace, protein clustering, WD protein Introduction A superposition of the protein domains for the seven-bladed WD40-repeat β-propeller family is shown in Figure 1B. In this report we use structural rather than just sequence information to classify seven-bladed WD β-propellers. While the sequence data certainly gives some information, 3D structures are usually required to gain detailed biological and functional characterization (Chakravarty and Sanchez, 2004). The value gained from the comparative modelling of proteins over sequence alone has been demon- strated previously (Chakravarty and Sanchez, 2004; Chakravarty et al. 2005). Here we carried out large-scale modelling of the seven-bladed WD β-propellers followed by comparative structural analyses using the evolutionary trace (ET) method (Lichtarge et al. 1996), examination of surface cavities (Tsodikov et al. 2002) and electrostatics analyses (Blomberg et al. 1999). g The WD motif has been extensively studied in sequence space, over 900 repeats have been aligned in order to reﬁ ne the pattern (Smith et al. 1999). The relationship between amino acid sequence and function for WD repeat containing proteins has been addressed by Yu et al. (Yu et al. 2000). This analysis relies entirely on sequence similarity and does not use information from the 3D (three- dimensional) structures of the proteins. The out- come of the Yu et al. (Yu et al. 2000) study was the separation of 278 protein sequences into different groups, with each group possessing potentially unique functions. Sequences with unassigned func- tions were predicted to have the same function as other proteins in the same group. A second study on functional annotation of WD proteins by Yu et al. (Yu et al. 2000) deﬁ ned 32 functional groups amongst the WD proteins with various numbers of blades. This result was based on the assumption that surface similarity deﬁ nes a common function for WD repeat proteins (Smith et al. 1999). Only three seven-bladed β-propeller families G protein, ET uses a phylogenetic tree of a protein family and identiﬁ es evolutionary conserved residues in branches of the tree. Many studies report that resi- dues predicted by ET to be evolutionary privileged signiﬁ cantly overlap with functional sites (Lichtarge et al. 1996; Lichtarge et al. 1996; Lichtarge et al. 1997; Lichtarge and Sowa, 2002; Lichtarge et al. 2002; Lichtarge et al. 2003). It has also been shown that such positions form statistically signiﬁ cant clusters (Madabushi et al. 2002). Introduction This second family is thought by the SCOP authors to be possibly related to the WD-repeat family, but both sequence similarity between the blades and the WD40 repeat signature are very weak. 203 Evolutionary Bioinformatics 2008:4 203–216 Valeyev et al Top View Side View Top Bottom A C B a b c d N- C- 1 7 6 5 4 3 2 Figure 1. Seven-bladed β-propeller proteins. A. Seven-bladed β-propeller (1got) coloured according to the WD repeats. B. Backbone representation of the superimposed WD repeat template structures (1erj—red, 1got—green, 1gxr—blue, 1k8k—yellow, 1pgu—magenta, 1p22—cyan, 1sq9—grey). C. A cartoon representation of a β-propeller. The structure is different in radius on the “top” and “bottom”. The narrower side is deﬁ ned as the “top” region in this report. Both A and B show the top surface. The strands a-d form seven blades of the propeller structure. Top View Side View Top Bottom C B A a b c d N- C- 1 7 6 5 4 3 2 B C A Figure 1. Seven-bladed β-propeller proteins. A. Seven-bladed β-propeller (1got) coloured according to the WD repeats. B. Backbone representation of the superimposed WD repeat template structures (1erj—red, 1got—green, 1gxr—blue, 1k8k—yellow, 1pgu—magenta, 1p22—cyan, 1sq9—grey). C. A cartoon representation of a β-propeller. The structure is different in radius on the “top” and “bottom”. The narrower side is deﬁ ned as the “top” region in this report. Both A and B show the top surface. The strands a-d form seven blades of the propeller structure. Groucho, and Tup1 were identiﬁ ed amongst those functional clusters. The seven-bladed protein domains found in the WD40-repeat family are listed in Table 1 with annotated functions and available PDB (1got (Lambright et al. 1996), 1erj (Sprague et al. 2000), 1gxr (Pickles et al. 2002), 1k8k (Robinson et al. 2001), 1p22 (Wu et al. 2003), 1pgu (Voegtli et al. 2003), 1sq9 (Madrona and Wilson, 2004)) struc- tures. A superposition of the protein domains for the seven-bladed WD40-repeat β-propeller family is shown in Figure 1B. The seven-bladed protein domains found in the WD40-repeat family are listed in Table 1 with annotated functions and available PDB (1got (Lambright et al. 1996), 1erj (Sprague et al. 2000), 1gxr (Pickles et al. 2002), 1k8k (Robinson et al. 2001), 1p22 (Wu et al. 2003), 1pgu (Voegtli et al. 2003), 1sq9 (Madrona and Wilson, 2004)) struc- tures. Introduction We have applied this technique to explore the functional surface interface in combination with experimentally deter- mined key surface residues found to be important for some seven-bladed WD β-propeller families. Evolutionary Bioinformatics 2008:4 204 Electrostatics and Functions of Seven-Bladed WD β-Propellers Another important factor determining how proteins function is their surface cavities or clefts. It has been reported that small surface cavities could be important for binding of allosteric effec- tors (DesJarlais et al. 1988). It has also been pro- posed (Kuntz et al. 1982; DesJarlais et al. 1988) that enzymatic active sites are usually situated in large surface clefts. Rigorous analysis of the impor- tance of the cleft size for protein-protein interac- tions has been carried out previously (Laskowski et al. 1996). modules. In the work described here, we carry out functional protein characterization on a larger scale. We have created a large set of unique homology models (∼700) from which the smaller subset of models (∼170) was used to carry out the functional classiﬁ cation of β-propellers containing seven WD repeats. We have performed large-scale modelling of seven bladed WD β-propeller proteins. The models were electrostatically characterized by the PIPSA program (Blomberg et al. 1999) with default parameters, and then analysed using the statistical package R (http://www.r-project.org/). We show that by using the surface electrostatics as a criterion, the members of the seven-bladed β-propeller WD40-repeat family can be classified into functional clusters. This classiﬁ cation is possible given high accuracy homology models. If the sequence identity between the template and the modelled sequence is too low, the classiﬁ cation becomes less reliable due to the increasing error. We argue that this method can predict biological role or function of uncharacterized protein sequences. The electrostatics-based classiﬁ cation differs from clusters based on sequence similarity. This result may suggest an important role of protein surface charge distribution for functional interactions. We also calculated the evolutionary trace (Lichtarge et al. 1996), and surface cavities (Tsodikov et al. 2002) of the seven-bladed WD40- repeat β-propeller proteins. The combination of residues predicted by these methods together with experimentally identiﬁ ed residues are given in Table 1. These residues are possibly important in the WD40 interactions with binding partners. It is clear from experimental data (Table 1) that seven- bladed WD β-propeller proteins have a wide variety of functions. Introduction This diversity is clearly shown to arise from multiple functional sites on the surface rather that adoption of a single site. Furthermore, in those subfamilies not yet experimentally characterized, these techniques also predict a multifunctional interface. Electrostatic potential is a protein property which can only be examined with structural knowledge. Recently, several studies have used electrostatic surface potential as a tool for predict- ing protein functions and for predicting function- ally similar groups in protein families. Livesay et al. looked at the electrostatics near the active site of 54 experimentally determined structures of four enzyme families and one superfamily (Livesay et al. 2003). In another example, Botti et al. studied cholinesterases and neural cell-adhe- sion proteins (12 experimentally determined structures in total) (Botti et al. 1998). The authors showed that in the absence of any sequence simi- larity these protein families have a common elec- trostatic motif at the catalytic site. Homology modelling of 104 Pleckstrin Homology (PH) domains (Blomberg and Nilges 1997) followed by electrostatic analysis (Blomberg et al. 1999) revealed a high degree of functional conservation across the superfamily. Electrostatic calculations have also been used to analyze RNAs recognition and stabilization properties (Chin et al. 1999). Chin et al. have calculated electrostatic properties of some structures of RNA molecules from Protein Data Bank and Nucleic Acid Database (Chin et al. 1999). The authors found that the sites with unusual electrostatic features correspond to the function- ally important regions. Large scale homology modelling work has also been done on comple- ment control protein (CCP) modules (Soares et al. 2005). In total, 136 models of CCP domains were generated and subjected to multiple methods of surface characterization. In particular, it was found that assignments to clusters based on the surface properties differed from assignments based on sequence similarity. This result was suggested to reﬂ ect a particular role of surface exposed residues crucial for protein-protein interactions. The applied surface characterization methods were indeed able to pinpoint functional sites within CCP Evolutionary Bioinformatics 2008:4 Alternative characterization methods Alternative characterization methods The ET method was used to identify residues con- served throughout evolution in each seven-bladed WD40-repeat β-propeller subfamily. ET identiﬁ es conserved residues in sets of sequences which are in a speciﬁ c region of the phylogenetic tree. If these residues are in statistically signiﬁ cant structural clusters, they are identiﬁ ed by ET and can be used for functional sites prediction. The sets of sequences are identiﬁ ed using “cut-off” lines. These are drawn on the phylogenetic tree and called “partitions”. For the ET analysis presented here, ten partitions were used with the 10th partition encompassing the most class-specific residues. Residues, predicted to be conserved within each partition are divided into four categories. Exposed class speciﬁ c residues (from partition 10) predicted by the method were mapped onto the surface of the representative experimental structures (1got (Lambright et al. 1996); 1erj (Sprague et al. 2000); 1gxr (Pickles et al. 2002); 1k8k (Robinson et al. PSI-PLAST search and modelling procedures A set of template structures for the seven-bladed WD40 family were extracted from the SCOP v1.69 database (Murzin et al. 1995). A BLAST (basic 205 Valeyev et al local alignment search tool) (http://www.ncbi.nlm. nih.gov) (Lipman et al. 1989; Altschul et al. 1997) search using each template as a query gave a rep- resentative sequence subset for each subfamily deﬁ ned in SCOP under WD40-repeat family sec- tion. BLAST was run with an E-value inclusion threshold equal to 0.005. Since the object of this study was to analyse the structure-function rela- tionship of seven-bladed WD β-propellers, all BLAST searches were performed using only pro- peller domains and extraneous protein sequence was not included. The sequences collected by BLAST were aligned against the experimental structures using ClustalW (Thompson et al. 1994). All unique hits from BLAST searches with greater than 20% sequence identity to the queries, were used to produce homology models with Modeller (Sali and Blundell, 1993), resulting in about ∼1400 unique models. This entire process was fully auto- mated. Each model was then inspected in order to identify those which were poorly built resulting from incorrect automated alignment or other errors. Incorrect models (structures with severe distortions from the propeller shape) were excluded from further analysis. In total 688 correctly built models were analysed. A smaller subset of models (166) with sequence identity cut-offs at 90% for 1got, 1gxr, 1k8k, 1pgu, 1p22 families, at 70% for the 1erj family, and at 40% for the 1sq9 family was used for testing in this study. The models of sequences meeting the cut-off criteria to the experimental structures from WD40-repeat family as deﬁ ned in SCOP v1.69 database were built using Modeller program. To carry out the electrostatic analysis, the models were superimposed using a script written in Perl and the CE program (Shindyalov and Bourne, 1998). simple C program. This program uses the coordinates of a given point on the outer blade of the superim- posed β-propellers as an input. These points (the middle of the outer strand) were chosen by visual inspection of each representative structure. Vectors are calculated from the centre of mass to the chosen points. These vectors are characteristic for each structure. (Fig. 2 in Supplementary Material). Electrostatics El The structurally superimposed WD propellers were used as input for PIPSA. This program computes the similarity indexes between pairs of proteins based on the monopole and dipole terms of the molecule’s electrostatics. The Hodgkin index is calculated to measure the similarity between two molecular potentials. This parameter encompasses the differences in sign, magnitude, and spatial behaviour of the potentials. The program calculates pairwise electrostatic indexes of multiple structures allowing comparisons of large datasets and subse- quent clustering analysis. The calculated electro- static indexes can range from −1 to 1. If the Hodgkin index equals 1, the two potentials are identical, if it equals 0 the potentials are fully uncorrelated, and −1, the potentials are anticorre- lated. These 3D electrostatic descriptions of the proteins were clustered using Agnes (Struyf et al. 1997) as coded in the R statistical package. WD proteins are a structurally conserved subfamily y In order to investigate the WD40-repeat family which is a part of the seven-bladed β-propeller fold we focused on the ‘global’ geometry of the fold and have examined the arrangement of blades within the ‘plane’ of the β-propeller. There are potentially two geometrical “regions” that one could analyse in the β-propeller type structures. One geometrically distinct part is the relatively rigid protein core composed of strands arranged into blades. The question to ask is whether or not these strands are arranged differently in different subfamilies within the WD40-repeat family. Another potentially interesting question is whether there is any difference between subfamilies in the inherently ﬂ exible regions of the β-propellers, such as loops connecting the blades. There are of course a variety of geometrical parameters that one can use to characterize symmetrical β-propeller struc- tures. For example, one could look at the charac- teristic distances between corresponding strands in the neighbouring blades to see if they are con- served or varied between subfamilies. Another potential option is to measure radius and height of the β-propellers. We calculated vectors (for representative experimental structures 1got (Lambright et al. 1996); 1erj (Sprague et al. 2000); 1gxr (Pickles et al. 2002); 1k8k (Robinson et al. 2001); 1p22 (Wu et al. 2003); 1pgu (Voegtli et al. 2003); 1sq9 (Madrona and Wilson, 2004)) from the centre of mass to a deﬁ ned point on the outer side of each blade, this simple parameter contains information about the radius and implicitly describes the height of the structures as well as angular distribution of the individual blades. Given that the blades are composed of four anti-parallel We next examined more general structural diversity within the WD40-repeat family of seven- bladed β-propellers. For example, it has not been previously established that seven-bladed β-propellers composed of WD-repeats are structur- ally distinct from other seven-bladed β-propellers and we addressed this point by determining the structurally similarity between the two sets of domains. High structural similarity was found within the WD40-repeat protein family which was not found in any other seven-bladed β-propeller superfamily. Figure 2 shows a comparison of per- centage sequence identity (A) and sequence similarity (B) with the corresponding RMSDs, for all experimentally determined seven-bladed β-propeller domains (some representative structures are shown in Figure 1 of the Supplementary Material). Structural comparison The β-propeller parts of all seven-bladed β-propel- ler containing structures defined in the SCOP database were extracted. Structural comparison of all seven-bladed β-propellers was performed automatically by pairwise superimposition using the CE program based on the Cα atom positions. The RMSD and sequence identity and the sequence similarity (using the BLOSUM62 matrix (Henikoff and Henikoff, 1992)) were calculated for each superimposition. The geometrical characterization of the seven- bladed β-propeller domains was performed using a Evolutionary Bioinformatics 2008:4 206 Electrostatics and Functions of Seven-Bladed WD β-Propellers 2001); 1p22 (Wu et al. 2003); 1pgu (Voegtli et al. 2003); 1sq9 (Madrona and Wilson, 2004)). 2001); 1p22 (Wu et al. 2003); 1pgu (Voegtli et al. 2003); 1sq9 (Madrona and Wilson, 2004)). beta strands, we used the middle of the outer strand as the characteristic measure of each blade. This approach allows analysis of how symmetrically the blades are arranged within each subfamily. The spatial distribution of the ‘middle point’ coordi- nates was mapped on the superimposed seven- bladed β-propeller, representative for different subfamilies (representative experimental structures listed in Material and Methods) (Fig. 2 in Supplemental Material). This analysis demonstrated that the spatial organization of the blades (the actual propeller) appears quite conserved across all the seven-bladed subfamilies within WD40- repeat family. This result distinguishes the core part of the β-propellers from the ﬂ exible loops, in which some variability has been observed between different subfamilies. The program Surface Racer (Tsodikov et al. 2002) was used to perform protein surface curva- ture analysis and to identify potential cavities, which serve as additional hints for functional regions. The probe radius was chosen to be slightly larger than a water molecule radius and was equal to 1.5 Ǻ. The experimentally determined functional residues were compared with the ET and the sur- face curvature program predictions. Evolutionary Bioinformatics 2008:4 WD proteins are a structurally conserved subfamily Overall, the pairwise comparison of WD-repeat containing domains produces signiﬁ cantly lower RMSDs relative to identical comparisons of pairs of domains from other β-propeller classes or between these classes and WD-repeat containing domains. This observation is rather intriguing, because there are no obvious factors explaining why the WD sequence motif would have such a high impact on the structure. In fact, the sequence identity (and the sequence similarity) amongst many of the WD proteins is as low as the sequence identity across non-WD pro- teins. Homology models of WD β-propellers To characterize the WD40-repeat protein family in more detail, we built models of sequences with Evolutionary Bioinformatics 2008:4 207 Valeyev et al 0 20 40 60 80 100 0 1 2 3 4 5 6 WD protein structures non WD propellers RMSD sequence identity, % B A 20 40 60 80 100 0 1 2 3 4 5 6 WD protein structures non WD propellers RMSD sequence similarity, % Figure 2. The variation of RMSDs and sequence identity between seven-bladed β-propeller proteins. The data points for the com- parison between WD-WD proteins are coloured as red, and the green circles represent superimpositions between two non-WD proteins as well as between WD and non-WD proteins. The data shows clear structural conservation of the WD40 superfamily amongst all other seven- bladed β-propellers. RMSD has been calculated based on Cα atom positions. 0 20 40 60 80 100 0 1 2 3 4 5 6 WD protein structures non WD propellers RMSD sequence identity, % A B 20 40 60 80 100 0 1 2 3 4 5 6 WD protein structures non WD propellers RMSD sequence similarity, % B A RMSD Figure 2. The variation of RMSDs and sequence identity between seven-bladed β-propeller proteins. The data points for the com- parison between WD-WD proteins are coloured as red, and the green circles represent superimpositions between two non-WD proteins as well as between WD and non-WD proteins. The data shows clear structural conservation of the WD40 superfamily amongst all other seven- bladed β-propellers. RMSD has been calculated based on Cα atom positions. on proteins affect the speciﬁ city of protein-ligand or protein-protein interactions (Honig and Nich- olls, 1995). Electrostatic potentials generated by molecules have a variety of different characteris- tic surface features that can only be calculated when a structure is known. WD proteins are a structurally conserved subfamily The surface electro- static pattern of any charged protein creates either attractive or repulsive forces. These, inevitably, make certain protein-protein interactions more or less favourable. We have used our models of the seven-bladed β-propeller subfamilies to assess whether electrostatics is applicable in this par- ticular case. As we use protein models rather than experimental structures, one can possibly expect some inaccuracy. 20% or greater sequence identity to the representa- tive structures. The models were inspected for any modelling artefacts and well built models were used in the later analysis. It was clear that with decreasing sequence identity to the template, the quality of modelling performance decreased. A set of highly similar models (166) were used to fully explore the potential of the value gained from homology modelling. 20% or greater sequence identity to the representa- tive structures. The models were inspected for any modelling artefacts and well built models were used in the later analysis. It was clear that with decreasing sequence identity to the template, the quality of modelling performance decreased. A set of highly similar models (166) were used to fully explore the potential of the value gained from homology modelling. Surface electrostatics of seven bladed WD structures is conserved The high structural conservation of the WD propel- lers despite their low sequence identity allows the building of homologous models of reasonable quality in an automated manner. The WD pattern observed in this family also improves the homol- ogy model building by allowing a high degree of conﬁ dence in the alignment of sequence with the template structure. y A random subset of WD protein homology models (ten from each subfamily) were selected and used initially to analyse surface electrostat- ics. From this we identiﬁ ed that surface electro- statics were highly conserved within a subfamily and different between WD40-repeat subfamilies. Figure 3 shows the surface electrostatics of the homology models in the G-beta protein subfam- ily. The surface pattern remains similar even though the sequence identity to the experimental template (1GOT) decreases to around 30%. This ﬁ nding indicates that it may be possible to clas- sify protein function based on the value gained from modelling and using electrostatics as a criterion. The difference in surface between the subfamilies of the WD domains can be seen in Figure 4. We studied which β-propeller protein structure properties might reveal differences in function between structurally similar subfamilies. Amongst various protein surface properties important for protein-protein interactions, such as cavities, hydrophobic residues, speciﬁ c interaction residue pockets, electrostatics is the one we identiﬁ ed to have high potential for functional protein classi- ﬁ cation. It is known that electrostatics plays an important role in molecular interactions (Honig and Nicholls, 1995). Surface electrostatic patches Evolutionary Bioinformatics 2008:4 208 Electrostatics and Functions of Seven-Bladed WD β-Propellers 90.4% 1GOT 98% 88% 29.6% 28.9% 72.2% 56.4% Figure 3. The surface of G-beta protein and its homology models coloured by their electrostatic potential. The percentages given below each structure are the sequence identity between the homology model and the structural template (1GOT). The negative charge is shown as red, positive charge is coloured blue. The conservation of the electrostatic surface in the models even those with relatively low sequence identity, is striking. All structures show the propeller’s top surfaces. 1GOT 98% 90.4% 88% 1GOT 90.4% 88% 88% 98% 29.6% 72.2% 56.4% 28.9% 29.6% 28.9% 56.4% 72.2% Figure 3. The surface of G-beta protein and its homology models coloured by their electrostatic potential. The percentages given below each structure are the sequence identity between the homology model and the structural template (1GOT). Surface electrostatics of seven bladed WD structures is conserved The negative charge is shown as red, positive charge is coloured blue. The conservation of the electrostatic surface in the models even those with relatively low sequence identity, is striking. All structures show the propeller’s top surfaces. 1K8K Arp2/3 protein 1GXR Groucho protein 1SQ9 Antiviral protein Ski8p 1ERJ Tup1 protein 1P22 F-box/WD-repeat protein 1K8K Arp2/3 protein 1PGU Actin interacting Protein I 1GOT G-beta protein Figure 4. Electrostatic based grouping of the seven bladed WD repeat β-propellers. Two dimensional plot of three dimentional data is shown for PIPSA calculated electrostatic indexes of seven experimentally determined WD repeat proteins from different families and homol- ogy models. The squares represent experimentally deﬁ ned structures (1erj—magenta, 1got—green, 1gxr—red, 1k8k—yellow, 1pgu—blue, 1p22—cyan, 1sq9 – grey). Electrostatics are shown for the top surfaces of proteins. 1GXR Groucho protein 1SQ9 Antiviral protein Ski8p 1ERJ Tup1 protein 1P22 F-box/WD-repeat protein 1K8K Arp2/3 protein 1PGU Actin interacting Protein I 1GOT G-beta protein Figure 4. Electrostatic based grouping of the seven bladed WD repeat β-propellers. Two dimensional plot of three dimentional data shown for PIPSA calculated electrostatic indexes of seven experimentally determined WD repeat proteins from different families and homo ogy models. The squares represent experimentally deﬁ ned structures (1erj—magenta, 1got—green, 1gxr—red, 1k8k—yellow, 1pgu—blu 1P22 F-box/WD-repeat protein 1GXR Groucho protein 1PGU Actin interacting Protein I 1PGU Actin interacting Protein I 1SQ9 Antiviral protein Ski8p Actin interacting Protein I Figure 4. Electrostatic based grouping of the seven bladed WD repeat β-propellers. Two dimensional plot of three dimentional data is shown for PIPSA calculated electrostatic indexes of seven experimentally determined WD repeat proteins from different families and homol- ogy models. The squares represent experimentally deﬁ ned structures (1erj—magenta, 1got—green, 1gxr—red, 1k8k—yellow, 1pgu—blue, 1p22—cyan, 1sq9 – grey). Electrostatics are shown for the top surfaces of proteins. 209 Evolutionary Bioinformatics 2008:4 Valeyev et al From the visual inspection of the electrostatic potentials it is evident that each WD subfamily has a unique isopotential surface (Fig. 4). To measure quantitatively the degree of similarity of electrostatic properties between the subfamilies, we calculated similarity indexes between all the models using the PIPSA program. Figure 4 shows the electrostat- ics-based grouping for the smaller set of 166 models. It follows from this picture that WD sub- families differ from each other despite the striking structural similarity within the WD40-repeat fam- ily. Surface electrostatics of seven bladed WD structures is conserved Based on these results one may argue that it is possible to predict functions of uncharacterized protein sequences (with no deﬁ ned function or solved structure) based on electrostatic clustering. For example, sequences that cluster with Tup1 are transcription factors, the ones clustering with Gβ can be involved in signal transduction. However, these predictions are prone to some potential errors due to: i) homology models represent protein structures with limited accuracy; ii) electrostatic analysis is still a new approach for protein function classiﬁ cation. While one can clearly observe certain electro- statics based clustering on Figure 4, it is important to be able to quantify this result using a formal criterion. The observed electrostatic grouping was clustered in the R statistical package. All 166 models were clustered but in order to visualize electrostatics-based separation, we also performed this analysis on a representative subset, of up to 10 randomly picked models from each subfamily (Fig. 5). The degree of clustering shown in Figure 5 is very similar to that shown by all 166 models. 1GXR g g ( ) 1GOT 1ERJ 1PGU 1K8K 1P22 1SQ9 1ERJ 1GOT 1K8K 1P22 1GXR 1PGU 1SQ9 Dendrogram of agnes (X=X,dist) Figure 5. Electrostatic based clustering of the seven bladed WD repeat β-propellers. The different seven-bladed WD β-propeller sub- familes are clearly in separate clusters. Five (1GOT, 1SQ9, 1ERJ, 1K8K, 1P22) out of seven subfamilies appear to have good clustering in electrostatic space. The partial overlap of 1GXR and 1PGU families indicates that their electrostatic potentials are similar. Electrostatics are shown for the top surfaces of proteins. g g ( ) Dendrogram of agnes (X=X,dist) g g ( ) Dendrogram of agnes (X=X,dist) 1P22 1GOT 1ERJ 1P22 1K8K Figure 5. Electrostatic based clustering of the seven bladed WD repeat β-propellers. The different seven-bladed WD β-propeller sub- familes are clearly in separate clusters. Five (1GOT, 1SQ9, 1ERJ, 1K8K, 1P22) out of seven subfamilies appear to have good clustering in electrostatic space. The partial overlap of 1GXR and 1PGU families indicates that their electrostatic potentials are similar. Electrostatics are shown for the top surfaces of proteins. Evolutionary Bioinformatics 2008:4 210 Electrostatics and Functions of Seven-Bladed WD β-Propellers the experimentally determined functionally important residues on to the protein structures. The clustering shows separate branches that reasonably distinguish models from different subfamilies. Surface electrostatics of seven bladed WD structures is conserved Homology models of ﬁ ve out of the seven subfamilies are clustered on the same branch as their representative experimental structure, whereas models of two subfamilies (1GXR and 1PGU) do not cluster as tightly. This might be due to the limited accuracy of comparative models as well as similar electrostatics of different subfami- lies which leads to the ‘mixing’ of subfamilies in the electrostatic space. These data suggest that electrostatics provides sufficient functional distinction and can potentially have predictive power. The applied method can suggest whether or not a structure built from an uncharacterized sequence belongs to a particular functional group. It, therefore, appears possible to classify protein sequences based on the information gained from homology modelling and electrostatics. Table 1 in supplementary material shows. ET is a powerful tool for prediction of protein functional sites. The method uses a set of aligned sequences and the corresponding phylogenetic tree. Here the evolutionary privileged residues predicted as exposed class-speciﬁ c in the 10th partition (see Methods) are highlighted on the surface (Fig. 6). These residues may be functionally important, but in order to identify others or conﬁ rm these we have additionally applied a program developed by Tsodikov et al. (Tsodikov et al. 2002) to search for regions forming cavities on the surface, because these may also suggest possible binding sockets. It has not been suggested that any of the seven- bladed WD β-propeller families have an enzymatic activity, we therefore analyse the small cavities on the surface as these may suggest possible allosteric interaction sites. The surface residues, creating small cavities that are shown on the representative structures (Fig. 6), suggest potential binding pock- ets. One of the best ways to characterize the bind- ing areas is obviously experimental. We have done a literature search to ﬁ nd surface residues reported to be involved in interactions in seven-bladed WD β-propellers. The residues experimentally deter- mined to be functionally important in each seven- bladed WD β-propeller subfamily (Table 1), were mapped onto the corresponding structures. Figure 6 shows the continuum of potential functional sites for all seven subfamilies. Although the predictions reveal wide distribution, it appears there is a higher probability for functional interaction sites on the “top” side of the WD β-propeller subfamilies. Previous experimental studies also report that functional residues are located mainly on the “top” interface (Pﬂ ugrad et al. 1997; Ford et al. 1998). Surface electrostatics of seven bladed WD structures is conserved pp y The observed electrostatic differences between the seven-bladed WD β-propeller protein subfamilies may imply that the surface electrostatic pattern diverged with the protein function during evolution while the β-propeller fold remained conserved throughout the WD40-repeat family. The RMSD and sequence identity as well as sequence similar- ity comparisons (Fig. 2) show that the sequence identity between seven-bladed WD β-propeller representative structures is as low as 10%. This is an interesting observation because RMSD between different WD β-propellers is low, in the range from 1 to 3 Å (Fig. 2). While high sequence identity may suggest similarity of function, very low sequence identity or similarity does not rule out similar func- tion, especially if the fold is very conserved. Here our results show that protein function can be dis- tinguished based on the electrostatic properties of protein subfamilies with very low sequence identity, but strikingly similar fold. ( g ) The ET method, surface cavities mapping and even the experimental mutations by no means per- fectly indicate interaction sites. However, the regions of residues overlap or predicted neighbour- ing patches are strong indicators of functional importance. The two theoretical techniques in combination with experimental mutagenesis data (where available) suggest that all seven-bladed WD β-propeller subfamilies have multifunctional inter- faces, a property also observed in PH domains (Shaw, 1996). Based on our results, we suggest that these residues are likely to be important for protein function: i) G-beta protein subfamily: K89, S98, W99, L117, N119, D186, D228 are experimentally determined functional residues that are also pre- WD propellers exhibit a continuum of functional sites Unfor- tunately this overlap is incomplete as can be seen from Table 1. There are experimentally determined functionally important residues not found by evo- lutionary trace. This trend is seen in most of the subfamilies. S97, D118, G141, T164, G185, A206 are predicted by the ET and surface curvature pro- grams and located next to the residues identiﬁ ed by both experimental mutations and ET ii) Tup1 pro- tein subfamily: Y489, N673 are the experimentally determined functional residues that are also pre- dicted to be functionally important by ET; R652, H671 are predicted by the surface curvature pro- gram and located next to residues identiﬁ ed by experimental mutations and ET; Q486, R465, D464, V346, L547 are predicted by the ET and surface curvature programs and located next to residues identiﬁ ed by both experimental mutations and ET; iii) Groucho protein subfamily: H646 is the only residue (of the three possible ones) identiﬁ ed by experimental mutations and ET; iv) F-box/WD- repeat protein subfamily: R285, S325, R474 are identiﬁ ed by experimental mutations and ET; T266, K268, N287, T307, S327 are predicted by the ET dicted to be functionally important by ET. Unfor- tunately this overlap is incomplete as can be seen from Table 1. There are experimentally determined functionally important residues not found by evo- lutionary trace. This trend is seen in most of the subfamilies. WD propellers exhibit a continuum of functional sites Studies of the WD40-repeat family reveal that it has multiple binding partners and it has also been suggested that it serves as a scaffold for protein-protein interactions (Paoli, 2001). To investigate this functional divergence further we have analyzed the spatial distribution of the functionally important amino acids by a combina- tion of ET, surface curvature analysis and mapping 211 Evolutionary Bioinformatics 2008:4 Valeyev et al C R X G 1 B J R E 1 Figure 6. The seven-bladed WD protein families reveal a continuum of functional sites. The residues proposed to be functionally important are mapped on to experimental structures. Blue—residues identiﬁ ed by mutational experiments, Yellow – exposed class-speciﬁ c residues classiﬁ ed by ET in the trace 10, Red shows the overlap between ET and experimental mutations, Cyan – residues proposed to form cavities on the surface. (A) shows the WD part of G-beta protein (1GOT), (B) Transcriptional repressor Tup1 (1ERJ), (C) Transcriptional repressor Groucho (1GXR), (D) F-box/WD-repeat protein 1 (1P22), (E) Arp2/3 complex (1K8K), (F) Actin interacting protein 1 (1PGU), (G) Ski8p, mRNA degradation regulating protein (1SQ9). All proteins are oriented in an identical fashion. A B C D 2 2 P 1 R X G 1 J R E 1 T O G 1 E F G 1K8K 9 Q S 1 U G P 1 A T O G 1 D 2 2 P 1 C B D T O G 1 2 2 P 1 J R E 1 F U G P 1 E 1K8K G 9 Q S 1 E G F U G P 1 U G P 1 9 Q S 1 9 Q S 1 1K8K Figure 6. The seven-bladed WD protein families reveal a continuum of functional sites. The residues proposed to be functionally important are mapped on to experimental structures. Blue—residues identiﬁ ed by mutational experiments, Yellow – exposed class-speciﬁ c residues classiﬁ ed by ET in the trace 10, Red shows the overlap between ET and experimental mutations, Cyan – residues proposed to form cavities on the surface. (A) shows the WD part of G-beta protein (1GOT), (B) Transcriptional repressor Tup1 (1ERJ), (C) Transcriptional repressor Groucho (1GXR), (D) F-box/WD-repeat protein 1 (1P22), (E) Arp2/3 complex (1K8K), (F) Actin interacting protein 1 (1PGU), (G) Ski8p, mRNA degradation regulating protein (1SQ9). All proteins are oriented in an identical fashion. dicted to be functionally important by ET. Conclusions In this study we have analysed the seven-bladed WD40-repeat protein family a member of the seven-bladed β-propeller fold. We have investi- gated a variety of characteristics of the experimen- tal structures and the set of homologous models representing seven different seven-bladed WD β- propeller subfamilies that form the family. The models were built in a fully automated manner, to investigate the functional similarities and differ- ences of the seven-bladed WD β-propeller proteins. We have shown that it is possible to classify WD β-propeller protein subfamilies based on the value gained from large scale homology modelling. Blomberg, N. and Nilges, M. 1997. Functional diversity of PH domains: an exhaustive modelling study. Fold Des., 2:343–55. Botti, S.A., Felder, C.E. et al. 1998. Electrotactins: a class of adhesion proteins with conserved electrostatic and structural motifs. Protein Eng., 11:415–20. Chakravarty, S. and Sanchez, R. 2004. Systematic analysis of added-value in simple comparative models of protein structure. Structure, 12:1461–70. Chakravarty, S., Wang, L. et al. 2005. Accuracy of structure-derived properties in simple comparative models of protein structures. Nucleic Acids Res., 33:244–59. Chin, K., Sharp, K.A. et al. 1999. Calculating the electrostatic properties of RNA provides new insights into molecular interactions and Nat. Struct Biol., 6:1055–61. DesJarlais, R.L., Sheridan, R.P. et al. 1988. Using shape complementa- rity as an initial screen in designing ligands for a receptor binding site of known three-dimensional structure. J. Med. Chem., 31:722–9. g g gy g We found that the WD40-repeat family is struc- turally distinct from all other superfamilies form- ing the seven-bladed β-propeller fold. This difference we believe is due to the speciﬁ c WD sequence motif, which must lead to distinct struc- tural differences. In fact the sequence identity amongst the WD subfamilies is as low as sequence identity between non WD and other families form- ing the fold. Our ﬁ nding allows us to suggest that the WD motif translates into the higher structural similarity and makes the WD40-repeat family distinct in structural as well as sequence terms from other superfamilies of the fold. Ford, C.E., Skiba, N.P. et al. 1998. Molecular basis for interactions of G protein betagamma subunits with effectors. Science, 280:1271–4. Henikoff, S. and Henikoff, J.G. 1992. Amino acid substitution matrices from protein blocks. Proc. Natl. Acad. Sci., U.S.A., 89:10915–9. Honig, B. and Nicholls, A. 1995. Classical electrostatics in biology and chemistry. Science, 268:1144–9. Kuntz, I.D., Blaney, J.M. et al. 1982. Funding Funding NVV is supported by ORS, The Hill Foundation, and The Wellcome Trust funded Prize Studentship (070417), AKD (057725) is supported by The Wellcome Trust. Funding NVV is supported by ORS, The Hill Foundation, and The Wellcome Trust funded Prize Studentship (070417), AKD (057725) is supported by The Wellcome Trust. p It has already been suggested earlier that the WD fold can serve as a scaffold for other protein- protein interactions. Here we support that hypothesis by demonstrating the continuum of potential binding sites on the surface of all analyzed seven-bladed WD subfamilies. References Altschul, S.F., Madden, T.L. et al. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res., 25:3389–402. Blomberg, N., Gabdoulline, R.R. et al. 1999. Classiﬁ cation of protein sequences by homology modeling and quantitative analysis of electrostatic similarity. Proteins, 37:379–87. WD propellers exhibit a continuum of functional sites S97, D118, G141, T164, G185, A206 are predicted by the ET and surface curvature pro- grams and located next to the residues identiﬁ ed by both experimental mutations and ET ii) Tup1 pro- tein subfamily: Y489, N673 are the experimentally determined functional residues that are also pre- dicted to be functionally important by ET; R652, H671 are predicted by the surface curvature pro- gram and located next to residues identiﬁ ed by experimental mutations and ET; Q486, R465, D464, V346, L547 are predicted by the ET and surface curvature programs and located next to residues identiﬁ ed by both experimental mutations and ET; iii) Groucho protein subfamily: H646 is the only residue (of the three possible ones) identiﬁ ed by experimental mutations and ET; iv) F-box/WD- repeat protein subfamily: R285, S325, R474 are identiﬁ ed by experimental mutations and ET; T266, K268, N287, T307, S327 are predicted by the ET and surface curvature programs and located next to residues identiﬁ ed by experimental mutations and ET; G408, R410, N450, E471 are predicted by the ET and surface curvature programs and located next to residues identiﬁ ed by experimental mutations; v) Ski8p, mRNA degradation regulating protein superfamily: F89, R237 are identified by both experimental mutations and ET. p The residues predicted by the ET do not appear to signiﬁ cantly overlap with the experimentally determined functional residues, suggesting that the method may not be as robust as would be hoped. However, this might be due to the fact that WD proteins have a diverse interaction interface and only a limited number of residues important for functional interactions are reported in the literature. This also demonstrates the signiﬁ cance of the results with regards to electrostatic clustering, on a family as functionally diverse as the WD proteins. It is clear that sequence based methods (even those using some structure) such as ET are not able to capture the complexity of the family. We believe, that additional experimental studies of the functional interaction sites might reveal better Evolutionary Bioinformatics 2008:4 Evolutionary Bioinformatics 2008:4 212 Electrostatics and Functions of Seven-Bladed WD β-Propellers correspondence between the ET predictions and the experimental data. Evolutionary Bioinformatics 2008:4 Conclusions A geometric approach to macromolecule-ligand interactions. J. Mol. Biol., 161:269–88. Lambright, D.G., Sondek, J. et al. 1996. The 2.0 A crystal structure of a heterotrimeric G protein. Nature (London), 379:311–9. Laskowski, R.A., Luscombe, N.M. et al. 1996. Protein clefts in molecular recognition and function. Protein Sci., 5:2438–52. Lichtarge, O., Bourne, H.R. et al. 1996. Evolutionarily conserved Galphabetagamma binding surfaces support a model of the G protein- receptor complex. Proc. Natl. Acad. Sci., U.S.A., 93:7507–11. We have analysed the surface electrostatics of the seven-bladed WD β-propeller subfamilies and found that it is highly conserved within each sub- family, but differs between subfamilies. The analysis of the surface electrostatics of homology models with sequence identity to the template structure as low as 30% reveals a high degree of electrostatics conservation. The comparison of 166 homology models showed that it is possible to discriminate between the seven-bladed WD β-propeller subfamilies using electrostatics, if the homology models are reasonable. Clustering analysis of the electrostatic results allowed quan- tiﬁ cation of the differences and similarities between the seven-bladed WD β-propeller subfamiles. Lichtarge, O., Bourne, H.R. et al. 1996. An evolutionary trace method deﬁ nes binding surfaces common to protein families. J. Mol. Biol., 257:342–58. Lichtarge, O. and Sowa, M.E. 2002. Evolutionary predictions of binding surfaces and interactions. Curr. Opin. Struct Biol., 12:21–7. Lichtarge, O., Sowa, M.E. et al. 2002. Evolutionary traces of functional surfaces along G protein signaling pathway. Methods Enzymol, 344:536–56. Lichtarge, O., Yamamoto, K.R. et al. 1997. Identiﬁ cation of functional surfaces of the zinc binding domains of intracellular receptors. J. Mol. Biol., 274:325–37. Lichtarge, O., Yao, H. et al. 2003. Accurate and scalable identiﬁ cation of functional sites by evolutionary tracing. J. Struct Funct Genomics, 4:159–66. Lipman, D.J., Altschul, S.F. et al. 1989. A tool for multiple sequence alignment. Proc. Natl. Acad. Sci., U.S.A., 86:4412–5. Livesay, D.R., Jambeck, P. et al. 2003. Conservation of electrostatic properties within enzyme families and superfamilies. Biochemistry, 42:3464–73. 213 Valeyev et al Madabushi, S., Yao, H. et al. 2002. Structural clusters of evolutionary trace residues are statistically signiﬁ cant and common in proteins. J. Mol. Biol., 316:139–54. Smith, T.F., Gaitatzes, C. et al. 1999. The WD repeat: a common architecture for diverse functions. Trends Biochem. Sci., 24:181–5. Soares, D.C., Gerloff, D.L. et al. 2005. Large-scale modelling as a route to multiple surface comparisons of the CCP module family. Protein Eng. Des. Sel., 18:379–88. Madrona, A.Y. Evolutionary Bioinformatics 2008:4 Conclusions and Wilson, D.K. 2004. The structure of Ski8p, a protein regulating mRNA degradation: Implications for WD protein structure. Protein Sci., 13:1557–65. Sprague, E.R., Redd, M.J. et al. 2000. Structure of the C-terminal domain of Tup1, a corepressor of transcription in yeast. Embo. J., 19:3016–27. Murzin, A.G. 1992. Structural principles for the propeller assembly of beta-sheets: the preference for seven-fold symmetry. Proteins, 14:191–201. Struyf, A., Hubert, M. et al. 1997. Integrating Robust Clustering Techniques in S-PLUS. Computational Statistics and Data Analysis, 24:17–37. Murzin, A.G., Brenner, S.E. et al. 1995. SCOP: a structural classiﬁ cation of proteins database for the investigation of sequences and structures. J. Mol. Biol., 247:536–40. Thompson, J.D., Higgins, D.G. et al. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-speciﬁ c gap penalties and weight matrix choice. Nucleic Acids Res., 22:4673–80. Paoli, M. 2001. Protein folds propelled by diversity. Prog Biophys. Mol. Biol., 76:103–30. Tsodikov, O.V., Record Jr, M.T. et al. 2002. Novel computer program for fast exact calculation of accessible and molecular surface areas and average surface curvature. J. Comput. Chem., 23:600–9. Pﬂ ugrad, A., Meir, J.Y. et al. 1997. The Groucho-like transcription factor UNC-37 functions with the neural speciﬁ city gene unc-4 to govern motor neuron identity in C. elegans. Development, 124:1699–709. van der Voorn, L. and Ploegh, H.L. 1992. The WD-40 repeat. FEBS Lett., 307:131–4. y g Pickles, L.M., Roe, S.M. et al. 2002. Crystal structure of the C-terminal WD40 repeat domain of the human Groucho/TLE1 transcriptional corepressor. Structure, 10:751–61. Voegtli, W.C., Madrona, A.Y. et al. 2003. The structure of Aip1p, a WD repeat protein that regulates Coﬁ lin-mediated actin depolymerization. J. Biol. Chem., 278:34373–9. Robinson, R.C., Turbedsky, K. et al. 2001. Crystal structure of Arp2/3 complex. Science, 294:1679–84. Wu, G., Xu, G. et al. 2003. Structure of a beta-TrCP1-Skp1-beta-catenin complex: destruction motif binding and lysine speciﬁ city of the SCF(beta-TrCP1) ubiquitin ligase. Mol. Cell., 11:1445–56. Sali, A. and Blundell, T.L. 1993. Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol., 234:779–815. Yu, L., Gaitatzes, C. et al. 2000. Thirty-plus functional families from a single motif. Protein Sci., 9:2470–6. Shaw, G. 1996. The pleckstrin homology domain: an intriguing multifunctional protein module. Bioessays, 18:35–46. Shindyalov, I.N. and Bourne, P.E. 1998. Protein structure alignment by incremental combinatorial extension (CE) of the optimal path. Protein Eng., 11:739–47. 214 Evolutionary Bioinformatics 2008:4 Electrostatics and Functions of Seven-Bladed WD β-Propellers Electrostatic and Functional Analysis of the Seven-Bladed WD β-Propellers Najl V Valeyev1,4, A Kristina Downing1, John Sondek2 and Charlotte Deane3 Najl V Valeyev1,4, A Kristina Downing1, John Sondek2 and Charlot Evolutionary Bioinformatics 2008:4 Supplementary Material Supplementary Material 1L0Q 1GOT A B C D 1GOF 1QNI E F G H 1A12 1C9L 1M1X 1QFM 1JOF 1RI6 1OLZ L I K J 1K32 Figure S1. Representative experimental structures for all seven-bladed WD β-propeller superfamilies. The structures shown reveal some variability mostly observed in loops. (A) Galactose Oxidase, (B) Surface layer protein, (C) Nitrous oxide reductase, (D) G-beta protein, (E) Regulator of chromosome condensation RCC1, (F) Clathrin, (G) Integrin, (H) Prolyl oligopeptidase, (I) Tricorn protease, (J) 3-carboxy-cis, cis-mucoante lactonizing enzyme, (K) Putative isomerase YbhE, (L) Sema domain (found in proteins involved in development, tissue regeneration and cancer), (M) Backbone representation of the G-beta protein. A 1GOF 1L0Q B 1GOT D C 1QNI A B D C 1GOF 1L0Q 1GOT G 1M1X H 1QFM F 1C9L F 1C9L 1JOF J E 1A12 F E G 1A12 1QFM 1C9L 1M1X 1M1X 1C9L 1JOF J 1RI6 K I 1K32 1OLZ L I J 1OLZ 1RI6 1JOF 1K32 Figure S1. Representative experimental structures for all seven-bladed WD β-propeller superfamilies. The structures shown reveal some variability mostly observed in loops. (A) Galactose Oxidase, (B) Surface layer protein, (C) Nitrous oxide reductase, (D) G-beta protein, (E) Regulator of chromosome condensation RCC1, (F) Clathrin, (G) Integrin, (H) Prolyl oligopeptidase, (I) Tricorn protease, (J) 3-carboxy-cis, cis-mucoante lactonizing enzyme, (K) Putative isomerase YbhE, (L) Sema domain (found in proteins involved in development, tissue regeneration and cancer), (M) Backbone representation of the G-beta protein. 215 Evolutionary Bioinformatics 2008:4 Valeyev et al Figure S2. Geometrical characterization of the experimental seven-bladed β-propeller structures. Each sphere represents the middle position on the outer strand of the β-propeller blades. The representative experimental structures from Figure 1 B were superimposed and are each shown in a different colour. Despite some degree of structural variability observed across seven-bladed β-propellers (Fig. 1 in Supplementary Material), these data suggest that the β-propeller blades are spatially conserved across the superfamily. Figure S2. Geometrical characterization of the experimental seven-bladed β-propeller structures. Each sphere represents the middle position on the outer strand of the β-propeller blades. The representative experimental structures from Figure 1 B were superimposed and are each shown in a different colour. Despite some degree of structural variability observed across seven-bladed β-propellers (Fig. 1 in Supplementary Material), these data suggest that the β-propeller blades are spatially conserved across the superfamily. Figure S2. Geometrical characterization of the experimental seven-bladed β-propeller structures. Supplementary Material Each sphere represents the middle position on the outer strand of the β-propeller blades. The representative experimental structures from Figure 1 B were superimposed and are each shown in a different colour. Despite some degree of structural variability observed across seven-bladed β-propellers (Fig. 1 in Supplementary Material), these data suggest that the β-propeller blades are spatially conserved across the superfamily. e experimental seven-bladed β-propeller structures. Table SI. Protein sequences used for the electrostatics analysis 216 Evolutionary Bioinformatics 2008:4
https://openalex.org/W2011382435	https://link.springer.com/content/pdf/10.1140/epjc/s10052-015-3411-z.pdf	English	null	The effective Planck mass and the scale of inflation	European physical journal. C, Particles and fields	2,015	cc-by	11,748	The effective Planck mass and the scale of inﬂation Ignatios Antoniadis1,2,a, Subodh P. Patil3,b 1 Albert Einstein Center for Fundamental Physics, Institute for Theoretical Physics, Bern University, Sidlerstrasse 5, 3012 Bern, Switzerland 2 Ecole Polytechnique, 91128 Palaiseau, France 3 Theory Division, PH-TH Case C01600, CERN, 1211 Geneva, Switzerland Ignatios Antoniadis1,2,a, Subodh P. Patil3,b 1 Albert Einstein Center for Fundamental Physics, Institute for Theoretical Physics, Bern University, Sidlerstrasse 5, 3012 Bern, Switzerland 2 Ecole Polytechnique, 91128 Palaiseau, France 3 Theory Division, PH-TH Case C01600, CERN, 1211 Geneva, Switzerland Received: 12 March 2015 / Accepted: 14 April 2015 / Published online: 1 May 2015 © The Author(s) 2015. This article is published with open access at Springerlink.com Abstract Observable quantities in cosmology are dimen- sionless, and therefore independent of the units in which they are measured. This is true of all physical quantities associ- ated with the primordial perturbations that source cosmic microwave background anisotropies such as their amplitude and spectral properties. However, if one were to try and infer an absolute energy scale for inﬂation—a priori, one of the more immediate corollaries of detecting primordial tensor modes—one necessarily makes reference to a partic- ular choice of units, the natural choice for which is Planck units. In this note, we discuss various aspects of how infer- ring the energy scale of inﬂation is complicated by the fact that the effective strength of gravity as seen by inﬂation- ary quanta necessarily differs from that seen by gravitational experiments at presently accessible scales. The uncertainty in the former relative to the latter has to do with the unknown spectrum of universally coupled particles between laboratory scales and the putative scale of inﬂation. These intermediate particles could be in hidden as well as visible sectors or could also be associated with Kaluza–Klein resonances associated with a compactiﬁcation scale below the scale of inﬂation. We discuss various implications for cosmological observables. quantum corrections effect the effective strength of gravity depending on the characteristic energy of the process probing it.2 Massive particles are particularly interesting for the threshold effects they impart once we start to probe energies above their mass M, i.e. at distances below M−1. This can be understood via a simple thought experiment [5]: consider scattering a test particle off a very heavy point mass. The inverse Fourier transform of the scattering amplitude yields the gravitational potential generated by the source. The effective Planck mass and the scale of inﬂation Once the inter-particle separation approaches x ∼M−1, M being the mass of some heavy particle, virtual pairs of these parti- cles are created, the positive/ negative energy virtual quanta of which are attracted/ repelled by the source, creating a grav- itational dipole distribution that effectively anti-screens the source, strengthening its gravitational ﬁeld. Therefore, the strength of gravity is increased by this effective ‘vacuum polarization’ far enough away from the threshold induced by a particle of mass M j that couples to gravity,3 i.e. as we probe increasingly shorter distances x ≪M−1 j . j One can quantitatively understand this effective strength- ening through the computation of the graviton propagator with loops of the massive ﬁelds contributing to the gravi- ton self-energy insertions. We trace through the details of this computation in Appendix A following the treatment of [6]; however, a quick understanding of this can be arrived at through the argument presented in [7]. Consider the correc- tion to the graviton propagator induced by loops of various particles—suppressing all index structure, we ﬁnd that the leading correction will have the form Eur. Phys. J. C (2015) 75:182 DOI 10.1140/epjc/s10052-015-3411-z Regular Article - Theoretical Physics Regular Article - Theoretical Physics 2 See [1,2] for reviews of treating gravity as an effective theory. In the following discussion, we steer clear of potentially problematic aspects of the notion of running gravitational couplings [3,4] by focusing only on physically observable quantities such as amplitudes and cross sec- tions. b e-mail: subodh.patil@cern.ch a e-mail: Ignatios.Antoniadis@cpht.polytechnique.fr b 3 This is true regardless of whether these massive particles couple directly to the sector that contains the probe particle (e.g. the Standard Model) or not. 2 See [1,2] for reviews of treating gravity as an effective theory. In the following discussion, we steer clear of potentially problematic aspects of the notion of running gravitational couplings [3,4] by focusing only on physically observable quantities such as amplitudes and cross sec- tions. 3 This is true regardless of whether these massive particles couple directly to the sector that contains the probe particle (e.g. the Standard Model) or not. a e-mail: Ignatios.Antoniadis@cpht.polytechnique.fr b e-mail: subodh.patil@cern.ch 4 And is thus unitarized by the appearance of new degrees of freedom at M∗∗from some UV completion, such as string theory. 5 We are grateful to Sergey Sibiryakov for discussions concerning this point. 1 Preliminaries However, when one tries to infer an absolute energy scale for inﬂation, one ﬁnds that it is undetermined commensurate with (5) up to the unknown spectrum of uni- versally coupled species between laboratory scales and the inﬂationary scale, the details of which we elaborate upon in the following. In what follows, we work out the consequences of this scale dependence of the strength of gravity for inferring various quantities during inﬂation, which we take to be driven by a single ﬁeld for economy of discussion and because the data does not compel us to consider otherwise [11,12].6 As is to be expected, all dimensionless observables, such as the amplitude and spectral properties of the perturbations, are unaffected by the changing strength of gravity at inﬂation- ary energies. However, when one tries to infer an absolute energy scale for inﬂation, one ﬁnds that it is undetermined commensurate with (5) up to the unknown spectrum of uni- versally coupled species between laboratory scales and the inﬂationary scale, the details of which we elaborate upon in the following. ⟨T (−p)T (p)⟩∼ c 16π2 p4 log p2 μ2 (2) (2) where μ is some arbitrary renormalization scale, and where c is the central charge of the theory that effectively counts the number of degrees of freedom running through the loop, i.e. where μ is some arbitrary renormalization scale, and where c is the central charge of the theory that effectively counts the number of degrees of freedom running through the loop, i.e. c ≈N [7]. As an illustrative example, in four-dimensional Minkowski space, the central charge of a non-interacting the- ory containing Nφ scalars, Nψ Dirac fermions and NV vector bosons is given by [8–10]: c ≈N [7]. As an illustrative example, in four-dimensional Minkowski space, the central charge of a non-interacting the- ory containing Nφ scalars, Nψ Dirac fermions and NV vector bosons is given by [8–10]: c := N = 4 3 Nφ + 8Nψ + 16NV . (3) (3) Comparison with the free propagator 1/(p2M2 pl) implies that the perturbative expansion fails at the scale p = M∗∗, where M∗∗∼Mpl N , (4) (4) 8 The comoving (or unitary) gauge is deﬁned as the foliation where inﬂaton ﬁeld ﬂuctuations have been locally gauged away. In words, it is the time slicing where the inﬂaton itself is the clock. 7 In what follows, we assume that all of the extra species have sufﬁ- ciently suppressed couplings to the inﬂaton during inﬂation (e.g. either through derivative couplings or as Planck suppressed interactions), so that isocurvature perturbations are not signiﬁcantly generated. This is trivially true for hidden sector ﬁelds. 2 The scale of inﬂation According to the inﬂationary paradigm, the primordial per- turbations observed in the CMB were created at horizon crossing during the quasi-de Sitter phase of early acceler- ated expansion sourced by the inﬂaton ﬁeld. Therefore, all quantities that enter calculations of primordial correlation functions (which we subsequently relate to observables in the CMB) refer to quantities at the scale at which inﬂation occurred. We denote all quantities measured at the scale of inﬂation with a starred subscript. The dominant contribution to the temperature anisotropies comes from adiabatic pertur- bations7 sourced by the comoving curvature perturbation R, deﬁned as the conformal factor of the 3-metric hi j in comov- ing gauge8: which is when gravity becomes strongly coupled. That is, M∗∗is the effective cut-off of gravity at short distances. How- ever, one must take care to distinguish between the scale of strong gravity M∗∗from the strength of gravity at a particu- lar energy scale, which we denote M∗. Whereas the former sets the scale at which unitarity starts to break down in the effective theory,4 the latter determines the strength of grav- itationally mediated processes at any particular scale below M∗∗. As detailed in Appendix B, although every massive speciescontributestoloweringthescaleatwhichstronggrav- ity effects become important, one has to distinguish between species that universally couple directly to the matter energy- momentum tensor at tree level [such as massive Kaluza– Klein (KK) gravitons, non-minimally coupled scalars, and U(1) gauge ﬁelds] from ordinary four-dimensional ﬁelds that couple at one loop, in terms of their effects on the strength of gravity as one crosses the threshold set by the mass M of the species, but are still far below the scale M∗∗. Whereas the former immediately affect the strength of gravity, the latter do not make their effects known until very close to M∗∗.5 Therefore, for the rest of our discussion we denote use N as hi j(t, x) = a2(t)e2R(t,x) ˆhi j; ˆhi j := exp[γi j] (6) (6) 6 See also the related studies [13,14], which explore how additional ﬁelds and non-adiabaticity further complicates inferring the scale of inﬂation from the detection of primordial tensors. 6 See also the related studies [13,14], which explore how additional ﬁelds and non-adiabaticity further complicates inferring the scale of inﬂation from the detection of primordial tensors. 7 In what follows, we assume that all of the extra species have sufﬁ- ciently suppressed couplings to the inﬂaton during inﬂation (e.g. 6 See also the related studies [13,14], which explore how additional ﬁelds and non-adiabaticity further complicates inferring the scale of inﬂation from the detection of primordial tensors. 1 Preliminaries The strength of the gravitational force depends on the scale at which it is measured.1 At laboratory scales, the strength of gravity is characterized by the reduced Planck mass Mpl = 2.435 × 1018 GeV which determines Newton’s con- stant G N = M−2 pl . However, like all other interactions, 1 E.g. via Cavendish type experiments where we have precise knowl- edge of two masses (one of which could be a test mass), or equivalently in principle through gravitational scattering experiments. 3 This is true regardless of whether these massive particles couple directly to the sector that contains the probe particle (e.g. the Standard Model) or not. 12 3 182 Page 2 of 12 182 Page 2 of 12 Eur. Phys. J. C (2015) 75 :182 1 M4 pl 1 p2 ⟨T (−p)T (p)⟩1 p2 , (1) a shorthand for the weighted index that effectively counts the number of universally coupled degrees of freedom below the energyscaleof interest correspondingtothegeneralizationof (3), such that the strength of gravity at that scale, henceforth taken to be the scale of inﬂation, is given by (1) where T (p)schematicallyrepresentsthetotal energymomen- tum tensor of the theory. We further consider the limit where the external momentum satisﬁes p2 ≫M2 where M2 is the mass of the heaviest particle that can run through the loops. In this limit, the theory becomes conformal, which ﬁxes the ﬁnite part of the loop integral to be where T (p)schematicallyrepresentsthetotal energymomen- tum tensor of the theory. We further consider the limit where the external momentum satisﬁes p2 ≫M2 where M2 is the mass of the heaviest particle that can run through the loops. In this limit, the theory becomes conformal, which ﬁxes the ﬁnite part of the loop integral to be M∗∼Mpl √ N . (5) (5) In what follows, we work out the consequences of this scale dependence of the strength of gravity for inferring various quantities during inﬂation, which we take to be driven by a single ﬁeld for economy of discussion and because the data does not compel us to consider otherwise [11,12].6 As is to be expected, all dimensionless observables, such as the amplitude and spectral properties of the perturbations, are unaffected by the changing strength of gravity at inﬂation- ary energies. ﬁelds and non-adiabaticity further complicates inferring the scale of inﬂation from the detection of primordial tensors. 7 In what follows, we assume that all of the extra species have sufﬁ- ciently suppressed couplings to the inﬂaton during inﬂation (e.g. either through derivative couplings or as Planck suppressed interactions), so that isocurvature perturbations are not signiﬁcantly generated. This is trivially true for hidden sector ﬁelds. 8 The comoving (or unitary) gauge is deﬁned as the foliation where inﬂaton ﬁeld ﬂuctuations have been locally gauged away. In words, it is the time slicing where the inﬂaton itself is the clock. 2 The scale of inﬂation (8) (8) Taking the ratio of the above with (7), we ﬁnd the tensor to scalar ratio Taking the ratio of the above with (7), we ﬁnd the tensor to scalar ratio V 1/4 ∗ ∼r1/4 ∗ √ N 3.28 × 1016 GeV. (13) (13) r∗:= Pγ PR = 16ϵ∗. (9) (9) Presuming a range for r∗such that 0.001 ≲r∗≲0.1, it is amusing to infer that in order to have an energy scale of inﬂation around 10 TeV, one requires N ∼1026 univer- sally coupled species directly to the matter stress-tensor with masses less than that energy. Presumably any such particles in the visible sector would have started to appear in collider events accessed at the LHC. Note that as one lowers the scale of strong gravity, the maximum reheating temperature Ti is necessarily lowered as well, since it cannot be higher than (13). Conservatively, Ti cannot be too far below the TeV scale without spoiling the standard scenarios of big bang cosmology—in particular, mechanisms for leptogenesis and baryogenesis which can occur no lower than the electroweak scale [18,19]. Therefore any determination of r∗, either through direct mea- surements of the stochastic background of primordial gravi- tational waves or through their secondary effects on the polar- ization of the CMB [15–17] allows us in principle to ﬁx the scale of inﬂation. Speciﬁcally, by re-expressing (7) as Presuming a range for r∗such that 0.001 ≲r∗≲0.1, it is amusing to infer that in order to have an energy scale of inﬂation around 10 TeV, one requires N ∼1026 univer- sally coupled species directly to the matter stress-tensor with masses less than that energy. Presumably any such particles in the visible sector would have started to appear in collider events accessed at the LHC. Note that as one lowers the scale of strong gravity, the maximum reheating temperature Ti is necessarily lowered as well, since it cannot be higher than (13). Conservatively, Ti cannot be too far below the TeV scale without spoiling the standard scenarios of big bang cosmology—in particular, mechanisms for leptogenesis and baryogenesis which can occur no lower than the electroweak scale [18,19]. Therefore any determination of r∗, either through direct mea- surements of the stochastic background of primordial gravi- tational waves or through their secondary effects on the polar- ization of the CMB [15–17] allows us in principle to ﬁx the scale of inﬂation. 9 So that (12) denotes a tree level relation. 10 In addition to quantum corrections to the effective action itself being set by the ratio H2 ∗/M2 ∗. 11 Note that this would require a desert not only in the sector in which the standard model resides, but in all other hidden sectors as well. 2 The scale of inﬂation either through derivative couplings or as Planck suppressed interactions), so that isocurvature perturbations are not signiﬁcantly generated. This is trivially true for hidden sector ﬁelds. 8 The comoving (or unitary) gauge is deﬁned as the foliation where inﬂaton ﬁeld ﬂuctuations have been locally gauged away. In words, it is the time slicing where the inﬂaton itself is the clock. 12 3 Page 3 of 12 182 Eur. Phys. J. C (2015) 75 :182 Page 3 of 12 182 182 the energy scale of inﬂation, deﬁned as V 1/4 ∗ . The reason for this distinction is that H∗deﬁnes (among other things) the scale above or below which massive particles respond to the background expansion irrespective of any direct couplings to the inﬂaton10 whereas V 1/4 ∗ deﬁnes the physical energy density in the inﬂaton ﬁeld as seen by particles that couple to it, such as all decay products produced in (p)reheating. We take this distinction for granted in what follows. with ∂iγi j = γii = 0 deﬁning transverse traceless gravi- ton perturbations. The temperature anisotropies are charac- terized by the dimensionless power spectrum for R, whose amplitude is given by PR := H2 ∗ 8π2M2∗ϵ∗ = A × 10−10, (7) (7) where ϵ∗:= −˙H∗/H2 ∗, H∗being the Hubble factor during inﬂation. Given that R is conserved on super-horizon scales (in the absence of entropy perturbations), this immediately relates to the amplitude of the late time CMB anisotropies, which ﬁxes A ∼22.15 [11]. The tensor anisotropies are characterized by the tensor power spectrum where ϵ∗:= −˙H∗/H2 ∗, H∗being the Hubble factor during inﬂation. Given that R is conserved on super-horizon scales (in the absence of entropy perturbations), this immediately relates to the amplitude of the late time CMB anisotropies, which ﬁxes A ∼22.15 [11]. The tensor anisotropies are characterized by the tensor power spectrum In a universe where there is a true desert between labo- ratory scales and the onset of inﬂation,11 M∗= Mpl. How- ever, given our ignorance of particle physics between collider scales and the scale of inﬂation in addition to all hidden sec- tor physics, M∗is in general lower than Mpl according to (12), where N represents a model dependent parameter that obscures our ability to infer the actual energy scale of inﬂa- tion from observations of CMB temperature and polarization anisotropies. That is, Pγ := 2 H2 ∗ π2M2∗ . 11 Note that this would require a desert not only in the sector in which the standard model resides, but in all other hidden sectors as well. 10 In addition to quantum corrections to the effective action itself being set by the ratio H2 ∗/M2 ∗. 12 We presume for simplicity that there are no further hierarchies between the extra dimensions. Note that μc can be in general (hier- archically) different from the actual compactiﬁcation scale associated to their inverse size. 13 This will remain true for more general compactiﬁcations (up to fac- tors of order unity) provided again that there are no further hierarchies among the extra dimensions. 2 The scale of inﬂation Speciﬁcally, by re-expressing (7) as H∗= M∗ π2Ar∗ 2 · 1010 1/2 , (10) (10) one can determine the value of the potential during inﬂation in the slow roll approximation: V 1/4 ∗ = M∗ 3π2Ar∗ 2 · 1010 1/4 . (11) (11) We see that any measurements of r∗and A determines the scale of inﬂation up to our ignorance of the effective strength of gravity at the scale H∗, given by We see that any measurements of r∗and A determines the scale of inﬂation up to our ignorance of the effective strength of gravity at the scale H∗, given by We note, as a consistency check on the above considera- tions, that although additional species increase the strength of gravity, the ratio H2 ∗/M2 ∗is independent of N and is ﬁxed by observable quantities as M∗∼Mpl √ N (12) (12) H∗ M∗ = π2Ar∗ 2 · 1010 1/2 := ϒ = 1.05 √r∗× 10−4. ( where Mpl = 2.435 × 1018 GeV is the reduced Planck mass that deﬁnes the strength of gravitational interactions at labo- ratory scale wavelengths and longer. As noted above, N is a weighted index that effectively counts the number of all uni- versally coupled9 species up to the scale H∗—whether they exist in the visible sector or in any hidden sector. Presuming r∗= 0.1, Eq. (11) implies an energy scale for inﬂation of V 1/4 ∗ = 7.6 × 10−3M∗. (14) where Mpl = 2.435 × 1018 GeV is the reduced Planck mass that deﬁnes the strength of gravitational interactions at labo- ratory scale wavelengths and longer. As noted above, N is a weighted index that effectively counts the number of all uni- versally coupled9 species up to the scale H∗—whether they exist in the visible sector or in any hidden sector. Presuming r∗= 0.1, Eq. (11) implies an energy scale for inﬂation of V 1/4 ∗ = 7.6 × 10−3M∗. Therefore the effects of strong gravity are evidently neg- ligible during inﬂation even if M∗is much smaller than the macroscopic strength of gravity Mpl. Hence inﬂationary dynamics,inparticularthedynamicsofadiabaticﬂuctuations remain weakly coupled independent of N and the usual com- putation of adiabatic correlators can be implemented [20]. In order to keep track of concepts in the discussion to follow, we distinguish between what we henceforth refer to as the scale of inﬂation—deﬁned as H∗during inﬂation—and 12 3 182 Page 4 of 12 Eur. 2 The scale of inﬂation Phys. J. C (2015) 75 :182 3 Extra species as Kaluza–Klein states number of extra massive species is now given by (neglecting factors of order unity): It is an interesting exercise to work out the consequences of extra species associated with the Kaluza–Klein (KK) states of a particular compactiﬁcation. One of two scenarios are possible—that inﬂation occurs below (H∗< μc), or above (H∗> μc) the effective compactiﬁcation scale μc deﬁned as the mass scale associated with the moduli that ﬁx the size of the extra dimensions.12 In the former case, the moduli corre- sponding to the extra dimensions remain ﬁxed at their min- ima during inﬂation and we have available the usual relation between the fundamental gravity scale M∗∗below the effec- tive compactiﬁcation scale and the long wavelength strength of gravity (the Planck mass): N = (M∗∗/M)n ≡V∗∗, (18) (18) which corresponds to the number or lattice sites such that the condition i l2 i M2 ≤M2 ∗∗is satisﬁed. We note that one could also have inﬂation happening above the effective com- pactiﬁcation scale (H∗> μc). In general, this would involve having to track the full dynamics of the moduli ﬁelds on their way to stabilization, which does not permit any straightfor- ward generalizations. However, there are certain limits for which the moduli are effectively frozen in spite of not being ﬁxed at the minima of their effective potentials. As discussed in Appendix C, this occurs in the limit where the sum of the inﬂationary and moduli potentials satisfy an analog of the slow roll conditions. We will presume this to be the case when H∗> μc. Although the discussion to follow presumes H∗< μc, the results generalize straightforwardly were we to replace M∗∗with ¯M∗∗deﬁned as the effective cut-off when the compact dimensions have the (effectively frozen) volume ¯V∗∗during inﬂation. which corresponds to the number or lattice sites such that the condition i l2 i M2 ≤M2 ∗∗is satisﬁed. We note that one could also have inﬂation happening above the effective com- pactiﬁcation scale (H∗> μc). In general, this would involve having to track the full dynamics of the moduli ﬁelds on their way to stabilization, which does not permit any straightfor- ward generalizations. However, there are certain limits for which the moduli are effectively frozen in spite of not being ﬁxed at the minima of their effective potentials. 3 Extra species as Kaluza–Klein states As discussed in Appendix C, this occurs in the limit where the sum of the inﬂationary and moduli potentials satisfy an analog of the slow roll conditions. We will presume this to be the case when H∗> μc. Although the discussion to follow presumes H∗< μc, the results generalize straightforwardly were we to replace M∗∗with ¯M∗∗deﬁned as the effective cut-off when the compact dimensions have the (effectively frozen) volume ¯V∗∗during inﬂation. M D−2 ∗∗ Vn = M2 pl , where Vn is the volume of the compactiﬁed sub-manifold [21, 22]. Again, the double asterisked subscript is to differentiate M∗∗from M∗, the strength of gravity at the inﬂationary scale H∗. In D = 4 + n, this relation becomes M2+n ∗∗Vn = M2 pl. For the example of toroidal compactiﬁcations,13 Vn = M−n, so that 3.1 Extra KK species and the scale of inﬂation M2 ∗∗ Mn ∗∗ Mn := M2 ∗∗V∗∗= M2 pl , (15) (15) During inﬂation, all masses much lighter than H∗correct the graviton propagator and will contribute towards lowering the effective gravitational cut-off. If furthermore, these states are universally coupled (as are KK gravitons), they will also increase the effective strength of gravity now set by M∗. All heavier KK states do not correct the short range interactions (i.e. they decouple) and can safely be ignored. Therefore N, the number of massive species that correct the strength of gravity, is bounded by n2M2 = m2 n ≪H2 ∗in the case of one extra dimension. Hence where we have deﬁned V∗∗as the volume in units of M∗∗. Comparison with (4) implies where we have deﬁned V∗∗as the volume in units of M∗∗. Comparison with (4) implies N = V∗∗, (16) (16) 4 (P)reheating which depending on the number of extra particles between H∗and M∗∗implies that V 1/4 ∗ can be greater than14 M∗∗(of the same order or an order of magnitude higher for 2 ≤n ≤ 6), even though it is always less than the effective cut-off M∗at the scale H∗through (11). We note that this is never problematic, even though M∗∗is the cut-off induced by the underlying UV completion. This is because we remain in the perturbative regime with respect to corrections from the heavy states that UV complete the theory, which relies on derivatives being suppressed relative to this scale i.e. by the ratio H∗/M∗∗, guaranteed to be less than unity by (26). which depending on the number of extra particles between H∗and M∗∗implies that V 1/4 ∗ can be greater than14 M∗∗(of the same order or an order of magnitude higher for 2 ≤n ≤ 6), even though it is always less than the effective cut-off M∗at the scale H∗through (11). We note that this is never problematic, even though M∗∗is the cut-off induced by the underlying UV completion. This is because we remain in the perturbative regime with respect to corrections from the heavy states that UV complete the theory, which relies on derivatives being suppressed relative to this scale i.e. by the ratio H∗/M∗∗, guaranteed to be less than unity by (26). The big bang begins shortly after inﬂation ends. The mech- anism through which the inﬂaton dumps its energy density into the material content of the universe is known as reheating if this process occurs in thermal equilibrium, and preheating otherwise. During preheating, parametric resonance during the inﬂaton’s ﬁnal oscillations about its minimum results in bursts of particle production for any massive ﬁelds coupled to it (see [28] and references therein for details as regards the points discussed here). The latter is a very out of equi- librium process and requires a subsequent period of ther- malization. Since the primary mechanisms for generating parametric resonance have a purely particle physics origin, gravitational effects do not play any signiﬁcant role and the mechanisms for preheating proceed as they do in the stan- dard context regardless of the value of M∗. The exception being the special case of ‘geometric preheating’, wherein the inﬂaton φ couples very weakly or has no direct couplings to a non-minimally coupled ﬁeld χ with a non-minimal cou- pling parameter ξ. 14 For a large enough ratio N/N—guaranteed for n ≥2 through the hierarchy implied by (26). 3.2 Large number of species in string theory (24) (24) Now using (10), (11), and (14), we have Now using (10), (11), and (14), we have V 1/4 ∗ ≃31/4 ϒ−1/2 H∗, (25) (25) so that equivalently Apart from the possibility of having light KK modes of large extra dimensions, the fundamental gravity scale can be lowered due to a large number of species from hidden sectors (even coupled gravitationally to the Standard Model), or even from string excitations whose number increase exponentially with their mass. In the later case, the effective number of particle species which are not broad resonances, with width less than their mass is N ≃1/g2 s [23–27]. H∗≃M∗∗ϒ2/(n+2) . (26) (26) It follows that H∗is one to three orders of magnitude below the fundamental gravity scale M∗∗for the range 0.001 ≲ r∗≲0.1. The ratio H∗/M∗is of course ﬁxed by (14). Fur- thermore, we note that from (25) the energy scale of inﬂation is related to the scale M∗∗by It follows that H∗is one to three orders of magnitude below the fundamental gravity scale M∗∗for the range 0.001 ≲ r∗≲0.1. The ratio H∗/M∗is of course ﬁxed by (14). Fur- thermore, we note that from (25) the energy scale of inﬂation is related to the scale M∗∗by V 1/4 ∗ ≃31/4ϒ2/(n+2)−1/2M∗∗, (27) (27) (16) N = V∗∗, where we again distinguish N from N, the former of course being the total number of species up to the effective cut-off whereas the latter is the total number of species up to the scale H∗. To see this another way, we note that we could also have arrived at (16) through more direct reasoning. Consider ﬁrst for simplicity a tower of KK states on a single ﬂat, compact dimension of radius R = M−1. The KK modes are character- ized by their quantized momenta along the extra dimension, resulting in a tower of masses: N ≲H∗ M . (19) N ≲H∗ M . (19) Imagine we were to saturate this bound, m2 l = l2M2 l = 0, ±1, . . . (17) N ≈H∗ M∗ M∗ M ≈ϒ M∗ M ≈1.05 √r∗× 10−4 M∗ M (20) (17) (20) Clearly, the maximum number of resonances there can be before one hits the scale of strong gravity is set by the highest permissible momentum quantum number l2 maxM2 = M2 ∗∗. Hence with one extra dimension, the number of extra massive species is given by N = M∗∗/M. With n extra dimensions, we have m2 {li} = i l2 i M2 with i running from 1 to n. The Clearly, the maximum number of resonances there can be before one hits the scale of strong gravity is set by the highest permissible momentum quantum number l2 maxM2 = M2 ∗∗. Hence with one extra dimension, the number of extra massive species is given by N = M∗∗/M. With n extra dimensions, we have m2 {li} = i l2 i M2 with i running from 1 to n. The where the latter follows from the observationally determined quantity (14). For n extra dimensions, the number of massive species with masses less than Hubble will be given by N ≈ H∗ M n ≈ H∗ M∗ n M∗ M n ≈ϒn M∗ M n . (21) Furthermore, given that N = (M∗∗/M)n = (M∗∗/H∗)n (H∗/M)n, we arrive at the relation between the number of species that lower the effective cut-off during inﬂation N with N: N = N H∗ M∗∗ n ≡V∗∗ H∗ M∗∗ n . (22) (22) 3 Eur. Phys. J. C (2015) 75 :182 Page 5 of 12 182 182 3.2 Large number of species in string theory 3.2 Large number of species in string theory As we shall see shortly, since H∗/M∗∗< 1 we have N < N, implying that in general we must also have M∗∗< M∗as one can only cross additional mass thresholds from the scale of inﬂation to the scale of strong gravity. We note that (21) immediately translates the uncertainty in the energy scale of inﬂation in terms of an intermediate compactiﬁcation scale M in units of M∗through (13) and (14): In the framework of string theory, the effective higher- dimensional Planck mass M∗∗is proportional to the funda- mental string scale Ms, and Eq. (B18) becomes M2 pl = 1 g2s M2 s V∗∗, (28) (28) V 1/4 ∗ ≃31/4M∗γ 1/2 = 31/4 M ϒ M∗ n/2 ϒ1/2Mpl, (23) (23) where gs is the string coupling and the internal volume V∗∗ is now given in string units. The corresponding number of species is then N = V∗∗/g2 s , which is ﬁxed by the number of KK modes with mass lower than Ms for gs ≃O(1), as is the case of D-branes where gs is given by the gauge coupling. Again, we distinguish N, the number of KK modes below the effective cut-off around the compactiﬁcation scale from N, the number of states with masses less than H∗. Note that the lower bound for the string scale of few TeV is consistent with a reheating temperature around above the electroweak scale (see discussion at the end of the previous section). where gs is the string coupling and the internal volume V∗∗ is now given in string units. The corresponding number of species is then N = V∗∗/g2 s , which is ﬁxed by the number of KK modes with mass lower than Ms for gs ≃O(1), as is the case of D-branes where gs is given by the gauge coupling. Again, we distinguish N, the number of KK modes below the effective cut-off around the compactiﬁcation scale from N, the number of states with masses less than H∗. Note that the lower bound for the string scale of few TeV is consistent with a reheating temperature around above the electroweak scale (see discussion at the end of the previous section). or equivalently or equivalently V 1/4 ∗ ≃31/4 M∗∗ Mpl M∗∗ H∗ n/2 ϒ1/2 Mpl. 15 Also in the multi-ﬁeld context are the references [30–32]. In these studies, no species below the scale of inﬂation were considered, and so the strength of gravity at H∗was taken to be the usual Mpl. 5 Discussion (29) It is commonlypresumedthat detectionof aprimordial tensor mode background would allow us to determine the (energy) scale of inﬂation in the context of single ﬁeld inﬂation. The purpose of this note was to highlight the fact that, instead, one can only infer the (energy) scale of inﬂation from obser- vations up to our ignorance of the scale M∗= Mpl/ √ N, the precise value for which depends on the spectrum of all uni- versally coupled species with masses up to H∗, and for which ﬁeld content of the standard model alone suggests an N dif- ferentfromonethoughstilloforderunity.Theseobservations raise the possibility that the energy scale for inﬂation can be signiﬁcantly lowered by the presence of many gravitation- ally coupled species, an observation that has a particularly natural realization in extra-dimensional scenarios, although is equally pertinent in a four-dimensional context. As the inﬂaton oscillates around its minimum, the scalar cur- vature R = 12H2 + 2 ˙H oscillates as well. Upon time aver- aging we have the relation ⟨m2 φφ2⟩= ⟨˙φ2⟩, which implies R ∼m2 φφ2/M2 ∗, inducing an effective coupling to φ and which can produce parametric resonance for large enough ξ. By enhancing the strength of gravity, one enhances the effects of the geometric coupling term and thus widening the bands in which the Floquet index [28] is positive, assisting parametric resonance non-linearly the more M∗is reduced. Reheating on the other hand is an equilibrium process that produces quanta of matter ﬁelds through one body decays such as φ →χχ or φ →¯ψψ where χ, ψ are scalar and fermionic quanta, respectively. The interactions that can gen- erate such decays are Lφχχ = μφχ2 or Lφ ¯ψψ = yφ ¯ψψ, where μ has dimensions of mass and y is dimensionless. In the limit m2 φ ≫m2 ψ, m2 χ the decay rates can be estimated as [28]: Acknowledgments We wish to thank Robert Brandenberger, Cliff Burgess, Sergey Sibiryakov, and Michael Trott for many informative discussions and comments on the draft. During an earlier portion of this collaboration, SP was supported by a Marie Curie Intra-European Fel- lowship of the European Community’s 7th Framework Program under Contract No. PIEF-GA-2011-302817. φ→χχ = μ2 8πmφ , (30) φ→¯ψψ = y2mφ 8π . 5 Discussion (31) (30) Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecomm ons.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. Funded by SCOAP3. (31) Thermal equilibrium requires interactions to be efﬁcient enough to equipartition all available states in phase space. In an expanding universe this necessitates tot > H∗. Hence, the maximum temperature reheating can occur at is implied by the condition tot = H∗= ρ/(3M2∗). Assuming g∗rel- ativistic species in thermal equilibrium after reheating, we have Thermal equilibrium requires interactions to be efﬁcient enough to equipartition all available states in phase space. In an expanding universe this necessitates tot > H∗. Hence, the maximum temperature reheating can occur at is implied by the condition tot = H∗= ρ/(3M2∗). Assuming g∗rel- ativistic species in thermal equilibrium after reheating, we have 16 As we shall see shortly, the bare cosmological constant term is neces- sitated to ensure the satisfaction of the Slavnov–Taylor identities when expanding around ﬂat space. 4 (P)reheating For this scenario, we ﬁrst observe that on a background sourced by φ at the end of inﬂation, the mode functions for χ satisfy Furthermore, we stress that although extra dimensions (compactiﬁed at a scale below that of inﬂation) provide a natural context for the appearance of extra massive species, the relation (13) is also valid in a strictly four-dimensional context and illustrates an irreducible uncertainty in our abil- ity to infer a scale for inﬂation given our lack of knowledge of particle physics from collider energies up to the energy scale of inﬂation. 12 3 3 Eur. Phys. J. C (2015) 75 :182 182 Page 6 of 12 182 Page 6 of 1 ¨χk + 3H ˙χk + k2 a2 + ξ R χk = 0. (29) 17 Furthermore, the addition of the bare cosmological term in (A1) was necessitated by the non-vanishing tadpole in Fig. 1, which we have to cancel in order to consistently expand around ﬂat space—the remaining diagrams thus resulting in (A11)–(A13). Appendix A: Loop corrections to the graviton propagator C (2015) 75 :182 and to deﬁne the graviton ϕμν (note the departure from the which evaluates to and to deﬁne the graviton ϕμν (note the departure from the which evaluates to the departure from the which evaluates to and to deﬁne the graviton ϕμν (note the departure from the which evaluates to which evaluates to and to deﬁne the graviton ϕμν (note the departure from the usual deﬁnition) as and to deﬁne the graviton ϕμν (note the departure from the usual deﬁnition) as g ϕ ( p sual deﬁnition) as κ2 Qνσμλ = κ2 p4 T1 pν pσ pμ pλ −p2δμλ pν pσ −p2δνσ pμ pλ + p4δνσδμλ +T3 p4δμνδσλ + p4δνλδσμ −2p4δνσδμλ + 2p2δμλ pν pσ + 2p2δνσ pμ pλ −4p2 p(νδσ)(μ pλ) (A11) usual deﬁnition) as ˜gμν = δμν + κϕμν (A3) so that √g = det[gμν]1/(d−2) = exp 1 d −2Tr ln δμν + κϕμν . (A4) Qνσμλ = κ2 p4 T1 pν pσ pμ pλ −p2δμλ pν pσ −p2δνσ pμ pλ + p4δνσδμλ +T3 p4δμνδσλ + p4δνλδσμ −2p4δνσδμλ + 2p2δμλ pν pσ + 2p2δνσ pμ pλ −4p2 p(νδσ)(μ pλ) (A11) ˜gμν = δμν + κϕμν (A3) so that ˜gμν = δμν + κϕμν so that so that √g = det[gμν]1/(d−2) = exp 1 d −2Tr ln δμν + κϕμν . Appendix A: Loop corrections to the graviton propagator As a concrete example to illustrate how gravity can become more strongly coupled at lower energies due to loops of mat- terﬁelds,wereproducetheoneloopcorrectiontothegraviton propagator on a ﬂat Euclidean spacetime due to loops of a massive scalar ﬁeld, as calculated by Capper [6] (see also ref- erences therein). Beginning with the matter sector action16 ρ = g∗π2 30 T 4, (32) (32) and therefore Ti = 90 g∗π2 1/4 totM∗∼ 90 g∗π2 1/4 totMpl N 1/4 . (33) (33) S = −2 κ2 d4x√g (R −2) −1 2 d4x√g gμν∂μϕ∂νϕ + m2ϕ2 . (A1) That is, the maximum temperature that can reheat is corre- spondingly reduced, consistent with our discussion in Sect. 3. We mention in closing that there are rich phenomenologi- cal possibilities in considering hidden sector ﬁelds produced in reheating as possible dark matter candidates in scenarios with many extra species, certain aspects of which have been studied in the multi-ﬁeld inﬂationary context in [29].15 That is, the maximum temperature that can reheat is corre- spondingly reduced, consistent with our discussion in Sect. That is, the maximum temperature that can reheat is corre- spondingly reduced, consistent with our discussion in Sect. 3. We mention in closing that there are rich phenomenologi- cal possibilities in considering hidden sector ﬁelds produced in reheating as possible dark matter candidates in scenarios with many extra species, certain aspects of which have been studied in the multi-ﬁeld inﬂationary context in [29].15 (A1) It is particularly handy to deﬁne the tensor density ˜gμν = √ggμν, (A2) (A2) ˜gμν = √ggμν, 16 As we shall see shortly, the bare cosmological constant term is neces- sitated to ensure the satisfaction of the Slavnov–Taylor identities when expanding around ﬂat space. 12 3 Page 7 of 12 182 Eur. Phys. J. Appendix A: Loop corrections to the graviton propagator (A4) (A11) The matter Lagrangian can be expanded in powers of κ as The matter Lagrangian can be expanded in powers of κ as where 2ω = d is the parameter through which we implement dimensional regularization, μ is an arbitrary renormalization scale, and where L = k=2 κk−2Lk (A5) with L = k=2 κk−2Lk (A5) (A5) T1 = m2(ω−2) μ2(2−ω) 2(4π)ω (2 −ω) × 1 3 3F2 2 −ω, 1, 3; 2, 5/2; −p2 4m2 −1 2 3F2 2 −ω, 4, 1; 5/2, 3, −p2 4m2 +1 5 3F2 2 −ω, 5, 1; 3, 7/2, −p2 4m2 , (A12) with L3 = −1 2ϕμν∂μϕ∂νϕ + m2 2 ϕ2ϕμ μ d −2 (A6) and (A6) and L4 = m2ϕ2 d −2 −ϕμνϕμν + ϕμ μϕν ν d −2 , (A7) (A12) (A7) which implies the cubic and quartic interaction vertices Vαβ and Uαβ δγ : T3 = m2ω μ2(2−ω) 8(4π)ω (2 −ω) p4(ω −1) 1 1 −ω + 1 ω 2F1 × −ω, 1; 3/2; −p2 4m2 . (A13) Vα,β(p, k1, k2) = −k1(μk2ν) + m2δμν n −2 ; Uαβ γ δ(p1, p2, k1, k2) = m2 n−2 −δα(γ δβ(δ)+ δαβδγ δ n−2 . (A8) Vα,β(p, k1, k2) = −k1(μk2ν) + m2δμν n −2 ; Uαβ γ δ(p1, p2, k1, k2) = m2 n−2 −δα(γ δβ(δ)+ δαβδγ δ n−2 . (A8) Supplemented with the massive scalar and graviton propa- t (th l tt bt i d i th d D d [6]) Vα,β(p, k1, k2) = −k1(μk2ν) + m2δμν n −2 ; Uαβ γ δ(p1, p2, k1, k2) = m2 n−2 −δα(γ δβ(δ)+ δαβδγ δ n−2 . (A8) Supplemented with the massive scalar and graviton propa- gators (the latter obtained in the de Donder gauge [6]), (A13) Some remarks are in order at this stage. There are ﬁve possi- ble terms with the right tensor structure that could contribute to Tαβθτ, three of whose coefﬁcients can be eliminated by repeated applications of the Ward identities. Doing so results in (A11),17 for which it is easily veriﬁed that pν Qνσμλ = 0. Having dimensionally regularized the loop integrals, we ﬁnd the usual 1/(2 −ω) poles which require appropriate coun- terterms to subtract the divergences. Appendix B: Note on universally coupled massive species so that We wish to demonstrate that, although all massive species contribute to lowering the effective scale of strong gravity, the threshold effects imparted by universally coupled species (e.g. massive KK gravitons) differs qualitatively from that of massive, non-universally coupled species. Whereas the for- mer changes the strength of gravity by an order one effect as you cross the threshold M, the latter only changes the strength of gravity by a factor of M2/M2 pl, even as both con- tribute equally to lowering the scale at which strong gravity effects become important. This can be seen by considering the correction to the graviton propagator from loops of N ordinary massive ﬁelds in the limit of high momentum trans- fer p2 ≫M2: ϕμν(x) = 2πm dD−1 p (2π)D ei ⃗p·⃗x × D 00 μν (p) + Q 00 μν (p) \|p0=0. (B5) (B5) One can revert to the traditional dimensionless metric per- turbation hμν via the relation (A2), hμν = −1 Mpl ϕμν − ϕλ λ D −2δμν . (B6) (B6) Ignoring the second term and using the expression for the propagator in the de Donder gauge Dαβ μν = 1 2p2 δαμδβν + δανδβμ −δαβδμν 1 M2 pl p2 + 1 M4 pl p4 Np4 16π2 log p2/μ2 + · · · (B1) (B1) we ﬁnd ϕi j = −m 8πr δi j, ϕ00 = m 8πr , (B7) (B7) Repeated insertions of self-energy diagrams would result in a geometric series in the above of which we can compute the (leading log) resummed propagator: whichthrough(B6)impliestheusualNewtonpotential.From (A11) to (A13) the one loop correction results in three pos- sible ﬁnite contributions to the integrand (suppressing tensor structure and factors of order unity), 1 M2 pl p2 1 −Np2 M2 pl log p2/μ2 . (B2) (B2) 16π2∥Q∥∼ 1 M2 pl log M2/μ2 + 1 ⃗p2 M2 M2 pl log M2/μ2 + M2 ⃗p4 M2 M2 pl log M2/μ2 . (B8) Clearly from (B1) we can infer the usual scale of strong grav- ity M∗∗= Mpl/ √ N, however, if we wanted to interpret (B2) as a strengthening of gravity as you cross the mass threshold M, we see that the effect is not signiﬁcant until you come very close to M∗∗. That is, in the regime M2 ≪p2 ≪M2 pl/N, the usual Newton’s force would result with G N = M−2 pl . Appendix A: Loop corrections to the graviton propagator These are given (in Lorentzian signature) as Supplemented with the massive scalar and graviton propa- gators (the latter obtained in the de Donder gauge [6]), Lc.t. = − √−g 16π2 1 (2 −ω) m4 4 + m2 12 R + 1 120 R2 2 + Rμν Rμν . (A14) Supplemented with the massive scalar and graviton propa- gators (the latter obtained in the de Donder gauge [6]), D(p) = −1 p2 + m2 ; Dαβ μν = 1 2p2 δαμδβν + δανδβμ −δαβδμν , (A9) (A14) Given the asymptotic behavior of the hypergeometric and Gamma functions, it is straightforward to evaluate the remaining (ﬁnite) part of Qνσμλ(p) in the limit p2 ≫m2, where we ﬁnd (suppressing tensor structure), the expected (A9) one can sum up all the self-energy diagrams that contribute to the one loop corrected graviton propagator up to order κ2: Formally,theloopcorrectedpropagator Qνσμλ isobtained by inserting the sum of all self-energy diagrams: Qνσμλ = κ2D αβ νσ Tαβθτ Dθτ μλ, (A10) (A10) 12 3 Eur. Phys. J. C (2015) 75 :182 182 Page 8 of 12 Fig. 1 Diagrams contributing to the one loop correction to the graviton propagator Fig. 1 Diagrams contributing to the one loop correction to the graviton propagator ϕμν(x)= dD p (2π)D/2 eip·x D λβ μν (p)+Q λβ μν (p) Tλβ(p). (B3) conformal limit for N minimally coupled massive scalars [cf. the result of (2) plugged into (A10) and recalling that the usual deﬁnition of the metric perturbation hμν := κϕμν] (B3) Consider the energy-momentum tensor of a point mass at rest, given by Consider the energy-momentum tensor of a point mass at rest, given by (A15) Tλβ(x) = mδD−1(⃗x)δ0 λδ0 β; Tλβ(p) = 2πm (2π)D/2 δ(p0)δ0 λδ0 β, (B4) (B4) Appendix B: Note on universally coupled massive species 3 Correction to the gravitational interaction between conserved sources from crossing a massive KK graviton ϕαβ so that above the effective Higgs mass m H, in addition to the usual massless graviton exchange, one mediates an extra gravitational strength force, 1 M2 pl p2 → 1 M2 pl p2 + g2 i M2 pl(p2 + m2 H), (B13) with g2 i := c2 i v2/M2 pl. (B14) 1 M2 pl p2 → 1 M2 pl p2 + g2 i M2 pl(p2 + m2 H), (B13) with (B13) Fig. 3 Correction to the gravitational interaction between conserved sources from crossing a massive KK graviton ϕαβ g2 i := c2 i v2/M2 pl. g2 i := c2 i v2/M2 pl. g2 i := c2 i v2/M2 pl. (B14) (B14) diagrams of massive particles vanish in the long wavelength limit). Clearly the latter two terms have contributions that are M2/M2 pl suppressed as per the usual expectation [1,2]. Therefore crossing any particular threshold scale M does not result in a signiﬁcant strengthening of gravity around that scale, whose summed effects accumulate only close to the scale M∗∗as per our discussion around (B2). Just as in the case for massive KK gravitons, one ﬁnds that in the regime m2 H ≪p2 ≪M2 pl the effective strength of gravity is enhanced as Just as in the case for massive KK gravitons, one ﬁnds that in the regime m2 H ≪p2 ≪M2 pl the effective strength of gravity is enhanced as M2 pl → M2 pl 1 + i g2 i . (B15) (B15) The situation for massive KK gravitons is markedly dif- ferent, however, as these resonances couple universally to all conserved sources and can therefore correct their grav- itational interactions at tree level through the diagrams in Fig. 3. We realize that perhaps this should not be too surprising, as one can always ﬁeld redeﬁne the operators (B11) via the trace of the (two derivative) background equations of motion [33] R = −T μ μ /M2 pl so that the effective interactions are equivalent to Let us say there were n KK resonances with mass M, then it is straightforward to calculate the correction to the gravitational interaction, Leff ∼c{1,2}H†H R. Appendix B: Note on universally coupled massive species This can also be appreciated by directly computing the one loop corrected Newton’s potential between two conserved sources. Using (A11)–(A13), we can compute the gravita- tional ﬁeld generated by the source Tλβ given by (in the notation of Appendix A) Clearly from (B1) we can infer the usual scale of strong grav- ity M∗∗= Mpl/ √ N, however, if we wanted to interpret (B2) as a strengthening of gravity as you cross the mass threshold M, we see that the effect is not signiﬁcant until you come very close to M∗∗. That is, in the regime M2 ≪p2 ≪M2 pl/N, 2 (B8) The ﬁrst two terms contribute a contact (delta function) term and a correction to the usual Newton potential, respectively. the usual Newton’s force would result with G N = M−2 pl . This can also be appreciated by directly computing the one loop corrected Newton’s potential between two conserved sources. Using (A11)–(A13), we can compute the gravita- tional ﬁeld generated by the source Tλβ given by (in the notation of Appendix A) The third term contributes linear and logarithmic corrections that depend on r M, which are only to be understood in the regime where they are small corrections that get completed by higher order terms in the perturbative expansion such that the total amplitude satisﬁes the usual decoupling require- ments (i.e. that the corrections from insertion of self-energy 123 Eur. Phys. J. C (2015) 75 :182 Page 9 of 12 182 Page 9 of 12 182 tational strength can also be generated from universally cou- pled species such as the Higgs through higher-dimensional operators of the form tational strength can also be generated from universally cou- pled species such as the Higgs through higher-dimensional operators of the form Leff ∼c1 H†H M2 pl ∂μϕ∂μϕ+c2 H†H M2 pl ¯ψ /∂ψ ∼c{1,2} H†H M2 pl T μ μ , (B11) (B11) where the ci are generic Wilson coefﬁcients. Expanding the singlet operator H†H around some vev v as H†H = v2 + 2vh generates a vertex that contributes another channel to the diagram Fig. 3, Fig. 2 Correction to the gravitational interaction between conserved sources (denoted by a double line) from crossing the mass threshold of a massive particle Leff ∼ci v h M2 pl T μ μ , (B12) (B12) Fig. 18 Order unity non-minimal couplings will generically be generated through renormalization group (RG) ﬂow for the singlet component of the Higgs and any other massive scalars present in the early universe [34]. 19 In full generality we allow for the generation of a non-trivial the adiabatic sound speed cs < 1 as well as higher spatial derivative terms from the curvature squared corrections. Appendix B: Note on universally coupled massive species (B16) (B16) Therefore we see that it is not just the Higgs, but any non- minimally coupled massive scalar18 that can enhance the effective strength of gravity as (B15), where the Wilson coef- ﬁcients ci are now replaced with non-minimal couplings ξi. We stress that such additional effective interactions gener- ate extra gravitational strength scalar forces that violate the equivalence principle at very high energies; but this is not in conﬂict with any presently accessible observations. To brieﬂyrecap,thephysicalbasisofthedifferingeffectsofnon- universallycoupledcomparedtouniversallycoupledmassive species on the strength of gravity as you cross each threshold 1 M2 pl p2 → 1 M2 pl p2 + n M2 pl(p2 + M2). (B9) (B9) Unlike the case for (B2), we see that the regime M2 ≪p2 ≪ M2 pl/n the strength of gravity is modiﬁed immediately above p = M as 1 M2 pl p2 + n M2 pl p2 1 + M2/p2 →n + 1 M2 pl p2 , (B10) (B10) that is, as each threshold corresponding to a massive KK res- onance is crossed, the effective strength of gravity increases immediately as M2 pl/n →M2 pl/(n + 1), where n counts the numberofmassivespeciesthatcontributetothetreeleveldia- gram Fig. 3. We also note that effective interactions of gravi- 18 Order unity non-minimal couplings will generically be generated through renormalization group (RG) ﬂow for the singlet component of the Higgs and any other massive scalars present in the early universe [34]. 12 3 182 Page 10 of 12 Eur. Phys. J. C (2015) 75 :182 particles can only additively renormalize Newton’s constant such that at the scale of inﬂation, M2 R(μ) = M2(μref) + i dim2 i log(μ2/μ2 ref) where the di are spin dependent weights each suppressed by a factor of 1/(16π2). Impos- ing the renormalization conditions at macroscopic scales, M2(μref) = M2 pl so that can be readily understood through the former being a loop effect and the latter being a tree level effect; cf. Figs. 2 and 3. One can complement this understanding of how regular massivespeciesdonotaffectthestrengthofgravityasseenby curvature quanta during inﬂation through the simple exercise ofcomputingtheactionforR,giventhefactthattheeffectsof massive particles in Fig. 2 can be reproduced by the effective action M2 R(μ)= M2 pl 1+ i di m2 i M2 pl log(μ2/μ2 ref) ≈M2 pl (B22) S = M2 R 2 d4x√−g R+ d4x√−g c1R2+c2Rμν Rμν + d4x√−gL [φ0] (B17) within the domain of validity of our approximation. (B17) where the last term in the above is the action for the back- ground that sources the quasi-de Sitter phase, and the sub- script on φ0 anticipates that we will work in a gauge where all inﬂaton ﬂuctuations have been gauged away. The dimen- sionful prefactor M2 R emphasizes that the above is the net result of having subtracted the usual divergences that result after integrating out the ﬁelds in question and that are ﬁxed by renormalization conditions at some particular scale, necessi- tating the introduction of a bare cosmological constant term that is canceled by tadpole contributions. One can straight- forwardly deduce that the effect of the higher curvature terms will be to modify the usual action for the comoving curvature perturbation from where the last term in the above is the action for the back- ground that sources the quasi-de Sitter phase, and the sub- script on φ0 anticipates that we will work in a gauge where all inﬂaton ﬂuctuations have been gauged away. The dimen- sionful prefactor M2 R emphasizes that the above is the net result of having subtracted the usual divergences that result after integrating out the ﬁelds in question and that are ﬁxed by renormalization conditions at some particular scale, necessi- tating the introduction of a bare cosmological constant term that is canceled by tadpole contributions. One can straight- forwardly deduce that the effect of the higher curvature terms will be to modify the usual action for the comoving curvature perturbation from Appendix C: Moduli dynamics during inﬂation In this appendix, we consider the dynamics of moduli ﬁelds during inﬂation in the case where H∗> μc, where μc is the characteristic mass of the moduli. We wish to show that are regimes where the moduli are effectively frozen, even if they are dynamically displaced off their minima, allowing us to treat the extra-dimensional volume as effectively constant during inﬂation. We begin by considering the following D- dimensional action: S2 = M2 pl d4x a3ϵ ˙R2 −(∂R)2 a2 (B18) to S2 = M2 ∗ d4x a3ϵ ˙R2 c2s −(∂R)2 a2 + λ M2∗ (∂R)4 a4 + ... (B19) S2 = M2 pl d4x a3ϵ ˙R2 −(∂R)2 a2 (B18) (B18) (B18) S = M D−2 ∗ 2 dDx √ −G R(D) − dDx √ −G 1 2(∂φ)2 + V (φ) (C1) to S2 = M2 ∗ d4x a3ϵ ˙R2 c2s −(∂R)2 a2 + λ M2∗ (∂R)4 a4 + ... (B19) (C1) where R(D) isthe D-dimensionalRicciscalarconstructedout of the metric G AB. We now consider a D = 4 +n decompo- sition with Greek indices denoting four non-compact coordi- nates and lower case Latin indices denoting n periodic extra- dimensional coordinates that range from 0 ≤ya ≤2π R and where we furthermore presume the metric tensor G AB to be deﬁned through the block diagonal form, where R(D) isthe D-dimensionalRicciscalarconstructedout of the metric G AB. We now consider a D = 4 +n decompo- sition with Greek indices denoting four non-compact coordi- nates and lower case Latin indices denoting n periodic extra- dimensional coordinates that range from 0 ≤ya ≤2π R and where we furthermore presume the metric tensor G AB to be deﬁned through the block diagonal form, where we see from (B17) that the corrections from the cur- vature squared terms to the quadratic action for R must have twoderivativesactingonbackgroundquantities,19 sothat,for example, the operator (∂R)2 gets a correction of the form where we see from (B17) that the corrections from the cur- vature squared terms to the quadratic action for R must have twoderivativesactingonbackgroundquantities,19 sothat,for example, the operator (∂R)2 gets a correction of the form L2 ∼c1,2M2 R (∂R)2 a2 H2 M2 R (B20) (B20) ds2 = gμν(x)dxμdxν + e2ω(x)γabdyadyb. Appendix B: Note on universally coupled massive species There- fore in the context of (B21), we see that unless H2 ∼ M2 pl/ √ N, the correction to the effective strength of grav- ity as seen by the curvature perturbations is negligible and M∗∼Mpl for curvature quanta above the scale of any mas- sive (non-universally coupled) species. (B17) 21 With six extra dimensions for example, this factor equals 1/12. One may be tempted to redeﬁne ω in (C7) to absorb the factor of n(n + 2), however, this appropriately rescales the arguments of the potential energy contributions such that (C17) also results for the rede- ﬁned variables. Appendix C: Moduli dynamics during inﬂation (C2) where c1, c2 ∼O(N) so that the overall dimensionful coef- ﬁcient of the quadratic action (B19) becomes (C2) If we assume γab = δab, and if no other quantity depends on the ya, then If we assume γab = δab, and if no other quantity depends on the ya, then M2 ∗= M2 R 1 − ˜cN H2 16π2M2 R (B21) (B21) √ −G = √−g enω (C3) (C3) where ˜c is some positive number (by unitarity) of order unity. Furthermore, we note that integrating out massive and R(D)[G] = R(4)[g] −2ne−ωgμν∇μ∇νeω −n(n −1)gμν∇μω∇νω. (C4) (C4) 12 3 3 Eur. Phys. J. C (2015) 75 :182 Page 11 of 12 182 Substituting into the above, integrating over the compact dimensions and doing a few integrations by part result in where the denominator on the rhs is given by (C9). Deﬁning the total kinetic and potential energies where the denominator on the rhs is given by (C9). Deﬁning the total kinetic and potential energies S = M D−2 ∗ 2 Vn d4x√−g enω R(D) + n(n −1)(∂ω)2 −Vn d4x√−g enω 1 2(∂φ)2 + V (φ) , (C5) KT := M2 pl n(n + 2) 4 ˙ω2 + ˙φ2 2 ; VT := M2 pl U(ω) 2 + e−2ωV (φ), (C14) (C5) (C14) where Vn := dny. To bring the action above into the usual Einstein Hilbert form, we further make the conformal trans- formation we ﬁnd that in general we ﬁnd that in general ϵ = − ˙H H2 = 3KT KT + VT , (C15) (C15) gμν = e−nωgμν, (C6) gμν = e−nωgμν, (C6) so that ϵ ≪1 consistently if KT /VT ≪1, i.e. we require the potential terms to dominate the kinetic terms in the energy density, as usual. This can be re-expressed using (C10) and (C12) as a condition on the potential if one neglects the ¨φ and ¨ω terms, which is only possible if so that ϵ ≪1 consistently if KT /VT ≪1, i.e. we require the potential terms to dominate the kinetic terms in the energy density, as usual. 20 For a review of mechanisms to stabilize Kähler moduli in the context of type II and heterotic string theory, see [35,36]. See also [37,38] for complementary approaches utilizing the energetics of the string free energy around enhanced symmetry points. Appendix C: Moduli dynamics during inﬂation This can be re-expressed using (C10) and (C12) as a condition on the potential if one neglects the ¨φ and ¨ω terms, which is only possible if so that, ﬁnally, the action becomes so that, ﬁnally, the action becomes S = M2 pl 2 d4x −g R(D)[g]−n 2(n+2)(∂ω)2−U(ω) − d4x −g 1 2(∂φ)2 + e−2ωV (φ) (C7 (C7) 1 n(n + 2) V 2 T,ω V 2 T + M2 pl 2 V 2 T,φ V 2 T ≪1, (C16) (C16) where M2 pl := M(D−2) ∗ Vn and φ := √ V nφ, and where we where M2 pl := M(D−2) ∗ Vn and φ := √ V nφ, and where we have explicitly introduced by hand the Einstein frame poten- tial U, which is responsible for stabilizing the ω modulus.20 Restricting ourselves to spatially homogeneous solutions, we make the metric ansatz pl have explicitly introduced by hand the Einstein frame poten- tial U, which is responsible for stabilizing the ω modulus.20 Restricting ourselves to spatially homogeneous solutions, we make the metric ansatz wherethesecondtermaboveistheusualϵ parameter.Wenote from the deﬁnition (C14) that requiring U(ω)e2ω/V (φ) ≪ 1 (which is consistent with requiring inﬂation being above the effective compactiﬁcation scale H∗≫μc over a broad regime) results in ds2 = −dt2 + e2λδi jdxidx j (C8) (C8) and ﬁnd the Einstein constraint equation (dropping the tildes on φ in what follows), 4 n(n + 2) + M2 pl 2 V ′(φ)2 V (φ)2 ≪1, (C17) (C17) 3˙λ2 = n(n + 2) 4 ˙ω2 + U(ω) 2 + ˙φ2 2M2 pl + e−2ω M2 pl V, (C9) (C9) so that in addition we also need a sufﬁcient number of extra dimensions in order that the second numerical factor can also be neglected.21 One can understand why this is so— we see from (C7) that, for the canonically normalized vari- able ω = √n(n + 2)ω, the inﬂationary potential becomes e−2ω/√n(n+2)V (φ), and it therefore one ﬂattens the ω depen- dence of this contribution to the total potential more and more the greater n is, eventually allowing for the ‘slow-roll’ con- dition on the moduli ﬁeld to be satisﬁed. 20 For a review of mechanisms to stabilize Kähler moduli in the context of type II and heterotic string theory, see [35,36]. See also [37,38] for complementary approaches utilizing the energetics of the string free energy around enhanced symmetry points. References 19. S. Davidson, E. Nardi, Y. Nir, Leptogenesis. Phys. Rep. 466, 105 (2008). arXiv:0802.2962 [hep-ph] 20. C. Cheung, P. Creminelli, A.L. Fitzpatrick, J. Kaplan, L. Senatore, The effective ﬁeld theory of inﬂation. JHEP 0803, 014 (2008). arXiv:0709.0293 [hep-th] 1. J.F. Donoghue, General relativity as an effective ﬁeld theory: the leading quantum corrections. Phys. Rev. D 50, 3874 (1994). arXiv:gr-qc/9405057 21. N. Arkani-Hamed, S. Dimopoulos, G.R. Dvali, The hierarchy prob- lem and new dimensions at a millimeter. Phys. Lett. B 429, 263 (1998). arXiv:hep-ph/9803315 g q 2. C.P. Burgess, Quantum gravity in everyday life: general relativ- ity as an effective ﬁeld theory. Living Rev. Relativ. 7, 5 (2004). arXiv:gr-qc/0311082 22. I. Antoniadis, N. Arkani-Hamed, S. Dimopoulos, G.R. Dvali, New dimensions at a millimeter to a Fermi and superstrings at a TeV. Phys. Lett. B 436, 257 (1998). arXiv:hep-ph/9804398 3. M.M. Anber, J.F. Donoghue, M. El-Houssieny, Running couplings and operator mixing in the gravitational corrections to coupling constants. Phys. Rev. D 83, 124003 (2011). arXiv:1011.3229 [hep- th] 23. I. Antoniadis, B. Pioline, Low scale closed strings and their duals. Nucl. Phys. B 550, 41 (1999). arXiv:hep-th/9902055 4. M.M. Anber, J.F. Donoghue, On the running of the gravitational constant. Phys. Rev. D 85, 104016 (2012). arXiv:1111.2875 [hep- th] 24. I. Antoniadis, S. Dimopoulos, A. Giveon, Little string theory at a TeV. JHEP 0105, 055 (2001). arXiv:hep-th/0103033 25. I. Antoniadis, A. Arvanitaki, S. Dimopoulos, A. Giveon, Phe- nomenology of TeV little string theory from holography. Phys. Rev. Lett. 108, 081602 (2012). arXiv:1102.4043 5. G.R. Dvali, G. Gabadadze, M. Kolanovic, F. Nitti, Scales of gravity. Phys. Rev. D 65, 024031 (2002). arXiv:hep-th/0106058 6. D.M. Capper, On quantum corrections to the graviton propagator. Nuovo Cim. A 25, 29 (1975) 26. G. Dvali, D. Lust. arXiv:0912.3167 [hep-th] 27. G. Dvali, C. Gomez. arXiv:1004.3744 [hep-th] 7. G. Dvali, M. Redi, Black hole bound on the number of species and quantum gravity at LHC. Phys. Rev. D 77, 045027 (2008). arXiv:0710.4344 [hep-th] 28. B.A. Bassett, S. Tsujikawa, D. Wands, Inﬂation dynam- ics and reheating. Rev. Mod. Phys. 78, 537 (2006). arXiv:astro-ph/0507632 8. M.J. Duff, Observations on conformal anomalies. Nucl. Phys. B 125, 334 (1977) 29. G. Dvali, I. Sawicki, A. Vikman, Dark matter via many copies of the standard model. JCAP 0908, 009 (2009). arXiv:0903.0660 [hep-th] 9. H. Osborn, A.C. Petkou, Implications of conformal invariance in ﬁeld theories for general dimensions. Ann. Phys. 231, 311 (1994). arXiv:hep-th/9307010 30. A.R. Appendix C: Moduli dynamics during inﬂation the equation of motion ¨φ + 3˙λ ˙φ + e−2ωV,φ = 0, (C10) ¨φ + 3˙λ ˙φ + e−2ωV,φ = 0, (C10) and the Friedmann equations and the Friedmann equations ¨λ + n(n + 2) 4 ˙ω2 + ˙φ2 2M2 pl = 0, (C11) ¨ω + 3˙λ ˙ω + U,ω n(n + 2) − 4e−2ω n(n + 2)M2 pl V = 0. (C12) ¨λ + n(n + 2) 4 ˙ω2 + ˙φ2 2M2 pl = 0, (C11) (C11) Therefore although it is not generally true that we can have inﬂation without the moduli undergoing non-trivial excur- sions, we see that in certain limits, this can indeed be accom- plished consistently allowing us to treat them as effectively ﬁxed even though they are displaced from their minima. In this situation, the characteristic masses of the associated KK states remain almost constant during inﬂation and the usual analysis can be implemented. ¨ω + 3˙λ ˙ω + U,ω n(n + 2) − 4e−2ω n(n + 2)M2 pl V = 0. (C12) (C12) We now ask: is there a solution such that the background inﬂates, i.e. −˙H/H2 ≪1? From (C11), this clearly requires − ˙H H2 = −¨λ/˙λ2 = 3 3˙λ2 n(n + 2) 4 ˙ω2 + ˙φ2 2M2 pl ≪1 (C13) (C13) 12 3 182 Page 12 of 12 Eur. Phys. J. C (2015) 75 :182 References Liddle, A. Mazumdar, F.E. Schunck, Assisted inﬂation. Phys. Rev. D 58, 061301 (1998). arXiv:astro-ph/9804177 10. R. Rattazzi, S. Rychkov, A. Vichi, Central charge bounds in 4D conformal ﬁeld theory. Phys. Rev. D 83, 046011 (2011). arXiv:1009.2725 [hep-th] 31. S. Dimopoulos, S. Kachru, J. McGreevy, J.G. Wacker, N-ﬂation. JCAP 0808, 003 (2008). arXiv:hep-th/0507205 32. A. Ashoorioon, M.M. Sheikh-Jabbari, Gauged M-ﬂation, its UV sensitivity and spectator species. JCAP 1106, 014 (2011). arXiv:1101.0048 [hep-th] 11. P.A.R. Ade et al. [Planck Collaboration], Planck 2013 results. XVI. Cosmological parameters. Astron. Astrophys. (2014). arXiv:1303.5076 [astro-ph.CO] 33. S. Weinberg, Effective ﬁeld theory for inﬂation. Phys. Rev. D 77, 123541 (2008). arXiv:0804.4291 [hep-th] 12. P.A.R.Adeetal.[PlanckCollaboration],Planck2013results.XXII. Constraints on inﬂation. arXiv:1303.5082 [astro-ph.CO] 34. N.D. Birrell, P.C.W. Davies, Quantum Fields in Curved Space. Cambridge Monographs Mathematical Physics (Cambridge Uni- versity Press, Cambridge, 1982) 13. O. Zsoy, K. Sinha, S. Watson, How well can we really determine the scale of inﬂation? arXiv:1410.0016 [hep-th] 14. M. Mirbabayi, L. Senatore, E. Silverstein, M. Zaldarriaga, Gravi- tational waves and the scale of inﬂation. arXiv:1412.0665 [hep-th] 35. M. Grana, Flux compactiﬁcations in string theory: a comprehensive review. Phys. Rep. 423, 91 (2006). arXiv:hep-th/0509003 15. A.G. Polnarev, Polarization and anisotropy induced in the microwave background by cosmological gravitational waves. Sov. Astron. 29, 607 (1985) 36. B. de Carlos, S. Gurrieri, A. Lukas, A. Micu, Moduli stabilisa- tion in heterotic string compactiﬁcations. JHEP 0603, 005 (2006). arXiv:hep-th/0507173 16. M. Kamionkowski, A. Kosowsky, A. Stebbins, A probe of primor- dial gravity waves and vorticity. Phys. Rev. Lett. 78, 2058 (1997). arXiv:astro-ph/9609132 p 37. T. Catelin-Jullien, C. Kounnas, H. Partouche, N. Toumbas, Induced superstring cosmologies and moduli stabilization. Nucl. Phys. B 820, 290 (2009). arXiv:0901.0259 [hep-th] 17. U. Seljak, M. Zaldarriaga, Signature of gravity waves in polar- ization of the microwave background. Phys. Rev. Lett. 78, 2054 (1997). arXiv:astro-ph/9609169 38. S.P. Patil, R. Brandenberger, Radion stabilization by stringy effects in general relativity. Phys. Rev. D 71, 103522 (2005). arXiv:hep-th/0401037 18. M. Trodden, Electroweak baryogenesis. Rev. Mod. Phys. 71, 1463 (1999). arXiv:hep-ph/9803479 123 123
https://openalex.org/W2555158337	http://ejournal-balitbang.kkp.go.id/index.php/jra/article/download/1998/1615	Indonesian	null	KONDISI METEOROLOGI, KLIMATOLOGI, DAN PERIKANAN DI KAWASAN WADUK CIRATA, JAWA BARAT: Analisis Awal Kemungkinan Dampak Pemanasan Global terhadap Perikanan Budidaya	Jurnal riset akuakultur/Jurnal Riset Akuakultur	2,011	cc-by-sa	5,885	I Nyoman Radiarta), Anang Hari Kristanto), dan Adang Saputra*) ) Pusat Penelitian dan Pengembangan Perikanan Budidaya Jl. Ragunan 20, Pasar Minggu, Jakarta Selatan 12540 E-mail: radiarta@yahoo.com I Nyoman Radiarta), Anang Hari Kristanto), dan Adang Saputra*) ) Pusat Penelitian dan Pengembangan Perikanan Budidaya Jl. Ragunan 20, Pasar Minggu, Jakarta Selatan 12540 E-mail: radiarta@yahoo.com ) Balai Penelitian dan Pengembangan Perikanan Budidaya Air Tawar Jl. Sempur No. 1, Bogor 16154 ) Balai Penelitian dan Pengembangan Perikanan Budidaya Air Tawar Jl. Sempur No. 1, Bogor 16154 (Naskah diterima: 5 Mei 2011; Disetujui publikasi: 5 September 2011) (Naskah diterima: 5 Mei 2011; Disetujui publikasi: 5 September 2011) Kondisi meteorologi, klimatologi, dan perikanan ..... (I Nyoman Radiarta) Kondisi meteorologi, klimatologi, dan perikanan ..... (I Nyoman Radiarta) ABSTRAK Pemanasan global adalah perubahan iklim yang dapat teridentifikasi melalui perubahan rata-rata yang terjadi pada jangka waktu yang relatif panjang. Perubahan ini disebabkan baik karena variasi kondisi alam maupun akibat dari aktivitas manusia (antropogenic). Pemanasan global diproyeksikan akan berpengaruh terhadap kondisi ekosistem, kondisi sosial dan ekonomi, dan meningkatnya tekanan terhadap sumber mata pencaharian yang berimplikasi pada penyediaan pangan. Bagi perikanan budidaya, pemanasan global dapat berpengaruh pada perubahan tingkat produktivitas, distribusi, komposisi spesies, dan lingkungan perairan sehingga dapat menyebabkan perubahan dalam operasional perikanan budidaya. Berdasarkan perkembangan isu tentang pemanasan global ini, penelitian dampak pemanasan global terhadap perikanan budidaya dirasakan sangat penting. Tujuan dari penelitian ini adalah untuk melihat kemungkinan adanya dampak pemanasan global terhadap aktivitas perikanan budidaya (ikan air tawar) di kawasan Waduk Cirata, Jawa Barat. Data utama yang dikumpulkan dalam penelitian ini diperoleh dari berbagai sumber meliputi data klimatologi, meteorologi, tutupan lahan dan karakteristik waduk. Analisis data yang ditampilkan dalam tulisan ini masih merupakan analisis awal yang lebih banyak diuraikan secara deskriptif melalui tampilan grafik. Hasil dari penelitian ini diharapkan dapat memberikan gambaran umum mengenai kemungkinan adanya dampak pemanasan global bagi perikanan budidaya air tawar khususnya di Waduk Cirata, sehingga nantinya dapat membantu dalam pengelolaan waduk yang lebih baik. KATA KUNCI: meteorologi, klimatologi, budidaya ikan air tawar, pemanasan global, Waduk Cirata ABSTRACT: PENDAHULUAN fluktuasi iklim setiap tahunnya (Blenckner, 2005). Sehubungan dengan pemanasan global yang terjadi, masih adanya ketidak- pastian mengenai berapa besar, seberapa cepat dan di mana saja iklim akan berubah secara signifikan. Untuk melihat perubahan iklim yang terjadi, para ahli telah melakukan berbagai cara untuk mengkarakterisasi perubahan yang terjadi baik regional maupun global. Satu pendekatan yang umum di- gunakan adalah melalui zone indek. El Niño Southern Oscillation (ENSO) indek merupakan satu zone indeks yang sangat populer di wilayah Pasifik tropis, yang dapat berdampak pada perubahan lingkungan perairan dan daratan. Pendekatan menggunakan zone indeks ini secara umum sangat bermanfaat untuk melihat dampak perubahan lingkungan terhadap perubahan iklim. Hal ini disebabkan indek ini mengintegrasikan berbagai variabel iklim (misalnya suhu, curah hujan, dan tutupan awan) dan memungkinkan melihat variasinya secara tahunan untuk iklim regional. Intergovernmental Panel on Climate Change (IPCC, 2007) mendefinisikan pemanasan global adalah perubahan iklim yang terjadi pada jangka waktu yang panjang misalnya satu dekade ataupun lebih panjang lagi, dan dapat teridentifikasi dengan baik (misalnya menggunakan uji statistik). Perubahan ini dapat diakibatkan oleh variasi kondisi alam atau aktivitas manusia. IPCC dalam buku laporannya yang keempat telah memprediksi terjadinya perubahan suhu udara sampai pada akhir abad 21 (2009-2099), perubahan yang terjadi berkisar antara 1,8oC (estimasi untuk prediksi terendah-B1) sampai 4oC (estimasi untuk prediksi tertinggi-A1FI) (IPCC, 2007). Pemanasan global yang terjadi dapat mempengaruhi variasi suhu udara/air, cuaca, dan ketersediaan pasokan air (kualitas dan kuantitas). Dampak dari perubahan suhu dapat mempengaruhi tingkat produktivitas dari suatu ekosistem yang pada akhirnya dapat berdampak pada variasi waktu dan keberhasilan pola migrasi, pemijahan, dan kelimpahan (Walther et al., 2002; Beukema & Dekker, 2005; Saitoh et al., 2011). Pemanasan global diproyeksikan akan berpengaruh terhadap kondisi ekosistem, kondisi sosial dan ekonomi, dan meningkatnya tekanan terhadap sumber mata pencaharian yang berimplikasi pada penyediaan bahan pangan. Penelitian dampak pemanasan global terhadap perikanan budidaya masih sangat terbatas. Beberapa pustaka yang ada umumnya masih bersifat review tentang kemungkinan dampak yang ditimbulkan baik dampak secara langsung maupun tidak langsung (Bardach, 1989; Meisner & Shuter, 1992; Harley et al., 2006; Cochrane et al., 2009). Secara global, Handisyde et al. (2006) telah melakukan analisis kemungkinan dampak dari perubahan iklim global terhadap perikanan budidaya. Parameter utama yang digunakan dalam analisis tersebut meliputi kondisi klimatologi dan perairan (suhu udara, presipitasi, penaikan muka air (sea level rise), kondisi ekstrim, variasi iklim, dan arus laut), Kondisi iklim akan selalu bervariasi ber- dasarkan waktu dan tempat. meteorology, climatology, freshwater fish aquaculture, climate change, Cirata Reservoir KEYWORDS: ABSTRACT: ABSTRACT: Meteorology, climatology, and fisheries conditions in Cirata Reservoir, West Java: A preliminary analysis on possible impact of climate change to aquaculture. By: I Nyoman Radiarta, Anang Hari Kristanto, and Adang Saputra ABSTRACT: Meteorology, climatology, and fisheries conditions in Cirata Reservoir, West Java: A preliminary analysis on possible impact of climate change to aquaculture. By: I Nyoman Radiarta, Anang Hari Kristanto, and Adang Saputra ABSTRACT: Meteorology, climatology, and fisheries conditions in Cirata Reservoir, West Java: A preliminary analysis on possible impact of climate change to aquaculture. By: I Nyoman Radiarta, Anang Hari Kristanto, and Adang Saputra Global warming is the change of climate condition that can be identified through long term average of its variability. The change could be caused by either natural condition or antropogenic activities. Climate change will influence the surrouding natural ecosystem, socio-economic of afffected communities, and inflict to the availability of livelihood related with food security. In term of aquaculture, climate change could 495 J. Ris. Akuakultur Vol.6 No.3 Tahun 2011: 495-506 impact the system to level of productivity, species distribution and composition, and environmental condition, and finally change the practices of aquaculture activities. Taking to the account of issues on climate change, research on possible impact of climate change to aquaculture has become an important discussion topic. The aim of this study was to investigate the possible impacts of climate change to freshwater aquaculture in Cirata Reservoir, West Java. The main data used in this study was collected from different sources including climatology, meteorology, land cover, characteristic of the reservoir. All data were then analyzed in order to observe the conectivity among them in relation with the climate condition. The result from this study was a preliminary analysis based on the availability of secondary data. The finding from this study could give a general overview on the possible impact of climate change to freshwater aquaculture especially in Cirata Reservoir. PENDAHULUAN Sangat jelas perbedaannya antara perubahan iklim dan variasi iklim. Perubahan iklim adalah tren (fluktuasi) iklim selama kurun waktu yang cukup panjang, paling sedikit sekitar 20 tahun, sedangkan variasi iklim mengacu kepada 496 Kondisi meteorologi, klimatologi, dan perikanan ..... (I Nyoman Radiarta) dan kondisi perikanan budidaya (produksi, mata pencaharian, dan ketersediaan minyak ikan dan makanan ikan). Meskipun dilengkapi dengan contoh kasus di Bangladesh, penelitian ini hanya memberikan gambaran secara global kemungkinan dampak yang ditimbulkan dari perubahan iklim terhadap perikanan budidaya. Dari penelitian ini, di- sarankan agar melakukan kajian yang lebih spesifik, sehingga nantinya dapat memberikan kontribusi yang nyata terhadap pemahaman kita mengenai bagaimana pemanasan global berdampak terhadap sumberdaya perikanan budidaya. Dampak dari perubahan iklim khususnya pengaruh langsung dari zone indeks: El Niño dan La Niña terhadap budidaya kekerangan (Japanese scallop) telah di- demontrasikan oleh Baba et al. (2009). Mereka menyimpulkan bahwa, terjadinya El Niño dapat mempengaruhi rendahnya kelimpahan spat yang tersedia, sedangkan La Niña menunjukkan pengaruh yang sangat signifi- kan terhadap reproduksi dan pertumbuhan dari scallop. Hal ini menunjukkan bahwa penelitian lebih lanjut dari dampak pemanasan global terhadap perikanan budidaya merupakan suatu tantangan dan perlu dikaji lebih mendalam. Hasil kajian tersebut tentunya akan bermanfaat guna dapat diambil tindakan adaptasi terhadap aktivitas perikanan budi- daya yang pada akhirnya dapat berkontribusi pada usaha perikanan budidaya yang berkelanjutan. melihat keterkaitannya dengan kondisi iklim secara global. dan kondisi perikanan budidaya (produksi, mata pencaharian, dan ketersediaan minyak ikan dan makanan ikan). Meskipun dilengkapi dengan contoh kasus di Bangladesh, penelitian ini hanya memberikan gambaran secara global kemungkinan dampak yang ditimbulkan dari perubahan iklim terhadap perikanan budidaya. Dari penelitian ini, di- sarankan agar melakukan kajian yang lebih spesifik, sehingga nantinya dapat memberikan kontribusi yang nyata terhadap pemahaman kita mengenai bagaimana pemanasan global berdampak terhadap sumberdaya perikanan budidaya. Dampak dari perubahan iklim khususnya pengaruh langsung dari zone indeks: El Niño dan La Niña terhadap budidaya kekerangan (Japanese scallop) telah di- demontrasikan oleh Baba et al. (2009). Mereka menyimpulkan bahwa, terjadinya El Niño dapat mempengaruhi rendahnya kelimpahan spat yang tersedia, sedangkan La Niña menunjukkan pengaruh yang sangat signifi- kan terhadap reproduksi dan pertumbuhan dari scallop. Hal ini menunjukkan bahwa penelitian lebih lanjut dari dampak pemanasan global terhadap perikanan budidaya merupakan suatu tantangan dan perlu dikaji lebih mendalam. Hasil kajian tersebut tentunya akan bermanfaat guna dapat diambil tindakan adaptasi terhadap aktivitas perikanan budi- daya yang pada akhirnya dapat berkontribusi pada usaha perikanan budidaya yang berkelanjutan. PENDAHULUAN Secara umum dampak secara langsung yang mungkin ditimbulkan dengan perubahan suhu (udara/air) adalah kelayakan dari suatu jenis ikan untuk dibudidayakan di lokasi tertentu, sedangkan dampak tidak langsung berupa pengaruh terhadap kandungan oksigen, penyakit, dan terjadinya kelimpahan alga beracun (De Silva & Soto, 2009). Pemanasan global juga dapat berpengaruh terhadap fluktuasi muka air kawasan waduk/ danau disebabkan karena perubahan curah hujan, yang tentunya sangat berpengaruh bagi keberlangsungan kegiatan perikanan di kawasan waduk/danau. Melihat isu-isu yang ada tersebut, penelitian spesifik dampak pemanasan global terhadap perikanan budidaya (misalnya dampaknya terhadap timbulnya penyakit, produktivitas perikanan air tawar ataupun budidaya laut) sangat diperlukan guna dapat memberikan dukungan data dan informasi yang relevan mengenai dampak pemanasan global terhadap perikanan budidaya. Tujuan dari penelitian ini adalah untuk melakukan analisis kemungkinan adanya dampak pemanasan global terhadap aktivitas perikanan budidaya ikan air tawar di Waduk Cirata, Jawa Barat. Analisis dilakukan ber- dasarkan data sekunder yang dikumpulkan dengan melihat dua aspek (filter) yang dapat mempengaruhi besarnya dampak pemanasan global terhadap ekosistem waduk. Analisis yang disajikan dalam tulisan ini adalah analisis awal yang dibahas dengan melihat keterkaitan antara beberapa kondisi baik di dalam maupun luar waduk terhadap kemungkinan adanya dampak pemanasan global. Data klimatologi dan meteorologi (iklim) sangat berperan bagi kegiatan perikanan. Lama penyinaran dan suhu udara sangat mempengaruhi kondisi suhu perairan dan secara langsung akan berdampak terhadap tingkat produktivitas dari suatu perairan dan tingkat pertumbuhan dari biota yang di- budidayakan. Data iklim telah dimanfaatkan dibidang perikanan budidaya (Kapetsky, 2000). Namun, belum banyak upaya yang dilakukan sehubungan dengan akuisisi dan pem- bentukan aplikasi data iklim sebagai satu media untuk perikanan budidaya. Munculnya isu pemanasan global belakangan ini membuat perspektif yang cukup menarik dalam hal pemanfaatan data iklim. Data tersebut jika dikaji secara komprehensif dan dihubungkan dengan kondisi perairan tentunya akan memberikan gambaran yang lebih baik. Sebagai contoh terjadinya kematian ikan di Waduk Cirata, umumnya kajian yang ada bersifat parsial dari aspek kondisi kualitas perairan saja (Abery et al., 2005; Hayami et al., 2008), belum BAHAN DAN METODE Penelitian ini telah dilakukan di Waduk Cirata, Jawa Barat (Gambar 1). Lokasi penelitian terletak pada posisi 6o44’ LS; 107o18’ BT. Waduk ini merupakan deretan tiga waduk besar yang terletak di daerah aliran sungai (DAS) Citarum. Dua waduk lainnya adalah Waduk Jatiluhur dan Sanguling. Sejak pertama kali dioperasikan yaitu tahun 1988, aktivitas perikanan budidaya dengan menggunakan keramba jaring apung (KJA) telah berkembang di waduk ini (Soemarwoto et al., 1990). Data utama yang digunakan dalam penelitian ini adalah data sekunder. Data ini 497 J. Ris. Akuakultur Vol.6 No.3 Tahun 2011: 495-506 Gambar 1. Lokasi penelitian di Waduk Cirata, Jawa Barat Figure 1. Study area at Cirata Reservoir, West Java Gambar 1. Lokasi penelitian di Waduk Cirata, Jawa Barat Figure 1. Study area at Cirata Reservoir, West Java dikumpulkan dari instansi terkait, studi pustaka (publikasi ilmiah), dan sumber dari internet. Instansi terkait yang berhubungan dengan dengan pengelolaan waduk Cirata di antaranya Dinas Kelautan dan Perikanan (DKP), Badan Pengelola Waduk Cirata (BPWC), dan Unit Pembangkit Jawa-Bali (UPJB). Data yang dikumpulkan dalam penelitian ini meliputi: kondisi iklim (suhu udara minimum, suhu udara maksimum, suhu udara rata-rata, dan curah hujan), kondisi lahan waduk, dan kondisi aktivitas budidaya ikan. Data spasial klimatologi untuk kawasan DAS Citarum diperoleh dari World Climatology (Hijmans et al., 2005; http://www.worldclim.org/). waduk. Kombinasi aspek dari luar dan dalam waduk (Blenckner, 2005) akan memberikan perspektif yang komprehensif mengenai respons dari ekosistem waduk. Hasil yang ditampilkan dalam penelitian hanya merupakan suatu kajian awal dengan memperhatikan trend perkembangan lingkungan dan kondisi waduk yang kiranya dapat dikaitan dengan ber- kembangnya isu mengenai pemanasan global yang terjadi sekarang ini (IPCC, 2007). Karakteristik Lokasi (Kondisi Luar Waduk) Waduk Cirata merupakan waduk yang terletak di DAS Citarum, berlokasi sekitar 40 km dari arah Barat Laut Kota Bandung. Waduk ini berada pada ketinggian sekitar 221 m dari permukaan laut (dpl). Maksimum kedalaman waduk adalah 125 m (rata-rata kedalaman 35 m) dengan volume air sekitar 2.2 km3 (Hayami et al., 2008). Lokasi ketinggian waduk dari permukaan laut tentunya akan mempengaruhi volume air yang masuk. Waduk yang berlokasi cukup tinggi hanya memperoleh sumber air berasal dari curah hujan (precipitation) sedangkan waduk yang terletak di dataran rendah memperoleh air dari aliran sungai dan air tanah. Blenckner (2005) mengemukakan bahwa danau yang berlokasi di ketinggian umumnya berukuran kecil, memiliki kecerahan yang tinggi, jumlah spesies ikan terbatas, dan Analisis data dalam penelitian ini meng- adopsi metode yang dikemukakan oleh Blenckner (2005). Menurut Blenckner (2005), konsepsual model kemungkinan dampak perubahan/variasi iklim terhadap ekosistem waduk dapat dianalisis dari dua aspek (filter) penting yaitu aspek karakteristik lokasi dan karakteristik waduk (Gambar 2). Berdasarkan kedua aspek ini, data yang telah dikumpulkan kemudian dilihat hubungannya dan dibahas secara deskriptif yang disertai dengan gambar. Respon ekosistem danau/waduk Lake/reservoir ecosystem respond Respon ekosistem danau/waduk Lake/reservoir ecosystem respond Gambar 2. Model analisis data kemungkinan dampak perubahan/variasi iklim terhadap ekosistem waduk. Gambar 2. Model analisis data kemungkinan dampak perubahan/variasi iklim terhadap ekosistem waduk. Figure 2. Modeling approach of possible impact of climate variability on reservoir ecosystem. dari suatu kawasan waduk sangat terbatas ketersediaannya. Variasi suhu udara ini dapat dijadikan data proxy untuk menduga fluktuasi suhu air waduk. Seperti yang dilaporkan oleh Boyd & Pine (2006), bahwa fluktuasi rata-rata suhu udara bulanan identik dengan fluktuasi rata-rata suhu air. Hal ini berimplikasi bahwa perubahan yang terjadi pada suhu udara setiap musimnya akan mempengaruhi suhu air dan pertumbuhan dari biota air. Suhu udara bulanan di Waduk Cirata cukup berfluktuasi (Gambar 5a). Kisaran suhu udara hasil pemantauan stasiun klimatologi UPJB adalah antara 20oC- 30oC. Dari data histori yang ada (tahun 2003-2009), menunjukkan bahwa terjadi kondisi yang cukup ekstrim di tahun 2004 dan 2007. Pada tahun 2004, suhu udara meningkat secara signifikan mencapai lebih dari 35oC. Kondisi ini sangat berhubungan dengan terjadinya El Niño di kawasan tropis (http:// w w w . c p c . n o a a . g o v / p r o d u c t s / analysis_monitoring/ensostuff/ens). Sebaliknya tahun 2007 suhu udara menurun drastis mencapai sekitar 17oC. Kondisi ini jika dikaitkan dengan zone indek di kawasan tropis adalah bersamaan dengan terjadinya La Niña. Kedua contoh kondisi ekstrim ini, merupakan dari suatu kawasan waduk sangat terbatas ketersediaannya. Variasi suhu udara ini dapat dijadikan data proxy untuk menduga fluktuasi suhu air waduk. Seperti yang dilaporkan oleh Boyd & Pine (2006), bahwa fluktuasi rata-rata suhu udara bulanan identik dengan fluktuasi rata-rata suhu air. Hal ini berimplikasi bahwa perubahan yang terjadi pada suhu udara setiap musimnya akan mempengaruhi suhu air dan pertumbuhan dari biota air. Suhu udara bulanan di Waduk Cirata cukup berfluktuasi (Gambar 5a). Kisaran suhu udara hasil pemantauan stasiun klimatologi UPJB adalah antara 20oC- 30oC. Dari data histori yang ada (tahun 2003-2009), menunjukkan bahwa terjadi kondisi yang cukup ekstrim di tahun 2004 dan 2007. Pada tahun 2004, suhu udara meningkat secara signifikan mencapai lebih dari 35oC. Kondisi ini sangat berhubungan dengan terjadinya El Niño di kawasan tropis (http:// w w w . c p c . n o a a . g o v / p r o d u c t s / analysis_monitoring/ensostuff/ens). Sebaliknya tahun 2007 suhu udara menurun drastis mencapai sekitar 17oC. HASIL DAN BAHASAN Respons ekosistem waduk terhadap dampak pemanasan global akan berbeda-beda sesuai dengan karakteristik dari DAS dan 498 Kondisi meteorologi, klimatologi, dan perikanan ..... (I Nyoman Radiarta) Perubahan iklim (Climate change) Variasi iklim (Climate variability) Respon ekosistem danau/waduk Filter 1 (luar waduk): Karakteristik lokasi Filter 1 (outside reservoir): Location characteristic Filter 2 (kondisi dalam): Karakteristik waduk Filter 2 (inside reservoir): Characteristic reservoir Posisi geografis Geographic position Karakteristik DAS Cathment characteristic Meteorologi/klimatologi Climate condition Histori danau/waduk Lake/reservoir history Kondisi kualitas perairan Water quality condition Interaksi abiotik/biotik Biotic/abiotic interaction Perubahan iklim (Climate change) Variasi iklim (Climate variability) Perubahan iklim (Climate change) Variasi iklim (Climate variability) Posisi geografis Geographic position Karakteristik DAS Cathment characteristic Meteorologi/klimatologi Climate condition Filter 1 (luar waduk): Karakteristik lokasi Filter 1 (outside reservoir): Location characteristic Filter 1 (luar waduk): Karakteristik lokasi Histori danau/waduk Lake/reservoir history Filter 2 (kondisi dalam): Karakteristik waduk Filter 2 (inside reservoir): Characteristic reservoir Kondisi kualitas perairan Water quality condition Interaksi abiotik/biotik Biotic/abiotic interaction Respon ekosistem danau/waduk Lake/reservoir ecosystem respond Kondisi ini jika dikaitkan dengan zone indek di kawasan tropis adalah bersamaan dengan terjadinya La Niña. Kedua contoh kondisi ekstrim ini, merupakan rendahnya dampak dari aktivitas manusia dibandingkan dengan danau yang berlokasi di daratan rendah. Dilihat dari sisi ketinggian, Waduk Cirata dapat dikategorikan berada pada daratan yang tidak terlalu tinggi. Sumber air waduk ini umumnya berasal dari aliran Sungai Citarum, yang merupakan aliran sungai terbesar di Jawa Barat. Hasil analisis data curah hujan di kawasan DAS Citarum menunjukkan bahwa terdapat dua musim di kawasan ini yaitu musim kering pada bulan Mei-September dan musim penghujan pada bulan Oktober-April (Gambar 3). Rata-rata presipitasi bulanan adalah 67-155 mm pada musim kering dan 169-328 mm pada musim penghujan (Hayami et al., 2008). Dampak langsung dari adanya pemanasan global adalah terjadinya perubahan suhu udara. Perubahan suhu udara secara spasial di kawasan DAS Citarum cukup bervariasi (Gambar 4). Suhu udara terendah terjadi sekitar bulan Juli, sedangkan suhu udara maksimum terjadi bulan Agustus-September. Data suhu udara dari stasiun Badan Meteorologi Klimatologi dan Geofisik (BMKG) dan UPJB umumnya dapat diperoleh secara berkelan- jutan (time series), sedangkan data suhu air 499 Gambar 3. Kondisi presipitasi (mm) di kawasan DAS Citarum Figure 3. Precipitation (mm) condition in Citarum Watershed Rendah (Low) Tinggi (High) Presipitasi (Precipitation) (mm) 107o15’E 107o30’E 6o30’S 6o45’S 7o0’S Gambar 4. Kondisi suhu udara (oC) di kawasan DAS Citarum: (a) suhu minimum, (b) suhu maksimum, dan (c) suhu rata-rata Figure 4. Water temperature (oC) condition in Citarum Watershed: (a) minimum temperature, (b) maximum temperature, and (c) average temperature Rendah (Low) 6o30’S 107o15’E 107o30’E 6o45’S 7o0’S Tinggi (High) Suhu udara (Air temperature) (oC) Ris. Akuakultur Vol.6 No.3 Tahun 2011: 495-506 00 J. Ris. Akuakultur Vol.6 No.3 Tahun 2011: 495-506 Gambar 3. Kondisi presipitasi (mm) di kawasan DAS Citarum Figure 3. Precipitation (mm) condition in Citarum Watershed Rendah (Low) Tinggi (High) Presipitasi (Precipitation) (mm) 107o15’E 107o30’E 6o30’S 6o45’S 7o0’S Gambar 3. Kondisi presipitasi (mm) di kawasan DAS Citarum Figure 3. Precipitation (mm) condition in Citarum Watershed Gambar 4. Kondisi suhu udara (oC) di kawasan DAS Citarum: (a) suhu minimum, (b) suhu maksimum, dan (c) suhu rata-rata Figure 4. Water temperature (oC) condition in Citarum Watershed: (a) minimum temperature, (b) maximum temperature, and (c) average temperature Rendah (Low) 6o30’S 107o15’E 107o30’E 6o45’S 7o0’S Tinggi (High) Suhu udara (Air temperature) (oC) Gambar 4. Respon ekosistem danau/waduk Lake/reservoir ecosystem respond Kondisi suhu udara (oC) di kawasan DAS Citarum: (a) suhu minimum, (b) suhu maksimum, dan (c) suhu rata-rata Figure 4. Water temperature (oC) condition in Citarum Watershed: (a) minimum temperature, (b) maximum temperature, and (c) average temperature Rendah (Low) 6o30’S 107o15’E 107o30’E 6o45’S 7o0’S Tinggi (High) Suhu udara (Air temperature) (oC) Gambar 4. Kondisi suhu udara (oC) di kawasan DAS Citarum: (a) suhu minimum, (b) suhu maksimum, dan (c) suhu rata-rata Figure 4. Water temperature (oC) condition in Citarum Watershed: (a) minimum temperature, (b) maximum temperature, and (c) average temperature 500 Kondisi meteorologi, klimatologi, dan perikanan ..... (I Nyoman Radiarta) Gambar 5. Kondisi suhu udara (a) dan curah hujan (b) di Waduk Cirata dari tahun 2003-2009 Figure 5. Air temperature condition (a) and rain fall (b) in Cirata Reservoir from 2003-2009 Suhu udara Air temperature 40 (a) (b) 30 20 10 25 15 10 5 0 curah hujan Rain fall Bulan (Months) 2003 2004 2005 2006 2007 2008 2009 1 5 9 1 5 9 1 5 9 1 5 9 1 5 9 1 5 9 1 5 9 Maksimun (Maximum) Minimum (Minimum) Rataan (Average) 20 (a) (b) Gambar 5. Kondisi suhu udara (a) dan curah hujan (b) di Waduk Cirata dari tahun 2003-2009 Figure 5. Air temperature condition (a) and rain fall (b) in Cirata Reservoir from 2003-2009 Maksimun (Maximum) Minimum (Minimum) Rataan (Average) Gambar 5. Kondisi suhu udara (a) dan curah hujan (b) di Waduk Cirata dari tahun 2003 Figure 5. Air temperature condition (a) and rain fall (b) in Cirata Reservoir from 20 indikasi penting yang dapat dikaitkan dengan adanya dampak dari pemansan global yang terjadi di kawasan Waduk Cirata. Kondisi ini jika dikaitkan dengan kondisi perikanan budidaya tentunya akan memberikan prespektif yang berbeda. diakibatkan karena pasokan limbah aktivitas rumah tangga, industri, dan pertanian yang terbawa dari aliran sungai masuk ke dalam waduk. Indeks kesesuaian kualitas perairan di Waduk Cirata menunjukkan kondisi yang lebih baik dibandingkan dengan Waduk Saguling (Soemarwoto et al., 1990). Hal ini disebabkan karena pasokan limbah yang berasal dari Kota Bandung, Cimahi, dan Padalarang masuk di Waduk Saguling melalui Sungai Citarum sangat besar. Kondisi curah hujan yang terjadi di kawasan Waduk Cirata hasil pemantauan stasiun UPJB menunjukkan fluktuasi yang cukup tinggi (Gambar 5b). Secara umum, curah hujan yang tinggi terjadi sekitar bulan Oktober- April setiap tahunnya, dengan curah hujan maksimum ditemukan pada tahun 2003. Respon ekosistem danau/waduk Lake/reservoir ecosystem respond Tingginya curah hujan yang terjadi dapat mempengaruhi terjadinya penurunan suhu air yang jika dibarengi dengan terjadinya hembusan angin yang kencang akan memicu terjadinya pengadukan kolom air (terjadinya umbalan, upwelling) yang sering berimplikasi pada terjadinya kematian ikan (Krismono et al., 2001). Karakteristik Waduk (Kondisi Dalam Waduk) Aktivitas yang terdapat dalam kawasan waduk juga akan memberikan respons yang cukup signifikan terhadap kemungkinan dampak pemanasan global terhadap ekosistem waduk. Waduk Cirata yang di- bangun pada tahun 1988 bertujuan untuk memenuhi tenaga listrik dan pengendalian banjir. Namun sejalan dengan perkembangan waktu, aktivitas penunjang semakin berkembang di antaranya perikanan dan pariwisata. Kegiatan perikanan budidaya di waduk ini telah dilakukan dengan meng- gunakan KJA (Costa-Pierce, 1998). Ikan yang umumnya dibudidayakan adalah ikan mas (Cyprinus carpio) dan nila (Oreochromis niloticus) (Abery et al., 2005). Pengelolaan waduk untuk kegiatan perikanan ditujukan Kondisi tutupan lahan di sekitar area resapan (catchmen area) merupakan aspek luar waduk yang berperan penting bagi kondisi lingkungan perairan dalam waduk (Gambar 6). Masing-masing jenis tutupan lahan akan memberikan pengaruh berbeda terhadap kondisi perairan waduk (Xiaoyan et al., 2002). Padatnya pemukiman dan aktivitas pertanian yang ada di sekitar Waduk Cirata dapat memicu terjadinya peningkatan polusi perairan yang 501 J. Ris. Akuakultur Vol.6 No.3 Tahun 2011: 495-506 Gambar 6. Tutupan lahan di kawasan Waduk Cirata (Sumber: Radiarta & Ardi, 2009) Figure 6. Land cover condition around Cirata Reservoir area (Source: Radiarta & Ardi, 2009) 6o42’S Pertanian (Agriculture field) 6o44’S 6o46’S 107o15’E 107o18’E 107o21’E 107o15’E 107o18’E 107o21’E Tutupan lahan (Land cover) Perkebunan (Plantation) Pemukiman (Settlement) Hutan (Forest) Tanah kosong (Land) Waduk (Reservoir) Sungai (River) 6o46’S 107o18’E 107o18’E 107o15’E 107o21’E Gambar 6. Tutupan lahan di kawasan Waduk Cirata (Sumber: Radiarta & Ardi, 2009) Figure 6. Land cover condition around Cirata Reservoir area (Source: Radiarta & A Gambar 6. Tutupan lahan di kawasan Waduk Cirata (Sumber: Radiarta & Ardi, 2009) Gambar 7. Distribusi KJA di Waduk Cirata pada waktu pemantauan yang berbeda: (a) Landsat ETM+ akuisisi tanggal 29 April 2002 dan (b) ALOS AVNIR-2 akuisisi tanggal 27 September 2008 (Sumber: Radiarta et al., 2005; Radiarta & Ardi 2009) Figure 7. Cage culture distribution in Cirata Reservoir at different observation time: (a) Landsat ETM+ aquired on April 29, 2002 and (b) ALOS AVNIR-2 aquired on September 27, 2008 (Source: Radiarta et al., 2005; Radiarta & Ardi 2009) (a) LANDSAT ETM+ 29 April 2002 (b) ALOS AVNIR-2 27 September 2008 (b) ALOS AVNIR-2 27 September 2008 (a) LANDSAT ETM+ 29 April 2002 Gambar 7. Distribusi KJA di Waduk Cirata pada waktu pemantauan yang berbeda: (a) Landsat ETM+ akuisisi tanggal 29 April 2002 dan (b) ALOS AVNIR-2 akuisisi tanggal 27 September 2008 (Sumber: Radiarta et al., 2005; Radiarta & Ardi 2009) Figure 7. Karakteristik Waduk (Kondisi Dalam Waduk) Cage culture distribution in Cirata Reservoir at different observation time: (a) Landsat ETM+ aquired on April 29, 2002 and (b) ALOS AVNIR-2 aquired on September 27, 2008 (Source: Radiarta et al., 2005; Radiarta & Ardi 2009) 502 Kondisi meteorologi, klimatologi, dan perikanan ..... (I Nyoman Radiarta) untuk mengoptimalkan produksi ikan dan menjaga kelestarian sumberdaya perikanan serta lingkungan sehingga dapat dimanfaatkan secara berkelanjutan oleh masyarakat sekitar kawasan waduk. Perkembangan jumlah KJA yang ada di Waduk Cirata menunjukkan jumlah yang telah melebihi daya dukung waduk tersebut (Gambar 7). Hasil analisis citra satelit menunjukkan peningkatan tutupan KJA yang sangat signifikan (Radiarta & Ardi, 2009). Penempatan KJA yang ada sekarang ini juga sudah tidak sesuai lagi dengan zonasi pemanfaatan lahan yang telah ditetapkan sebelumnya (Radiarta et al., 2005). Kondisi ini dapat disebabkan karena lemahnya managemen pengelolaan waduk yang ada (Krismono & Wahyudi, 2002). variasi jumlah KJA, produksi ikan secara total dan per KJA dan frekuensi terjadinya kematian ikan dari tahun 1988-2002. Produksi total maksimum tercatat tahun 1997, sedangkan produksi maksimum per KJA tercatat tahun 1994. Peningkatan KJA setiap tahunnya tidak mendorong peningkatan produksi namun sebaliknya, produksi ikan semakin menurun (Gambar 8). Hal ini dapat disebabkan karena kondisi lingkungan dalam waduk sudah tidak dapat mendukung secara maksimal untuk kegiatan perikanan budidaya dengan KJA. Frekwensi kejadian kematian ikan selalu ditemukan setiap tahunnya dengan tingkat kematian tersebar ditemukan tahun 1995. Tahun tersebut merupakan periode La Niña. Tantangan Penelitian Ke Depan Peningkatan jumlah KJA ini dapat menyebabkan penurunan mutu lingkungan di dalam waduk. Beberapa hasil penelitian menunjukkan bahwa kandungan oksigen yang masih layak untuk aktivitas budidaya ikan di Waduk Cirata hanya tersedia sampai pada kedalaman maksimum sekitar 7 m sedangkan perubahan suhu secara drastis (thermocline) ditemukan sekitar kedalaman 5-7 m (Hayami et al., 2008). Kondisi ini menunjukkan jika terjadi perubahan iklim yang ekstrim akan cepat memacu terjadinya pengadukan yang mem- bawa gas-gas beracun dari dasar waduk kepermukaan sehingga dapat menyebabkan kematian ikan. Gambar 8 menyajikan data Dengan memperhatikan dua aspek (dari luar dan dalam) yang dapat memicu respon ekosistem waduk terhadap dampak pema- nasan global yang terjadi, masih terbuka peluang yang sangat besar untuk melakukan kajian-kajian yang lebih spesifik dan ber- kelanjutan. Sehingga nantinya ketersediaan data kondisi lingkungan perairan dapat dipadukan dengan data iklim yang tersedia. Kajian lingkungan perairan sehubungan dengan kondisi iklim global ini telah pula dilakukan oleh Balai Penelitian dan Pengembangan Perikanan Air Tawar mulai tahun 2010 (Adang Saputra, komunikasi Gambar 8. Variasi perubahan jumlah KJA, produksi per KJA, produksi total dan frekuensi kematian ikan di Waduk Cirata, Jawa Barat (Sumber: Abery et al., 2005) Figure 8. Variation on number of cage culture, cage culture production, total production, and fish kill frequency in Cirata Reservoir, West Java (Source: Abery et al., 2005) Jumlah KJA (Number of cages) Produksi (Production) (ton) Produksi per KJA (Production per cages) (kg) Kematian ikan (Fish kill ) (ton) Jumlah KJA/produksi Number of cages/production (ton) 60000 0 Produksi per KJA (kg)/kematian ikan (ton) Production per cages (kg)/fish kill (ton) 2500 50000 40000 30000 20000 10000 2000 1500 1000 500 0 Jumlah KJA (Number of cages) Produksi (Production) (ton) Produksi per KJA (Production per cages) (kg) Kematian ikan (Fish kill ) (ton) Jumlah KJA/produksi Number of cages/production (ton) 60000 0 Produksi per KJA (kg)/kematian ikan (ton) Production per cages (kg)/fish kill (ton) 2500 50000 40000 30000 20000 10000 2000 1500 1000 500 0 Variasi perubahan jumlah KJA, produksi per KJA, produksi total dan frekuensi kematian ikan di Waduk Cirata, Jawa Barat (Sumber: Abery et al., 2005) Figure 8. Variation on number of cage culture, cage culture production, total production, and fish kill frequency in Cirata Reservoir, West Java (Source: Abery et al., 2005) 503 J. Ris. Akuakultur Vol.6 No.3 Tahun 2011: 495-506 pribadi). UCAPAN TERIMA KASIH Penulis mengucapkan terima kasih kepada tim survei pemanasan global Pusat Penelitian dan Pengembangan Perikanan Budidaya yang telah membantu kelancaran pengumpulan data. Penelitian ini merupakan bagian dari penelitian “dampak pemanasan global ter- hadap perikanan budidaya” Pusat Penelitian dan Pengembangan Perikanan Budidaya T.A. 2010. (1) Ketersediaan data yang berkelanjutan (time series) yang kadang kala sulit diperoleh terutama data kondisi lingkungan perairan. Kalaupun data perikanan tersedia, umumnya masih bersifat parsial. (2) Koordinasi lintas sektoral antar berbagai bidang ilmu masih terhambat dalam pelaksanaannya. DAFTAR ACUAN Abery, N.W., Sukadi, F., Budhiman, A.A., Kartamihardja, E.S., Koeshendrajana, S., Buddhiman, & De Silva, S.S. 2005. Fisher- ies and cage culture of three reservoirs in West Java, Indonesia: a case study of ambi- tious development and resulting interac- tions. Fisheries Management and Ecology, 12: 315–330. (3) Hasil-hasil penelitian yang kurang signifikan kadangkala susah untuk ditemukan, yang kemungkinan hasil tersebut dapat dikaitkan dengan kondisi iklim yang terjadi. (4) Kombinasi pemanfataan model ekosistem dengan data histori masih belum banyak dianalisis. Baba, K., Sugawara, R., Nitta, H., Endou, K., & Miyazono, A. 2009. Relationship between spat density, food availability, and growth of spawners in cultured Mizuhopecten yessoensis in Funka Bay: concurrence with ENSO. Canadian Journal of Fisheries and Aquactic Sciences, 12: 6-17. Selain keempat hambatan tersebut, tentunya masih terdapat hambatan yang lebih spesifik, namun kondisi ini menjadikan tantangan tersendiri dan membuka peluang penelitian pemanasan global. Metode yang sangat mungkin dilakukan untuk bidang budidaya perikanan yaitu dengan pemodelan, seperti yang telah berkembang dibidang lainnya (pertanian, perikanan tangkap, dll) Bardach, J.E. 1989. Global warming and the coastal zone. Climatic Change, 15: 117- 150. Tantangan Penelitian Ke Depan Kegiatan ini mengumpulkan data kondisi kualitas perairan secara vertikal dan horizontal yang direncanakan selama 5 kali pengambilan data lapangan dalam satu tahun dan direncanakan selama 4 tahun berjalan, sampai tahun 2014. Ketersediaan data ini tentunya akan sangat membantu dalam memberikan gambaran secara nyata kondisi Waduk Cirata guna mendukung pengelolaan waduk yang lebih baik. dampak pemanasan global. Analisis awal ini memberikan gambaran yang cukup menarik terhadap isu pemanasan global yang terjadi. Namun untuk dapat memberikan hasil analisis yang lebih spesifik masih diperlukan tambahan histori data baik dari aspek iklim dan perikanan. Hasil dari penelitian ini diharapkan dapat menambah wawasan sehingga nantinya dapat diimplementasi dilapangan guna mencegah akibat yang lebih buruk dari adanya dampak pemanasan global. Untuk memperoleh hasil kajian yang komprehensif tentang kemungkinan dampak pemanasan global terhadap ekosistem waduk masih dirasakan sangat kurang dalam pelaksanaannya, hal ini disebabkan karena beberapa alasan yaitu (Blenckner, 2005): KESIMPULAN Beukema, J.J. & Dekker, R. 2005. Decline of re- cruitment success in cockles and other bivalve in the Wadden Sea: possible role of climate change, predation on postlarvae and fisheries. Marine Ecology Progress Se- ries, 287: 149-167. Kajian kemungkinan dampak pemanasan global terhadap ekosistem waduk tidak hanya dipengaruhi oleh kondisi luar ekosistem, namun juga harus memperhatikan interaksi dalam waduk itu sendiri. Dengan memperhati- kan kedua aspek tersebut tentunya akan memberikan analisis yang lebih komprehensif mengenai respon waduk terhadap pemanasan global. Penelitian ini dilakukan untuk men- sintesis kondisi Waduk Cirata dilihat baik aspek dari luar dan dalam terhadap kemungkinan Blenckner, T. 2005. A cenceptual model of cli- mate-related effects on lake ecosystems. Hydrobioilogia, 533: 1-14. Byod, C.E. & Pine, H. 2006. Application of agrometeorology to aquaculture and fish- eries. WMO/CAgM Guide to Agricultural 504 Kondisi meteorologi, klimatologi, dan perikanan ..... (I Nyoman Radiarta) Cambridge, United Kingkom and New York, NY, USA. Meteorological Practices (GAMP) Chapter 14. Didownload tanggal28 Juli 2011: http:/ /www.agrometeorology.org/files-folder/ repository/gamp_chapt14final.pdf. Kapetsky, J.M. 2000. Present applications and future needs of meteorology and climatol- ogy data in inland fisheries and aquacul- ture. Agricultural and Forest Meteorology, 103: 109-117. Cochrane, K., De Young, C., Soto, D., & Bahri, T. (eds). 2009. Climate change implication for fisheries and aquaculture: overview of cur- rent scientific knowledge. FAO Fisheries and Aquaculture Technical Paper. No. 530. Rome, FAO, 212 pp. Krismono & Wahyudi, N.A. 2002. Analisis kebijakan pengelolaan KJA sebagai salah satu kegiatan pengelolaan danau dan waduk. Dalam Heruwati et al. (eds.). Analisis Kebijakan pembangunan perikanan 2001. Pusat Riset Pengolahan Produk dan Sosial Ekonomi Kelautan dan Perikanan, hlm. 75- 85. Costa-Pierce, B.A. 1998. Constraints to the sustainability of cage aquaculture for re- settlement from hydropower dams in Asia: an Indonesian case study. J. of Environ- ment and Development, 7: 333-363. Krismono, A.S.N., Krismono, & Kartamihardja, E.S. 2001. Dampak budidaya ikan dalam keramba jaring apung terhadap pening- katan unsur N dan P di perairan Waduk Saguling, Cirata dan Jatilihur. J. Penelitian Perikanan Indonesia, 7: 22-30. De Silva, S.S. & Soto, D. 2009. Climate change and aquaculture: potential impact, adapta- tion and mitigation. In Cochrane, K., De Young, C., Soto, D., & Bahri, T. (eds). Cli- mate change implication for fisheries and aquaculture: overview of current scientific knowledge. FAO Fisheries and Aquaculture Technical Paper. No. 530. Rome, FAO, p. 151-212. Meisner, J.D. & Shuter, B.J. 1992. Assessing potential effect of global climate change on tropical freshwater fishes. tics of non-point source pollution in the Watershed of Miyun Reservoir, Bejing, China. Chinese Journal of Geochemistry, 21: 89-95. Xiaoyan, W., Yixun, W., Tingfang, L., Wei, H., Qiuju, H., & Hongfen, Z. 2002. Characteris- Ecological responses to recent climate change, Nature, 416: 389-395. Xiaoyan, W., Yixun, W., Tingfang, L., Wei, H., Qiuju, H., & Hongfen, Z. 2002. Characteris- J. Ris. Akuakultur Vol.6 No.3 Tahun 2011: 495-506 KESIMPULAN Geo Journal, 28(1): 21-27. Harley, C.D.G., Hughes, A.R., Hultgren, K.M., Miner, B.G., Sorte, C.J.B., Thornber, C.S., Rodriguez, L.F., Tomanek, L., & Williams, A.L., 2006. The impact of climate change in coastal marine systems. Ecology Letters, 9: 228-241. Radiarta, I N. & Ardi, I. 2009. Pemetaan distribusi keramba jaring apung ikan air tawar di Waduk Cirata, Jawa Barat dengan multi tem- poral data ALOS AVNIR-2. J. Ris. Akuakultur, 4: 439-446. Radiarta, I N., Prihadi, T.H., & Sunarno, T. 2005. Pemantauan perikanan budi daya berbasis KJA di Waduk Cirata dengan menggunakan multi-temporal data landsat 7. Warta Penelitian Perikanan Indonesia, 11: 2-8. Handisyde, N.T., Ross, L.G., Badjeck, M-C., & Allison, E.H. 2006. The effects of climate change on world aquaculture: a global per- spective. Available online at www. aquacultur.stir.ac.uk/GISAP/gis-group/ climate.php. Saitoh, S-I., Mugo, R., Radiarta, I N., Asaga, S., Takahashi, F.,Hirawake, T., Ishikawa, Y., Awaji, T., & Shima, S. 2011. Some opera- tional uses of satellite remote sensing and marine GIS for sustainable fisheries and aquaculture. ICES Journal of Marine Science, 68: 687-695. Hayami, Y., Ohmori, K., Yoshino, K., & Garno, Y.S., 2008. Observation of anoxic water mass in a tropical reservoir: the Cirata Res- ervoir in Java, Indonesia. Limnology, 9: 81- 87. Hijmans, R.J., Cameron, S.E., Parra, J.L., Jones, P.G., & Jarvis, A. 2005. Very high resolution interpolated climate surfaces for global land areas. International Journal of Clima- tology, 25: 1965-1978. Soemarwoto, O., Roem, C.M., Herawati, T., & Costa-Pierce, B.A. 1990. Water quality suit- ability of Saguling and Cirata reservoirs for development of floating net cage aquac- ulture. In: B.A. Costa-Pierce, O. Soemarwoto (eds) Reservoir Fisheries and Aquaculture Development for Resettlement in Indone- sia. Manila, Philippines: ICLARM, Technical Report, 23: 18–111. IPCC. 2007. Summary for policymakers. In: Cli- mate change 2007: the physical science basis. Contribution of working group I to the fourth assessment report of the inter- governmental panel of climate change [Solomon, S., D. Qin, M. Manning, Z. Chen, M. Marquis, K.B. Averyt, M. Tignor and H.L. Miller (eds.)]. Cambridge University Press, Walther, G-R., Post, E., Convey, P., Menzel, A., Parmesan, C., Beebee, T.J.C., Fromentin, J- M., Hoegh-Guldberg, O., & Barlein, F. 2002. 505 J. Ris. Akuakultur Vol.6 No.3 Tahun 2011: 495-506 Ecological responses to recent climate change, Nature, 416: 389-395. tics of non-point source pollution in the Watershed of Miyun Reservoir, Bejing, China. Chinese Journal of Geochemistry, 21: 89-95. 506
https://openalex.org/W2537071554	https://cityterritoryarchitecture.springeropen.com/track/pdf/10.1186/s40410-016-0048-7	English	null	Urban disorder and vitality	City, territory and architecture	2,016	cc-by	5,852	Abstract The concepts of order and disorder applied to urban and territorial issues involve complex definitions. Why do cities, in spite of the effort made to give them order, even though from time to time this order has been codified in different ways, end up being untidy? In the context in which the two concepts will be used in this paper it is not possible to imagine them as alternatives, but they are taken as dialectically constituting territorial reality. “Order” and “disorder” oppose each other but do not clash with each other. In the hectic organisation of urban and territorial reality one stimulates the other and each, in opposing, determines change. The inclination towards order (or tendency towards order) tends, on the one hand, to “repair” the disorder but, on the other, brings out the conditions for disorder to show itself again and materialise with its problems but with the vitality implicit in change. Change brings disorder but public commit- ment through institutions cannot but aspire to recover a level of order, hopefully, more advanced. Order and disorder are closely linked with each other, one producing the other in a circular process. The urban, precisely because of its constituent construction (social, productive and economic variability; clash between powers and options of models of society) cannot be stable, but the continuous recovery of “order” responds not only to functional needs, but also to ethical options: we should not consider all urban “order” as positive, compared with negative disorder; there are experiences of oppressive and coercive urban order. It is always disorder that determines better levels and quality of order. Though it may be the dynamic factor of every urban condition, we must remember that it always requires new order from which to start out again. Disorder, however it is identified, constitutes a permanent fact, inherent in the urban condition; it is neither the result of wrong planning (sometimes also this), nor of a perverse will, but rather of the dynamic mechanisms of the city itself. Keywords: Alternation between order and disorder, Urban space organisation, Vitality Urban development, the work of planning and organising the city and territory, is characterised by a strong voca- tion to impose order. © The Author(s) 2016. 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. Urban disorder and vitality Francesco Indovina* Indovina City Territ Archit (2016) 3:18 DOI 10.1186/s40410-016-0048-7 *Correspondence: indovina@iuav.it Department of Architecture, Design & Urban Planning, University of Sassari, Palazzo del Pou Salit, Piazza Duomo 6 Alghero, 07041 Sassari, Italy Abstract When an urban planning instru- ment defines what, where, when and how it is possible to “realise” some change in the organisation of space it ends up modifying what exists and imposing a new kind of “order”. We tend to think of “physical” order but actually, it is a case of order that has a variety of aspects, includ- ing the indications of “meaning” space organisation produces. from an activity without a code that responded to social, economic and life requirements and ones based on func- tionality and representation, to a discipline often based on self-determination. In the cultural history of the discipline certain traits can be picked out in which an interest for functional order seemed to prevail, though in fact the attention for the city and territory that this activity has adopted is the overall one involving functionality but also social struc- ture, co-habiting, power, culture, and sacred and profane rites. Each kind of urban order, in its divisions and con- struction, may have been the source of conflicts, oppres- sion, liberation, marginalisation and exaltation (Indovina 2016).hl Urban development, giving order to settlement, is extremely ancient and arose with the first settlements, even though the discipline was codified in modern times: The question that forces us to reflect is very simple: why do cities, in spite of the effort made to give them order, even though from time to time this order has been codified in different ways, end up being untidy? Indovina City Territ Archit (2016) 3:18 Page 2 of 7 social, cultural, political, institutional, etc.) able to leave a footprint on urban organisation. The concepts of order and disorder applied to urban and territorial issues involve complex definitions, and to understand them it is worth referring to some facts. If each function were free to leave its own “footprint” on the urban territory, it is clear that the result could not but be a sort of marsh where footprints overlapped and depended on the weight of the bodies (the interested par- ties) creating them. Presumably, however, footprints have to follow rules (we might say the “plans” or any other instrument managing the territory), but it is only an assumption that rules can accurately steer the bodies and therefore the respective footprints. Abstract Spatial order in the country, often admired, is the result of a need; it satisfies that expressed by production which presents very limited alternatives. Change is not excluded (crop variation, agrarian science achievements, use of new technologies, etc.); the point may even be reached of building a new agrarian landscape with order that is dif- ferent. Cultural movements linked with tradition some- times oppose these changes, harming the intrinsic nature of the agrarian landscape, which is destined to change. Needs and reduced alternatives may be considered key categories in determining the agrarian landscape, but we are not dealing with two categories that could be imag- ined to be playing the same role in determining the urban landscape. Contemplation that is not always consistent, not always progressive, and not always attentive to the general interest.h In the city it is not possible to pick out such a bind- ing, decisive need, or, rather, there is a great number of highly diversified needs and they express inhomogene- ous content and justification; not only, but they are needs that are not compelling like in the country, as each one is compatible with various alternatives, therefore with dif- ferent models of urban organisation. The urban condition is characterised not by the absence of production but, rather, by the organisation of this production, by a greater presence of functions, greater unnaturalness of the con- text, the existence of functions integrated with each other and, at the same time, of functions that are antagonistic or incompatible, but above all by the high concentration of people with characteristics, projects, needs and desires that are different from each other. Differences that are also manifested as an expression of power (economic, The history of the city teaches how changeable it can be, in its shape and organisation, and how the change is the fruit, mainly, of change in social structure, produc- tion organisation and power organisation itself. Urban space organisation appears as a “passive” outcome of these changes, not as being determined by a factor that imposes change. The dynamics (the change) of the city and its organisation have to take into account its “hard” part: the built city, its history and its works: each new order does not wipe out the past but fits into an exist- ing context (hopefully, respecting it), perhaps giving it a purpose (from various points of view, including social) in the new reality. Abstract The doubt is not sug- gesting the fact that rules can be slack (this, too), or that bodies move deliberately opposing the rules (this, too), but the fact that the “needs” of the single bodies change over time in a process of interrelation between them, and that the possibilities offered account for the needs of the single body and the encounter of various bodies. Cultivated land shows us a tidy territory, and it is immediately understood that this comes directly down from the organisation of agricultural production, from the techniques used and the social relations character- ising farming work. The orography of the territory, too, with the crops compatible with the different heights above sea-level, serves the purpose of spatial design; its appearance, colours and scents change with the change of seasons or the change in production and technology adopted. Basically, there is a “strength”, agricultural pro- duction, with very limited alternatives, giving particular spatial solutions. “Dry walls”, which surround fields or enclosures and which we marvel at, are also a rational and reasonable solution for the use of stones unearthed by the plough; land reclamation gives substance to spatial construction. Just as canals and ditches serve the purpose of taking water where needed, while rows of trees con- solidate their banks, and lanes and farm-roads aim at the movement of men, animals and machinery, depending on the need. It should be pointed out once more that while for the country order is imposed by production, for the city, as well as production, the multiplicity of social subjects are important, as is the prominence of the service functions, events, needs, etc. that are projected onto the organisa- tion of space. Urban space organisation requires a spon- taneous directive; agricultural order, we might say, is given, while urban order has to be constructed. Urban order contemplates: the need to guarantee hygiene and the best exposition as regards the climatic situations of the place; the possibility of carrying out and developing productive and economic activities; “power” representa- tion; the citizens’ life needs; the intention to differentiate places by the presence of different social strata; the devel- opment and localisation of services; the need to avoid mixing functions little or not at all compatible with each other, and so on. Abstract On the one hand, imposed order presupposes within itself a principle of stability; we could say that the enforcement of order does not have aims that are just immediate but also future ones, but, as has been seen, this claim appears to be contradicted by the character of the territorial processes that are by nature dynamic, featuring changes and movements not always predict- able. The economic, social, demographic, cultural, tech- nological and power mechanisms involving the city and territory are of such an innovative impact that they can- not but cause change, i.e. a challenge to the pre-existing order. But there is more, the irruption of change, namely disorder, should be taken as positive or at least inevitable. If we were to assume that the enforcement of order were aimed at improving the organisation of the city and territory and ultimately at improving the living condi- tions of those inhabiting and “using” that territory, we would need to consider any challenge to order as a pro- spective worsening of those living conditions. But it does not seem to be like this, not only because disorder arises round those who have settled in the city or territory and use it, with the prospect of improving their condition, but On the one hand, imposed order presupposes within itself a principle of stability; we could say that the enforcement of order does not have aims that are just immediate but also future ones, but, as has been seen, this claim appears to be contradicted by the character of the territorial processes that are by nature dynamic, featuring changes and movements not always predict- able. The economic, social, demographic, cultural, tech- nological and power mechanisms involving the city and territory are of such an innovative impact that they can- not but cause change, i.e. a challenge to the pre-existing order. But there is more, the irruption of change, namely disorder, should be taken as positive or at least inevitable. Each change, however, rapid or slow, does not wipe out the past organisation of space, but there are phenom- ena of continuous transformation and adaptability. The organisation of space is a continuous task of conserva- tion-destruction-innovation-construction: what is pre- served is not always appreciated; often what is destroyed produces criticism, regret and the quest for revival; inno- vation, though real, is presented not rarely as an illusion; what is built seldom gains general approval. Abstract Change is at the same time destructive and protective; the present also has elements of the past and, fortunately, elements of the future (which we are not perhaps able to recognise). Indovina City Territ Archit (2016) 3:18 Page 3 of 7 Urban organisation actually undergoes the effects of continuous alternation between order and disorder. In the context in which the two concepts will be used in this paper it is not possible to imagine them as alternatives, but they are taken as dialectically constituting territo- rial reality. “Order” and “disorder” oppose each other but do not clash with each other. In the hectic organisation of urban and territorial reality one stimulates the other and each, in opposing, determines change. What we mean to say is that the inclination towards order (or ten- dency towards order) tends, on the one hand, to “repair” the disorder but, on the other, brings out the conditions for disorder to show itself again and materialise with its problems but with the vitality implicit in change. above all because the inevitable innovation, may, in gen- eral, be considered ameliorative. Highly complex economic, social, political and cultural designs are projected onto the city and its transforma- tions. It is impossible that the city be domesticated and dominated by a single interest, even when this appears as strong, and when it avails itself of the strength of eco- nomic and political power. The overall design always seems incoherent or, rather, is the outcome of compro- mises between different interests and strong points. But from these strengths, their compromise, their agree- ment or disagreement, from the conflicts, the city takes shape and gives itself organisation, a type of order. Order that seconds evolution but, at the same time, creates contradictions. Order and disorder are closely linked with each other, one producing the other in a circular process. They are like Siamese twins: one takes pains to find the most comfortable position for itself, but this does not mean it is comfortable for the other, too; the latter reacts and achieves a more comfortable position for itself but uncomfortable for the other. Abstract This metaphor might make clear how the dynamic relation between order and disor- der is continuous, not linear and may never find a point of stability.h Fundamentally, urban order is not independent from the requirements of various kinds (economic, social, cul- tural and of life) that the city produces; but none of these needs tends to fully prevail, precisely due to the opposing dialectics of the various requirements; urban order is not the outcome of mediation between the various require- ments but, rather, gives rise to a solution that includes spaces for the interests of each to be realised, whilst simultaneously leaving antagonistic spaces. Whatever meaning we wish to give to these statements, we cannot ignore the fact that men and women occupy “space” and organise it according to material and intel- lectual needs. The reality that springs from it is neither fortuitous or spontaneous; its content is different in pro- portion to the growth of knowledge and the type of co- existence chosen (or imposed), to the regime regulating production relations, but also the phase of culture. What is seen around us, from the cities to the country, repre- sents what has come down to our times from thousands of years of work on constructing, adapting, destroying, reconstructing, modifying and enlarging, to which the species has applied itself so as to overcome all obstacles that, on the one hand, “stepmother nature” has placed against the achievement of the welfare of the species and, on the other, power (economic, but not only) that has continued to hinder the achievement of freedom. The dialectic of the Siamese twins applied to the rela- tion order/disorder is easy to share, but in fact presents a large number of aporias and many consequences. A com- fortable position for one of the children, as we said, risks being uncomfortable for the other, and when the former achieves a better position, it affects that of the other child. No position exists that meets the demands of both. child. No position exists that meets the demands of both. Abstract In modernity the instruments for giving order to space, apart from being refined, are a way of reducing discre- tion, namely the will to overcome, or more simply the achievement of the interests of one party to the detri- ment of those of the collectivity or of other single indi- viduals. It is obvious that discretion is founded on power, and also in advanced democracies power (especially economic power) is not eliminated, but the “rules” that dictate space organisation, which should be valid for eve- ryone, certainly put ties on or at least hinder the success of single powers. At least, this is their intention (doubts and criticisms may be put forward on the respective realisation, but everything depends on political will or weakness). By this we do not mean to ignore the strength and importance of the individual and along this path the pos- sibility of partial processes of self-organisation (well-tem- pered individualism can be good for space organisation, too), but just to emphasise once more that space organi- sation responds to requirements of a collective, social, functional and cultural nature, and that as such, space cannot but be organised according to collective (planned) political processes. And that if self-organisation pro- cesses go beyond aspects of little importance they deter- mine, even if not intended for this purpose, breakdowns in the collective organisation of space. We mean to maintain that in order to achieve urban order (however defined) the existence of a set of rules is taken for granted, which indicate the ways in which the city is organised so as to enable its efficient and effective functioning, with the satisfaction of everyone or prefer- ring some (this is a variable of politics). While it is obvi- ous that these rules vary over time and in space, it should be emphasised that their application is a manifestation of politics or, if preferred, a way of governing. It seems mis- leading, in effect, to believe that the existence of “good rules” can guarantee space organisation coherent with the (abstract) aims of the said rules. The rules relating to space organisation have ambiguous features, they are both perceptive and programmatic, and this ambigu- ity can in no way be eliminated given the way they are applied. Abstract It is certain, however, that a static state does not exist; were this mani- fest it would be the sign of a remoteness from the con- tinuous flow of evolution and innovation. If we were to assume that the enforcement of order were aimed at improving the organisation of the city and territory and ultimately at improving the living condi- tions of those inhabiting and “using” that territory, we would need to consider any challenge to order as a pro- spective worsening of those living conditions. But it does not seem to be like this, not only because disorder arises round those who have settled in the city or territory and use it, with the prospect of improving their condition, but Indovina City Territ Archit (2016) 3:18 Page 4 of 7 The field of spatial order is not one of “rational” choices (depending on epochs) but rather of “political” choices. The city and territory are a field of tensions and conflicts; however much the species is civilised it remains divided by interests, often contrasting, that aspire to “appropriat- ing” space, and spatial organisation that bears witness to their power (or absence of power). This is social “typol- ogy” that is projected onto the space: its conflictive- democratic or rather pacified-authoritarian features (or, more to the point, where the conflicts are repressed) find their configuration in space organisation (Fregolent and Savino 2013). time is a very important feature; density, also understood as the concentration of change phenomena. In recent times, with the establishment of liberalism also at the level of organisation of the territory, so that organised spaces may seem like a conflict between indi- viduals (the other side of the coin is collaboration), it has not been possible to deny the existence of processes of exchange, integration, collaboration and competition, and conflicts between individual interests. At the same time, it must be acknowledged that stakeholders (very powerful groups) exist and that the struggle for space appropriation is not only interpretable as the result of “competition” between individuals but, rather, as a politi- cal clash between more or less codified interested parties, while social conflicts appear as the initiative of the social forces (economically the weakest) to defend the general interests (Belli 2016). Abstract The city (and the territory) are no more than an exer- cise field for various options maintained by different forces, often with alternative objectives; it is indeed due to the presence of opposed forces that the city, in gen- eral, and up to now, has never completely responded to the interests of one party. Just as the Siamese twins seek but do not find a position that is comfortable for both, the city moves continuously between order and disorder, between ruptures and recomposition. It is possible to return to the dialectic of the Siamese twins: the movement of one of the two children stimu- lates the other to change its place; the intervention of a “third” party (in the case of the city, politics) may arrange the two children in such a way that each achieves a com- fortable situation, even if it is known that it will be of brief duration as very soon they will move. Perception of order and its advantages and of disor- der and its disadvantages is not only not immediately clear, but can only be the outcome of a reflective pro- cess, which cannot but bypass individuals to take on the complete, complex urban society and its values. If it were true, as we have mentioned more than once previously, that (functional, aesthetic, social) order did not, in the past evolution of the human species, constitute a sponta- neous event but the outcome of a project, then we would need to devote some thought to this. If urban dynam- ics, however defined, are on one side a challenge (partial and/or total) for this project, then, on the other side, they actually expect a new project at the same time. Planning is an instrument that in a “coercive” way defines urban order; generally, in its application it has to overcome obstacles of meaning that end up limiting its action, or to contrast the work of power groups that attempt to make it ineffective, or, again, keep account of the behaviour of citizens who evade its rules.h The statement that planning is the suitable instrument to give answers to the problems of the city and territory nowadays does not meet with much support. The prev- alent cultural, ideological and professional tendencies assert the need for a drastic review of it, or even its effec- tive elimination. Abstract The guarantee of the results proves closely linked with the expression of a specific political will (an inten- tion) and the management of transformation processes (another aspect of political will). But the ordered condi- tion is only temporary; disorder tends to emerge from the tidy mesh. As we have already said, disorder, as considered here, can take on different aspects but is nevertheless charac- terised by the “rupture” of an order that corresponds to a society’s systems. It cannot be picked out as an exclu- sively negative element, for it actually represents positive distortions, too, of the systems set up, and can introduce elements of dynamism and innovation and translate social transformations into spatial terms also; in a cer- tain sense it corresponds to the vitality of a specific urban condition. From this point of view it should not only be considered the establishment (against the collectivity) of specific, easily identified individual interests (often spec- ulative on the economic plane, “degrading” on the social plane, and frustrating from a cultural standpoint) but, rather, a “composition” of many things, the expressions of the (precarious) equilibrium that society has achieved in a particular phase. We thus need to think that the phenomena that encourage disorder belong both to the exogenous and the endogenous categories: new technology may require changes in the organisation of the city (namely: new order), but “requests” may be seen to emerge directly from the citizens and the organised social forces that require changes in urban order to improve their specific When the variation (the urban dynamics) is complex, of considerable size and speed, this cannot but cause dis- order (with respect to the previous order). In short, the characteristics of urban transformations that determine disorder are: quantity, i.e. the size of the changes com- pared with the size of the city; speed, i.e. transformation Indovina City Territ Archit (2016) 3:18 Page 5 of 7 (wherever located) who would like to “subject” order to their own interests or points of view, to assert, on the contrary, principles of freedom, coexistence, solidarity and equality, though still leaving space for change (Nel. lo 2012). urban life condition. The political nature of this demand places it on the terrain of the comparison and strength relations. Abstract On one side, it does not seem possible to oppose the request for a review and modernisation of planning systems to take changes in society into account while, on the other, we forget that attention to the changes in society and, therefore, the respective adapta- tion of the instrument of intervention, is a constituent element of planning itself (even though in practice this attention has not always been shown). One might ask what drives a collectivity to a sort of Sisyphean torment, namely to the continuous repetition of an action that is useless: to set up, work for, commit oneself to the achievement of urban order that is not last- ing. One might maintain that this need is the only one compatible with the possibility of survival of the spe- cies, which has on the one hand to oppose the forces of nature that would like to blow it away, but must, on the other, fight the stimulus towards the disintegration of coexistence with a counter-stimulus consisting of rules, of norms and values that are not stable but continuously renewed, though ever present. We are referring to good planning, which we would define as that which pays great attention to the collec- tive interest and an improvement in the quality of life of inhabitants, and is organised using suitable instruments, having tackled the democratic encounter in its approach, taken into account processes underway and qualified and quantified the (negative and positive) effects of its action.ihi To design and achieve urban order is fundamentally an option in favour of coexistence. A project that consti- tutes an attempt, not always perfect, to respond both to demand expressed or implicit, not just of functions but also meaning. As can be observed, even though ideas and specific situations may drive us towards individual- ist exaltation that tends to be unbridled, the species has an imprinting that urges it towards coexistence; contem- porary life, increasingly complex, effectively imposes greater and better regulation (in spite of different appear- ances), entrusting each individual or social group once more with full freedom in belonging to spheres previ- ously regulated and taking on the need to regulate new, different spheres not considered before. if But good planning also finds obstacles ahead. The first is a certain “doubling” that characterises the attitude of the “citizen” and his “common sense” as regards the city. Abstract This “common sense” is loaded with positive expecta- tions and requests: the territory is to be safeguarded, land consumption reduced, the landscape protected, hydro- geological reclamation to be carried out, the city must function, suburban decay must be eliminated, limits and rules for growth need to be introduced, and the environ- ment must be healthy, etc. Any plan seeking to realise these expectations and meet the requests expressed, would assert the “val- ues” contained in them and should be welcomed with great approval and enthusiasm. But things are not like this. For every public decision on urban organisation f We have thus reached a crucial knot: urban order design. Design that will resist pressure from the “powers” Indovina City Territ Archit (2016) 3:18 Page 6 of 7 Page 6 of 7 predefined objectives (an adjustment able to take into account environmental dynamics). and reorganisation, for all regulatory action, each tie or limit imposed, opposition and differentiation with vary- ing degrees of importance arise. These refusals do not have their origin in the defence of private and individual interests, which would be understandable, but are justi- fied by collective action to protect the general interest. Considerable distrust has matured over time in any pub- lic decision or choice that may also generate “apathy” or indifference; the notable errors, programmatic inconsist- ency, not to mention the spread of corruption of public workers, explain this attitude, but it also originates in the prevalence of a political culture that denies public action true consistency and utility. It should also not be forgotten that at the moment in which the great options in principles turn into specific decisions, private interests tend to emerge. Subjects do not contest the great options, they would happily see them achieved, but possibly with- out harming their own interests. Political decision-mak- ing, and therefore planning, finds itself in the situation of being appealed to, but at the same time rejected. It may be stated that planning finds it enjoys generic consensus, but also equally strong opposition. It is my personal experience that each action that is accomplished, even if well targeted, may determine unforeseen outcomes, some of which can assume a nega- tive value, “perverse effects”, not only negative in them- selves but also contrary to all expectations and forecasts. Abstract Unforeseen effects, also when perverse, suggest a careful analysis of the mechanisms activated and the responses of the interested parties involved, and not the impossibil- ity of public action. Unforeseen effects, moreover, can in some cases determine innovations.l It has been maintained that the conflict represents a positive element in that it expresses at the same time uneasiness and a request, provided actions exist and are put in place to solve such conflicts.l l However, in the present historic phase the conflict appears also to show a selfish effect: Not In My Back Yard (or NIMBY). A work (an oil pipeline, a landfill, wind power station, street, refugee camp, etc.) deemed in some way necessary is not wanted on one’s own territory, but preferred on “another” territory. What seems to prevail in these attitudes, very often justified, but not always, by the effects these structures could cause in the local situation, is a sort of local egoism, the child of a prevailing identity attitude that is extremely mean and utterly short-sighted. What has previously been observed as regards urban dynamics, the speed of change, and the increased com- plexity of the city and territory, challenges planning in itself, according to some (interest groups and scholars), or at least the current form of planning; what is criticised is the compulsory nature of the choice of plan.h Obstacles to improving and renewing means and pro- cesses of territorial planning are not thought to exist, provided improvements and renovation are able to increase the efficiency and public effectiveness on the management of urban and territorial transformations. It is not a case of stating an ideological position, but of the awareness that the organisation of the city and territory cannot but have a public/collective guide, able to improve the inhabitants’ living conditions.i The idea that society’s dynamism is much more rapid and does not easily endure the ties of the plan does not justify denying the need for the plan; greater and better planning is actually required. Abstract The request put forward by various parties to respond to the dynamism of society is to develop a flexible plan; this request only apparently seems to side with the need for planning; it is actually the complete denial of any form of planning.lf Disorder, however it is identified, constitutes a perma- nent fact, inherent in the urban condition; it is neither the result of wrong planning (sometimes also this), nor of a perverse will, but rather of the dynamic mechanisms of the city itself. Change brings disorder but public commit- ment through institutions cannot but aspire to recover a level of order, hopefully, more advanced. The urban, pre- cisely because of its constituent construction (social, pro- ductive and economic variability; clash between powers and options of models of society) cannot be stable, but the continuous recovery of “order” responds not only to func- tional needs, but also to ethical options: only the “strong- est” (from all points of view) know how to take advantage of disorder; the weakest usually pay a high price. But at the same time we should not consider all urban “order” as positive, compared with negative disorder; there are expe- riences of oppressive and coercive urban order. A flexible planis effectively an antithesis, for it presup- poses objectives that have not been defined or that may be continuously redefined according to needs; but plan- ning, obviously, is such because it defines objectives and a strategy to achieve them. If we were to consider the plan process as formed of defined objectives and “poli- cies” (specific public actions) suitable to enable these objectives to be achieved, in a highly dynamic situation with unforeseen changes, continuous reconsideration or updating of these policies—namely the means to achieve the objectives—would be necessary and useful. These are policies that could be considered flexible and modified, according to needs, to achieve defined and unchanged objectives. What is suggested, and would seem adequate to respond to many specific situations is, therefore, not a flexible plan but, rather, flexible instruments to achieve Indovina City Territ Archit (2016) 3:18 Page 7 of 7 Indovina City Territ Archit (2016) 3:18 Indovina City Territ Archit (2016) 3:18 It is disorder that breaks up all oppressive order, and it is always disorder that determines better levels and quality of order. References Belli G (2016) A colloquio con l’urbanistica Italiana. Interviste a B. Secchi, F. Indovina, L. Mazza e PL. Crosta. Clean, Napoli Fregolent L, Savino M (eds) (2013) Economia, società, territorio. Riflettendo con Francesco Indovina. FrancoAngeli, Milano Indovina F (2016) Ordine e disordine (in press) Nel.lo O (2012) Francesco Indovina Del analisi del territorio al gobierno de la ciudad. Icaria Editorial, Barcellona Abstract Though it may be the dynamic factor of every urban condition, we must remember that it always requires new order from which to start out again. Received: 27 September 2016 Accepted: 12 October 2016 Competing interests Competing interests The author declares that he has no competing interests. p g The author declares that he has no competing interests. Received: 27 September 2016 Accepted: 12 October 2016 Received: 27 September 2016 Accepted: 12 October 2016
https://openalex.org/W4238630295	https://journals.unisba.ac.id/index.php/JRA/article/download/54/30	Indonesian	null	Pengaruh Akuntansi Dana Desa dan Partisipasi Masyarakat terhadap Pengelolaan Dana Desa	Jurnal Riset Akuntansi	2,021	cc-by	2,678	ERROR: type should be string, got "https://doi.org/10.29313/jra.v1i1.54 https://doi.org/10.29313/jra.v1i1.54 Journal Riset Akuntansi Yustin Novia Pratiwi* \nProdi Akuntansi, Fakultas Ilmu Ekonomi dan Bisnis, Universitas Islam \nBandung, Indonesia. yustinnoviapratiwi@gmail.com Corresponding Author \nEmail: yustinnoviapratiwi@gmail.com Pengaruh Akuntansi Dana Desa dan Partisipasi Masyarakat terhadap \nPengelolaan Dana Desa Yustin Novia Pratiwi \nProdi Akuntansi, Fakultas Ilmu Ekonomi dan Bisnis, Universitas Islam \nBandung, Indonesia. Yustin Novia Pratiwi* Yustin Novia Pratiwi* \nProdi Akuntansi, Fakultas Ilmu Ekonomi dan Bisnis, Universitas Islam \nBandung, Indonesia. *yustinnoviapratiwi@gmail.com Abstract. Today village funds are one of the topics being discussed hotly, in their implementation of the many \ndeviations of priority development funds carried out because they increase understanding of village funds and \nincrease community participation in village fund management. This research was made with the aim to study \nthe effect of village funds and community participation on village fund management. The test tool in this study \nis a simple and multiple linear regression analysis using SPSS software. This research uses quantitative \nmethods using surveys. In this study, the technique used in collecting data consisted of 62 questionnaires \ndistributed by 62 people to villages and village communities in the area of West Bandung Regency. The results \nshow that the village financial statements have gone well against the system, community participation has gone \nwell and involves accounting of village funds and community participation in the management of village funds. Keywords: Accounting, Contribution , Management , Village Fund Keywords: Accounting, Contribution , Management , Village Fund Abstrak. Dewasa ini dana desa adalah salah satu topik yang sedang hangat dibicarakan, pada pelaksanaanya \nseringkali banyak penyimpangan dana maupun prioritas pelaksanaan pembangunan yang terjadi karena \nkurangnya pemahaman atas akuntansi dana desa dan kurangnya partisipasi masyarkat dalam pengelolaan dana \ndesa. Penelitian ini dibuat dengan tujuan untuk mengetahui pengaruh akuntansi dana desa dan partisipasi \nmasyarakat terhadap pengelolaan dana desa . Alat uji dalam penelitian ini ialah analisis regresi linier sederhana \ndan berganda dengan menggunakan software SPSS. Penelitian ini menggunakan metode kuantitatif dengan \npendekatan survey . Dalam penelitian ini, teknik yang digunakan dalam pengumpulan data berupa kuisioner \nyang disebarkan sebanyak 62 kepada para aparatur desa dan masyarakat desa di wilayah Kabupaten Bandung \nBarat . Hasil penelitian menunjukan bahwa pada umumnya akuntansi dana desa sudah berjalan dengan baik \ndisertai dengan sistemnya, partisipasi masyarakat sudah berjalan dengan baik dan terdapat pengaruh akuntansi \ndana desa dan partisipasi masyarakat terhadap pengelolaan dana desa. Kata Kunci: Akuntansi, Partisipasi ,Pengelolaan, Dana Desa. Kata Kunci: Akuntansi, Partisipasi ,Pengelolaan, Dana Desa. Corresponding Author \nEmail: yustinnoviapratiwi@gmail.com Corresponding Author \nEmail: yustinnoviapratiwi@gmail.com 24 24 25 Pengaruh Akuntansi Dana Desa dan Partisipasi Masyarakat ...\| 25 A. \nPendahuluan Indonesia pernah berada pada sistem perekonomian sentralistik yaitu pada zaman rezim \nSoeharto (1967-1998). Kemudian seiring perkembangan waktu indonesia beralih pada sistem \ndesentralisasi. Desentralisasi sendiri menurut Undang-Undang No 32 Tahun 2004 adalah \npenyerahan wewenang dari pmerintah pusat kepada daerah otonom untuk mengatur dan \nmengurus urusan pemerintahan dalam sistem Negara Kesatuan Republik Indonesia. Seiring \nberjalannya waktu perekonomian di indonesia semakin gencar melakukan langkah-langkah \nperubahan dalam rangka mencapai tingkat kesejahteraan terutama dalam program kabinet kerja \nPresiden Joko Widodo yang berkaitan dengan representasi unit terkecil tonggak pembangunan \nInsfrastuktur Indonesia. Salah satu langkahnya adalah dengan dibentuknya Undang-Undang \nRepublik Indonesia Nomor 6 Tahun 2014 Dan Peraturan Mentri Dalam Negri Republik \nIndonesia Nomor 113 pasal 1 ayat 6 Tahun 2014 yang berkaitan dengan pengelolaan dana desa \nmenyatakan bahwa Pengelolaan Keuangan Desa adalah seluruh kegiatan yang meliputi \nperencanaan, pelaksanaan, penatausahaan, pelaporan, dan pertanggungjawaban keuangan desa. Peraturan tersebut menjadi angin segar bagi desa-desa di Indonesia sekaligus memberikan bukti \nketegasan dan keseriusan pemerintah dalam membangun ekonomi Indonesia serta sebagai \nsarana perlindungan secara yuridis dan pemberdayaan desa agar menjadi lebih mandiri, \nsejahtera dan demokratis . Dana desa di kucurkan dengan jumlah yang fantantis disetiap \ntriwulannya dengan tujuan untuk memeratakan kesejahteraan dan kualitas sumber daya manusia \ndi Indonesia. Pengelolaan dana desa diharapkan dapat berjalan secara optimal namun, tak jarang \ndana desa dikelola dengan sistem tidak baik. Dana desa nampaknya menjadi kesempatan atau \nlahan bagi para oknum desa untuk melakukan penyimpangan salah satunya dilansir dari \nKompas.com. Kabul santoso terkena operasi tangkap tangan KPK yang bahkan merugikan \nnegara sebesar 137.9 juta rupiah. Kemudian , dilansir dari Liputan6.com mentri keuangan Sri \nmulyani menyebutkan karena adanya transfer tetap dari anggaran pendapatan dan belanja \nnegara. Sekarang banyak muncul desa-desa fiktif yang bahkan tidak ada penduduk didalamnya. Berdasarkan latar belakang tersebut maka peneliti tertarik untuk meneliti lebih lanjut prihal dana \ndesa dengan rumusan masalah sebagai berikut: (1) Bagaimana pengaruh akuntansi dana desa \nterhadap pengelolaan dana desa? (2) Bagaimana pengaruh partisipasi masyarakat terhadap \npengelolaan dana desa? Rumusan masalah tersebut , ditujukan untuk mengetahui (1) tujuan akuntansi dana desa \nterhadap pengelolaan dana desa (2)tujuan partisipasi masyarakat terhadap pengelelolaan dana \ndesa. JRA is licensed under Creative Commons Attribution- \nNonCommercial-ShareAlike 4.0 International License B. \nLandasan Teori Teori Keagenan Menurut Jansen dan Meckling (1976) dalam Syarifudin (2014:30) dapat di \ndefinisikan sebagai suatu hubungan yang terdapat pada sebuah kontrak dimana satu orang atau \nlebih (Principal) memerintah orang lain (Agent) untuk membuat keputusan yang terbaik bagi \nPrincipal. Dalam hal ini aparatur desa menjadi agen danmendapatkan mandat untuk mengelola \ndana desa bagi masyarakat yang berperan sebagai pricipal atau sasaran adanya dana desa. Dalam \nmengelola dana desa tentunya sangat dibutuhkan laporan keuangan sebagai bentuk tanggung \njawab aparatur desa kepada masyarakat dan pemerintah pusat. Laporan keuangan dibentuk dari \nproses akuntansi atau pencatan dari setiap kegiatan selama dana desa. Akuntansi dana desa \nmenurut Sujarweni (2015:17) adalah pencatatan dari proses transaksi yang terjadi di desa, \ndibuktikan dengan nota-nota kemudian dilakukan pencatatan dan pelaporan keuangan sehingga \nakan menghasilkan informasi dalam bentuk laporan keuangan yang digunkan pihak-pihak yang \nberhubungan dengan desa. Dalam hal ini pengelolaan bukan hanya dilakukan oleh aparatur desa \nsaja tetapi seharusnya dilakukan oleh masyarakat juga. Partisipasi masyarakat sangat \ndibutuhkan untuk memastikan bahwa dana desa dilaksanakan sesuai dengan prioritas atau \nkebutuhan desa dan masyarakatnya serta untuk mengawasi jalannya dana desa . karakteristik \nlaporan keuangan menurut Ratmono dan Solihin (2015:15) sebagai berikut: Riset Akuntansi Riset Akuntansi 26 \| Yustin Novia Pratiwi. 1. Relevan 1. Relevan 2. Andal 3. Dapat Dibandingkan 4. Dapat Dipahami 4. Dapat Dipahami Partisipasi masyarakat diartikan oleh Adi (2007:27) sebagai keikutsertaan masyarakat \ndalam proses mengidentifikasi masalah dan potensi yang ada di masyarakat, pemilihan dan \npengambilan keputusan tentang alternatif solusi untuk menangani masalah, pelaksanaan upaya \nmengatasi masalah, dan keterlibatan masyarakat dalam proses mengevaluasi perubahan yang \nterjadi”. prinsip partisipasi sendiri menurut Bastian (2015:119) adalah: 1. Ketepatan waktu 2. Akuntabilitas 3. Inklusif 4. Metodologi yang Tanggap dan Sesuai 5. Keberlanjutan Proses Partisipasi 6. Sumber Daya yang Memadai Dengan demikian , Teori agensi memberikan gambaran bahwa seharunya aparatur desa \n(agent) dan masyarakat sebagai (principal) saling bekerja sama dalam pengelolaan dana desa. Aparatur desa memberikan yang terbaik bagi masyarakat lewat pemahamannya terhadap \nakuntansi dana desa dan masyarakat membantu mewujudkan pengoptimalaan dana desa lewat \npartisipasinya. Agar dana desa dapat dijalan kan sesuai dengan prosedur seharusnya. C. \nHasil Penelitian dan Pembahasan Berikut ini pengaruh antara akuntansi dana desa terhadap pengelolaan dana desa yang diuji \nmenggunakan analisis regresi sederhana Tabel 1. Uji pengaruh antara akuntansi dana desa terhadap pengelolaan dana desa Tabel 1. Uji pengaruh antara akuntansi dana desa terhadap pengelolaan dana desa Variabel \nKoefesien \nregresi \nT \nhitung \nSig \nKonstanta 17.318 \n4.526 \n0.000 \nADD-\nPDD \n0.563 \n4.472 \n0.000 \nR \n0.563 \n \nr2 \n0.317 \n \nSumber : data yang diolah 2020 Hasil dari pengolahan data diatas dapat diketahui besarnya koefisien korelasi adalah \n0.563. dan Nilai r2. atau koefisien determinasi Sebesar 0.317 berarti sebesar pengelolaan dana \ndesa dipengaruhi sebesar 31,7% oleh akuntansi dana desa dengan signifikansi sebesar 0.000. Maka dapat disimpulkan bahwa terdapat pengaruh akuntansi dana desa terhadap pengelolaan \ndana desa. Hasil tersebut juga didapatkan dari beberapa faktor yang mempengaruhi salah \nsatunya Laporan keuangan desa yang lahir dari pemahaman aparatur desa terkait Akuntansi dana \ndesa. Pemahaman aparatur desa dapat menghasilkan laporan keuangan yang bukan hanya sesuai \nstandar saja tapi informasi yang dihasilkan nya juga akan mudah dipahami. Dalam hal ini \naparatur desa dituntut untuk patuh dan paham . karena sebagai agent aparatur desa juga \nberkewajibkan menghasilkan output atau keputusan yang terbaik bagi principal (masyarakat). Jalannya akuntansi dana desa juga dipengaruhi oleh tingkat pengawasan . Semakin baiknnya \npengawasan jalannya proses akuntansi dana desa , maka akan baik juga pengelolaan dana desa \nyg dihasilkan. Pengawasan dana desa dilakukan oleh pihak internal(Badan Permusyawaratan \nDesa) dan eksternal (Inspektorat Kabupaten dan Badann Pengawas Keuangan). Berdasarkan \nuraian diatas maka hipotesis dari penelitian ini sejalan atau diterima sesuai dengan penelitian ISSN: 2798-6438 Volume 1, No. 1, Tahun 2021, Hal: 24-29 27 Pengaruh Akuntansi Dana Desa dan Partisipasi Masyarakat ...\| 2 yang dilakukan oleh Sri fadilah dkk (2019:1) yang berjudul pengaruh penerapan sistem \npengendalian intern pemerintahan (SPIP) dan kompetensi sumber daya manusia terhadap \nlaporan keuangan pemerintah daerah (survey pada pemerintah daerah bandung raya) \nbahwa laporan keuangan pemerintah di bandung raya pada umunya sudah berkualitas . hal tersebut berarti bahwa pemahaman akuntansi dana desa juga sudah baik . JRA is licensed under Creative Commons Attribution- \nNonCommercial-ShareAlike 4.0 International License C. \nHasil Penelitian dan Pembahasan Selain itu, \npada saat wawancara salah satu aparatur desa Dadi mengungkapkan bahwa: ”Pelaporan dana desa diusahkan selalu tepat waktu, namun terkadang informasi prihal \npembangunan sulit didapat, ada kendala seperti hilang bon dan macam-macam karena kan ketka \npembangunan masyarakat yang membuat tim untuk itu” Hal tersebut bermakna bahwa bukan hanya aparatur desa saja yang sangat \nmembutuhkan pelatihan dana desa tetapi juga masyarakat karena masyarakat juga sudah mulai \nterlibat dalam pelaksanaan dana desa dan menentukan seberapa besar ketepatan waktu dalam \npenyampaian laporan keuangan desa. p\ny\np\np\ng\nBerikut ini pengaruh antara partisipasi masyarakat terhadap pengelolaan dana desa: Tabel 2. Uji pengaruh antara partisipasi masyarakat terhadap pengelolaan dana desa Tabel 2. Uji pengaruh antara partisipasi masyarakat terhadap pengelolaan dana desa Variabel \nKoefesien \nregresi \nT \nhitung \nSig \nKonstanta 4.768 \n0.940 \n0.000 \nPM-PDD \n0.665 \n5.833 \n0.000 \nR \n0.665 \n \nr2 \n0.442 \n \nSumber data : data yang telah diolah Hasil dari pengolahan data partisipasi masyarakat ialah partisipasi masyarakat \nberpengaruh positif terhadap pengelolaan dana desa kemudian r2 yang dihasilkan adalah 0.442 \nberarti sebesar 44,2 % perubahan yang terjadi pada pengelolaan dana desa disebabkan oleh \npartisipasi masyarakat dengan segnifikansi sebesar 0.000. para aparatur desa bersama-sama \ndengan masyarakat memutuskan apa yang harus diprioritaskan dalam musyawarah dusun , \nmusyawarah desa hingga musyawarah perencanaan pembangunan desa (musrenbang des). Masyarakat dilibatkan bukan hanya pada saat rapat saja , tapi pada pelaksanaanya masyarakat \ndilibatkan dengan cara membuat tim untuk proyek pembangunan desa . Antusiasme masyarakat \nterhadap pengelolaan dana desa bahkan semakin meningkat dari tahun ketahun . Masyarakat \nbukan hanya ikut berperan didalamnya tapi mereka juga ikut mencari tahu bagaimana distribusi \ndana desa, apakah sudah turun atau belum. Berdasarkan uraian diatas maka hipotesis dari \npenelitian ini sejalan atau diterima sesuai dengan penelitian yang dilakukan oleh Julianto \n(2019:35) yang menyatakan bahwa partisipasi berpengaruh secara positif dan signifikan \nterhadap keberhasilan pengelolaan dana desa dengan nilai signifikansi sebesar 0.048 . Berikut ini pengaruh akuntansi dana desa dan partisipasi masyarakat terhadap \npengelolaan dana desa : Riset Akuntansi Riset Akuntansi 28 \| Yustin Novia Pratiwi. Tabel 3. E. \nSaran Berdasarkan hasil kesimpulan diatas , maka dapat dierikan saran mengenai Pengaruh akuntansi \ndana desa dan partisipasi masyarakat terhadap pengelolaan dana desa di Kabupaten Bandung \nBarat yaitu: Saran Teoritis Sebaiknya untuk penelitian selanjutnya dianjurkan untuk menambahkan jumlah kabupaten yang \ndiambil untuk memperdalam lagi penelitian. Saran Praktis \nSebaiknya aparatur desa lebih terbuka pada masyarakat dengan mengadakan sosialisasi lebih C. \nHasil Penelitian dan Pembahasan Uji pengaruh akuntansi dana desa dan partisipasi masyarakat terhadap pengelolaan\ndana desa \nCoefficientsa \nModel \nUnstandardized \nCoefficients \nStandardized \nCoefficients \n \n \nB \nBeta \nT \nSig \n1 \n(Constant) \n1.139 \n \n0.235 \n0.815 \nTOTAL_ADD \n0.333 \n0.339 \n2.908 \n0.006 \nTOTAL_PM \n0.617 \n0.518 \n4.437 \n0.000 \n \nR \n0.732 \n \n \n \n \n \nR2 \n0.535 Berdasarkan r hitung pada variabel X1 sebesar 0.333 maka setiap penambahan nilai 1 \npada variabel X1 akan meningkatkan variabel Y sebesar 0.333 (33.3 %) kemudian sisanya \ndijelaskan atau dipengaruhi oleh variabel lain sebesar 66.7 %. Sedangkan r hitung pada variabel \nX2 sebesar 0.617 yang berarti setiap penambahan 1 poin pada variabel X2 akan meningkatkan \nvariabel Y sebesar 0.617 (61.7 %) dan sisanya dipengaruhi oleh variabel lain sebesar 38.3 %. Sejalan dengan itu, ultafiah (2017:145) yang menyatakan bahwa akuntabilitas dan \npartisipasi masyarakat berpengaruh secara positif dan signifikan terhadap pengelolaan \ndana desa dengan Sig. sebesar 0.000. Maka dapat disimpulkan bahwa pengaruh akuntansi dana desa dan partisipasi \nmasyarakat terhadap pengelolaan dana desa di sebabkan oleh berbagai faktor selain dari laporan \nkeuangan nya pengelolaan dana desa juga dipengaruhi oleh partisipasi masyarakat baik lewat \naspirasi fikirannya maupun sumbangan tenaga dalam pelaksanaan programnya . hal tersebut \nsejalan dengan teori agensi yang menyatakan bahwa aparatur desa sebagai agen penerima \nmandat dari pihak masyarakat mempunyai tugas untuk melaksanakan program dana desa secara \noptimal dan mengkomunikasikan dengan masyarakat lewat melibatkannya dengan rapat yang \ndi adakan oleh dasa. D. \nKesimpulan Berdasarkan hasil penelitian yang telah disampaikan pada bab sebelumnya ,yaitu tentan\nPengaruh Akuntansi Dana Desa dan Partisipasi Masyarakat terhadap pengelolaan dana desa d\nK b\nB\nd\nB\nk d\ndi\nik k\ni\nl\nb\ni b ik Berdasarkan hasil penelitian yang telah disampaikan pada bab sebelumnya ,yaitu tentang \nPengaruh Akuntansi Dana Desa dan Partisipasi Masyarakat terhadap pengelolaan dana desa di \nKabupaten Bandung Barat maka dapat ditarik kesimpulan sebagai berikut: Pengaruh Akuntansi Dana Desa dan Partisipasi Masyarakat terhadap pengelolaan dana desa di \nKabupaten Bandung Barat maka dapat ditarik kesimpulan sebagai berikut: 1. Terdapat pengaruh Akuntansi dana desa terhadap pengelolaan dana desa. Dengan adanya \nlaporan keuangan maka para aparatur desa harus memahami peristiwa akuntansi yang \nterjadi didalamnya sebagai bentuk tanggung jawab dan pembuktian bahwa aparatur desa \nakan memberikan yang terbaik bagi pihak prinsipal (masyarakat). 2. Terdapat pengaruh partisipasi masyarakat terhadap pengelolaan dana desa \nDengan adanya musyawarah dusun dan desa maka terjadi komunikasi yang baik dan \nprogram-program yang dihasilkan sesuai dengan prioritas desa. Saran Praktis Saran Praktis \nSebaiknya aparatur desa lebih terbuka pada masyarakat dengan mengadakan sosialisasi lebih Volume 1, No. 1, Tahun 2021, Hal: 24-29 ISSN: 2798-6438 29 Pengaruh Akuntansi Dana Desa dan Partisipasi Masyarakat ...\| 2 lanjut terkait dana desa. Kemudian sebaiknya informasi desa juga dapat diakses oleh pihak \neksternal lainnya . Aparatur desa juga sebaiknya mengadakan kerjasama dengan instansi yang \nterkait agar mendapatkan binaan teknologi maupun yang terkait dengan pencatatan laporan \nkeuangan hingga pelaporan pengelolaan dana desa . Agar mendapatkan pembinaan secara \nmenyeluruh. Pemberdayaan sumber daya manusia juga sebaiknya bekerja sama dengan instansi \nyang lain agar tidak selalu bergantung pada dana desa JRA is licensed under Creative Commons Attribution- \nNonCommercial-ShareAlike 4.0 International License Daftar Pustaka [1] Bastian, indra. 2015, Akuntansi untuk Kecamatan dan Desa. Jakarta: Erlangga [2] Headline : misteri desa siluman salah data \natau modu sedot dana desa, Tersedia di:\n https://www.liputan6.com/news/re \nd/4108390/headline-misteri-desa\n siluman-salah-data-atau-modus sedot-dana-desa [2] Headline : misteri desa siluman salah data \natau modu sedot dana desa, Tersedia di:\n https://www.liputan6.com/news/re \nd/4108390/headline-misteri-desa\n siluman-salah-data-atau-modus sedot-dana-desa [3] Jensen and Meckling. 1976. Theory of the \nFirm: Managerial Behavior, \nAgency Cos\nand Ownership Structure. Journal of Financial Economics . Vol. 3 Iss 4 pp 308 [4] Julianto, dewi, 2019 . Pengaruh partisipasi \nmasyarakat , penggunaan sistem keuangan \ndaerah , kompetensi \npendamping desa terhadap \nkeberhasilan pengelolaan dana\n desa . Bali : Jurnal ilmiah akuntansi \nVol 4 No 1 [5] Korupsi Dana Desa Rp 137,9 Juta, \nSeorang Kades Ditahan, tersedia \nhttps://regional.kompas.com/read/017/05/23/11545871/korupsi.dana \ndesa.rp.137.9.juta.seorang.kades.diahan [5] Korupsi Dana Desa Rp 137,9 Juta, \nSeorang Kades Ditahan, tersedia \nhttps://regional.kompas.com/read/017/05/23/11545871/korupsi.dana \ndesa.rp.137.9.juta.seorang.kades.diahan di: [6]Peraturan Menteri Dalam Negri No.113 pasal 1 ayat 6 Tahun 2014 tentan\nPengelolaan Keuangan Desa [6]Peraturan Menteri Dalam Negri No.113 pasal 1 ayat 6 Tahun 2014 tentang \nPengelolaan Keuangan Desa [7] Putri, Rahma., Fadilah, Sri dan Nurhayati. 2016. Pengaruh Penerapan Sistem Pengendalian \nIntern Pemerintah (SPIP) dan Kompetensi Sumber Daya Manusia terhadap Kualitas \nPelaporan Keuangan Daerah (Survey pada Pemerintah Daerah di Wilayah Bandung \nRaya). Prosiding Akuntansi. Vol. 2., No. 2 [8] Ratmono, Dwi & Solihin , Mahfud.. 2015. Akuntansi Keuangan Daerah Berbasis Akrual \n.Yogyakarta. UPP STIM YKPN. [8] Ratmono, Dwi & Solihin , Mahfud.. 2015. Akuntansi Keuangan Daerah Berbasis Akrual \n.Yogyakarta. UPP STIM YKPN. [9] Ultafiah weny, 2017. Pengaruh akuntabilitas, transparansi, partisipasi terhadap\n pengelolaan dana desa untuk mewujudkan Good corporate \ngovernance. Palembang: \nUMP [10] Undang-Undang No 32 Tahun 2004 tentang Desentralisasi [10] Undang-Undang No 32 Tahun 2004 tentang Desentralisasi [11] Undang-Undang No.6 Tahun 2014 tentang Desa. [12] Undang-Undang No.32 Tahun 2004 tentang Pemerintahan Daerah. [13] V Wiratna, Sujarweni 2015. Akuntansi Desa: Panduan Tata Kelola Keuangan Desa. Wonosari: Pustaka Baru Pers [14] V Wiratna, Sujarweni . 2014 . Akuntansi Dana Desa : Panduan Tata Kelola Keuangan Desa. Wonosari: Pustaka Baru Pers JRA is licensed under Creative Commons Attribution- \nNonCommercial-ShareAlike 4.0 International License Riset Akuntansi Riset Akuntansi"
https://openalex.org/W3097210587	https://hal.science/hal-02948210/document	English	null	Alleviation of an arctic sea ice bias in a coupled model through modifications of the subgrid scale orographic parameterization	null	2,020	cc-by	12,955	To cite this version: Guillaume Gastineau, François Lott, Juliette Mignot, Frédéric Hourdin. Alleviation of an Arctic Sea Ice Bias in a Coupled Model Through Modifications in the Subgrid-Scale Orographic Parame- terization. Journal of Advances in Modeling Earth Systems, 2020, 12 (9), 10.1029/2020MS002111. hal-02948210 Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-02948210 https://hal.science/hal-02948210v1 Submitted on 19 Apr 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. Distributed under a Creative Commons Attribution 4.0 International License Citation: Gastineau, G., Lott, F., Mignot, J., & Hourdin, F. (2020). Alleviation of an Arctic sea ice bias in a coupled model through modiﬁcations in the subgrid‐scale orographic parameterization. Journal of Advances in Modeling Earth Systems, 12, e2020MS002111. https://doi.org/ 10.1029/2020MS002111 Plain Language Summary Some of the processes responsible for the impacts of orography on the mean ﬂow, such as low‐level ﬂow blocking, or mountain waves, are unresolved in climate models at standard horizontal resolution. Such processes are accounted for using subgrid‐scale orography parameterization in climate models. Adjusting such parameterization is well known to improve the simulation of the mean climate in midlatitudes and to increase the skill of operational forecasts. In this study, the impact on the Arctic climate is studied in a climate model. It is found that adjusting the subgrid‐scale orography parameterization modulates both the atmospheric variability and mean state, with a large impact on the atmospheric momentum, heat, and moisture transport from the midlatitude to the Arctic. In particular, increasing the low‐level ﬂow blocking leads to decreased atmospheric transport to the Arctic. Such impacts are found in both atmosphere‐only and coupled ocean‐atmosphere sensitivity simulations designed to investigate the inﬂuence of the parametrized orography. The coupled climate simulations further illustrate the impact of the subgrid‐scale orography adjustment for the sea ice and oceanic circulation. Increasing low‐level ﬂow blocking is found to increase substantially the winter sea ice growth, while it reduces the Atlantic meridional overturning circulation. Received 21 MAR 2020 Accepted 29 JUL 2020 Accepted article online 7 AUG 2020 ©2020. The Authors. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Key Points: • The adjustment of the parametrized orography in climate models can alleviate the near‐surface winter biases over the Arctic Abstract In climate models, the subgrid‐scale orography (SSO) parameterization imposes a blocked ﬂow drag at low levels that is opposed to the local ﬂow. In IPSL‐CM6A‐LR, an SSO lift force is also applied perpendicular to the local ﬂow to account for the effect of locally blocked air in narrow valleys. Using IPSL‐CM6A‐LR sensitivity experiments, it is found that the tuning of both effects strongly impacts the atmospheric circulation. Increasing the blocking and reducing the lift lead to an equatorward shift of the Northern Hemisphere subtropical jet and a reduction of the midlatitude eddy‐driven jet speed. It also improves the simulated synoptic variability, with a reduced storm‐track intensity and increased blocking frequency over Greenland and Scandinavia. Additionally, it cools the polar lower troposphere in boreal winter. Transformed Eulerian Mean diagnostics also show that the low‐level eddy‐driven subsidence over the polar region is reduced consistent with the simulated cooling. The changes are ampliﬁed in coupled experiments when compared to atmosphere‐only experiments, as the low‐troposphere polar cooling is further ampliﬁed by the temperature and albedo feedbacks resulting from the Arctic sea ice growth. In IPSL‐CM6A‐LR, this corrects the warm winter bias and the lack of sea ice that were present over the Arctic before adjusting the SSO parameters. Our results, therefore, suggest that the adjustment of SSO parameterization alleviates the Arctic sea ice bias in this case. However, the atmospheric changes induced by the parametrized SSO also impact the ocean, with an equatorward shift of the Northern Hemisphere oceanic gyres and a weaker Atlantic meridional overturning circulation. • Increasing low‐level drag reduces Northern Hemisphere stationary wave amplitudes and shifts the subtropical jet equatorward • Increasing low‐level drag increases the Arctic sea ice coverage and reduces the Atlantic meridional overturning circulation • Increasing low‐level drag increases the Arctic sea ice coverage and reduces the Atlantic meridional overturning circulation Supporting Information: • Supporting Information S1 Supporting Information: i f i • Supporting Information S1 Correspondence to: G. Gastineau, guillaume.gastineau@locean-ipsl. upmc.fr Correspondence to: G. Gastineau, guillaume.gastineau@locean-ipsl. upmc.fr Alleviation of an Arctic Sea Ice Bias in a Coup Through Modiﬁcations in the Subgrid‐Scale Orographic Parameterization Special Section: The IPSL Climate Model Used in CMIP6 Special Section: The IPSL Climate Model Used in CMIP6 Guillaume Gastineau1 , François Lott2 , Juliette Mignot1 , and Frederic Hourdin2 1UMR LOCEAN, Sorbonne Université/IRD/MNHN/CNRS, IPSL, Paris, France, 2UMR LMD, ENS/Sorbonne Université/CNRS/Ecole Polytechnique, IPSL, Paris, France Journal of Advances in Modeling Earth Systems 10.1029/2020MS002111 only included mountain wave drags at upper levels that are oriented against the low‐level ﬂow, more recent schemes start to take into account directional effects (Bacmeister, 1993; Baines & Palmer, 1985; Garner, 2005). In such schemes, the gravity wave drag is in an intermediate direction between the low‐level winds and the minor axis of the SSO ridges, depending on the degree of anisotropy. Progress was also made in the late 1990s, with the inclusion of low‐level blocked ﬂow drag. In most schemes, its inten- sity is also a function of anisotropy, but its direction is often assumed to be opposed to the low‐level winds (Lott & Miller, 1997). Although the inclusion of directional effects was never thoroughly tested for the grav- ity waves in the aforementioned studies, it soon appeared that applying low‐level drag alone was not sufﬁ- cient to improve the simulated stationary planetary waves (Lott, 1999). Lott (1999) then proposed to implement additionally the effect of lift forces perpendicular to the local ﬂow. Based on an analogy with the effect of the envelope orography (Wallace et al., 1983), the lift force represents the dynamical separation of the air in narrow valleys from the large‐scale ﬂow. To some extent, it demonstrates that direction matters: The component of the forces perpendicular to the winds does not decelerate the ﬂow directly, but it still dis- torts it efﬁciently when applied regionally. The lift force mimics the vortex stretching effect over large‐scale mountains and yields a realistic planetary wave with little zonal‐mean ﬂow deceleration (Lott, 1999). As model resolution increases, one could have expected that these issues become less critical. Yet it happens not to be the case. The spectrum of unresolved and resolved processes is currently still not well understood, and much care is needed to evaluate the inﬂuence of parameterized orography (van Niekerk et al., 2016). For instance, Zadra (2015) found that the parametrized surface stress is highly model dependent, with impacts at all time scales. Furthermore, how the ocean is impacted by SSO parameterizations remains not well understood. Based on the similarity between Climate Model Intercomparison Project Phase 5 (CMIP5) model midlatitude biases and changes simulated while suppressing SSO effects, Pithan et al. (2016) suggested that much of the CMIP5 climate model biases could be alleviated by increasing the parametrized drag. van Niekerk et al. Journal of Advances in Modeling Earth Systems (2017) also found that the CMIP5 model biases in the position of the North Atlantic and North Paciﬁc jets found can be linked to the parametrized low‐level drag. Another relevant example of the impact of SSO parametrizations concerns the tuning of the GFDL model where different SSO schemes were tested (Zhao et al., 2018). It was noted that increasing orographic drag was associated with a cooling of Arctic surface air temperature. However, the physics and feedbacks related to air‐sea cou- pling behind these corrections need to be analyzed according to Held et al. (2019). Such analysis is indeed essential: since mountain wave drags are often introduced to reduce cold biases (Palmer et al., 1986) through downward control, we have to understand how low‐level parametrized drag can result in opposite effects. Following on from these studies, we investigate here the effect of SSO parameterization in the Arctic region. The intention is also to reduce a warm winter bias in the lower troposphere over the Arctic sea ice. Such bias, previously linked to the poor simulation of the planetary boundary layer (Tjernström & Graversen, 2009) or clouds (Walsh et al., 2009), is indeed present in many models (Graham et al., 2019). The present study addresses these issues with the IPSL‐CM6A‐LR model (Boucher et al., 2020), with a focus on its atmospheric component, LMDZ6A (Hourdin et al., 2020). The IPSL‐CM6A‐LR model was used to perform the Coupled Model Intercomparison Project Phase 6 (CMIP6; Eyring et al., 2016) simu- lations. The study was motivated by a difﬁculty encountered during the tuning of this model conﬁgura- tion, namely, a systematic underestimation of the Arctic sea ice at the end of winter. This deﬁciency was in part attributed to a bad representation of the stationary planetary waves. In our case, this produces an overestimation of warm air advection from low latitudes to the Arctic in winter, thereby inhibiting win- ter sea ice growth. This motivated a tuning of the SSO parameterization, which indeed appeared to play a crucial role in the representation of Arctic sea ice. The simulations presented in this paper reassess this particular tuning step through sensitivity experiments starting from the ﬁnal version of the model, using the atmospheric model component LMDZ6A both in stand‐alone atmospheric mode and coupled to the ocean. 1. Introduction The representation of subgrid‐scale orography (SSO) in global climate models is still considered to be a major challenge (Sandu et al., 2019). Although the large‐scale orography inﬂuence is partly resolved in standard resolution models (~100 km), processes like gravity waves, the blocking effect of small‐scale mountains and hills, and the associated turbulence indeed require the use of very high resolution models (<1 km). Most global climate models use SSO parameterizations to capture the missing effect of orographic gravity waves and low‐level blocking (Lott & Miller, 1997; Palmer et al., 1986). Although early parameterizations ©2020. The Authors. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. 1 of 19 GASTINEAU ET AL. GASTINEAU ET AL. 2.1. Atmosphere‐Only Experiments This study uses the land‐atmosphere components of the IPSL‐CM6A‐LR model used for CMIP6, called LMDZOR6, in stand‐alone mode, forced by sea surface temperature (SST) and sea ice concentration. LMDZOR6 is based on the atmospheric model LMDZ version 6, which is described in a companion paper of the same Special Collection (Hourdin et al., 2020). It has a resolution of 2.5° × 1.25° and 79 vertical levels that extend up to 80 km (~1.5 Pa). It is coupled to the ORCHIDEE (Boucher et al., 2020) land surface model. In LMDZ6, the convective and planetary boundary layer scheme was revisited (Hourdin et al., 2020). A reﬁnement of the vertical grid and a new adjustment of the thresholds of stability functions were implemented for a better representation of the very stable atmo- spheric boundary layer (Vignon et al., 2017). The scheme producing SSO gravity waves drag is also used to produce a shear production term in the prognostic turbulent kinetic equation of the planetary boundary layer scheme. This produces a turbulent orographic form drag, which was carefully validated over the Antarctica ice sheet (see details in the appendix of Cheruy et al., 2020). In LMDZ, the SSO parameterization applies gravity wave drag at upper levels and low‐level drag and lift forces at the model levels that intersect the SSO. The low‐level drag force represents the blocking effect of orography. It is opposed to the local wind (Lott & Miller, 1997). The lift represents the effect of blocked air in narrow valleys intensifying the vortex compression (Lott, 1999). Among others, the low‐level drag and lift effects depend on Cd and Cl, respectively, which are two dimensionless scaling parameters that need to be carefully adjusted. Cd directly controls the blocked ﬂow component of the drag, while Cl controls the amplitude of the lift force. 1 30 0.2 0.1 1 30 0.6 0.0 1 30 0.6 1.0 1 200 0.6 0.1 5 80 0.2 0.25 We integrate several LMDZOR6 simulations (see Table 1) using a repeated annual cycle of SST and sea ice concentration as boundary conditions, calculated from a climatology of the 1979–2008 forcings designed for the CMIP6 Atmospheric Model Intercomparison Project (AMIP; Durack & Taylor, 2018). The simulations are performed over 30 yr, with ﬁxed present‐day external forcings, using the CMIP6 (Eyring et al., 2016) plant functional type maps, greenhouse gases, ozone, aerosols, and solar forcing of the year 2000. 2.1. Atmosphere‐Only Experiments The control simulation, referred to as Atm‐6A, uses the standard value for Cd and Cl from the IPSL‐CM6A‐LR CMIP6 conﬁguration. We also use the ensemble of 10 AMIP simulations produced for CMIP6 with the same atmo- spheric model (Boucher et al., 2020). These simulations are identical to Atm‐6A, but they used interannual SST, sea ice, and external forcings. We also focus on the 1979–2008 period in this ensemble. We also integrate simulations identical to Atm‐6A, but using increased (decreased) values of Cd in Atm‐6A‐ Drg+ (Atm‐6A‐Drg−) and similarly for Cl in Atm‐6A‐Lft+ (Atm‐6A‐Lft−). The exact values are given in Table 1. In case Cl is reduced to 0, the orographic lift parametrization is deactivated. Lastly, a simulation combining these two changes with an increased Cd and a decreased Cl is referred to as Atm‐5DL. This cor- responds to the setup of the previous version of the atmospheric model, named LMDZ5A (Hourdin et al., 2006) and used for CMIP5. Hereafter, the signiﬁcance level for the difference of any variable between two simulations is given by the p‐value of a Student's t test assuming equal variances. The number of degrees of freedom used in the t tests is n −2, with n the number of years or seasons considered for computing the average value. Journal of Advances in Modeling Earth Systems Another goal of this study is to assess the performance of IPSL‐CM6A‐LR regarding Northern Hemisphere climate characteristics, as the CMIP6 simulations produced by IPSL‐CM6A‐LR will be used next in many studies. We will explore the sensitivity of this model to the SSO drag and lift effect, and we will illustrate why and how the Arctic and midlatitude climate is modiﬁed by adjusting both effects. 2 of 19 2 of 19 GASTINEAU ET AL. GASTINEAU ET AL. Journal of Advances in Modeling Earth Systems GASTINEAU ET AL. Journal of Advances in Modeling Earth Systems 10.1029/2020MS002111 Table 1 Presentation of the Main Simulation Discussed in This Study Name Model Members Length (in yr) Parameters Cd Cl Atm‐6A LMDZOR‐6A 1 30 0.6 0.1 Atm‐5DL LMDZOR‐6A 1 30 0.2 0.25 Atm‐6A‐Drg+ LMDZOR‐6A 1 30 1.2 0.1 Atm‐6A‐Drg− LMDZOR‐6A 1 30 0.2 0.1 Atm‐6A‐Lft− LMDZOR‐6A 1 30 0.6 0.0 Atm‐6A‐Lft+ LMDZOR‐6A 1 30 0.6 1.0 AO‐6A IPSL‐CM6A‐LR 1 200 0.6 0.1 AO‐5DL IPSL‐CM6A‐LR 5 80 0.2 0.25 This manuscript is organized as follows: The model and the methodology are presented in section 2. Sensitivity atmosphere‐only experiments are analyzed in section 3, and coupled ones in section 4. Conclusions are given in section 5. 3.1. Mean State The inﬂuence of the SSO parameters on the Arctic climate is ﬁrst assessed in atmosphere‐only experiments. Although the atmospheric component of LMDZOR6 includes a series of physical updates as compared to previous versions (see the previous section), stationary planetary wave errors over Northern America and Northern Atlantic remain when using the SSO parameters of the CMIP5 version. More speciﬁcally, the sta- tionary planetary wave is much more pronounced than in reanalysis, with the three troughs visible in the 700‐hPa geopotential height, located over North America, western Europe, and eastern Asia being deeper than in ERA‐Interim (Figure 1c). This can result in meridional exchanges, for instance, from enhanced (reduced) advection of warm air from the midlatitudes to the polar regions where most of the Arctic sea ice forms in winter. The zonally asymmetric changes (Figure 1d) also show that the stationary wave is shifted west when compared to ERA‐Interim over eastern Asia, North Paciﬁc, and North America. The overestimated stationary wave amplitude might be corrected by imposing more orographic drag and, therefore, decelerating the ﬂow (Sandu et al., 2016). For instance, such an effect of increasing low‐level drag was found by van Niekerk et al. (2017), although opposed changes were found north of 60°N. Furthermore, as the lift force leads to more vortex stretching over large‐scale mountains, reducing the lift effect may also reduce the planetary wave with little impacts on the zonal ﬂow, as discussed in Lott (1999). We, therefore, chose to reduce Cl (from 0.25 to 0.1) and increase Cd (from 0.2 to 0.6) between Atm‐5DL and Atm‐6A (see Table 1). Figures 1e and 1f show that doing so, the errors on both the planetary waves and the zonally sym- metric part of the low‐level jet are reduced. The improvement is quantiﬁed in Figure 1 by the root mean square error (RMSE) in the 20–90°N latitude band, which is reduced for both the 700‐hPa geopotential height (from 48.8 to 36.3 m) and its asymmetric component (from 29.4 to 23.2 m). Journal of Advances in Modeling Earth Systems 10.1029/2020MS002111 simulations. This motivates the use of a 200‐yr period of the preindustrial simulation as a control for our sen- sitivity study, to ensure that this variability does not affect our results. We arbitrarily chose to focus here on the 1990–2189 model years. This simulation is referred to as AO‐6A. Although preindustrial external for- cings are quite different from present‐day ones, the results presented next are likely unchanged in present‐day conditions. simulations. This motivates the use of a 200‐yr period of the preindustrial simulation as a control for our sen- sitivity study, to ensure that this variability does not affect our results. We arbitrarily chose to focus here on the 1990–2189 model years. This simulation is referred to as AO‐6A. Although preindustrial external for- cings are quite different from present‐day ones, the results presented next are likely unchanged in present‐day conditions. Starting from this preindustrial conﬁguration, we integrate a ﬁve‐member ensemble, called AO‐5DL, using the values of Cd and Cl from the previous CMIP5 IPSL model version (increased Cd and decreased Cl, as pre- viously described, see Table 1). The setup is otherwise identical to AO‐6A. The members last 80 yr and start at dates sampled every 40 yr in the given 200‐yr period. The ﬁrst 30‐yr period of each ensemble is discarded. The integration of such ensemble ensures an accurate estimation of the SSO inﬂuence so that the important centennial variability present in IPSL‐CM6A‐LR does not affect too much the results. 2.3. Observations Monthly and/or daily sea ice concentration, sea level pressure (SLP), geopotential height, air temperature, and zonal and meridional wind are retrieved from the ERA‐Interim reanalysis interpolated onto a 2° grid (Dee et al., 2011) over the 1979–2014 period. 2.2. Coupled Experiments We also use the IPSL‐CM6A‐LR (Boucher et al., 2020) atmosphere‐ocean general circulation model (AOGCM), which consists of LMDZOR6 coupled to the NEMO ocean model using a nominal horizontal resolution of about 1° with reﬁnement at the equator and poles (eORCA1 grid), 75 vertical levels, and the LIM3 sea ice module. The Northern Hemisphere climate of the preindustrial CMIP6 control simulation of this model shows a marked centennial variability linked to Atlantic meridional overturning circulation (AMOC) ﬂuctuations (Boucher et al., 2020). This variability is also visible in CMIP6 historical GASTINEAU ET AL. GASTINEAU ET AL. 3 of 19 GASTINEAU ET AL. 3.1. Mean State (e) and (f) are the same as (a) and (b), but for the difference Atm‐6A minus ERA‐Interim. In (c)–(f) panels, the root mean square (RMS) of the difference over 20–90°N is also given on top of each panel; only grid points with statistical signiﬁcance lower than 10% are colored. Lastly, we note that the inﬂuence of varying SST does not change the overall standing planetary wave pat- tern. Indeed, the ensemble mean of AMIP CMIP6 experiments using interannual forcings shows 700‐hPa geopotential height asymmetries largely similar to the simulation Atm‐6A using climatological surface boundary conditions (see Supporting Information Figure S1). Lastly, we note that the inﬂuence of varying SST does not change the overall standing planetary wave pat- tern. Indeed, the ensemble mean of AMIP CMIP6 experiments using interannual forcings shows 700‐hPa geopotential height asymmetries largely similar to the simulation Atm‐6A using climatological surface boundary conditions (see Supporting Information Figure S1). 3.1. Mean State To illustrate how the lift and drag can be combined to modify the planetary wave and the zonal‐mean ﬂow, Figure 2 shows the differences between Atm‐6A and Atm‐5DL (Figures 2a and 2b) as well as the difference between runs where the drag is enhanced by a factor of 6 (Cd increased from 0.2 to 1.2; Figures 2c and 2d) and differences between a run with strong lift and a run with no lift (Cl parameter decreased from 1.0 to 0.0; Figures 2e and 2f). In these sensitivity simulations, we see that the drag alone can well decelerate the global ﬂow (cf. Figures 2a and 2c), with a weakening of the tropospheric polar jet. This effect of the SSO on the zonal‐mean ﬂow is consistent with the effect expected from mountain drags onto the zonal‐mean atmospheric mass distribution (Lott & D'andrea, 2005; Lott et al., 2004) and is consistent with the results of previous studies (Sandu et al., 2016; Zadra et al., 2003). The meridional pressure gradient produced is con- sistent with an anomalous geostrophic westward zonal ﬂow due to the low‐level blocking. The drag also reduces the trough over north‐eastern America and tends to produce a strong ridge to the west of the Alaska peninsula. The lift force is less efﬁcient in producing an axisymmetric response (cf. Figures 2c and 2e) but much more efﬁcient in producing a planetary wave (Figures 2d and 2f). 4 of 19 GASTINEAU ET AL. GASTINEAU ET AL. 10.1029/2020MS002111 f Advances in Modeling Earth Systems 10.1029/20 Journal of Advances in Modeling Earth Systems 10.1029/2020MS002111 Journal of Advances in Modeling Earth Systems Figure 1. (a) Geopotential height at 700 hPa averaged over the winter months (DJFM) in ERA‐Interim (1979–2014) and (b) its zonally asymmetric component. (c) and (d) are the same as (a) and (b), but for the difference Atm‐5DL minus ERA‐Interim. (e) and (f) are the same as (a) and (b), but for the difference Atm‐6A minus ERA‐Interim. In (c)–(f) panels, the root mean square (RMS) of the difference over 20–90°N is also given on top of each panel; only grid points with statistical signiﬁcance lower than 10% are colored. Figure 1. (a) Geopotential height at 700 hPa averaged over the winter months (DJFM) in ERA‐Interim (1979–2014) and (b) its zonally asymmetric component. (c) and (d) are the same as (a) and (b), but for the difference Atm‐5DL minus ERA‐Interim. GASTINEAU ET AL. 3.2. Atmospheric Variability Although changes in the direction and intensity of the climatological westerlies can have a large inﬂuence on the Arctic climate, a large fraction of the low‐troposphere transport of heat and moisture toward the Arctic is also related to the transient eddies. To measure how they are modiﬁed, we next evaluate the winter daily 500‐hPa geopotential height standard deviation, band‐pass ﬁltered at 2.5–6 days (Blackmon, 1976). The GASTINEAU ET AL. GASTINEAU ET AL. Journal of Advances in Modeling Earth Systems 10.1029/2020 ournal of Advances in Modeling Earth Systems 10.1029/2020MS002111 Figure 2. Difference of the simulated DJFM (a) 700‐hPa geopotential height and (b) its zonally asymmetric component, in Atm‐6A minus Atm‐5DL. (c) and (d) are the same as (a) and (b), but for Atm‐6A‐Drg+ minus Atm‐6A‐Drg−. (e) and (f) are the same as (a) and (b), but for Atm‐6A‐Lft−minus Atm‐6A‐Lft+. Only grid points with statistical signiﬁcance lower than 10% are colored. Figure 2. Difference of the simulated DJFM (a) 700‐hPa geopotential height and (b) its zonally asymmetric component, in Atm‐6A minus Atm‐5DL. (c) and (d) are the same as (a) and (b), but for Atm‐6A‐Drg+ minus Atm‐6A‐Drg−. (e) and (f) are the same as (a) and (b), but for Atm‐6A‐Lft−minus Atm‐6A‐Lft+. Only grid points with statistical signiﬁcance lower than 10% are colored. geopotential height standard deviation of the model is quite realistic (Figures 3a–3c), with the Paciﬁc and Atlantic storm tracks located at 50°N over both basins. Nevertheless, Atm‐6A and Atm‐5DL tend to slightly underestimate the variance over both storm tracks, while the variance is overestimated over land (Figures 3e and 3f), especially over northwestern America. In Atm‐6A, the variance is reduced almost every- where around the globe in the polar and midlatitudes compared to Atm‐5DL (Figure 3d). The reduction of the overestimated variance over land explains the overall reduction of the 20–90°N RMSE from 4.72 m in Atm‐5DL to 3.81 m in Atm‐6A. The decreased variance in Atm‐6A is consistent with the weaker polar vortex described in Figure 2a if we assume that a weaker amplitude vortex is more stable. To understand the impact on the midlatitude synoptic variability, we also investigate the blocking character- istics. The blockings are closely linked to the main mode of atmospheric variability (Davini et al., 2012; Woollings et al., 2008) and are usually not well represented in climate models, with underestimated GASTINEAU ET AL. GASTINEAU ET AL. 6 of 19 6 of 19 Journal of Advances in Modeling Earth Systems Figure 3. Daily band‐pass (2.5–6 days) DJFM 500‐hPa geopotential height standard deviation, in m, for (a) ERA‐Interim, (b) Atm‐5DL, (c) Atm‐6A, (d) Atm‐6A minus Atm‐5DL, (e) Atm‐5DL minus ERA‐Interim, and (f) Atm‐6A minus ERA‐Interim. In (e) and (f), the mean root mean square (RMS) 20–90°N difference with ERA‐Interim is given on top right. In (d), the change of the root mean square difference with ERA‐Interim (ΔRMS) is indicated. Journal of Advances in Modeling Earth Systems In (d), the red contours provide the Atm‐5DL daily band‐pass DJFM 500‐hPa geopotential height standard deviation, in m. In (e) and (f), the red contours provide the ERA‐Interim daily band‐pass DJFM 500‐hPa geopotential height standard deviation, in m. Only grid points with statistical signiﬁcance lower than 10% are colored. Figure 3. Daily band‐pass (2.5–6 days) DJFM 500‐hPa geopotential height standard deviation, in m, for (a) ERA‐Interim, (b) Atm‐5DL, (c) Atm‐6A, (d) Atm‐6A minus Atm‐5DL, (e) Atm‐5DL minus ERA‐Interim, and (f) Atm‐6A minus ERA‐Interim. In (e) and (f), the mean root mean square (RMS) 20–90°N difference with ERA‐Interim is given on top right. In (d), the change of the root mean square difference with ERA‐Interim (ΔRMS) is indicated. In (d), the red contours provide the Atm‐5DL daily band‐pass DJFM 500‐hPa geopotential height standard deviation, in m. In (e) and (f), the red contours provide the ERA‐Interim daily band‐pass DJFM 500‐hPa geopotential height standard deviation, in m. Only grid points with statistical signiﬁcance lower than 10% are colored. blocking frequencies over Northern Europe (Davini & Cagnazzo, 2014). Pithan et al. (2016) attributed this underestimation to a lack of SSO drag in most models. A blocking index is deﬁned following Scherrer et al. (2006), using the meridional gradient of daily geopotential height at 500 hPa and considering only blocking events lasting more than ﬁve consecutive days. When comparing with ERA‐Interim, the blocking frequency simulated by Atm‐6A is overestimated over the Urals and far eastern Siberia, while it is underes- timated over the British Isles (see Figure 4). The SSO adjustment in Atm‐6A has however contributed to increasing the frequency of blocking over Greenland and Scandinavia that was largely underestimated in Atm‐5DL. From Atm‐5DL to Atm‐6A, the blocking frequency RMSE is reduced by 0.44% over the North Atlantic section (Figure 4). However, the blocking frequency has been degraded in far eastern Siberia, with an increased RMSE of 0.22%. Journal of Advances in Modeling Earth Systems 10.1029/2020MS002111 Journal of Advances in Modeling Earth Systems Figure 4. DJFM blocking frequency, in %, for (a) ERA‐Interim 1979–2014, (b) Atm‐6A, and (c) Atm‐6A minus Atm‐5DL. The contour interval is 1% for all panels. In (b), the mean root mean square (RMS) difference of ATM‐6A minus ERA‐Interim is given on top in three boxes (global 35–75°N/North Atlantic 100°E–40°W, 35–75°N/North Paciﬁc‐Eurasia 60°W–120°E, 35–75°N). In (c), only grid points with statistical signiﬁcance lower than 10% are colored. The change of the root mean square difference with ERA‐Interim (ΔRMS) for the same boxes as (b) is given on top. Fi 4 DJFM bl ki f i % f ( ) ERA I t i 1979 2014 (b) At 6A d ( ) At 6A i At 5 Figure 4. DJFM blocking frequency, in %, for (a) ERA‐Interim 1979–2014, (b) Atm‐6A, and (c) Atm‐6A minus Atm‐5DL. The contour interval is 1% for all panels. In (b), the mean root mean square (RMS) difference of ATM‐6A minus ERA‐Interim is given on top in three boxes (global 35–75°N/North Atlantic 100°E–40°W, 35–75°N/North Paciﬁc‐Eurasia 60°W–120°E, 35–75°N). In (c), only grid points with statistical signiﬁcance lower than 10% are colored. The change of the root mean square difference with ERA‐Interim (ΔRMS) for the same boxes as (b) is given on top. frictions can easily adapt to enforce quasi‐steady states. In the case of the SSO modiﬁcations tested here, the surface drags are imposed in the lower troposphere, and the “downward control” argument is not easy to adapt. Seminal papers like Eliassen (1951) show that in principle, a drag applied near the surface can cause direct cells above where the drag is applied, which is a northward low‐level ﬂow yielding by mass conservation an upward ﬂow north and hence adiabatic cooling. According to past literature, one nevertheless needs to be extremely careful with such conclusions and test the changes in surface friction and upper‐level forcing by the resolved waves. Although the momentum budget equation can be used to interpret directly the changes of the mean meridio- nal circulation resulting from changes in momentum ﬂuxes, TEM quantities provide a more complete description of the atmospheric changes. To disentangle the feedbacks, Figure 5 presents zonal‐mean diagnos- tics of TEM quantities derived following Andrews et al. (1987). 3.3. Zonal‐Mean Changes Increasing orographic drag to cool the polar regions poses a challenge since, in the past, orographic gravity wave drags were often introduced to warm the upper troposphere and low stratosphere (Palmer et al., 1986). The arguments involve downward control principles (Haynes et al., 1991), where an upper‐level drag is balanced via the Coriolis torque by a poleward Transformed Eulerian Mean (TEM) meridional velocity (called v) that corresponds to the upper branch of an indirect circulation cell. In the poleward branch of the cell and below where the drag is applied, the TEM vertical velocity (called w) is downward (w* < 0) caus- ing adiabatic warming. A key aspect of the downward control argument is that the vertical integration used to predict the meridional circulations starts at z ¼ ∞to use the boundary condition ρw* ¼ 0. Integration from the surface is systematically disregarded (“upward control”) based on the argument that the surface GASTINEAU ET AL. 7 of 19 GASTINEAU ET AL. Journal of Advances in Modeling Earth Systems GASTINEAU ET AL. Journal of Advances in Modeling Earth Systems First in Figure 5a, one sees that the SSO drags in Atm‐6A and difference in SSO drags from Atm‐6A to Atm‐5DL are both negative at low levels in the Northern Hemisphere midlatitudes and polar regions as expected. The tendencies due to SSO lift are much weaker for the zonal ﬂow (not shown). The zonal‐mean zonal wind in Figure 5b presents a subtropical jet with center around (28°N, 12 km) that is in agreement with observations. It is tilted poleward when altitude decreases, and the lower‐troposphere jet maximum (i.e., the eddy driven jet) is around 35–40°N. The impact of the changes in the zonal‐mean winds is consistent with Lott (1999), as the jet decreases above where the drag is applied, reducing the intensity of the eddy‐driven jet. Besides, the zonal wind increases in the subtro- pical regions shifting the subtropical jet equatorward. Importantly, the response to the changes in SSO drag is almost barotropic, consistent with the fact that the low‐level mountain drag is balanced by northward mass ﬂuxes where it is applied, increasing the surface pressure northward and decreasing it southward. This is consistent with the changes in mass distribution due to mountains (Lott & D'andrea, 2005; Lott et al., 2004). The reduction in the baroclinic part of the jet, as indicated by the difference of zonal‐mean zonal wind between 300 hPa and 850 Pa at 35°N, is not signiﬁcant at the 10% level (−0.46 m s−1). 8 of 19 GASTINEAU ET AL. GASTINEAU ET AL. Journal of Advances in Modeling Earth Systems 10.1029/2020MS002111 The jet changes strongly impact the total drag in return. This is because above where the jet is decelera turbulent friction drag calculated by the boundary layer scheme is weaker (less negative) and vice ve Figure 5. DJFM zonal‐mean circulation illustrated by (contour) the climatological ﬁelds in Atm‐6A and difference of Atm‐6A minus Atm‐5DL (contour interval [CI] provided on top of each panel): (a) zonal‐mean zo wind tendency due to orographic drag, in m s−1 day−1; (b) zonal‐mean zonal wind, in m s−1; (c) zonal‐mean zon tendency due to atmospheric physics, in m s−1 day−1; (d) residual vertical velocity, in mm s−1; (e) eddy zonal ﬂux, in m2 s−2; (f) eddy temperature meridional ﬂux, in K m s−1; (g) zonal wind tendency implied by the Eliasse ﬂux divergence, in 102 m s−1 day−1; and (h) zonal‐mean temperature, in K. Journal of Advances in Modeling Earth Systems 10.1029/2020MS002111 balanced as much as elsewhere, and we suggest two reasons for this. The ﬁrst is that the changes in the near‐surface winds are not as large as at lower latitudes; the second is that in these regions the near‐surface air is so stratiﬁed that the boundary layer does not develop well enough to efﬁciently balance the SSO drag. balanced as much as elsewhere, and we suggest two reasons for this. The ﬁrst is that the changes in the near‐surface winds are not as large as at lower latitudes; the second is that in these regions the near‐surface air is so stratiﬁed that the boundary layer does not develop well enough to efﬁciently balance the SSO drag. To a certain extent, the TEM vertical velocity in Figure 5d responds to the near‐surface force in Figure 5c consistent with the case of Eliassen (1951) where drag is applied at the surface: North of 70°N, the residual vertical velocity is upward (w* > 0) above the surface and in the troposphere, consistent with the fact that the negative anomaly in low‐level drag is almost centered at 70°N and drives a direct cell aloft. Nevertheless, as this interpretation challenges downward control principles, it is important to investigate the associated upper‐level changes in eddy forcing. In the classical “downward control” description of the mer- idional circulations, the meridional wind response to eddy‐driven forces is “supposedly” equilibrated by an opposing response due to the adjustment of the boundary layer. Such an equilibration is needed when one does long temporal average because the absence of equilibration yields a meridional transfer of mass and then a non‐stationary change in the zonal‐mean surface pressure ﬁeld. As we adopt a more “upward con- trolled” view, one should test if our surface forces are in part compensated by changes in upper‐level eddy forces. To some extent, we have begun to address this in Figure 3, where we found that the eddy activity was reduced in Atm‐6A. To evaluate this more precisely, Figures 5e and 5f show the zonal wind and temperature meridional ﬂuxes due to the eddies, v0u0 and v0T0, respectively. We see that both decay in Atm‐6A compared to Atm‐5DL and also that near the surface between 50°N and 75°N, v0T0 is smaller in Atm‐6A than in Atm‐5DL. This could well explain the polar cooling, with smaller meridional poleward heat ﬂux decreasing the near‐surface temperature directly. Journal of Advances in Modeling Earth Systems What is also important, nevertheless, is the eddy forcing, which is the zonal wind tendency due to the divergence of the Eliassen‐Palm (EP) ﬂux in Figure 5g. Note that in the upper troposphere, the EP ﬂuxes converge and decelerate the zonal wind (Figure 5g, contours and vectors), while the EP ﬂuxes diverge in the lower troposphere inducing the formation of the eddy‐driven jet. The difference in eddy forcing between Atm‐6A and Atm‐5DL is positive in the midtroposphere north of 50°N (Figure 5h, colors), so that the zonal wind is accelerated by eddies in this zone in Atm‐6A. If we equilibrate this positive difference in forcing by a negative difference in TEM meridional wind v*, according to downward control, it is associated with a direct anomaly in meridional circulation below, with reduced polar subsidence in Atm‐6A, and decreased near‐surface temperature north of 60°N. Journal of Advances in Modeling Earth Systems In (g), the vectors show the climat Eliassen‐Palm ﬂux (vector, with a typical magnitude of 150 m2 s−1 day−1), using the scaling of Edmon et al. (1 Figure 5. DJFM zonal‐mean circulation illustrated by (contour) the climatological ﬁelds in Atm‐6A and (color) difference of Atm‐6A minus Atm‐5DL (contour interval [CI] provided on top of each panel): (a) zonal‐mean zonal wind tendency due to orographic drag, in m s−1 day−1; (b) zonal‐mean zonal wind, in m s−1; (c) zonal‐mean zonal wind tendency due to atmospheric physics, in m s−1 day−1; (d) residual vertical velocity, in mm s−1; (e) eddy zonal wind ﬂux, in m2 s−2; (f) eddy temperature meridional ﬂux, in K m s−1; (g) zonal wind tendency implied by the Eliassen‐Palm ﬂux divergence, in 102 m s−1 day−1; and (h) zonal‐mean temperature, in K. In (g), the vectors show the climatological Eliassen‐Palm ﬂux (vector, with a typical magnitude of 150 m2 s−1 day−1), using the scaling of Edmon et al. (1980). The jet changes strongly impact the total drag in return. This is because above where the jet is decelerated the turbulent friction drag calculated by the boundary layer scheme is weaker (less negative) and vice versa. In our model, this more than balances the extra SSO drag between 30°N and 60°N where the total drag in Atm‐6A is weaker than in Atm‐5DL (Figure 5c). Interestingly, north of 65°N, the SSO drag is not The jet changes strongly impact the total drag in return. This is because above where the jet is decelerated the turbulent friction drag calculated by the boundary layer scheme is weaker (less negative) and vice versa. In our model, this more than balances the extra SSO drag between 30°N and 60°N where the total drag in Atm‐6A is weaker than in Atm‐5DL (Figure 5c). Interestingly, north of 65°N, the SSO drag is not 9 of 19 GASTINEAU ET AL. 9 of 19 GASTINEAU ET AL. 3.4. Air Temperature Changes To evaluate how the upper air diagnostics translate in the boundary layer, Figure 6b shows the 2‐m tempera- ture difference between Atm‐6A and Atm‐5DL. In Atm‐6A, North America is warmer, but most of the other regions are cooler, that is, Eurasia, and most importantly for sea ice, a large part of the Arctic. In the other sensitivity experiments, a similar warming, but with larger amplitude over North America, is reproduced for a large decrease of the lift (Figure 6c), as well as a cooling in western Eurasia. These surface temperature changes are consistent with the modiﬁed standing wave pattern (Figure 2f), with the anomalous southerly ﬂow over North America and anomalous easterly ﬂow in western Eurasia. However, as the lift is only slightly decreased in Atm‐5DL when compared to Atm‐6A (see Table 1), the effect of the lift is likely not dominant in Atm‐6A minus Atm‐5DL over Eurasia and the Arctic. The cooling simulated in Atm‐6A over the Arctic and Eurasia is somewhat similar to the one simulated when increasing the drag (Figure 6a, also given in Cheruy et al., 2020, their Figure 9). The standing wave pattern is only modiﬁed over America by the increasing drag, with the anomalous southerly (northerly) ﬂow in eastern (western) America, thereby pro- ducing surface warming (cooling). The cooling produced over Eurasia and the Arctic is likely dominant in the zonal‐mean temperature changes illustrated previously (Figure 5h). As discussed in Cheruy et al. (2020), as the atmospheric model has a warm winter bias over the Northern Hemisphere midlatitude, the SSO changes in Atm‐6A partly reduce the bias over Eurasia but increase it over North America. The Arctic cooling is occurring only during the winter in Atm‐6A (from November to March; Figure 6d, bot- tom, black curve) and is consistent with a dominant effect of the increasing drag (red curve), while little air temperature changes are simulated during the other seasons. Although in the TEM diagnostics we insisted on the role of the increased drag, the decreased lift (green curve) may also attenuate the dominant GASTINEAU ET AL. GASTINEAU ET AL. 10 of 19 10.1029/2020MS002111 Journal of Advances in Modeling Earth Systems Journal of Advances in Modeling Earth Systems Figure 6. (a) DJFM 2‐m air temperature difference, in K, of Atm‐6A‐D+ minus Atm‐6A‐D−. Only grid points with statistical signiﬁcance lower than 10% colored. The latitude 60°N is shown with a dashed circle. 3.4. Air Temperature Changes (b) Same as (a), but for Atm‐6A minus Atm‐5DL. (c) Same as (a), but for Atm‐6A‐L−minus Atm‐6A‐L+. (d) Mean 2‐m air temperature changes over the polar cap (60–90°N) induced by SSO modiﬁcations; black: Atm‐6A minus Atm‐5DL; red: Atm‐6A‐ + minus Atm‐6A‐Drg−; green: Atm‐6A‐Lft−minus Atm‐6A‐Lft+; blue: AO‐6A minus AO‐5DL. The error bars indicate the standard errors of the mean. Figure 6. (a) DJFM 2‐m air temperature difference, in K, of Atm‐6A‐D+ minus Atm‐6A‐D−. Only grid points with statistical signiﬁcance lower than 10% are colored. The latitude 60°N is shown with a dashed circle. (b) Same as (a), but for Atm‐6A minus Atm‐5DL. (c) Same as (a), but for Atm‐6A‐L−minus Atm‐6A‐L+. (d) Mean 2‐m air temperature changes over the polar cap (60–90°N) induced by SSO modiﬁcations; black: Atm‐6A minus Atm‐5DL; red: Atm‐6A‐Drg + minus Atm‐6A‐Drg−; green: Atm‐6A‐Lft−minus Atm‐6A‐Lft+; blue: AO‐6A minus AO‐5DL. The error bars indicate the standard errors of the mean. drag‐induced near‐surface cooling in March or in November. This again demonstrates the importance of the eddy forcing, the lift being important for the planetary waves. 4.1. Atmospheric Circulation Changes The planetary standing wave of the ocean‐atmosphere coupled experiments based on the atmospheric model component studied previously is shown in Figure 7. The overall biases of the 700‐hPa geopotential height in AO‐5DL resemble the biases illustrated previously in the atmosphere‐only experiments: a too deep polar depression and three anomalous troughs over north‐eastern America, northern Europe, and eastern Asia (see Figure 7c). The 700‐hPa geopotential height biases are larger in the coupled model (cf. Figures 1c and 7c), with a maximum bias of ~100 m in AO‐5DL and ~80 m in Atm‐5DL. The 700‐hPa height changes (Figures 7e and 7f) in AO‐6A relative to AO‐5DL are qualitatively similar to that illustrated previously in the atmosphere‐only experiments (Figures 2a and 2b), with a strengthening of the geopotential height over the Arctic in AO‐6A when compared to AO‐5DL and a weakening over the 20–40°N latitude band, especially over the North Atlantic. When compared to AO‐5DL, two dominant 11 of 19 GASTINEAU ET AL. GASTINEAU ET AL. 10.1029/2020M ournal of Advances in Modeling Earth Systems 10.1029/2020MS002111 Figure 7. (a) Geopotential height at 700 hPa averaged over the winter months (DJFM) for AO‐6A minus ERA‐Interim and (b) its zonally asymmetric component. (c) and (d) are the same as (a) and (b), but for AO‐5DL minus ERA‐Interim. (e) and (f) are the same as (a) and (b), but for AO‐6A minus AO‐5DL. In (a)–(d), the root mean square (RMS) 20–90°N difference with ERA‐Interim is indicated on top. In all panels, only grid points with statistical signiﬁcance lower than 10% are colored. Figure 7. (a) Geopotential height at 700 hPa averaged over the winter months (DJFM) for AO‐6A minus ERA‐Interim and (b) its zonally asymmetric component. (c) and (d) are the same as (a) and (b), but for AO‐5DL minus ERA‐Interim. (e) and (f) are the same as (a) and (b), but for AO‐6A minus AO‐5DL. In (a)–(d), the root mean square (RMS) 20–90°N difference with ERA‐Interim is indicated on top. In all panels, only grid points with statistical signiﬁcance lower than 10% are colored. Figure 7. (a) Geopotential height at 700 hPa averaged over the winter months (DJFM) for AO‐6A minus ERA‐Interim and (b) its zonally asymmetric component. (c) and (d) are the same as (a) and (b), but for AO‐5DL minus ERA‐Interim. (e) and (f) are the same as (a) and (b), but for AO‐6A minus AO‐5DL. 4.1. Atmospheric Circulation Changes In (a)–(d), the root mean square (RMS) 20–90°N difference with ERA‐Interim is indicated on top. In all panels, only grid points with statistical signiﬁcance lower than 10% are colored. ridges are simulated, one downstream of the Rockies over north‐eastern America and another one over northern Europe. A smaller ridge is also simulated in eastern Asia, downstream of the Tibetan Plateau. Furthermore, two troughs are simulated upstream of the two major Northern Hemisphere mountain ranges. As in stand‐alone atmospheric simulations, the SSO modiﬁcation in AO‐6A alleviates the atmospheric cir- culation biases for the asymmetric component as compared to AO‐5DL (Figures 7c and 7d, RMSE from ridges are simulated, one downstream of the Rockies over north‐eastern America and another one over northern Europe. A smaller ridge is also simulated in eastern Asia, downstream of the Tibetan Plateau. Furthermore, two troughs are simulated upstream of the two major Northern Hemisphere mountain ranges. As in stand‐alone atmospheric simulations, the SSO modiﬁcation in AO‐6A alleviates the atmospheric cir- culation biases for the asymmetric component as compared to AO‐5DL (Figures 7c and 7d, RMSE from 12 of 19 GASTINEAU ET AL. GASTINEAU ET AL. Journal of Advances in Modeling Earth Systems 10.1029/2020MS002111 atmosphere‐only case (Figures Figure 8. (a) DJFM difference of zonal‐mean zonal wind (in m s−1) o AO‐6A minus AO‐5DL. (b) Same as (a), but for the zonal‐mean temperature (in K). (c) DJFM 2‐m air temperature difference, in K, of AO‐6A minus AO‐5DL. In all panels, only grid points with statistical signiﬁcance lower than 10% are colored. 24.7 to 20.6 m), but the response is weaker in the coupled model case, except over Northern Europe. For example, the same SSO modiﬁcation (i.e., 6A minus 5DL) in the atmospheric experiment led to changes of up to 50 m over north‐eastern America (Figure 2a), while the changes are of the order of 30 m in the same region in the AOGCM experiments (Figure 7e). The resulting geopotential height in AO‐6A (Figure 7a) remains too strong over the Arctic compared to ERA‐Interim (Figure 1a) and too weak over the midlatitudes, yet less than in AO‐5DL (Figure 7e). The biases of the 700‐hPa geopotential height are nevertheless larger than AO‐5DL in AO‐6A (RMSE increase from 49.3 to 50.5 m) as the geopotential height decreases in the latitudinal band 20–40°N. 4.1. Atmospheric Circulation Changes The difference in duration between the coupled and atmospheric experi- ments might explain the larger changes simulated in the 30‐yr atmosphere‐only experiments, as the internal variability is presumably better removed in the coupled experiment (duration ≥200 yr). Nevertheless, a comparison of the pairwise differences reveals that the changes are indeed signiﬁcantly weaker in the coupled model experi- ments (Figure S2). The zonal‐mean zonal wind anomalies in AO‐6A relative to AO‐5DL, in the coupled simulations (Figure 8a), are also similar to that shown in the analogous atmosphere‐only simulations (Figure 5b). Both show a bar- otropic enhancement of the subtropical jet in its equatorward ﬂank and a weakening of the eddy‐driven jet at 50°N. Nevertheless, consistently with the geopotential height response, the changes of AO‐6A minus AO‐5DL are about half of Atm‐6A minus Atm‐5DL. The associated zonal‐mean temperature changes are much larger in the coupled model (Figure 8b). Indeed, the lower‐troposphere cooling is quite intense, with a cooling of more than 2 K north of 60°N. A clear cooling is also simulated elsewhere in the troposphere, with values of −0.2 to −0.4 K in the tropics, and ampli- ﬁed values in the upper troposphere, as expected from the adjustment of the moist adiabat. On the other hand, warming is simulated in the polar stratosphere, and the stratospheric polar vortex weakens. The surface air temperature (Figure 8c) is about 3 K cooler over the whole Arctic, with a maximum cooling up to 8 K occurring over the Barents and Okhotsk Seas where the sea ice cover is thin and particularly sensitive to climate ﬂuctuations. The cooling also extends over the Eurasian continent and, to a lesser extent, into the North Paciﬁc and Atlantic. The atmospheric variability in the coupled model also shows a decreasing 500‐hPa geopotential height variance similar to that of the atmosphere‐only simulations, but with weaker amplitude (Figure S3a). The blocking frequency also increases over Scandinavia (Figure S3b). Such an increase is larger than the one simulated in the atmospheric experiments (Figure 4), with a blocking frequency RMSE reduced by 0.77% over the North Atlantic. Over northern Siberia, the RMSE is almost unchanged. In the upper troposphere, the meridional zonal wind and temperature transports are also similar in the coupled model and the atmosphere‐only case (Figures S3c and S3d). 4.1. Atmospheric Circulation Changes However, the lower‐troposphere meridional temperature trans- port at 30–60°N increases in the coupled experiments as a result of the larger meridional temperature gradi- ent. Nevertheless, the anomalous residual vertical velocity is still found to be ascending (negative) north of 60°N for the AOGCM case (Figure S3f), as the lower‐tropospheric lapse rate increases. nal‐mean zonal wind (in m s−1) of a), but for the zonal‐mean temperature difference, in K, of only grid points with statistical ed. Figure 8. (a) DJFM difference of zonal‐mean zonal wind (in m s−1) of AO‐6A minus AO‐5DL. (b) Same as (a), but for the zonal‐mean temperature (in K). (c) DJFM 2‐m air temperature difference, in K, of AO‐6A minus AO‐5DL. In all panels, only grid points with statistical signiﬁcance lower than 10% are colored. We conclude that in the coupled model the overall dynamical changes due to the SSO modiﬁcation are simi- lar to the ones inferred from the atmospheric model but weaker. However, these changes in the coupled GASTINEAU ET AL. GASTINEAU ET AL. GASTINEAU ET AL. 13 of 19 10.1029/2020MS002111 l of Advances in Modeling Earth Systems Journal of Advances in Modeling Earth Systems Figure 9. Arctic sea ice concentration, in %, in (a) March and (b) September. The black contour provides the observed sea ice concentration of 50% from 1979 to 2014. The blue contour illustrates the same contour for AO‐6A (dashed line) and AO‐5DL (full line). Arctic sea ice thickness, in m, in (c) March and (d) September. The gray contours give the mean value in AO‐5DL. In all panels, the color illustrates AO‐6A minus AO‐5DL. Figure 9. Arctic sea ice concentration, in %, in (a) March and (b) September. The black contour provides the observed sea ice concentration of 50% from 1979 to 2014. The blue contour illustrates the same contour for AO‐6A (dashed line) and AO‐5DL (full line). Arctic sea ice thickness, in m, in (c) March and (d) September. The gray contours give the mean value in AO‐5DL. In all panels, the color illustrates AO‐6A minus AO‐5DL. model are superimposed onto a lower‐tropospheric cooling over the polar cap. The next subsection focuses on the associated sea ice extension and thickness. 4.2. Ocean and Arctic Sea Ice The Arctic sea ice extent is increased in AO‐6A as compared to AO‐5DL in both summer and winter. In win- ter, the increase is mostly located over the Northern Paciﬁc and the Barents Sea (Figure 9a), while the sea ice concentration decreases locally over the Labrador Sea. The Arctic sea ice thickness also shows a large increase of ~0.8 m in the central Arctic (Figure 9c): It is ~3 m in AO‐5DL, and it raises up to ~3.8 m in AO‐6A. In summer, the sea ice extent increases especially along the coast of Russia in the eastern Arctic (Figure 9b). The multiyear ice thickness also increases by about 1 m off Greenland (Figure 9d). Our interpre- tation is that the colder winter temperature induced by the modiﬁed SSO (see Figure 6a) has led to enhanced Arctic sea ice growth in the coupled model. The resulting larger sea ice volume can favor a colder Arctic with a larger summer sea ice extent, as found, for example, in model experiments designed to study the inﬂuence of sea ice initialization (Blanchard‐Wrigglesworth et al., 2011; Holland et al., 2011), or when assimilating sea ice thickness in models (Blockley & Peterson, 2018). Besides, the summer sea ice changes may be ampliﬁed by the sea ice‐albedo feedback. In summary, the impact of SSO modiﬁcations over the Arctic is largely mod- iﬁed by the ocean‐atmosphere coupling, leading to a larger thermodynamic response when compared to the atmosphere‐only model. As the sea ice insulates the ocean from the atmosphere, the more extended sea ice inhibits the heat release from the ocean to the atmosphere in winter, thereby reinforcing the winter cooling. This feedback explains the maximum cooling in November and December (see Figure 6d, blue line). The ice‐albedo feedback may contribute to the smaller summer cooling. Lastly, we note that the SSO 14 of 19 GASTINEAU ET AL. GASTINEAU ET AL. 10.1029/2020MS002111 Journal of Advances in Modeling Earth Systems Journal of Advances in Modeling Earth Systems 10.1029/2020MS002111 modiﬁcation has corrected the underestimated summer sea ice extent simulated present in AO Annual mean wind stress difference between (a) AO‐6A and ERA‐Interim and (b) AO‐5DL and ERA‐Interim. The color indicates the magnit n 10−2 Pa−1, while the vectors indicate the difference in Pa. (c) Annual mean SST (contour interval ¼ 0.2 K) difference of AO‐6A minus (c), but for the SSH (contour interval ¼ 2 cm). 4.2. Ocean and Arctic Sea Ice The mean SSH (in cm) in AO‐6A is indicated in black contour. (e) Same as (c), but for the erval ¼ 0.1 psu). (f) Yearly Atlantic meridional overturning stream function (in Sv) changes for AO‐6A minus AO‐5DL. The mean Atlantic m stream function (in Sv) in AO‐6A is indicated in gray contour. In (c)–(e), only grid points with statistical signiﬁcance lower than 10% are Figure 10. Annual mean wind stress difference between (a) AO‐6A and ERA‐Interim and (b) AO‐5DL and ERA‐Interim. The color indicates the magnitude of the difference, in 10−2 Pa−1, while the vectors indicate the difference in Pa. (c) Annual mean SST (contour interval ¼ 0.2 K) difference of AO‐6A minus AO‐5DL. (d) Same as (c), but for the SSH (contour interval ¼ 2 cm). The mean SSH (in cm) in AO‐6A is indicated in black contour. (e) Same as (c), but for the SSS (contour interval ¼ 0.1 psu). (f) Yearly Atlantic meridional overturning stream function (in Sv) changes for AO‐6A minus AO‐5DL. The mean Atlantic meridional overturning stream function (in Sv) in AO‐6A is indicated in gray contour. In (c)–(e), only grid points with statistical signiﬁcance lower than 10% are colored. modiﬁcation has corrected the underestimated summer sea ice extent simulated present in AO‐5DL, illustrated in Figures 9a and 9b by the observed and simulated 50% contour for the sea ice concentration The oceanic changes are not restricted to the Arctic. The lower‐tropospheric westerlies are overestimated in AO‐5DL over the eastern North Atlantic and the Kuroshio extension in the Paciﬁc (Figure 10a). The simula- tion AO‐6A (Figure 10b) shows a reduction of these two biases, even if the underestimation of the wind stress in the eastern Paciﬁc becomes more pronounced. This reduction of the westerlies is associated with a south- ward shift of the Northern Hemisphere western boundary oceanic currents, namely, the Gulf Stream and Kuroshio. This can be seen through the maximum cooling located in the western Paciﬁc and Atlantic at Journal of Advances in Modeling Earth Systems Journal of Advances in Modeling Earth Systems 10.1029/2020MS002111 Figure 11. Total (black line), atmospheric (red line), and oceanic (blue line) annual mean meridional energy transport difference, in 1015 W, for AO‐6A minus AO‐5DL. Figure 11. Total (black line), atmospheric (red line), and oceanic (blue line) annual mean meridional energy transp difference, in 1015 W, for AO‐6A minus AO‐5DL. 40–45°N (Figure 10c). This is also consistent with the sea surface height (SSH) reduction at the same locations (Figure 10d). In AO‐6A minus AO‐5DL, the sea surface salinity is also reduced in the subpolar North Atlantic (Figure 10e), which is consistent with a decreasing northward salt transport related to the southward shift of the North Atlantic current. As discussed in Boucher et al. (2020), IPSL‐CM6A‐LR (identical to AO‐6A here) has an important cold (~3°C) and fresh (~1 psu) bias in the North Atlantic. It also shows a cold (~1°C) bias off Japan. As the anomalies indicated by AO‐6A minus AO‐5DL are smaller but consistent with such biases, SSO changes have contributed to amplify these biases. We also note that the warm bias in the Bering sea has been reduced by the SSO modiﬁcation. 40–45°N (Figure 10c). This is also consistent with the sea surface height (SSH) reduction at the same locations (Figure 10d). In AO‐6A minus AO‐5DL, the sea surface salinity is also reduced in the subpolar North Atlantic (Figure 10e), which is consistent with a decreasing northward salt transport related to the southward shift of the North Atlantic current. As discussed in Boucher et al. (2020), IPSL‐CM6A‐LR (identical to AO‐6A here) has an important cold (~3°C) and fresh (~1 psu) bias in the North Atlantic. It also shows a cold (~1°C) bias off Japan. As the anomalies indicated by AO‐6A minus AO‐5DL are smaller but consistent with such biases, SSO changes have contributed to amplify these biases. We also note that the warm bias in the Bering sea has been reduced by the SSO modiﬁcation. Cooling is also simulated in the equatorial Paciﬁc and the Indian Ocean in AO‐6A as compared to AO‐5DL. It might be explained by the global response to increased sea ice cover. Many previous studies indeed found that sea ice loss causes a tropical warming in coupled models, called “a mini‐global warming” (Blackport & Kushner, 2017; Deser et al., 2014), by analogy with the warming induced by increasing greenhouse gases. 4.3. Meridional Energy Transport In the coupled simulations, the atmospheric and oceanic meridional energy transports change as a response to the new surface and top‐of‐atmosphere energy budgets. The atmospheric and oceanic energy transports are calculated using the top of the atmosphere radiative budget and the net surface heat ﬂux integrated from 90°S. As the energy non‐conservation is stationary (not shown), we remove the mean non‐conservation term before calculation. In the coupled experiments, the extension of Arctic sea ice in AO‐6A relative to AO‐5DL leads to a decrease of incoming shortwave radiation over the Arctic, caused by the increased surface albedo. This implies an increase of the total northward meridional energy transport, as illustrated in Figure 11 (black line). The atmospheric meridional energy transport (AMET; red line) accounts for most of this increase. The AMET increase is consistent with the lower‐tropospheric meridional temperature transport in midlatitudes (Figure S3c). In the tropics, the AMET changes are consistent with the Hadley cells modiﬁcations expected from the Arctic cooling (Yoshimori et al., 2018), with a direct anomalous cross‐equatorial cell. The anoma- lous cell leads to northward meridional geopotential transport in its upper branch and increasing southward heat and moisture transport in its lower branch (Figure S4). However, the northward oceanic meridional energy transport (OMET; blue line) is reduced, which damps the inﬂuence of the AMET increase. The OMET reduction is consistent with the weaker Atlantic meridional overturning stream function (Figure 10f). In AO‐6A minus AO‐5DL, the decreasing subpolar North Atlantic salinity (Figure 10e) weakens the seawater density in the subpolar gyre, which likely leads in turn to the AMOC weakening. Journal of Advances in Modeling Earth Systems Such a tropical impact is explained by the water vapor feedback and ocean circulation changes (Deser et al., 2016). The tropical cooling produced by the sea ice increase in our experiments is very comparable to the results in these previous studies, but with an opposite sign. We will illustrate next the changes in the meridional energy transports. 4.3. Meridional Energy Transport GASTINEAU ET AL. GASTINEAU ET AL. 15 of 19 GASTINEAU ET AL. Journal of Advances in Modeling Earth Systems 10.1029/2020MS002111 to the local ﬂow. The lift was designed to represent the dynamical separation from the large‐scale ﬂow of the air in narrow valleys (Lott, 1999). The SSO changes implemented cause a reduced polar depression, as well as a better simulation of the Northern Hemisphere stationary wave pattern. Furthermore, we noticed a lower‐tropospheric cooling at 60–90°N over the Arctic. These changes are mainly due to the increased lower‐tropospheric drag. This effect is counter‐intuitive, as previous works found that enhanced drag gener- ally warms the midlatitudes and polar regions (Palmer et al., 1986). Using TEM diagnostics in atmosphere‐only experiments, we showed that the cooling is driven by the weaker eddy activity, which decreases the northward heat and momentum transport. In the coupled model, the same SSO modiﬁcation is found to have a large impact on Arctic sea ice, as the lower‐tropospheric atmospheric cooling is ampliﬁed by the winter sea ice growth and a reduced oceanic heat loss. Nevertheless, the changes in the standing wave or zonal winds are weaker than in the atmosphere‐only experiments. The adjustment of the SSO parameterization in IPSL‐CM6A‐LR has therefore contributed to restoring the Arctic sea ice cover, which was initially too sparse. In our case, the Arctic sea ice bias was associated with a warm winter air temperature bias, which was thus also reduced. Nevertheless, several other negative impacts are also found, so that caution is needed before applying such SSO modiﬁcations. In particular, increasing the SSO drag and decreasing the lift have led to a reduction of the AMOC, which is rather weak in this model (about 13 Sv; Boucher et al., 2020). We suggest that the AMOC changes are here induced by the weaker westerlies in the Eastern Atlantic, shifting southward the North Atlantic current, and decreasing the salinity transport toward the subpolar gyre. The wind‐induced southward shift of the North Atlantic current has also degraded the cold and fresh bias present in the central Atlantic (Boucher et al., 2020). This bias is a common feature in many models using a low‐resolution ocean. The surface air temperature impact was also speciﬁcally investigated outside the Arctic. In the atmospheric simulations, the SSO modiﬁcation is found to modulate the contrast of air temperature between North America and Eurasia. This reﬂects the inﬂuence of SSO drag on the planetary stationary wave. Acknowledgments Acknowledgments This research was supported by the Blue‐Action project (European Union's Horizon 2020 research and innovation program, Grant Number 727852) and by the ARCHANGE project of the “Make our planet great again” program (ANR‐18‐MPGA‐0001, France). F. L. acknowledges funding from the JPI‐ Climate/Belmont Forum project GOTHAM (ANR‐15‐JCLI‐0004‐01). This work used the HPC resources of TGCC under the allocations 2016‐ A0030107732, 2017‐R0040110492, and 2018‐1293R0040110492 (project gen- cmip6) provided by Grand Équipement National de Calcul Intensif (GENCI). This study beneﬁted from the Ensemble de Services Pour la Recherche à l'IPSL (ESPRI) computing and data center (https://mesocentre.ipsl.fr) which is supported by CNRS, Sorbonne Université, Ecole Polytechnique, and CNES and through national and inter- national grants. Data Availability Statement The data of the sensitivity experiments supporting the conclusions of the study can be obtained online (https://doi.org/10.5281/zenodo.3714902). Andrews, D. G., Leovy, C. B., & Holton, J. R. (1987). Middle atmosphere dynamics (Vol. 40). New York: Academic Press. Bacmeister, J. T. (1993). Mountain‐wave drag in the stratosphere and mesosphere inferred from observed winds and a simple mountain‐wave parameterization scheme. Journal of the Atmospheric Sciences, 50(3), 377–399. Baines, P. G., & Palmer, T. N. (1985). Rationale for a new physically‐based parametrization of subgrid‐scale orographic effects, ECMWF Technical Memoranda, 169, 11. https://doi.org/10.21957/h4h36b3u Blackmon, M. L. (1976). A climatological spectral study of the 500 mb geopotential height of the Northern Hemisphere. Journal of the Atmospheric Sciences, 33(8), 1607–1623. https://doi.org/10.1175/1520-0469(1976)033<1607:ACSSOT>2.0.CO;2 Blackport, R., & Kushner, P. J. (2017). Isolating the atmospheric circulation response to Arctic sea ice loss in the coupled climate system. Journal of Climate, 30(6), 2163–2185. https://doi.org/10.1175/JCLI-D-16-0257.1 Blanchard‐Wrigglesworth, E., Bitz, C. M., & Holland, M. M. (2011). Inﬂuence of initial conditions and climate forcing on predicting Arctic sea ice. Geophysical Research Letters, 38, L18503. https://doi.org/10.1029/2011GL048807 Blockley, E. W., & Peterson, K. A. (2018). Improving Met Ofﬁce seasonal predictions of Arctic sea ice using assimilation of CryoSat‐2 thickness. The Cryosphere, 12, 3419–3438. https://doi.org/10.5194/tc-12-3419-2018 Boucher, O., Servonnat, J., Albright, A. L., Aumont, O., Balkanski, Y., Bastrikov, V., et al. (2020). Presentation and evaluation of the IPSL‐ CM6A‐LR climate model. Journal of Advances in Modeling Earth Systems, 12, e2019MS002010. https://doi.org/10.1029/2019MS002010 Journal of Advances in Modeling Earth Systems The SSO drag also cools Eurasia, as it weakens the air advection from the warm Atlantic toward the land. The air tempera- ture modiﬁcation is also partly caused by the lift, which directly modiﬁes the Northern Hemisphere standing wave pattern. The results shown are likely sensitive to the model. However, the CMIP5 models all have biases of the North Atlantic storm track and European blockings. These biases were found to be quite similar to those produced in a simulation with a deactivated low‐level orographic blocking effect (Pithan et al., 2016). Furthermore, the low‐level winter warm biases over Arctic sea ice are also common to many other models (Graham et al., 2019). This suggests that a deﬁcit of low‐level drag is also present in other climate models, and more work might therefore be needed to understand the implications for Arctic sea ice and the oceanic circulation biases in the other AOGCMs. 5. Discussions and Conclusion During the tuning of the IPSL‐CM6A‐LR model, the parametrized orography was modiﬁed to alleviate the biases of the atmospheric circulation resulting from the updated model physics. We increased the orographic lower‐tropospheric blocking effect (so‐called drag). We also decreased the lift, which is a force perpendicular GASTINEAU ET AL. GASTINEAU ET AL. 16 of 19 Journal of Advances in Modeling Earth Systems Slow thermally or frictionally controlled meridional circulation in a circular vortex. Astrophysica Norvegica, 5, 19 Eyring, V., Bony, S., Meehl, G. A., Senior, C. A., Stevens, B., Stouffer, R. J., & Taylor, K. E. (2016). Overview of the Coupled Model Intercomparison Project Phase 6 (CMIP6) experimental design and organization. Geoscientiﬁc Model Development, 9(5), 1937–1958. https://doi.org/10.5194/gmd-9-1937-2016 Garner, S. T. (2005). A topographic drag closure built on an analytical base ﬂux. Journal of the Atmospheric Sciences, 62(7), 2302–231 https://doi.org/10.1175/JAS3496.1 p g Graham, R. M., Cohen, L., Ritzhaupt, N., Segger, B., Graversen, R. G., Rinke, A., et al. (2019). Evaluation of six atmospheric reanalyses o Arctic sea ice from winter to early summer. Journal of Climate, 32(14), 4121–4143. https://doi.org/10.1175/JCLI-D-18-0643.1 Haynes, P. H., McIntyre, M. E., Shepherd, T. G., Marks, C. J., & Shine, K. P. (1991). On the “downward control” of extratropical diabatic circulations by eddy‐induced mean zonal forces. Journal of the Atmospheric Sciences, 48(4), 651–678. https://doi.org/10.1175/1520- 0469(1991)048<0651:OTCOED>2.0.CO;2 ( ) ; Held, I. M., Guo, H., Adcroft, A., Dunne, J. P., Horowitz, L. W., Krasting, J., et al. (2019). Structure and performance of GFDL's CM4.0 climate model. Journal of Advances in Modeling Earth Systems, 11, 3691–3727. https://doi.org/10.1029/2019MS001829 Holland, M. M., Bailey, D. A., & Vavrus, S. (2011). Inherent sea ice predictability in the rapidly changing Arctic environment of the Community Climate System Model version 3 Climate Dynamics 36(7 8) 1239 1253 https://doi org/10 1007/s00382 010 0792 4 Holland, M. M., Bailey, D. A., & Vavrus, S. (2011). Inherent sea ice predictability in the rapidly changing Arctic environment of the Community Climate System Model, version 3. Climate Dynamics, 36(7–8), 1239–1253. https://doi.org/10.1007/s00382-010-0792-4 Hourdin, F., Musat, I., Bony, S., Braconnot, P., Codron, F., Dufresne, J.‐L., et al. (2006). The LMDZ4 general circulation model: Climate performance and sensitivity to parametrized physics with emphasis on tropical convection. Climate Dynamics, 27(7–8), 787–813. https:// doi.org/10.1007/s00382-006-0158-0 g Hourdin, F., Rio, C., Grandpeix, J.‐Y., Madeleine, J.‐B., Cheruy, F., Rochetin, N., et al. (2020). LMDZ6A: The atmospheric component of the IPSL climate model with improved and better tuned physics. Journal of Advances in Modeling Earth Systems, 12, e2019MS001892. https://doi.org/10.1029/2019MS001892 Lott, F. (1999). Alleviation of stationary biases in a GCM through a mountain drag parameterization scheme and a simple repre- sentation of mountain lift forces. Monthly Weather Review, 127(5), 788–801. https://doi.org/10.1175/1520-0493(1999)127<0788: AOSBIA>2.0.CO;2 Lott, F., & D'andrea, F. (2005). References Boucher, O., Servonnat, J., Albright, A. L., Aumont, O., Balkanski, Y., Bastrikov, V., et al. (2020). Presentation and evaluation of the IPSL‐ CM6A‐LR climate model. Journal of Advances in Modeling Earth Systems, 12, e2019MS002010. https://doi.org/10.1029/2019MS002010 GASTINEAU ET AL. 17 of 19 Journal of Advances in Modeling Earth Systems Mass and wind axial angular‐momentum responses to mountain torques in the 1–25 day band: Links with the Arctic Oscillation. Quarterly Journal of the Royal Meteorological Society, 131(608), 1483–1500. https://doi.org/10.1256/ qj.03.168 Lott, F., & Miller, M. J. (1997). A new subgrid‐scale orographic drag parametrization: Its formulation and testing. Quarterly Journal of the Royal Meteorological Society, 123(537), 101–127. https://doi.org/10.1002/qj.49712353704 Lott, F., Robertson, A. W., & Ghil, M. (2004). Mountain torques and Northern Hemisphere low‐frequency variability. Part II: Region aspects. Journal of the Atmospheric Sciences, 61(11), 1272–1283. https://doi.org/10.1175/1520-0469(2004)061<1272:MTANHL>2.0.C tt, F., Robertson, A. W., & Ghil, M. (2004). Mountain torques and Northern Hemisphere low‐frequency variability. Part II: Regional t J l f th At h i S i 61(11) 1272 1283 htt //d i /10 1175/1520 0469(2004)061 1272 MTANHL 2 0 CO 2 Lott, F., Robertson, A. W., & Ghil, M. (2004). Mountain torques and Northern Hemisphere lo aspects. Journal of the Atmospheric Sciences, 61(11), 1272–1283. https://doi.org/10.1175/152 Lott, F., Robertson, A. W., & Ghil, M. (2004). Mountain torques and Northern Hemisphere low‐frequency variability. Part II: Regional aspects. Journal of the Atmospheric Sciences, 61(11), 1272–1283. https://doi.org/10.1175/1520-0469(2004)061<1272:MTANHL>2.0.CO;2 Palmer, T. N., Shutts, G. J., & Swinbank, R. (1986). Alleviation of a systematic westerly bias in general circulation and numerical weather prediction models through an orographic gravity wave drag parametrization. Quarterly Journal of the Royal Meteorological Society, 112(474), 1001–1039. https://doi.org/10.1002/qj.49711247406 Palmer, T. N., Shutts, G. J., & Swinbank, R. (1986). Alleviation of a systematic westerly bias in general circulation and numerical weather prediction models through an orographic gravity wave drag parametrization. Quarterly Journal of the Royal Meteorological Society, 112(474), 1001–1039. https://doi.org/10.1002/qj.49711247406 ( ) p g qj Pithan, F., Shepherd, T. G., Zappa, G., & Sandu, I. (2016). Climate model biases in jet streams, blocking and storm tracks resulting fr missing orographic drag. Geophysical Research Letters, 43, 7231–7240. https://doi.org/10.1002/2016GL069551 Sandu, I., Bechtold, P., Beljaars, A., Bozzo, A., Pithan, F., Shepherd, T. G., & Zadra, A. (2016). Impacts of parameterized orographic drag on the Northern Hemisphere winter circulation. Journal of Advances in Modeling Earth Systems, 8, 196–211. https://doi.org/10.1002/ 2015MS000564 Sandu, I., van Niekerk, A., Shepherd, T. G., Vosper, S. B., Zadra, A., Bacmeister, J., et al. (2019). Impacts of orography on large‐scale atmospheric circulation. Climate and Atmospheric Science, 2(1), 10. https://doi.org/10.1038/s41612-019-0065-9 Scherrer, S. C., Croci‐Maspoli, M., Schwierz, C., & Appenzeller, C. (2006). Two‐dimensional indices of atmospheric blocking and their statistical relationship with winter climate patterns in the Euro‐Atlantic region. Journal of Advances in Modeling Earth Systems 10.1029/2020MS002111 Cheruy, F., Ducharne, A., Hourdin, F., Musat, I., Vignon, E., Gastineau, G., et al. (2020). Improved near surface continental climate in IPSL‐CM6 by combined evolutions of atmospheric and land surface physics. Journal of Advances in Modeling Earth Systems, 12, e2019MS002005. https://doi.org/10.1029/2019MS002005 y, , , , , , , , g , , , , ( ) p IPSL‐CM6 by combined evolutions of atmospheric and land surface physics. Journal of Advances in Modeling Earth Systems, 12, e2019MS002005. https://doi.org/10.1029/2019MS002005 Davini P & Cagnazzo C (2014) On the misinterpretation of the North Atlantic Oscillation in CMIP5 models Climate Dynamics 43(56) e2019MS002005. https://doi.org/10.1029/2019MS002005 Davini, P., & Cagnazzo, C. (2014). On the misinterpretation of the North Atlantic Oscillation in CMIP5 models. Climate Dynamics, 43(5–6), 1497 1511 https://doi org/10 1007/s00382 013 1970 y p g Davini, P., & Cagnazzo, C. (2014). On the misinterpretation of the North Atlantic Oscillation in CMIP5 models. Climate Dynamics, 43(5 1497–1511. https://doi.org/10.1007/s00382-013-1970-y g Davini, P., Cagnazzo, C., Neale, R., & Tribbia, J. (2012). Coupling between Greenland blocking and the North Atlantic Oscillation patt Geophysical Research Letters, 39, L14701. https://doi.org/10.1029/2012GL052315 Dee, D. P., Uppala, S. M., Simmons, A. J., Berrisford, P., Poli, P., Kobayashi, S., et al. (2011). The ERA‐Interim reanalysis: Conﬁguration and performance of the data assimilation system. Quarterly Journal of the Royal Meteorological Society, 137(656), 553–597. https://doi.org/ 10.1002/qj.828 qj Deser, C., Sun, L., Tomas, R. A., & Screen, J. (2016). Does ocean coupling matter for the northern extratropical response to projected Arctic sea ice loss? Geophysical Research Letters, 43, 2149–2157. https://doi.org/10.1002/2016GL067792 Deser, C., Tomas, R. A., & Sun, L. (2014). The role of ocean–atmosphere coupling in the zonal‐mean atmospheric response to Arctic sea loss. Journal of Climate, 28(6), 2168–2186. https://doi.org/10.1175/JCLI-D-14-00325.1 Deser, C., Tomas, R. A., & Sun, L. (2014). The role of ocean–atmosphere coupling in the zonal‐ loss. Journal of Climate, 28(6), 2168–2186. https://doi.org/10.1175/JCLI-D-14-00325.1 Durack, P. J., & Taylor, K. E. (2018). PCMDI AMIP SST and sea‐ice boundary conditions version 1.1.4. Version 20180427. Earth System G Federation. http://doi.org/10.22033/ESGF/input4MIPs.2204 Durack, P. J., & Taylor, K. E. (2018). PCMDI AMIP SST and sea‐ic Federation. http://doi.org/10.22033/ESGF/input4MIPs.2204 Durack, P. J., & Taylor, K. E. (2018). PCMDI AMIP SST and sea‐ice Federation. http://doi.org/10.22033/ESGF/input4MIPs.2204 p g p Edmon, H. J. Jr., Hoskins, B. J., & McIntyre, M. E. (1980). Eliassen‐Palm cross sections for the troposphere. Journal of the Atmospheric Sciences, 37(12), 2600–2616. https://doi.org/10.1175/1520-0469(1980)037<2600:EPCSFT>2.0.CO;2 Eliassen, A. (1951). Journal of Advances in Modeling Earth Systems International Journal of Climatology: A Journal of the Royal Meteorological Society, 26(2), 233–249. https://doi.org/10.1002/joc.1250 Tjernström, M., & Graversen, R. G. (2009). The vertical structure of the lower Arctic troposphere analysed from observations and the ERA‐40 reanalysis. Quarterly Journal of the Royal Meteorological Society, 135(639), 431–443. https://doi.org/10.1002/qj.380 van Niekerk, A., Scinocca, J. F., & Shepherd, T. G. (2017). The modulation of stationary waves, and their response to climate change Tjernström, M., & Graversen, R. G. (2009). The vertical structure of the lower Arctic troposphere analysed from observations and the ERA‐40 reanalysis. Quarterly Journal of the Royal Meteorological Society, 135(639), 431–443. https://doi.org/10.1002/qj.380 van Niekerk, A., Scinocca, J. F., & Shepherd, T. G. (2017). The modulation of stationary waves, and their response to climate change, by parameterized orographic drag. Journal of the Atmospheric Sciences, 74(8), 2557–2574. https://doi.org/10.1175/JAS-D-17-0085.1 p ( ) y p g parameterized orographic drag. Journal of the Atmospheric Sciences, 74(8), 2557–2574. https://doi.org/10.1175/JAS-D-17-0085.1 van Niekerk, A., Shepherd, T. G., Vosper, S. B., & Webster, S. (2016). Sensitivity of resolved and parametrized surface drag to changes in resolution and parametrization. Quarterly Journal of the Royal Meteorological Society, 142(699), 2300–2313. https://doi.org/10.1002/ qj.2821 qj Vignon, E., Hourdin, F., Genthon, C., Gallée, H., Bazile, E., Lefebvre, M.‐P., et al. (2017). Antarctic boundary layer parametrization in a general circulation model: 1‐D simulations facing summer observations at Dome C. Journal of Geophysical Research: Atmospheres, 122, 6818–6843. https://doi.org/10.1002/2017JD026802 Vignon, E., Hourdin, F., Genthon, C., Gallée, H., Bazile, E., Lefebvre, M.‐P., et al. (2017). Antarctic boundary layer parametrization in a general circulation model: 1‐D simulations facing summer observations at Dome C. Journal of Geophysical Research: Atmospheres, 122, 6818–6843. https://doi.org/10.1002/2017JD026802 18 of 19 GASTINEAU ET AL. Journal of Advances in Modeling Earth Systems 10.1029/2020MS002111 Wallace, J. M., Tibaldi, S., & Simmons, A. J. (1983). Reduction of systematic forecast errors in the ECMWF model through the introduction of an envelope orography. Quarterly Journal of the Royal Meteorological Society, 109(462), 683–717. https://doi.org/10.1002/ qj.49710946202 Wallace, J. M., Tibaldi, S., & Simmons, A. J. (1983). Reduction of systematic forecast errors in the ECMWF model through the introduction of an envelope orography. Quarterly Journal of the Royal Meteorological Society, 109(462), 683–717. https://doi.org/10.1002/ qj.49710946202 Walsh, J. E., Chapman, W. L., & Portis, D. H. (2009). Arctic cloud fraction and radiative ﬂuxes in atmospheric reanalyses. Journal of Climate, 22(9), 2316–2334. https://doi.org/10.1175/2008JCLI2213.1 ( ) p g oollings, T., Hoskins, B., Blackburn, M., & Berrisford, P. (2008). A new Rossby wave–breaking interpretation of the North Atlantic Oscillation. Journal of the Atmospheric Sciences, 65(2), 609–626. https://doi.org/10.1175/2007JAS2347.1 Yoshimori, M., Abe‐Ouchi, A., Tatebe, H., Nozawa, T., & Oka, A. (2018). The importance of ocean dynamical feedback for understanding the impact of mid–high‐latitude warming on tropical precipitation change. Journal of Climate, 31(6), 2417–2434. https://doi.org/ 10.1175/JCLI-D-17-0402.1 Zadra, A. (2015). WGNE drag project: An inter‐model comparison of surface stresses, technical report [Available at http://collaboration. cmc.ec.gc.ca/science/rpn/drag_project/documents/ wgne_drag_project_report01.pdf] Zadra, A. (2015). WGNE drag project: An inter‐model comparison of surface stresses, technical report [Available at http://collaboration. cmc.ec.gc.ca/science/rpn/drag_project/documents/ wgne_drag_project_report01.pdf] Zadra, A., Roch, M., Laroche, S., & Charron, M. (2003). The subgrid‐scale orographic blocking parametrization of the GEM model. Atmosphere‐Ocean, 41(2), 155–170. https://doi.org/10.3137/ao.410204 Zadra, A., Roch, M., Laroche, S., & Charron, M. (2003). The subgrid‐scale Atmosphere‐Ocean, 41(2), 155–170. https://doi.org/10.3137/ao.410204 Atmosphere‐Ocean, 41(2), 155–170. https://doi.org/10.3137/ao.410204 p p g Zhao, M., Golaz, J.‐C., Held, I. M., Guo, H., Balaji, V., Benson, R., et al. (2018). The GFDL global atmosphere and land model AM4.0/LM4.0: 2. Model description, sensitivity studies, and tuning strategies. Journal of Advances in Modeling Earth Systems, 10, 735–769. https://doi. org/10.1002/2017MS001209 g Zhao, M., Golaz, J.‐C., Held, I. M., Guo, H., Balaji, V., Benson, R., et al. (2018). The GFDL global atmosphere and land model AM4.0/LM4.0: 2. Model description, sensitivity studies, and tuning strategies. Journal of Advances in Modeling Earth Systems, 10, 735–769. https://doi. org/10.1002/2017MS001209 Zhao, M., Golaz, J.‐C., Held, I. M., Guo, H., Balaji, V., Benson, R., et al. (2018). The GFDL global atmosphere and land model AM4.0/LM4.0: 2. Model description, sensitivity studies, and tuning strategies. Journal of Advances in Modeling Earth Systems, 10, 735–769. https://doi. org/10.1002/2017MS001209 19 of 19 GASTINEAU ET AL.
W4393278549.txt	https://journals.wlb-stuttgart.de/ojs/index.php/zh/article/download/11156/11027	de		Hofkapellmeister Thomas Täglichsbeck	Zollerheimat	2,024	cc-by	3,113	ZOIERHEIMAT UrG DER HONEN« BLATTER ZUR FORD ZOLLERESCMED HEIMAT« HMD •OLUUNOE NUMMER 9 Hechingen, 15. September 1937 6. JAHRGANG Hhofkapellmetfter Thomas Cägltthsbeck 1828_ 1857 Dirigent der Hofkapelle des Fürsten Konstantin von Hohenzollern-Hechingen Von S. F l a d (Schluß) Als Fürst Konstantin 1836 persönlich die Leitung der Regierung übernahm, stellte er zur Förderung des Kunst lebens an seinem Hofe größere Mittel zur Verfügung. In verstärktem Maße wurden nunmehr hervorragende Virtuosen und Komponisten zur Mitwirkung bei den Konzerten herangezogen. Unter der stattlichen Zahl der Gäste rechnen wohl die Aufenthalte von Hektor Berlioz und Franz Liszt zu den bedeutendsten. In seinen Reisebriefen schreibt Berlioz über den Fürsten folgendermaßen: „Der Herr dieser malerischen Land schaft ist ein geistreicher, lebhafter junger Herr, den auf der Welt nur zweierlei zu beseelen scheint: der Wunsch, seine Untergebenen so glücklich wie möglich zu machen und die Liebe zur Musik. Gibt es wohl ein schöneres Da sein als das seine? Rings um sich her erblickt er nur Zu friedenheit, seine Untertanen vergöttern ihn, und die Tonkunst, die er als Dichter und Musiker durchdringt, hat ihn zu ihrem Liebling erkoren. Er selbst komponiert reizende Lieder, von denen zwei: „Der Fischerknabe“ und „Schiffers Abendlied“ mich wahrhaft ergriffen haben.“ Am Neujahrstage 1842 war Berlioz auf eine Ein ladung der Fürstin hier in Hechingen eingetroffen. In Eile wurde innerhalb von drei Tagen ein Konzert mit Werken von Berlioz vorbereitet. Schon nach 5 Tagen waren die Ouvertüre zu „König Lear“, der Pilgermarsch aus der „Harold-Symphonie“, die Ballszene aus der „Phantastischen Symphonie“ und Bruchstücke aus an deren, bisher ganz unbekannten Kompositionen einstu diert. In dem großen Konzert zu Ehren Berlioz spielte Täglichsbeck auf dem Flügel die Harfenstimme und in der Haroldsymphonie das große Bratschensolo. Die be achtlichen Leistungen der Kapelle stellten den an sehr hohe Anforderungen gewöhnten Komponisten zufrieden, vor allem fanden die ersten Kräfte des Orchesters die größte Anerkennung. Nach der Veranstaltung hatte der Fürst in der Villa Eugenia aus Anlaß des hohen Künst lerbesuches zu einem Essen gebeten. In zwangslosem Bei sammensein wurde hier noch am Abend musiziert, wobei Berlioz als Solocellist, Täglichsbeck am Flügel und der stimmbegabte Fürst mit seinen eigenen Kompositionen aufwarteten. Im Dezember 1843 und wiederholt im folgenden Jahre weilte auch Franz Liszt in Hechingen. Verschiedene Kon zerte fanden während seines vierwöchigen Aufenthaltes statt, in denen der große Meister von den Einheimischen und zahlreichen Fremden immer mit außerordentlicher Begeisterung gefeiert wurde. Durch die häufigen Be suche bahnten sich zwischen dem Fürsten und Liszt freundschaftliche Beziehungen an, die später in Löwen berg zu einem noch engeren persönlichen Zusammen schlusse führen sollten. Trotz der anspruchsvollen Dienstverpflichtungen wid mete sich Täglichsbeck mit Eifer seinen eigenen künst lerischen Neigungen. Schon im ersten Jahre seiner Anstellung in Hechingen (1828) unternahm er in den Herbstmonaten eine größere Konzertreise, die ihn mit allerbestem Erfolge nach Oesterreich und in die bedeu tendsten Städte Deutschlands führte. Neben seinem Auf treten in München, Wien und Berlin ist vor allen Dingen sein Aufenthalt in Leipzig hervorzuheben, wo ihm bei seinem Konzerte im Gewandhaus besonders begeisterte Aufnahme zuteil wurde. Leider konnte Täglichsbeck in den nächsten Jahren seine Reisen als konzertierender Künstler nicht wieder holen. Die vielseitigen Berufsgeschäfte erforderten seinen ganzen Kräfteeinsatz. 1833 nahm er aber mit neuer Be geisterung seine kompositorische und solistische Tätigkeit wieder auf. Neben einer Anzahl kleinerer Violin- und Klavierwerke beschäftigte er sich vorwiegend mit der Komposition seiner ersten Symphonie, durch deren 65 Aufführung in den Pariser Konservatoriumskonzerten er sich höchste Ehrungen erwerben sollte. Das Musikleben in Paris, das sich zur tonangebenden Hegemoniestellung emporgeschwungeri hatte, stand zu jener Zeit in seiner höchsten Blüte. Alle Meister von Ruf und Rang strebten nach dieser Metropole, um hier vor dem strengsten Gerichte einer Weltpresse das Siegel der Bestätigung ihrer künstlerischen Qualität zu erringen. Als vortrefflicher Violinist hatte er sich dort bereits einen beifälligen Eingang erringen können. Mit besonderem Interesse sahen die Pariser Kunstrichter seinem ersten Auftreten als Komponist entgegen. Ein überraschend starker und nachhaltiger Erfolg war denn auch der ersten Aufführung seiner Es-dur Symphonie op 10 beschieden, die darauf mehrfach noch in den Konzerten des Konservatoriums erklang. Als Ausdruck für die überaus herzliche Aufnahme, die seine erste Symphonie gefunden hatte, schrieb er eine zweite in e-moll. Er wurde von der Kon zertgesellschaft zum ersten Januar 1837 zur Aufführung beider Werke nach der französischen Hauptstadt ein geladen. Leider blieb es diesmal nur bei der Einladung. Auch seine zweite Symphonie wurde gut ausgenommen und erklang darnach in vielen deutschen Konzertsälen, besonders in Leipzig. • Nach den Erfolgen mit seinem symphonischen Erst lingswerke eröffneten sich weitere Möglichkeiten zu aus wärtigen Konzertreisen. So errang er sich noch ähnlich große Erfolge in Belgien, Holland, Böhmen, Schweden und Dänemark. Nicht ohne Bedeutung für sein Schaffen war seine Vermählung (1838) mit Therese Landauer, der Tochter einer angesehenen Stuttgarter Familie. Mehrere Konzert stücke für Violine und Blasinstrumente, eine S y m p h o nie, das große Oratorium „Salomos Thron besteigun g“, sowie einige Streichquartette und Duette wurden von ihm damals komponiert. Auch meh rere Lieder verdanken dieser Zeit ihre Entstehung. Im Herbst 1839 konzertierte er wieder in den Nieder landen und in Belgien. In Amsterdam, Rotterdam. Haag, Fürst Friedrich Wilhelm Konstantin von Hohenzollern Hechingen nach einem alten Stich 66 Middelburg, Vlissingen, Antwerpen und Brüssel fand Täglichsbeck begeisterte Aufnahme. Große Verehrung erregte sein Spiel in Utrecht. Im nächsten Jahre, 1840, unternahm er eine längere Konzertreise, die ihn nach Schweden und Dänemark führte. Ueber Brandenburg, Rostock, Wismar, Lübeck, wo er überall „brillante Kon zerte“ gegeben hatte, fuhr er nach Stockholm. Sowohl hier wie auch am Hofe zu Kopenhagen erhielt er den ehrenvollen Auftrag, vor der Königsfamilie zu spielen. Nachdem er noch in Hamburg und Kassel großen Bei fall errungen hatte, kehrte er nach seinem Wohnsitz zurück. Erst im Herbst 1843 verließ er wieder für längere Zeit den fürstlichen Hof, um in Böhmen seine bisherigen Aus landserfolge fortzusetzen. Auf seiner Hinreise verweilte er auch einige Tage in seiner Heimatstadt Hof und wurde dort herzlich gefeiert. Ueber Eger und Karlsbad reiste er dann nach Prag, wo er sich mehrere Wochen aushielt. Ueberall fanden seine Konzerte lebhaftesten Beifall. Auch seine Messe wurde in Prag aufgeführt, und mit der Annahme einer Oper schienen sich günstige Aus sichten zu eröffnen. Selbst eine Berufung an das dortige Konservatorium wurde ihm angetragen, was er jedoch ablehnte. Auf direktem Wege kehrte er von Prag wieder nach Hechingen zurück. Die Verdienste, die sich Täglichsbeck durch seine großen künstlerischen Leistungen erworben hatte, be zeugte der Fürst mit der Verleihung des Hausordens, als einem sichtbaren Beweis seines persönlichen Wohl wollens. Nach den fehlgeschlagenen Bemühungen um die An nahme einer Oper am Prager Theater unternahm er 1844 eine Reise nach München. Anscheinend bemühte er sich dort erneut um das Aufführungsangebot seiner beiden großen Opern „Enzio“ und „Kaiser Heinrich IV.“ Erstere hatte bereits in Karlsruhe eine beifällige Uraufführung erlebt. Leider sind seine Bemühungen an der Münchener Bühne nicht von Erfolg gekrönt gewesen. In das folgende Jahr (1845) fällt das große Musikfest der Stadt Bonn, an dem auch Täglichsbeck teilnahm. Mit zahlreichen ihm bekannten Musikgrößen des In- und Auslandes traf er dort zusammen, so z. B. Spohr, Fetis aus Brüssel, Bacher aus Wien u. a. m. Dieses bedeutungs volle Bonner Musikfest war das letzte große Ereignis, das er von Hechingen aus miterleben durfte. Eine ehren volle Aufforderung zur tätigen Mitwirkung bei einer Ver anstaltung des Salzburger Dom-Musik-Vereins 1846 — des Gründers des Mozarteum — wäre aus dieser Zeit noch nennenswert. In Löwenberg Nach der Februarrevolution 1848 war ein gedeihliches Weiterbestehen der Hechinger Hofkapelle unmöglich ge worden. Täglichsbeck und die Orchestermitglieder wur den deshalb auf unbestimmte Zeit und mit vollem Ge halte beurlaubt. Nach Abtretung des Landes an den König von Preußen siedelte der Fürst auf seine schlesi schen Besitzungen über, um nach Rückkehr ruhigerer Verhältnisse wieder seinen eigenen musikalischen Nei gungen nachgehen zu können. Bevor Löwenberg durch die Uebersiedelung der Hof kapelle des Fürsten Konstantin sich zu dem hervorragen den künstlerischen Mittelpunkte entwickelte, hatte hier in Schlesien die Tonkunst schon liebevolle Pflege gefun den. In Plagwitz, unweit von Löwenberg, unterhielt der Baron v o n H ohberg eine ansehnliche Kapelle. Als Pianistin pflegte die Baronin von Hohberg selbst in der Kapelle mitzuwirken. Gneisenau, der berühmte Feld marschall der Freiheitskriege, gehörte zu jener Zeit einem Löwenberger Regiment als Leutnant an und war damals ein ständiger Gast dieser Konzerte. Auf Schloß Hohlstein hatte Graf Röder durch großzügige künstlerische Veranstaltungen wiederum eine Stätte ech ter Musikpflege geschaffen. Haydn und Mozart erfreuten sich bei der Kapelle besonderer Pflege. Aus unbekannten Gründen wurde sie einst aufgelöst, bald darauf aber unter der nächsten Schloßbesitzerin, der Fürstin Pauline von Hohenzollern-Hechingen, geborenen Prinzessin von Kurland, wieder übernommen. Persön lichkeiten wie der Berliner Kapellmeister Himmel, der schon bejahrte Z e 11 e r und der Freiheitsdichter Theo dor Körner zählten zu ihren ständigen Gästen. Auf dem gleichen Schlosse sollte durch den Sohn dieser kunstsinnigen Fürstin, eben den Fürsten Fr. W. Konstantin, wieder eine musikalische Blütezeit an brechen, die in ihrer überragenden Bedeutung diesen Ort zu einem Stützpunkte deutscher Kultur und neuer musikalischer Kunstbestre bungen werden ließ. Fast 4 Jahre lang war in zwischen die Kapelle in Hechingen untätig zurückgeblie ben. Im Dezember 1852 konnte das gesamte Orchester nach Schloß Hohlstein bei Löwenberg einberufen werden. Bereits vorher hatte der Fürst einige ausgezeichnete Kam mermusiker seiner ehemaligen Kapelle zu privaten Hausmusiken herangezogen. Unter der Leitung „des in der musikalischen Welt wohlbekannten Kapellmeisters Täglichsbeck" begannen dann im folgenden Jahre wieder die öffentlichen Kon zerte. Zu dem bewährten Stamm der bisherigen Orchestermitglieder waren weitere 9 ausgezeichnete Kräfte verpflichtet worden. Noch im Dezember 1852 fand das erste Konzert am fürstlichen Hofe in Löwenberg statt. Durch das 1851 erfolgte morganatische Ehebündnis mit Amalie Schenk von Geyern zu Syburg erfuhr das Hof leben durch das vergnügungsfrohe Wesen seiner Gemah lin einen besonders regen Auftrieb. Für die großen Fest lichkeiten genügten die beschränkten Schloßverhältnisse auf Hohlstein nicht mehr. Der Fürst ließ daher ein neues geräumiges Schloß in Löwenberg erbauen. An der Gartenseite dieses Gebäudes wurde ein großer Saal errichtet, der vorwiegend für die wöchentlichen musika lischen Abendveranstaltungen bestimmt war. Durch den Fürsten, der den militärischen Rang eines preußischen Generals innehatte, und die heimatliche Garnison man gelte es den Konzerten auch an äußerem prächtigen Glanze nicht. Am 7. Dezember 1853 wurde der neue Konzertsaal mit einer Wiedergabe der C-dur Symphonie von L. von Beethoven feierlichst geweiht. Die Konzerte nahmen in der Regel im November ihren Anfang und Fürstin Eugenie von Hohenzollern-Hechingen geb. Prinzessin von Leuchtenberg-Beauharnais Zum 90. Todestag am 1. September 1937 Mach einem Stich in Hechinger Privatbesitj. der die Fürstin in Florentiner Tracht zeigt dauerten bis Ende April. Bei freiem Eintritt wurden gegen Lösung einer Einlaßkarte, die jedem musi kalisch Interessierten ohne jeglichen Standesunterschied verabreicht wurde, diese wahren Volkskonzerte all wöchentlich gegeben. Als Beschützer und Vermittler ech ter und hochstehender Kunst zeigte sich hier der Fürst von größter Uneigennützigkeit und weitherziger Opfer freudigkeit. In den historischen Forschungsergebnissen des Lehrers Gondolatsch in Görlitz lesen wir in einem Aufsatze über „Fürst Konstantin von Hohenzollein und die Löwenberger Hofkapelle*: „Ich habe selbst, als ich am Anfang meiner Berufstätigkeit in der Nähe von Löwenberg angestellt war, alte Kantoren mit leuchten den Augen und beredten Worten von jener köstlichen Zeit erzählen hören. Bei dem Mangel anderer Verkehrs mittel pilgerten die begeisterten Kunstfreunde im schwar zen Anzug, der für die Herren vorgeschrieben war, stun denweit von ihren entlegenen Dörfern nach Löwenberg, vertauschten in einem Gasthause ihre festen Wander stiefel gegen ein Paar mitgebrachte salonfähige Schuhe, reinigten sich vom Schnee und Schmus der Straße und genossen ein paar Stunden die herrlichen Gaben der Kunst, die damals vollendeter und moderner an wenigen Orten des weiten Vaterlandes geboten wurden, um dann 67 in der Nacht, voll der erhabensten Eindrücke, an ihre stillen Wirkungsstätten zurückzuwandern. Dankbarere Zuhörer wird selten eine Kapelle gehabt haben, und noch nach einem Menschenalter mußte jedem empfänglichen Menschen das Herz warm werden, wenn er von der „fürstlichen Zeit“ erzählen hörte.“ Mit stiller Wehmut und verhaltenem Neid liest man die Berichte über die Feiern und Konzerte, die in Löwen berg veranstaltet wurden. Wieviel an Kulturgut und musikalischer Erziehung und Beeinflussung ist unserem Zollerstädtchen durch den Wegzug des Fürsten nach Schlesien verloren gegangen! Unter der Leitung Täglichsbeck’s hatte sich die fürst liche Kapelle zu einer ungeahnten hohen künstlerischen Leistungsfähigkeit erhoben. Der äußerst kritisch veran lagte Hans von Bülow schrieb seinerzeit, daß „die Löwenberger Kapelle getrost mit den renommiertesten Hoforchestern, wenn auch nicht quantitativ, doch was mehr sagen will, qualitativ zu rivalisieren vermöge“. Berlioz, der die berühmtesten europäischen Orchester vereinigungen persönlich geleitet hatte, schrieb nach einer Aufführung seiner Harold-Symphonie in Löwenberg nieder: „Ich kann wahrhaftig sagen, daß ich den Harold nie in unwiderstehlicherer Weise habe spielen hören.“ Leider wurden der Hofkapelle diese höchsten Lob sprüche und Anerkennungen erst nach Täglichsbecks Fort gang zuteil, als sie unter der Leitung seines Schülers Max S e i f r i z stand, allergrößtenteils waren sie aber dem großen erzieherischen Verdienste des jahrelangen und steten Arbeitseifers Täglichsbecks zu verdanken. Mit UJßilheim 68 einer 30 Mann starken Kapelle war sie im Laufe der Zeit auf 45 Künstler vergrößert worden. Werke der Klassik und der Gegenwart wurden gepflegt. Ein ge mischter Chor und die Verpflichtung namhafter Solisten trugen zu einer abwechslungsreichen Programmgestal tung bei. Eine ganz besondere künstlerische Note wiesen die Konzerte auf, die zum Namens- und Geburtstag des Fürsten, des Königs und zur Neujahrsfeier veranstaltet wurden. Auch wurde jährlich zum Besten der Löwen berger Wohltätigkeitsvereine gegen ein Eintrittsgeld von 712 Sgr. ein Konzert gegeben. Infolge der großen dienstlichen Verpflichtungen durch die Hofkapelle konnte Täglichsbeck kaum an seine eigene musikalische Schaffenstätigkeit mehr denken. Trotz der Gewährung vollkommener Ungebundenheit in den Som mermonaten fühlte er sich allmählich als strebender Künstler innerlich eingeengt und unzufrieden. Was Täglichsbeck als Leiter der Kapelle geleistet hat, das sprechen die noch vorhandenen ca. 400 Konzertpro gramme aus. Die zahlreichen Symphonien Haydns, das umfangreiche Schaffen Mozarts, die genialen Schöpfun gen Beethovens, dessen Erika und Pastoral-Symphonie in keiner Spielzeit fehlten, bildeten das Rückgrat seines musikalischen Wollens. Auch den Werken fortschritt licher Elemente brachte er Unterstützung entgegen. Karl Maria von Weber, Felix Mendelssohn-Bartholdi, Franz Schubert, Meyerbeer, Spohr, Lachner, Kalliwoda, Spontini, Kreuder, Bellini, Auber und noch andere bedeu tende Meister spielten eine wichtige und einflußreiche Rolle. Daß auch die Kompositionen der leitenden Persön Foto: Heinz Holzinger, Hechingen lichkeiten und der Kapellmitglieder, wie von Täglichsbeck, Seifrit, dem Fürsten, Wichtl und anderer zur Auf führung gelangten,zeugt von dem guten Kameradschafts geiste, der in dieser Künstlergemeinschaft herrschte. Abdankung und Nachfolge Leider scheint das innige Freundschaftsverhältnis zwi schen dem Fürsten und seinem Hofkapellmeister durch irgendwelche persönlichen Verhältnisse getrübt worden zu sein, sodaß er im Juni 1857 um seine Versetzung in den Ruhestand bat. Als Hauptgrund seines Entlassungs gesuches gab Täglichsbeck neben dem auffallend ver änderten Verhalten des Fürsten seine durch das klein städtische Leben allzusehr gehemmte künstlerische Frei heit an. Mit schwerem Herzen mußte er nun dieses Kon zertinstitut verlassen, das unter ihm entstanden war. Nach Täglichsbecks Abdankung wurde sein ehemaliger Schüler Max Seifriz zum Intendanten und Dirigen ten der fürstlichen Hofmusik berufen. Die engen Bezie hungen dieses Künstlers zu Wagner und Liszt ließen dieses Hoforchester zu einem Stützpunkt der neudeutschen Kunstbestrebungen werden. Die zahlreich vorhandenen Konzertzettel zeigen, wie stark jetzt die Kompositionen von Liszt, sowie die von Richard Wagner und Hektor Berlioz in den Vordergrund traten. Bereits vor ihrer Ur aufführung gelangten von „Tristan und Isolde“ das Vor spiel, der Schlußsatz dieses Musikdramas und das „Meistersinger“-Vorspiel in den Konzerten zu Löwenberg zu Gehör. Berlioz und Wagner weilten sogar persönlich dort und bildeten den künstlerischen Höhepunkt der Saison; auch fehlten die berühmtesten Solisten und Sänger nicht unter den Gästen. Im Ruhestand Nachdem Täglichsbeck mit dem dekretmäßig zukom menden lebenslänglichen Gehalte in den Ruhestand ver setzt worden war, verließ er Löwenberg und suchte in Dresden die Verwirklichung seiner Wünsche und Hoffnungen. Aussichten auf Anstellung in Magdeburg und Aachen eröffneten sich ihm, doch schlug er die An gebote aus. Da ihm die Möglichkeiten auf zufriedenstel lende Beschäftigung fehlten und seine Bemühungen um die Aufführungen seiner Opernwerke am dortigen Thea ter fehlschlugen, wandte er sich bereits nach einem Jahre wieder seinem geliebten München zu, der Stadt seiner ersten Erfolge. In ruhiger Zurückgezogenheit lebte er ganz seiner Familie und leitete die geistige und künst lerische Ausbildung seiner geliebten Tochter. Zum größ ten Weh der Eltern wurde sie ihnen im Alter von 20 Jahren durch den Tod entrissen. Das war für Täglichs beck der härteste Schlag und erst nach Jahren fand er in der Kunst wieder erhebenden Trost. Innere Genugtuung sollte ihm durch seinen Fürsten widerfahren, der ihn zum Frühjahr 1862 zu sich nach Löwenberg einlud. In überaus herzlicher Weise wurde er vom Fürsten empfangen und gleich groß war auch die Freude des Wiedersehens bei den Mitgliedern der Hof kapelle. Hochgeehrt als Mensch und Künstler schied Täg lichsbeck nach mehrwöchentlichem Aufenthalte in seeli scher Harmonie vom Fürsten und der Stätte seiner einsti gen Wirksamkeit. Ausklang in Löwenberg Der Glanz, der die kleine schlesische Stadt Löwenberg zu einem Mittelpunkte musikalisch bedeutungsvollster Ereignisse werden ließ, sollte nach Täglichsbecks Fort ganz nicht mehr allzulange bestehen. Nachdem durch die modernen Bestrebungen der Neudeutschen das dortige Musikleben noch einen besonderen Auftrieb und Zuzug namhafter Künstler erfahren hatte, versank mit dem Hinscheiden des Fürsten plötzlich das ganze künstlerische Leben. „Mit ihm verlor die Menschheit eine Persönlich keit, die in ihrer edlen und großen Gesinnung während ihrer segensreichen Tätigkeit sich als Diener, Förderer und Kämpfer der Kunst unermeßliche Verdienste erwor ben hat.“ Die letzten Lebenstage Täglichsbeck unternahm nach seiner Rückkehr aus Lö wenberg von München aus mit seiner Gattin noch einige Reisen nach Italien und der Schweiz. Aus klimatischen Rücksichten siedelte er 1866 nach Baden-Baden über, wo er am 5. Oktober 1867 an Typhus und einer hinzugetretenen Lungenlähmung verschied. Der Leichnam wurde dem Wunsch des Verstorbenen entsprechend nach München überführt und auf dem Südfriedhof an der Seite seines Kindes zur ewigen Ruhe bestattet. Literaturangabe: „Thomas Täglichsbeck und seine Instrumental kompositionen.“ Ein Beitrag zur Geschichte der Instrumentalmusik des 19. Jahrhunderts von Dr. Edgar Burmester, Inaugural-Dissertation zur Erlangung der Doktorwürde der Universität zu München. Aus her Seichichte der Waldungen der früheren Sraffchaft Sollern Von F. G ä ß 1 e r - Thanheim 2. Fortsetzung. Waldgeschichte ist zunächst Bestandsgeschichte. Die Feststellung und Kennzeichnung der Reihenfolge der Holzarten, die Vorläufer unseres heutigen Waldes waren, führt weiter zurück, als alle menschliche Kultur reicht. Durch die pollenanalytischen Untersuchungen der Hochmoore des Albgebietes sind wir über die Wachs tumsvorgänge der Hochfläche unterrichtet, durch die natürlich auch die Hanglagen entscheidend beeinflußt wurden. Ueber diese Vorgänge schreibt Dr. K u h n - Hechingen in seinem Werk: „Die Pflanzengesellschaften im Neckargebiet der Schwäbischen Alb“ Seite 233 folgen des: In der Eiszeit waren von der Alb-Hochfläche sämtliche Bäume verschwunden, während sich in den tie feren Lagen am Fuße der Alb Bergkiefer, Birke und Weide gehalten haben. Die einzigen Holzarten, die in der Eiszeit sicher noch auf der Albhochfläche standen, sind Zwergbirken und Heidekrautgewächse. Nach der Eiszeit wanderten zuerst die Bergkiefer, später die Weiß 69
https://openalex.org/W4391677705	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0297954&type=printable	English	null	How previous experience shapes future affective subjective ratings: A follow-up study investigating implicit learning and cue ambiguity	PloS one	2,024	cc-by	14,245	PLOS ONE PLOS ONE RESEARCH ARTICLE Introduction Funding: The study was supported by a grant from Ministero dell’Istruzione Università e Ricerca (MIUR) (Dipartimenti di Eccellenza DM 11/05/2017 n. 262) to the Department of General Psychology. The funders had no role in study design, data How previous experience shapes future affective subjective ratings: A follow-up study investigating implicit learning and cue ambiguity Fiorella Del Popolo CristaldiID1, Giulia Buodo1, Filippo Gambarota2, Suzanne OosterwijkID3,4, Giovanni Mento1,5 1 Department of General Psychology, University of Padua, Padua, Italy, 2 Department of Developmental Psychology and Socialization, University of Padua, Padua, Italy, 3 Department of Social Psychology, University of Amsterdam, Amsterdam, The Netherlands, 4 Amsterdam Brain and Cognition Centre (ABC), Amsterdam, The Netherlands, 5 Scientific Institute, IRCCS E. Medea, Conegliano, Treviso, Italy a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 fiorella.delpopolocristaldi@unipd.it Editor: Fang Wang, Wilfrid Laurier University, CANADA Received: April 19, 2023 Accepted: January 11, 2024 Published: February 9, 2024 Received: April 19, 2023 Accepted: January 11, 2024 Published: February 9, 2024 Copyright: © 2024 Del Popolo Cristaldi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All materials, data and analysis code reported in this manuscript have been made publicly available on OSF and can be accessed at the links https://osf.io/gdr3b/ and https://osf.io/z5esb/. OPEN ACCESS People use their previous experience to predict future affective events. Since we live in ever- changing environments, affective predictions must generalize from past contexts (from which they may be implicitly learned) to new, potentially ambiguous contexts. This study investi- gated how past (un)certain relationships influence subjective experience following new ambiguous cues, and whether past relationships can be learned implicitly. Two S1-S2 para- digms were employed as learning and test phases in two experiments. S1s were colored cir- cles, S2s negative or neutral affective pictures. Participants (Experiment 1 N = 121, Experiment 2 N = 116) were assigned to the certain (CG) or uncertain group (UG), and they were presented with 100% (CG) or 50% (UG) S1-S2 congruency during an uninstructed (Experiment 1) or implicit (Experiment 2) learning phase. During the test phase both groups were presented with a new 75% S1-S2 paradigm, and ambiguous (Experiment 1) or unam- biguous (Experiment 2) S1s. Participants were asked to rate the expected valence of upcom- ing S2s (expectancy ratings), or their experienced valence and arousal (valence and arousal ratings). In Experiment 1 ambiguous cues elicited less negative expectancy ratings, and less unpleasant valence ratings, independently of prior experience. In Experiment 2, both groups showed similar expectancies, predicting upcoming pictures’ valence according to the 75% contingencies of the test phase. Overall, we found that in the presence of ambiguous cues subjective affective experience is dampened, and that implicit previous experience does not emerge at the subjective level by significantly shaping reported affective experience. People use their previous experience to predict future affective events. Since we live in ever- changing environments, affective predictions must generalize from past contexts (from which they may be implicitly learned) to new, potentially ambiguous contexts. This study investi- gated how past (un)certain relationships influence subjective experience following new ambiguous cues, and whether past relationships can be learned implicitly. Two S1-S2 para- digms were employed as learning and test phases in two experiments. S1s were colored cir- cles, S2s negative or neutral affective pictures. Participants (Experiment 1 N = 121, Citation: Del Popolo Cristaldi F, Buodo G, Gambarota F, Oosterwijk S, Mento G (2024) How previous experience shapes future affective subjective ratings: A follow-up study investigating implicit learning and cue ambiguity. PLoS ONE 19(2): e0297954. https://doi.org/10.1371/journal. pone.0297954 Editor: Fang Wang, Wilfrid Laurier University, CANADA Editor: Fang Wang, Wilfrid Laurier University, CANADA * fiorella.delpopolocristaldi@unipd.it Emotions as predictions People use previous experience to predict future affective events. For example, imagine that your neighbor has a medium-sized dog with brown spotted fur that always snarls at you, PLOS ONE \| https://doi.org/10.1371/journal.pone.0297954 February 9, 2024 1 / 24 PLOS ONE Implicit learning, cue ambiguity and affective predictions causing you fear and annoyment. Imagine now that you are having lunch with your best friend, who is introducing you to their recently adopted little dog. As the dog enters the room running briskly, you seem to see that his fur is brownish, and perhaps spotted. Does your past annoying experience with your neighbor’s dog make you think that your best friend’s new dog is extremely unfriendly, just because the two dogs look alike? When situations like this happen in real life, predictions play a crucial role. Based on your past experiences your brain general- izes the concept of unfriendliness, developed on your neighbor’s dog, also to new, perceptually similar dogs, predisposing you to feel fear and annoyment. All these computations occur quickly and without conscious awareness. In other words, affective predictions (and the associ- ated subjective experience) must generalize from the specific features of past contexts (from which they are implicitly learned) to new and potentially ambiguous contexts (i.e., contexts that look alike past ones, but have different perceptual features), to be effective in promoting survival and allostatic balance with the environment [1,2]. However, the specific mechanisms by which this process occurs remain unclear. collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. According to predictive models of emotion [3,4], affective predictions are constructed along three distinct neurocomputational stages: prediction generation, in which prior experi- ence is combined with present information to construct affective predictions; prediction implementation, in which predictions are used to pre-arrange the best action plans to deal with the expected situation; and prediction updating, in which current environmental inputs are compared with predictions, and in case of mismatch the unexpected information (encoded as a prediction error) acts as a feedback to adjust subsequent predictions. PLOS ONE \| https://doi.org/10.1371/journal.pone.0297954 February 9, 2024 Emotions as predictions Predictive models are assumed to be (i) probabilistic, since they encode the statistical regularities within the observed inputs [1,2,5]; (ii) generative, because they generalize across sensory modalities, contexts, and time [1,2]; and (iii) implicit, since they act mostly outside of awareness, potentially emerging into it only in case of a prediction violation [6]. It follows that human brains do not merely react to affective stimuli at the time of their occurrence, but rather, as a spontaneous activity, they are constantly busy in generating-implementing-updating predictions in the service of allostasis [7,8]. Prediction construction is assumed to represent the core mechanism on which the brain relies, in order to provide the body with optimal resources for growth, adaptation to the envi- ronment, and survival [1,5,7–9]. Previous experience (i.e., knowledge derived from the extrac- tion of statistical regularities of stimuli occurrence in the past) plays a pivotal role in the construction of new affective predictions: it interacts with momentary information (e.g., physi- cal properties of the environment, contingencies experienced between present stimuli) in con- straining and refining the pool of information used to generate predictions [1,3,5,6]. However, predictive processing’s assumptions about (i) whether contingency learning (i.e., extracting statistical regularities of stimuli co-occurrence) may develop implicitly and (ii) whether these learnings generalize to new, and potentially ambiguous contexts are to date mainly based on empirical evidence related to cognitive domains such as visual perception [10–12] or motor control [13,14]. Little experimental evidence about the application of these assumptions to the affective domain has been collected so far. To better understand how affective predictions are related to subjectively experienced affec- tive states, predictive models of emotion [3,4] have reconceptualized subjective affective expe- rience within their framework. Subjectively experienced affective states have been defined as the representation of valence (i.e., pleasantness/unpleasantness), arousal (i.e., activation/calm) or even discrete emotions (e.g., anger, fear) in subjective awareness, on which an individual can verbally self-report [15]. Predictive models of emotion [3,4] assume that affective experi- ence derives from the brain’s spontaneous predictive activity, and have redefined it as the active process of making meaning of present stimuli by predicting and categorizing them on PLOS ONE \| https://doi.org/10.1371/journal.pone.0297954 February 9, 2024 2 / 24 PLOS ONE Implicit learning, cue ambiguity and affective predictions the basis of past experiences [3]. Emotions as predictions When this process draws on conceptual emotion knowledge, the resulting predictive models and their associated affective responses can be subjectively experienced as emotions [3,16,17]. It follows that subjective affective experience may be sensi- tive to statistical regularities experienced in the past. For example, the more reliable the regu- larities, the more expectancies about the valence of upcoming stimuli will draw on them to predict upcoming stimuli [18]. However, it is still unknown whether previously learned proba- bilities may influence subjective affective experience when facing new, potentially ambiguous cues (i.e., ambiguous signals preceding the occurrence of an affective stimulus). Also, it is not clear if affective environmental contingencies might be inferred from past experience at an implicit level (i.e., without explicit awareness, and without focusing attention on contingencies themselves at the time of their occurrence), and if this may ultimately shape subjective affective experience during future predictions. The present research aimed to test whether these two crucial assumptions of predictive processing models apply to the affective domain by employ- ing a methodological replication approach. We draw on our previous work [18] to investigate (i) if being exposed to past (un)certain contingencies might influence future subjective affec- tive ratings to new and potentially ambiguous cues (i.e., cues with different perceptual proper- ties from the ones previously experienced); and (ii) if people are able to implicitly extract (un) certain probabilistic information, and use it later to subjectively predict new affective events. PLOS ONE \| https://doi.org/10.1371/journal.pone.0297954 February 9, 2024 A novel experimental paradigm To study the effects of previous experience on affective predictions we recently developed a novel experimental paradigm [18], which integrates the logic of traditional emotional S1-S2 paradigms (see [19] for a review) with an uninstructed learning component. In this novel para- digm two separate emotional S1-S2 paradigms are employed as a learning and test phase, respectively. As in typical emotional S1-S2 paradigms, a sequential presentation of two stimuli is implemented (both in the learning and test phases): the S1 (or cue) is a symbolic stimulus (manipulated at two levels, i.e., red or blue circles) preceding the occurrence of an affective stimulus, while the S2 (or target) is an emotional stimulus (manipulated at two levels, i.e., neg- ative or neutral affective pictures). The sequence of events in the S1-S2 paradigm allows to tar- get the three stages of affective prediction construction: the S1 reflects the generation stage, the inter-stimulus interval (ISI) between S1 and S2 the implementation stage, and the S2 the updat- ing stage [20]. The contribution of our new paradigm is that it manipulates actual previous experience through uninstructed certain vs. uncertain probabilistic contingencies between S1 and S2, experienced during a separate learning phase. This approach is markedly different from extant S1-S2 paradigms that manipulate explicitly labeled probabilistic information while participants perform the task. During the learning phase of this paradigm, previous experience is manipulated between subjects by dividing participants into two experimental groups: the certain group (CG) and the uncertain group (UG). According to the group, they are presented with a 100% (CG) or 50% (UG) S1-S2 congruency, namely the probabilistic ratio between S1 color and S2 valence they are exposed to. During the test phase, all participants are then presented with a new S1-S2 par- adigm with a fixed 75% S1-S2 congruency (see [18] and below for a detailed explanation of the choice to use the 75% S1-S2 ratio in the learning phase). In the test phase, they are asked to rate their subjective affective experience in terms of either the expected valence of upcoming S2s (expectancy ratings), or the experienced valence and arousal to S2s (valence and arousal ratings). Participants are left uninstructed about the probabilistic ratios they are exposed to during the whole paradigm. 3 / 24 PLOS ONE \| https://doi.org/10.1371/journal.pone.0297954 February 9, 2024 PLOS ONE Implicit learning, cue ambiguity and affective predictions Inconsistent evidence on subjective affective experience Extant literature has collected inconsistent evidence with regard to the effect of (un)certain stimulus predictability on subjective affective ratings. As for expectancy measures related to the generation-implementation stages, our previous study [18] showed that experiencing cer- tain contingencies (100%) during the learning phase subsequently elicited more extreme expectancy ratings (i.e., participants predicted the valence of future stimuli according to previ- ously learned contingencies), and this effect generalized from the visual to the auditory sensory modality. Other studies implementing typical S1-S2 paradigms found negatively-biased expec- tancies (i.e., an overestimation of negative S2s occurrence) in the uncertain (50%) condition [21–25]; and that ambiguous cues (i.e., cues with uninstructed 50% predictive value) elicited less negative expectancy ratings than unambiguous cues [26]. Regarding valence and arousal ratings measured during the updating stage, we did not find any effect of previous experience [18], consistently with some studies implementing typical S1-S2 paradigms that found no effect of current (un)certainty on valence ratings [21– 24,27,28]. Other traditional S1-S2 studies, instead, showed a more intense subjective experi- ence either in the certain (100%) [27,29–32] or in the uncertain (50%) condition [33], and also that ambiguous cues elicited more unpleasant mood ratings [26]. Another study [23] com- pared an explicit anticipation condition (in which participants were asked for expectancy rat- ings) with an implicit anticipation condition (in which participants were asked for a target detection task): no effects of cue predictive meaning (100% vs. 50%) emerged on accuracy to the target detection task, faster reaction times (RTs) were found in the certain condition, and no significant results were found on S2-valence ratings. PLOS ONE \| https://doi.org/10.1371/journal.pone.0297954 February 9, 2024 Method Experiment 1 investigated whether (un)certain past experience might influence future subjec- tive affective ratings as a function of cue ambiguity. We pre-registered the study on Open Sci- ence Framework (OSF) (https://osf.io/gdr3b/). According to predictive models of emotion [3,4], we can formulate the hypothesis (H1) that participants exposed to reliable contingencies in the learning phase (i.e., the CG) would generalize the learned information to the ambiguous cues of the test phase, thus showing more negative expectancy ratings after the cues that are more perceptually similar to those previ- ously paired with negative pictures. As no extant study directly investigated the interaction between past (un)certainty and cue ambiguity, we tested whether cue predictive meaning modulated expectancies as an exploratory analysis. Moreover, according to previous literature [26] and to what specified in the pre-registration, we expected that ambiguous cues would elicit (H2) less strong generalization effects on expectancy ratings as compared to unambigu- ous cues and (H3) more unpleasant valence ratings. We also tested (H4) whether cue ambigu- ity modulates arousal ratings as a function of previous experience. Participants. We used the provider platform Prolific (Prolific, Oxford, UK; www.prolific. co) to recruit 125 adult participants in August 2021. Participants were screened for vision diffi- culties, including color blindness. Researchers had access only to the following participant’s personal information: Prolific IDs, age, gender, country of birth, country of residence, nation- ality, employment status and languages spoken. Thus, no identifying information was pro- vided and collected. We estimated the required sample size through an a priori pre-registered (https://osf.io/gdr3b/) simulation-based power analysis for generalized linear mixed-effects models (GLMMs) (R package: simr; [34]). We estimated parameters from data of our previous study (Experiment 1, N = 185 [17]). Pre-registered exclusion criteria were the following: scor- ing lower than 75% accuracy on attention check items (see below) (N of discarded partici- pants = 0), reporting experienced technical issues in more than 25% of the experimental trials (N = 1). We also excluded data from 3 participants because of data collection failure. The final sample included 121 participants (58 males, age: M = 25.03, SD = 7.67, range = 18–58; CG N = 57, UG N = 52). All participants gave their written informed consent before starting the experiment, and were paid £1.93 for their participation. All experimental procedures were conducted in accordance with the Declaration of Helsinki, and approved by the local Ethical Committee (protocol no. 4177). A follow-up research Overall, evidence on the effects of past knowledge on subjective affective experience remains fragmentary, with our previous work [18] suggesting that a reliable (i.e., certain) previous experience affects future expectancies, and S1-S2 studies suggesting that either certain [27,29– 32] or uncertain [21–26,30,33] contingencies can lead to an intensification of the related affec- tive experience. Besides, little is known yet about the potential effects of cue ambiguity on new affective predictions, nor on the actual possibility to infer affective environmental contingen- cies implicitly. Both these factors are nonetheless crucial to construct efficient affective predic- tions. In fact, since our environments are characterized by frequent changes, affective predictions must be flexible in adapting to new, and potentially ambiguous, contextual features [3,6,8]. Moreover, in daily life people should be able to spontaneously learn contingencies from the environment, and to use them as priors for subsequent affective predictions, even in absence of explicit instructions and/or awareness of the contingencies themselves [6]. How- ever, despite the importance of both of these aspects for constructing efficient predictions, it still lacks solid experimental evidence on how they may influence subjective affective experience. Based on this, in the present research we implemented two follow-up experiments (pre-reg- istered on the Open Science Framework—OSF; Experiment 1: https://osf.io/gdr3b/, Experi- ment 2: https://osf.io/z5esb/), in which we modified some features of our former paradigm [18]. In particular, in order to investigate the construction of new affective predictions as a function of cue ambiguity, in Experiment 1 we introduced ambiguous cues in the test phase. Here, unbeknownst to participants, we presented two new reddish and bluish S1 colors (i.e., coral and turquoise, here defined as ambiguous) in addition to those already presented during the learning phase (i.e., red and blue, here defined as unambiguous). Further, to test if the probabilistic information available in the environment can be extracted and learned at an 4 / 24 PLOS ONE \| https://doi.org/10.1371/journal.pone.0297954 February 9, 2024 PLOS ONE Implicit learning, cue ambiguity and affective predictions implicit level (i.e., not explicitly focusing attention on the probabilistic relationships between stimuli), in Experiment 2 we engaged participants in a distracting task (i.e., a parity judgment task) during the learning phase. A follow-up research According to predictive models of emotion [3,4], we hypothesized that participants would generalize previously learned contingencies to new ambiguous cues in Experiment 1, and that they would infer affective environmental contingencies without explicitly focusing attention (and use them later to predict future stimuli) in Experiment 2. PLOS ONE \| https://doi.org/10.1371/journal.pone.0297954 February 9, 2024 Method Stimulus material and procedure. We employed two emotional S1-S2 paradigms as learning and test phase, respectively (as in [18]). In both phases, unambiguous S1s were red and blue 1-cm-diameter circles. In the test phase only, new ambiguous S1s were introduced unbeknownst to participants: same-sized reddish and bluish (i.e., coral- and turquoise-col- ored) circles. In both learning and test phases, S2s were colored 800 × 600 px pictures from Nencki Affective Picture System (NAPS) [35], whose valence was manipulated at two levels: negative (Neg; e.g., dead animals, injured people, human threat/war scenes) vs. neutral (Neu; 5 / 24 PLOS ONE \| https://doi.org/10.1371/journal.pone.0297954 February 9, 2024 PLOS ONE Implicit learning, cue ambiguity and affective predictions e.g., urban or natural landscapes, people jogging or sitting, animals standing still) (see S1 Table for a list of NAPS pictures employed as S2s). Negative and neutral pictures did not differ in luminance, contrast, complexity and color space indices (see S2 Table), and each picture con- tent (i.e., animals, faces, landscapes, objects, people) was equally represented within each valence level. In attention check trials, a 1-cm yellow circle was used as S1, while a 800 × 600 px picture of a black stripes pattern displayed on a transparent background was used as S2. The expected valence of upcoming S2s (expectancy ratings) and subjective affective responses to S2s (valence and arousal ratings) were assessed through three distinct Visual Analogue Scales (VASs) ranging from 0% to 100%. In the expectancy VAS 0% corresponded to “I defi- nitely expect to see a neutral picture”, 50% represented not knowing what to expect, and 100% corresponded to “I definitely expect to see a negative picture”. In the valence VAS 0% repre- sented “very negative” valence, 50% “neutral” valence, and 100% “very positive” valence. In the arousal VAS 0% meant “relaxed”, 50% represented an intermediate level of activation, and 100% meant “aroused”. We ran the experiment online, through OpenSesame [36] and the JATOS hosting server [37]. Participants were asked to run the study on a computer, to sit alone in a silent and private room, and to avoid distractions and interruptions, in order to ensure optimal conditions for participation. They were also asked to avoid that someone else could view their screen during the experiment, due to the involvement of emotionally salient material. Before the learning phase, participants were randomly assigned to the certain group (CG) or the uncertain group (UG). Method At the beginning of the learning phase, participants received the following instructions: they were asked to look at the screen and pay attention to the relation- ship between S1 color and S2 valence, and they were asked to press the ‘spacebar’ as fast as they could each time they saw a yellow circle (attention check trials). A practice session of 4 tri- als followed the instructions: here, participants received feedback on their performance to one attention check trial. After the practice, the learning session started. In each trial S1 was pre- sented first for 250 msec, and it was displayed on a gray background. This was followed by a fixed interstimulus interval (ISI) of 1000 msec, in which the screen remained gray. Then, the S2 was presented for 1000 msec. A white fixation cross was displayed in the center of the screen during the inter-trial interval (ITI), whose length randomly varied between 800 and 1200 msec. The total number of learning trials was 40, presented in random sequence. During this phase, CG participants were exposed to a certain predictive relationship between S1 and S2: each S1 color (i.e., red and blue) was paired with the same S2 valence in 100% of the trials (100% S1-S2 congruency). UG participants, instead, were presented with an uncertain rela- tionship: each S1 color was paired with negative S2s in 50% of the trials, and with neutral S2s in the other 50% (50% S1-S2 congruency). Color-valence pairings were counterbalanced between subjects, and participants were left uninstructed about the S1-S2 predictive ratios. After the learning phase, a 1-minute interval followed in which participants were asked to wait and relax. Then, both groups were introduced to the same test phase. Instructions of the test phase were to look at the screen and try to predict S2 valence based on S1 color. Partici- pants were told that in some trials they would be asked their expectancy (i.e., to rate how much they expected to see a negative picture after the S1) on a 0–100% scale; while in other trials they would be asked their subjective valence and arousal to the S2, on a 0–100% scale for each dimension. They were asked to give either their expectancy ratings during the ISI, or their valence and arousal ratings right after the S2. PLOS ONE \| https://doi.org/10.1371/journal.pone.0297954 February 9, 2024 Method VASs response times were self-paced, and the two rating conditions were balanced for the number of trials (50%:50%) and randomly deliv- ered. We presented the expectancy and the valence/arousal ratings in different trials to prevent the two kinds of ratings from influencing each other. Participants were lastly reminded to press the ‘spacebar’ as fast as they could each time they saw the yellow circle (attention check). 6 / 24 PLOS ONE \| https://doi.org/10.1371/journal.pone.0297954 February 9, 2024 PLOS ONE Implicit learning, cue ambiguity and affective predictions After the instructions, participants performed a practice session of 4 trials, in which they were trained to give their ratings, and received performance feedback on a single attention check. Then the test session started, with the same trial structure and timing as the learning phase, and a total number of 80 trials. The order of the trials was randomized. Cue color was manipu- lated within participants at four levels: red, blue, coral, and turquoise. Test trials in which we employed the same S1 colors as the learning phase were considered unambiguous (N = 40), whereas test trials in which we employed the new reddish and bluish colors were considered ambiguous (N = 40). Cue ambiguity, thus, included two levels: unambiguous (red and blue S1s) vs. ambiguous (coral and turquoise S1s). Participants were not warned about the exposure to new colors. In the test phase, S1 color was moderately predictive of S2 valence, since S1-S2 congruency was fixed at 75% (same S1 color-S2 valence pairings in 75% of the trials). Color- valence pairings were counterbalanced between subjects, and participants were left unin- structed about the S1-S2 predictive ratio. Notably, the new 75% ratio implied that CG partici- pants moved from a reliable predictive relationship (100%, experienced during the learning phase), to a new, more uncertain context (in which the previously learned predictive models were sometimes violated). UG participants, instead, moved from an unreliable predictive rela- tionship (50%), to a more predictive context. Moreover, the 75% is equidistant from the pre- dictive ratios experienced by the CG and the UG (it lies in the middle between 100% and 50%; see [18] for further details on this choice). Fig 1A shows a schematic representation of the experimental paradigm. After the test phase, participants were redirected to a Qualtrics survey (Qualtrics, Provo, UT; www.qualtrics.com), in which they were asked demographic information (age, gender) and some mood and trait measures. Method In particular, participants completed the Intolerance of Uncertainty Scale (IUS-12) [38] and the Depression, Anxiety and Stress Scale (DASS-21) [39], as intolerance of uncertainty as well as negative affectivity are known to be potential moderat- ing factors impacting affective predictions [40–44]. Also, participants were asked a forced- choice question about whether and in how many trials they had experienced any technical issues with the Internet connection and/or with pictures uploading (response options: “No, everything worked fine!”, “Yes, in less than 25% of the trials”, “Yes, between 25% and 50% of the trials”, “Yes, between 50% and 75% of the trials”, “Yes, in more than 75% of the trials”). At the end of the survey, participants were thanked, and redirected to Prolific to receive their pay- ment. The experiment lasted about 30 minutes. PLOS ONE \| https://doi.org/10.1371/journal.pone.0297954 February 9, 2024 Data analysis The study had a 2 (group, between-subjects: CG vs. UG) × 2 (cue ambiguity, within-subjects: ambiguous vs. unambiguous) × 2 (S2 valence, within-subjects: Neg vs. Neu) mixed design. The analysis plan was pre-registered on OSF (https://osf.io/gdr3b/). As pre-registered, univariate outliers (i.e., expectancy ratings) were detected through Median Absolute Deviation values (MAD > 3), and multivariate outliers (i.e., valence and arousal ratings) through the Mahalanobis-Minimum Covariance Determinant (MMCD, breakdown point 0.25) (R package: Routliers; [45]). We identified 12 univariate outliers, and we removed them from data analysis. We also identified 21 multivariate outliers. However, from the visual inspection of their ratings, they emerged as “potentially interesting outliers” (see [45]), since they showed only a slightly different relationship between valence and arousal ratings as compared to other participants. Thus, for this reason and given that none of them significantly impacted the models’ estimates (as assessed through Cook’s distance, see below), we chose to keep their data into the analysis. Overall, data from 109 participants were included in the analyses. 7 / 24 PLOS ONE \| https://doi.org/10.1371/journal.pone.0297954 February 9, 2024 PLOS ONE Implicit learning, cue ambiguity and affective predictions Fig 1. a) Example sequence of events and their duration for a trial of Experiment 1, according to the phase (learning, test), and the group (CG, UG). During the learning phase participants experienced a 100% (CG) vs. 50% (UG) affective contingency between S1 color (red or blue) and S2 valence (Neg or Neu), according to group assignment. During the test phase all participants were presented with unambiguous (i.e., red or blue) and ambiguous (i.e., coral or turquoise) S1s, and the S1-S2 affective contingency was fixed at 75%. Participants were asked to answer the VASs either during the ISI for ournal.pone.0297954 February 9, 2024 8 / 24 Fig 1. a) Example sequence of events and their duration for a trial of Experiment 1, according to the phase (learning, test), and the group (CG, UG). During the learning phase participants experienced a 100% (CG) vs. 50% (UG) affective contingency between S1 color (red or blue) and S2 valence (Neg or Neu), according to group assignment. During the test phase all participants were presented with unambiguous (i.e., red or blue) and ambiguous (i.e., coral or turquoise) S1s, and the S1-S2 affective contingency was fixed at 75%. Participants were asked to answer the VASs either during the ISI for Fig 1. PLOS ONE \| https://doi.org/10.1371/journal.pone.0297954 February 9, 2024 https://doi.org/10.1371/journal.pone.0297954.g001 To test our hypotheses (H1, H2, H3, H4), we fitted the following linear mixed-effects mod- els (LMMs) for each dependent variable (DV) (R package: lme4; [46]): 1. expectancy ratings (H1): group, cue color and their interaction as fixed factors, and random slopes for cue color within participants; 1. expectancy ratings (H1): group, cue color and their interaction as fixed factors, and random slopes for cue color within participants; 2. expectancy ratings (H2): group, cue ambiguity and their interaction as fixed factors, random slopes for cue ambiguity within participant; 3. valence ratings (H3): group, cue ambiguity, S2 valence and their interaction as fixed factors, random slopes for S2 valence within participant; 4. arousal ratings (H4): group, cue ambiguity, S2 valence and their interaction as fixed factors, random slopes for S2 valence within participant. The factor “cue ambiguity” codes for the ambiguity of the cue (within-subjects: ambiguous —that is, coral or turquoise S1 color vs. unambiguous—that is, red or blue S1 color). In the analysis script (shared in OSF https://osf.io/gdr3b/) this factor was called “cue”. Here, for the sake of clarity, we renamed it as “cue ambiguity”. The factor “cue color” codes for the predic- tive meaning of the cue according to the specific color-valence pairings presented to partici- pants in the test phase (within-subjects: red and coral—that is, a cue that preceded negative stimuli in the test phase vs. blue and turquoise—that is, a cue that preceded neutral stimuli in the test phase). In the analysis script (shared in OSF https://osf.io/gdr3b/) this factor was called “S1 color”. Here, for the sake of clarity, we renamed it as “cue color”. Please note that to com- pensate for the counterbalancing of S1 color–S2 valence pairings, in the analysis we re-coded as red all the unambiguous cues preceding negative pictures, as coral all the ambiguous cues preceding negative pictures, as blue all the unambiguous cues preceding neutral pictures, and as turquoise all the ambiguous cues preceding neutral pictures, irrespective of the actual color of the cue (see [18] for further details on the cue factor). The other pre-registered exploratory analyses regarding the effects of time, S2 congruency (expected vs. unexpected S2s), IUS, and DASS-21 questionnaires are reported in S7–S8 Tables. https://doi.org/10.1371/journal.pone.0297954.g001 Since the results of these analyses are mainly null, or redundant with respect to both the confir- matory models and the results of our previous work [18], these analyses will not be discussed further in the present manuscript. As pre-registered, for each model we evaluated influential cases through Cook’s distance (>1). No influential cases emerged. As pre-registered, models effects were tested by means of F-test and p-values, calculated via Satterthwaite’s degrees of freedom method (α = .05, R package: lmerTest; [47]). For each model we reported the esti- mated parameters with 95% confidence intervals (CI), marginal and conditional R2 (estimated as in [48]). Data analysis a) Example sequence of events and their duration for a trial of Experiment 1, according to the phase (learning, test), and the group (CG, UG). During the learning phase participants experienced a 100% (CG) vs. 50% (UG) affective contingency between S1 color (red or blue) and S2 valence (Neg or Neu), according to group assignment. During the test phase all participants were presented with unambiguous (i.e., red or blue) and ambiguous (i.e., coral or turquoise) S1s, and the S1-S2 affective contingency was fixed at 75%. Participants were asked to answer the VASs either during the ISI fo PLOS ONE \| https://doi.org/10.1371/journal.pone.0297954 February 9, 2024 8 / 24 PLOS ONE Implicit learning, cue ambiguity and affective predictions half of the trials (expectancy ratings), or right after the S2 for the other half of the trials (valence and arousal ratings). Response times were self-paced. b) Example sequence of events and their duration for a trial of Experiment 2, according to the phase (learning, test), and the group (CG, UG). During the learning phase participants experienced a 100% (CG) vs 50% (UG) affective contingency between S1 color (red or blue) and S2 valence (Neg or Neu), according to group assignment. Then, after the S2, they were presented with the parity judgment task. During the test phase the S1-S2 affective contingency was fixed at 75%. Participants were asked to answer the VASs either during the ISI for half of the trials (expectancy ratings), or right after the S2 for the other half of the trials (valence and arousal ratings). Response times were self-paced. ISI = inter-stimulus interval, ITI = inter-trial interval, VAS = visual analogue scale. The text and the pictures are not drawn to scale. half of the trials (expectancy ratings), or right after the S2 for the other half of the trials (valence and arousal ratings). Response times were self-paced. b) Example sequence of events and their duration for a trial of Experiment 2, according to the phase (learning, test), and the group (CG, UG). During the learning phase participants experienced a 100% (CG) vs 50% (UG) affective contingency between S1 color (red or blue) and S2 valence (Neg or Neu), according to group assignment. Then, after the S2, they were presented with the parity judgment task. During the test phase the S1-S2 affective contingency was fixed at 75%. Data analysis Participants were asked to answer the VASs either during the ISI for half of the trials (expectancy ratings), or right after the S2 for the other half of the trials (valence and arousal ratings). Response times were self-paced. ISI = inter-stimulus interval, ITI = inter-trial interval, VAS = visual analogue scale. The text and the pictures are not drawn to scale. Descriptive statistics are provided in Table 1. PLOS ONE Table 1. Descriptive statistics of Experiment 1. Group Cue ambiguity S2 valence Expectancy ratings Valence ratings Arousal ratings M SD M SD M SD CG Ambiguous Neg 52.59 28.74 35.51 29.41 54.98 25.63 Neu 48.23 28.64 56.31 29.9 42.1 26.49 Unambiguous Neg 57.18 30.62 31.62 27.36 58.01 25.07 Neu 48.95 30.5 56.36 28.47 41.17 26.12 UG Ambiguous Neg 52.74 25.38 35.56 27.26 55.08 23.16 Neu 48.76 26.4 54.07 26.47 44.89 23.64 Unambiguous Neg 54.03 26 32.96 25.96 57.99 22.84 Neu 50.13 25.88 55.73 24.99 43.16 22.74 For each group (CG, UG) and experimental condition (ambiguous/unambiguous cue, negative/neutral S2 valence) we report the mean (M) and standard deviation (SD) of expectancy, valence and arousal ratings. Table 1. Descriptive statistics of Experiment 1. For each group (CG, UG) and experimental condition (ambiguous/unambiguous cue, negative/neutral S2 valence) we repor of expectancy, valence and arousal ratings. For each group (CG, UG) and experimental condition (ambiguous/unambiguous cue, negative/neutral S2 valence) we report the mean (M) and standard deviation (SD) of expectancy, valence and arousal ratings. All the models are summarized in Table 2 and Fig 2. For the expectancy model testing H1 (R2 marginal = 0.190, R2 conditional = 0.452), we found a main effect of cue color (F(3, 107) = 33.24, p < .001), better specified by a significant interaction between group and cue color (F(3, 107) = 4.05, p = .009). Post-hoc contrasts confirmed our previous results [18], showing that participants in the CG reported significantly more extreme expectancy ratings as compared with participants in the UG, but only in the case of unambiguous cues. Indeed, the CG showed more negative expectancy ratings than the UG after red cues (i.e., unambiguous cues preced- ing negative pictures; CG—UG = 6.78, SE = 3.05, t(107) = 2.22, p = .028), but not after coral cues (i.e., ambiguous cues preceding negative pictures; CG—UG = 5.19, SE = 2.68, t(107) = 1.93, p = .056); and it showed less negative expectancy ratings than the UG after blue cues (i.e., unambiguous cues preceding neutral pictures; CG—UG = -10.07, SE = 3.10, t(107) = -3.25, p = .002), but not after turquoise cues (i.e., ambiguous cues preceding neutral pictures; CG—UG = -3.46, SE = 3.31, t(107) = -1.05, p = .297). Thus, contrary to H1, we did not find evidence for a strong generalization of the learned predictive meaning of cues to new, ambiguous ones. Results Descriptive statistics are provided in Table 1. 9 / 24 PLOS ONE \| https://doi.org/10.1371/journal.pone.0297954 February 9, 2024 Implicit learning, cue ambiguity and affective predictions PLOS ONE \| https://doi.org/10.1371/journal.pone.0297954 February 9, 2024 For each model, we reported the unstandardized regression coefficients, standard errors (SE), 95% confidence intervals (CI), and the associated t-test. For each model, we reported the unstandardized regression coefficients, standard errors (SE), 95% confidence intervals (CI) PLOS ONE For the expectancy model testing H2 (R2 marginal = 0.006, R2 conditional = 0.056), we found a main effect of cue ambiguity (F(1, 107) = 13.19, p < .001): ambiguous cues elicited less nega- tive expectancy ratings than unambiguous ones (ambiguous—unambiguous = -4.08, t(107) = -3.63, p < .001, 95% CI = [-6.31, -1.85]). Thus, the expectancy model supports H2, suggesting a reduced generalization on expectancy ratings to ambiguous cues within both groups. 2 2 For the valence model testing H3 (R2 marginal = 0.621, R2 conditional = 0.704), we found a main effect of cue ambiguity (F(1, 4138) = 8.26, p = .004), and a main effect of S2 valence (F(1, 107) = 997.35, p < .001), better specified by a significant interaction between cue ambiguity and S2 valence (F(1, 4138) = 7.45, p = .006). In particular, we found evidence for less unpleas- ant valence ratings to neutral pictures presented after ambiguous cues as compared to unam- biguous ones (Neu: ambiguous vs. unambiguous = 2.74, t(4138) = 3.96, p < .001, 95% CI = [1.38, 4.09]), while valence ratings to negative pictures were not affected by cue ambiguity (Neg: ambiguous–unambiguous = 0.071, t(4138) = 0.103, p = .918, 95% CI = [-1.28, 1.43]). Thus, cue ambiguity actually modulated valence ratings in both groups, but in the opposite direction as H3, with ambiguous cues leading subsequent neutral pictures to elicit less unpleas- ant subjective ratings. For the arousal model testing H4 (R2 marginal = 0.325, R2 conditional = 0.566), we found a main effect of S2 valence (F(1, 107) = 245.75, p < .001), with negative pictures eliciting higher arousal ratings than neutral pictures (Neg-Neu = 28.7, SE = 1.83, t(107) = 15.68, p < .001). We 10 / 24 PLOS ONE \| https://doi.org/10.1371/journal.pone.0297954 February 9, 2024 https://doi.org/10.1371/journal.pone.0297954.t002 https://doi.org/10.1371/journal.pone.0297954.t002 PLOS ONE Implicit learning, cue ambiguity and affective predictions PLOS ONE Table 2. Results of LMMs on expectancy, valence and arousal ratings in Experiment 1. Model Parameter Estimate SE Statistic df p 95% CI Expectancy (H1) Intercept 52.17 0.61 85.91 107.01 < 0.001 50.97 53.38 UG–CG 0.39 1.21 0.32 107.01 0.746 -2.01 2.80 Coral–Blue 19.00 2.39 7.96 107.00 < 0.001 14.27 23.72 Red–Blue 25.91 2.76 9.38 107.21 < 0.001 20.43 31.38 Turquoise–Blue -1.25 1.50 -0.83 107.05 0.407 -4.23 1.73 UG–CG x Coral–Blue -15.26 4.77 -3.20 107.00 0.002 -24.72 -5.80 UG–CG x Red–Blue -16.85 5.53 -3.05 107.21 0.003 -27.81 -5.90 UG–CG x Turquoise–Blue -6.61 3.01 -2.20 107.05 0.03 -12.58 -0.65 σ ID 5.40 σ Coral–Blue 23.06 σ Red–Blue 27.09 σ Turquoise–Blue 12.59 σ residual 20.82 Expectancy (H2) Intercept 52.17 0.61 85.91 107.00 < 0.001 50.97 53.38 UG–CG -0.39 1.21 -0.32 107.00 0.746 -2.80 2.01 Amb–Unamb -4.08 1.12 -3.63 107.00 < 0.001 -6.31 -1.85 group x cue 2.51 2.25 1.12 107.00 0.266 -1.94 6.97 σ ID 4.62 σ cue 7.89 σ residual 27.27 Valence (H3) Intercept 44.67 0.49 90.57 107.53 < 0.001 43.70 45.65 UG–CG 0.98 0.99 0.99 107.53 0.322 -0.97 2.94 Amb–Unamb 1.40 0.49 2.87 4,138.00 0.004 0.45 2.36 neg–neu -46.35 1.47 -31.58 107.23 < 0.001 -49.26 -43.44 group x cue 0.85 0.98 0.87 4,138.00 0.383 -1.06 2.77 valence x group -5.50 2.94 -1.87 107.23 0.064 -11.32 0.32 cue x valence -2.67 0.98 -2.73 4,138.00 0.006 -4.58 -0.75 group x cue x valence -2.40 1.95 -1.23 4,138.00 0.219 -6.23 1.43 σ ID 4.47 σ valence 14.43 σ residual 16.03 Arousal (H4) Intercept 49.59 0.86 57.89 107.19 < 0.001 47.89 51.29 UG–CG -1.62 1.71 -0.95 107.19 0.346 -5.02 1.78 Amb–Unamb -1.52 0.51 -2.97 4,138.00 0.003 -2.53 -0.52 neg–neu 28.71 1.83 15.68 107.17 < 0.001 25.08 32.34 group x cue -0.35 1.02 -0.34 4,138.00 0.733 -2.36 1.66 valence x group 4.67 3.66 1.27 107.17 0.205 -2.59 11.93 cue x valence -1.66 1.02 -1.62 4,138.00 0.105 -3.67 0.35 group x cue x valence 1.69 2.05 0.83 4,138.00 0.409 -2.32 5.70 σ ID 8.53 σ valence 18.34 σ residual 16.80 For each model, we reported the unstandardized regression coefficients, standard errors (SE), 95% confidence intervals (CI), and the associated t-test. Table 2. Results of LMMs on expectancy, valence and arousal ratings in Experiment 1. PLOS ONE \| https://doi.org/10.1371/journal.pone.0297954 February 9, 2024 11 / 24 PLOS ONE Implicit learning, cue ambiguity and affective predictions Fig 2. PLOS ONE Box-plot of expectancy, valence and arousal ratings in Experiment 1 according to the group (CG vs. UG), cue ambiguity (Amb-ambiguous vs. Unamb-unambiguous), and S2 valence (Neg vs. Neu, for valence and arousal ratings only). Points represent the mean estimated value for each participant and condition. ancy, valence and arousal ratings in Experiment 1 according to the group (CG vs. UG), cue ambiguity (Amb-ambiguous vs and S2 valence (Neg vs. Neu, for valence and arousal ratings only). Points represent the mean estimated value for each partic Fig 2. Box-plot of expectancy, valence and arousal ratings in Experiment 1 according to the group (CG vs. UG), cue ambiguity (Amb-ambiguous vs. Unamb-unambiguous), and S2 valence (Neg vs. Neu, for valence and arousal ratings only). Points represent the mean estimated value for each participant and condition. https://doi.org/10.1371/journal.pone.0297954.g002 https://doi.org/10.1371/journal.pone.0297954.g002 also found a main effect of cue ambiguity (F(1, 4138) = 8.85, p = .003): ambiguous cues elicited lower arousal ratings than unambiguous cues (ambiguous–unambiguous = -1.52, SE = 0.51, t (4138) = -2.98, p = .003). PLOS ONE \| https://doi.org/10.1371/journal.pone.0297954 February 9, 2024 Discussion The findings obtained in Experiment 1 suggest that no strong generalization of subjective expectancies from the specific features of the learning phase to the new ambiguous features of the test phase emerged. Indeed, when testing the effects of cue predictive meaning (i.e., cue color) on expectancy ratings, we replicated our previous results [18] showing that participants in the CG reported more extreme expectancies than the UG, but only for unambiguous cues. This may seem in contrast with predictive models of emotion [3,4] (see H1), since one may think that an efficient predictive model must be generalizable across different contexts. How- ever, recent contributions [49–51] have highlighted how affective predictions are inherently and indissolubly context-sensitive, being tight to the specific situation in which they develop. Thus, as an alternative explanation consistent with previous literature [26], it may be that the novelty of the stimuli employed as ambiguous cues in the testing phase (namely, current infor- mation) prevails over past information in shaping subjective affective ratings. Moreover, cue ambiguity per se (regardless of cue predictive meaning) did not interact with uncertainty of previous experience in shaping subjective affective ratings. In fact, no significant group by cue interaction was found in any of the models. We found instead that ambiguous cues elicited PLOS ONE \| https://doi.org/10.1371/journal.pone.0297954 February 9, 2024 12 / 24 PLOS ONE Implicit learning, cue ambiguity and affective predictions less negative expectancy ratings in the generation-implementation stage, less unpleasant valence to neutral stimuli and overall lower arousal in the updating stage, independently from previous learning. Thus, cue ambiguity appears to elicit a dampened subjective affective expe- rience at all stages. With regards to subjective expectancy (i.e., expectancy ratings) these results are consistent with our hypothesis (see H2, [26]), with regards to subjective reactions to new stimuli (i.e., valence and arousal ratings) they are instead partially in contrast (see H3, [26]). It must be noted, however, that in our paradigm we manipulated previous experience in a separate learn- ing phase and we asked for a trial-by-trial rating of experienced valence (and arousal) to S2s, whereas Chen and Lovibond [26] did not employ a learning paradigm and asked for a post- experiment mood rating. Thus, the different nature of the paradigm and the ratings requested to participants may be accountable for the opposing effects found. Discussion Moreover, another S1-S2 study [52] manipulating ambiguity of the target affective pictures, found that ambiguous pic- tures were rated as less unpleasant and less arousing than unambiguous pictures, coherently with what we found following ambiguous cues. These effects can be explained in light of the time-related distinction between ambiguity and uncertainty (as proposed in [53]). Ambiguity, on the one hand, refers to a static feature of the here and now, embedded in the present moment: an ambiguous situation or stimulus is characterized by novelty, unpredictability and also uncertainty [53]. Uncertainty, on the other hand, refers to a future-oriented feature, char- acterized by unpredictability but not necessarily also by ambiguity [53,54]. In Experiment 1, we manipulated uncertainty during the learning phase (where participants were not asked for any rating), and ambiguity during the test phase (where we asked for subjective affective rat- ings). As a consequence, and in line with the results of our previous research [18], it might be that current (i.e., ambiguous vs. unambiguous) information prevailed over past information (i.e., certain vs. uncertain) in shaping subjective affective experience. Moreover, it is interesting to note that the role of cue ambiguity in dampening subjective valence was exclusively expressed with regards to neutral stimuli. This may be due to a ceiling effect in valence ratings elicited by negative pictures, that may have covered any modulating contribution of cue ambi- guity. Neutral pictures, instead, elicited more variable valence ratings, allowing more subtle modulations of cue ambiguity to statistically emerge. PLOS ONE \| https://doi.org/10.1371/journal.pone.0297954 February 9, 2024 Participants We computed the required sample size through an a priori pre-registered (https://osf.io/z5esb/ ) power analysis for GLMMs (see 2.1.1), estimating parameters from pilot data (N = 18). We recruited 125 adult participants through the provider platform Prolific (Prolific, Oxford, UK; www.prolific.co) in August 2021. Participants were screened for vision difficulties, including color blindness. Researchers had access only to the following participant’s personal informa- tion: Prolific IDs, age, gender, country of birth, country of residence, nationality, employment status and languages spoken. Thus, no identifying information was provided and collected. In order to be included in Experiment 2, participants should not have participated in Experiment 1. Data from 9 participants were discarded according to the pre-registered exclusion criteria: scoring lower than -2 SD from the mean accuracy in the parity judgment task (N = 7), report- ing internet/uploading issues in more than 25% of the experimental trials (N = 2), reporting to have caught the exact probabilistic relationship between S1 color and S2 valence during the learning phase (N = 0). The final sample included 116 participants (58 males, age: M = 25.06, SD = 7.63, range = 18–55; CG N = 55, UG N = 54). All participants gave their written informed consent before starting the experiment, and were paid £2.13 for their participation. All experi- mental procedures were conducted in accordance with the Declaration of Helsinki, and approved by the local Ethical Committee (protocol no. 4177). Method Experiment 2 investigated whether experiencing implicit certain vs. uncertain probabilistic relationships between stimuli might influence subjective ratings to future affective predictions. More specifically, to ensure an implicit exposure to the probabilistic relationship between S1 and S2, during the learning phase we engaged participants in a distracting task (a parity judg- ment task, see Stimulus material and procedure below for details). Then, we tested if partici- pants were able to implicitly extract the (un)certain probabilistic information available during the learning phase, and to use it in the test phase to rate the expected valence of new affective events, or the subjective valence and arousal to new affective stimuli. We pre-registered the study on OSF (https://osf.io/z5esb/). We performed confirmatory analyses to test seven pre-registered hypotheses. The first two hypotheses concerned the behav- ioral performance to the parity judgment task. We hypothesized to find (H1a) faster RTs in the CG as compared to the UG, and (H1b) no differences in accuracy between the groups [22]. This would be in line with the predictive models’ assumption that the two contingencies (100% and 50%) can be experienced implicitly without any significant difference between them [6]. The remaining hypotheses regarded the test phase. As a third hypothesis (H2a), we PLOS ONE \| https://doi.org/10.1371/journal.pone.0297954 February 9, 2024 13 / 24 PLOS ONE Implicit learning, cue ambiguity and affective predictions expected to find more negative expectancy ratings in the UG as compared to the CG [21,22,24,55,56]. The fourth hypothesis (H2b), opposed to H2a, was that participants in the CG would show more negative expectancy ratings after the cues which were previously paired with a negative picture during the learning phase [18]. This hypothesis is coherent with predic- tive models of emotion [3,4], according to which we can expect that contingencies of the learn- ing phase would be implicitly inferred. The fifth hypothesis (H3a) was that participants in the UG would show higher arousal and more unpleasant valence ratings to S2s than participants in the CG [33]. The sixth hypothesis (H3b), opposed to H3a, was that participants in the CG would show higher arousal and more unpleasant valence ratings to S2s, as compared to partici- pants in the UG [27,29–32]. The last hypothesis (H3c), opposed to both H3a and H3b, was to find only a main effect of S2 valence, with significantly higher arousal and more unpleasant valence ratings to negative S2s independently from the experimental group [18,23]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0297954 February 9, 2024 Data analysis The study had a 2 (group, between-subjects: CG vs. UG) × 2 (S2 valence, within-subjects: Neg vs. Neu) mixed design. The analysis plan was pre-registered on OSF (https://osf.io/z5esb/). During data pre-processing, participants’ verbatim responses to the manipulation check question (see Stimulus Material and Procedure above) were qualitatively analyzed by the experimenter, and coded as “yes” or “no” according to their content. No response was coded as “yes”, suggesting that no participants reported to have caught the probabilistic relationship between S1 and S2. Following the pre-registered procedures for outliers detection and management, we detected 5 univariate outliers (MAD > 3) that were excluded from data analysis. We also detected 36 multivariate outliers (MMCD, breakdown point 0.25). From the visual inspection of their ratings it emerged that 2 of them had reversed the scales’ poles (“error outliers”, see [45]), and they were therefore removed from data analysis. The remaining multivariate outliers showed only a slightly different relationship between valence and arousal ratings as compared to other participants, thus we chose to keep them into data analysis (“potentially interesting outliers”, see [45]), since none of them impacted the models’ estimates (as assessed through Cook’s distance). Overall, data from 109 participants were included in the analyses. Before performing the analyses, we pre-processed RTs to the parity judgment task accord- ing to the following pre-registered steps: (i) trimming RTs between 100 and 1500 msec [57]; (ii) discarding RTs of incorrect trials; (iii) adjusting RTs for the speed-accuracy trade off by means of Inverse Efficiency Score (IES) transformation [58]; (iv) log-transforming IES to account for their skewed distribution [57,59]. In order to test our a-priori hypotheses (H1a and H1b on parity judgment task; H2a, H2b, H3a, H3b and H3c on test phase ratings), for each DV we fitted the following (G)LMMs (R package: lme4; [46]): 1. log-transformed IES (H1a): group as fixed factor, random intercept for participant; 2. accuracy (H1b): logistic regression with group as fixed factor, random intercept for participant; 3. expectancy ratings (H2a, H2b): group, cue (within-subjects: cueneg vs. cueneu) and their interaction as fixed factors, random slopes for cue within participant; 4. valence and arousal ratings (H3a, H3b, H3c): group, S2 valence and their interaction as fixed factors, random slopes for S2 valence within participant. Stimulus material and procedure Materials and procedures were the same as in Experiment 1 (see Stimulus Material and Proce- dure above), but in Experiment 2 we employed only unambiguous (i.e., red and blue) cues. Dur- ing the learning phase a parity judgment task was introduced. Single digits from 1 to 9 were employed as stimuli. Participants were informed that they would see a sequence of stimuli on the screen: a colored circle, followed by a picture, and then a number. They were instructed to look at the screen and judge if the number was odd or even, by pressing the ‘Z’ or ‘M’ keys. Response keys were counterbalanced between subjects. Instructions were followed by a practice session of 3 trials, in which participants were trained to give their parity judgments, and they received feedback on their performance. Then, the learning session started, with the same trials’ structure, timing and number as in Experiment 1. In each trial, after the S2, a second ISI with a random duration between 500 and 800 msec followed, in which the screen remained gray. Then, a random digit between 1 and 9 was presented in the center of the screen for 200 msec, and participants had up to 1500 msec to judge the digit’s parity by pressing the ‘Z’ or ‘M’ keys. Participants were left uninstructed about the S1-S2 ratios (100% for the CG, 50% for the UG). Fig 1B shows a schematic representation of the experimental paradigm. PLOS ONE \| https://doi.org/10.1371/journal.pone.0297954 February 9, 2024 14 / 24 PLOS ONE Implicit learning, cue ambiguity and affective predictions At the end of the test phase, participants were directed to the Qualtrics survey (see Stimulus Material and Procedure above). Here, we added an open question as a manipulation check: participants were asked whether they caught any relationship between the color of the circle and the affective valence of the pictures during the learning phase. Participants were then redi- rected back to Prolific to receive their payment. The experiment lasted about 30 minutes. PLOS ONE \| https://doi.org/10.1371/journal.pone.0297954 February 9, 2024 Data analysis The factor “cue” codes for the predictive meaning of the cue according to the specific color- valence pairings experienced by participants in the test phase (within-subjects: cueneg−that is, a cue that preceded negative stimuli in the test phase vs. cueneu−that is, a cue that preceded neu- tral stimuli in the test phase). In the analysis script (shared on OSF https://osf.io/z5esb/) this factor was called “S1 color”. Here, for the sake of clarity, we renamed it as “cue” (see [18] and above for further details on the cue factor, and on how we compensated for the counterbalanc- ing of S1 color–S2 valence pairings in the analysis). 15 / 24 PLOS ONE \| https://doi.org/10.1371/journal.pone.0297954 February 9, 2024 PLOS ONE Implicit learning, cue ambiguity and affective predictions The pre-registered exploratory analyses regarding the effects of time, S2 congruency (expected vs. unexpected S2s), IUS, and DASS-21 questionnaires are reported in S3–S6 Tables. Since the results of these analyses are mainly null, or redundant with respect to both the confir- matory models reported below and the results of our previous work [18], these analyses will not be discussed further in the present manuscript. Influential cases for each model, as well as the methods to test model’s effects were pre-reg- istered. For each model no influential cases emerged, as evaluated through Cook’s distance (>1). LMMs effects were tested by means of F-test and p-values, calculated via Satterthwaite’s degrees of freedom method (α = .05, R package: lmerTest; [47]), GLMMs effects were evalu- ated through Type II Analysis of Deviance (R package: car; [60]). For each model we reported the estimated parameters with 95% CI, marginal and conditional R2 (estimated as in [48]). For each group (CG, UG) and experimental condition (cueneg/cueneu, negative/neutral S2 valence) we report the mean (M) and standard deviation (SD) of expectancy, valence and arousal ratings. Results Descriptive statistics are provided in Table 3. All the models are summarized in Table 4 and Fig 3. For the IES model testing H1a (R2 marginal = 0.003, R2 conditional = 0.968), we did not find any effect of group (F(1, 105) = 0.33, p = .565). Thus, the IES model does not support the hypothesis of faster RTs in the CG as com- pared to the UG (H1a). We found the same result for the accuracy model testing H1b (R2 mar- ginal = 0.009, R2 conditional = 0.486), with no significant effect of group (χ2 = 1.71, p = .191). Therefore, we confirmed our hypothesis (H1b) of not finding group differences in accuracy scores to the parity judgment task. For the expectancy model testing H2a vs. H2b (R2 marginal = 0.068, R2 conditional = 0.267), we only found a main effect of cue (F(1, 107) = 54.98, p < .001): in both groups, participants showed significantly more negative expectancy ratings after the cueneg (i.e., cues preceding negative pictures in the test phase) than after the cueneu (i.e., cues preceding neutral pictures in the test phase) (cueneg vs. cueneu = 14.5, SE = 1.96, t(107) = 7.42, p < .001). Thus, the expec- tancy model does not support either of our two hypotheses (H2a, H2b). For both valence (R2 marginal = 0.596, R2 conditional = 0.673) and arousal (R2 mar- ginal = 0.352, R2 conditional = 0.52) models testing H3a vs. H3b vs. H3c, we found a main effect of S2 valence, with both groups reporting significantly greater unpleasantness (F(1, 107) = 1101.78, p < .001; Neg vs. Neu = -47.1, SE = 1.42, t(107) = -33.19, p < .001) and higher arousal (F(1, 107) = 384.03, p < .001; Neg vs. Neu = 30.9, SE = 1.57, t(107) = 19.6, p < .001) Table 3. Descriptive statistics of Experiment 2. Table 3. Descriptive statistics of Experiment 2. https://doi.org/10.1371/journal.pone.0297954.t003 For each group (CG, UG) and experimental condition (cueneg/cueneu, negative/neutral S2 valence) we report the mean (M) and standard deviation (SD) of expectancy, l d l ti https://doi.org/10.1371/journal.pone.0297954.t003 towards negative pictures. Thus, valence and arousal models support the hypothesis of no group differences in S2-ratings (H3c). towards negative pictures. Thus, valence and arousal models support the hypothesis of no group differences in S2-ratings (H3c). Results Group Cue S2 valence Expectancy ratings Valence ratings Arousal ratings M SD M SD M SD CG cueneg Neg 56.78 25.98 32.56 28.44 56.64 25.02 Neu 57.93 25.2 58.51 28.48 39.35 25.95 cueneu Neg 48.65 26.38 36.21 28.24 55.4 24.91 Neu 49.34 25.73 56.28 28.23 41.3 24.76 UG cueneg Neg 57.1 29 33.23 28.84 56.16 25.89 Neu 58.08 28.8 59.38 29.49 39.89 25.55 cueneu Neg 48.44 30.67 35.87 28.22 54.2 25.53 Neu 49.8 29.35 58.3 28.4 40.65 25.02 PLOS ONE \| https://doi.org/10.1371/journal.pone.0297954 February 9, 2024 PLOS ONE \| https://doi.org/10.1371/journal.pone.0297954 February 9, 2024 16 / 24 Implicit learning, cue ambiguity and affective predictions PLOS ONE \| https://doi.org/10.1371/journal.pone.0297954 February 9, 2024 PLOS ONE PLOS ONE Table 4. Results of confirmatory (G)LMMs on IES, accuracy, expectancy, valence and arousal ratings in Experiment 2. Model Parameter Estimate SE Statistic df p 95% CI IES (H1a) Intercept 6.61 0.08 84.06 105.24 < 0.001 6.46 6.77 UG—CG -0.09 0.16 -0.58 105.24 0.565 -0.40 0.22 σ ID 0.82 σ residual 0.15 Accuracy (H1b) Intercept 2.35 0.18 12.95 < 0.001 2.00 2.71 UG—CG 0.47 0.36 1.31 0.191 -0.23 1.18 σ ID 1.75 Expectancy (H2a, H2b) Intercept 53.36 0.85 62.75 107.01 < 0.001 51.68 55.05 UG—CG -0.17 1.70 -0.10 107.01 0.922 -3.54 3.20 cueneg—cueneu 14.55 1.96 7.42 107.00 < 0.001 10.66 18.44 cue x group -0.24 3.92 -0.06 107.00 0.952 -8.02 7.54 σ ID 8.03 σ cue 19.04 σ residual 23.92 Valence (H3a, H3b, H3c) Intercept 44.99 0.55 82.53 107.02 < 0.001 43.91 46.07 UG—CG 0.24 1.09 0.22 107.02 0.83 -1.93 2.40 Neg—Neu -47.10 1.42 -33.19 107.02 < 0.001 -49.91 -44.28 valence x group 0.79 2.84 0.28 107.02 0.782 -4.84 6.41 σ ID 4.98 σ valence 13.75 σ residual 17.45 Arousal (H3a, H3b, H3c) Intercept 48.95 0.76 64.77 106.96 < 0.001 47.45 50.45 UG—CG -0.49 1.51 -0.33 106.96 0.745 -3.49 2.50 Neg—Neu 30.86 1.57 19.60 107.01 < 0.001 27.74 33.98 valence x group 1.47 3.15 0.47 107.01 0.641 -4.77 7.72 σ ID 7.36 σ valence 15.42 σ residual 18.03 For each model we reported the estimates (unstandardized regression coefficients for LMMs, odds ratio for GLMMs), SE, 95% CI, and the associated statistics (t-test for LMMs, χ2 for GLMMs). atory (G)LMMs on IES, accuracy, expectancy, valence and arousal ratings in Experiment 2. Table 4. Results of confirmatory (G)LMMs on IES, accuracy, expectancy, valence and arousal ratings in Experiment 2. For each model we reported the estimates (unstandardized regression coefficients for LMMs, odds ratio for GLMMs), SE, 95% CI, and the associated statistics (t-test for LMMs, χ2 for GLMMs). https://doi.org/10.1371/journal.pone.0297954.t004 Discussion Experiment 2 replicated our previous findings [18] for valence and arousal, but not for expec- tancy ratings. Indeed, both groups showed similar expectancies, predicting upcoming pictures’ valence according to the 75% contingencies of the test phase rather than to previously (and implicitly) experienced contingencies. Thus, the results did not support the hypothesis that probabilistic information can be implicitly extracted and subsequently used to modulate sub- jective affective ratings, as it can be expected according to predictive models of emotion [3,4,6]. They are however consistent with alternative explanations derived from some recent evidence [61], according to which awareness seems to be a necessary precursor for learning, especially in the case of repetition learning. Remarkably, we can reasonably exclude any potential difference in the way the two groups experienced the implicit contingencies during the learning phase. In fact, no significant group 17 / 24 PLOS ONE \| https://doi.org/10.1371/journal.pone.0297954 February 9, 2024 PLOS ONE Implicit learning, cue ambiguity and affective predictions Fig 3. Box-plot of expectancy, valence and arousal ratings in Experiment 2 according to the group (CG vs. UG), and the cue (CueNeg vs. CueNeu, for expectancy ratings) or the S2 valence (Neg vs. Neu, for valence and arousal ratings). Points represent the mean estimated value for each participant and condition. Fig 3. Box-plot of expectancy, valence and arousal ratings in Experiment 2 according to the group (CG vs. UG), and the cue (CueNeg vs. CueNeu, for expectancy ratings) or the S2 valence (Neg vs. Neu, for valence and arousal ratings). Points represent the mean estimated value for each participant and condition. https://doi.org/10.1371/journal.pone.0297954.g003 https://doi.org/10.1371/journal.pone.0297954.g003 https://doi.org/10.1371/journal.pone.0297954.g003 differences emerged on the parity judgment task, as measured by RTs (see H1a) and accuracy (see H1b), suggesting that the distracting task acted similarly in the two groups. Demonstrat- ing this is crucial, as judgments among low levels of contingency (e.g., 50%) are more difficult than judgments among high levels of contingency (e.g., 100%) [62], and this could have (but did not) exert confounding effects on how contingency learning occurred in the two groups. PLOS ONE \| https://doi.org/10.1371/journal.pone.0297954 February 9, 2024 General discussion In spite of this, our result rea- sonably suggests that, without any explicit focus of attention on previous experience, the effect of the latter does not emerge at the subjective level significantly shaping the reported affective experience. Future investigations should clarify whether implicit learning can shape affective prediction construction at other levels. Overall, our results advance the understanding of the mechanisms underlying subjective affective experience as constructed from prior knowledge. Crucially, we fully replicated our previous results [18], collecting additional evidence that subjective expectancies are sensitive to uninstructed (but not implicit) statistical regularities experienced in the past. With this, we further supported predictive models of emotion [3,4]: certain past learning experiences are used to construct highly reliable predictions, thus leading to a coherent pre-activation of the associated subjective experience. However, our results suggest some caution when applying predictive processing theoretical principles to the affective domain, especially with regards to the updating stage. Indeed, we did not find evidence for a generalization effect to new and potentially ambiguous stimuli, nor evidence of implicitness. Predictive processing theories, instead, assume that prediction updating should be modulated by stimuli predictability [11,13,20,65]. Nonetheless, in recent studies it was consistently found that affective processing during the updating stage is not (or is only weakly) influenced by stimulus predictability both at the subjective [18] and at the neural levels [20,41]. Thus, when it comes to updating, more weight seems to be attributed to the affective nature of stimuli themselves and to the contextual information available in the current moment, rather than to top-down predictions based on past experiences. This may be due to the evolutionary relevance of affective stimuli, in that a quick bottom-up evaluation of present inputs may cover a more efficient role in promoting survival (and thus, it must be prioritized). As a selected broader implication of our study, consistent with predictive models of emo- tion [3,4], we argue that a paradigm shift is needed in the study of emotions, in the sense that we further supported the idea that emotions cannot be studied independently from the specific context in which they are constructed [3,49,66]. We found, indeed, that previous experience failed to generalize to new contexts in an experimental setting which is rather artificial as com- pared to everyday life and which implies the use of symbolic and simple stimuli. General discussion The studies reported in the present paper attempted to shed further light on the construction of subjective affective experience as conceived within the predictive framework [3,4]. Indeed, little is known about how subjective experience is shaped by new and potentially ambiguous environmental cues, or whether it can be influenced by implicit previous learnings, despite both being considered crucial factors for constructing efficient predictions [1,2]. Regarding cue ambiguity (Experiment 1), we found that ambiguous cues elicited a damp- ened subjective affective experience in all the prediction stages, independently from previous learnings. Interestingly, when presented with unambiguous cues, participants formerly exposed to certain contingencies (i.e., the CG, that constructed a reliable prediction) showed more extreme expectancy ratings than participants exposed to uncertainty (i.e., the UG). This fully replicates our previous results [18], and may be due to a pre-activation of the expected affective experience, as suggested by predictive models of emotion [3,4]. When faced with PLOS ONE \| https://doi.org/10.1371/journal.pone.0297954 February 9, 2024 18 / 24 PLOS ONE Implicit learning, cue ambiguity and affective predictions ambiguous cues, instead, participants were unable to implement a reliable prediction and coherently pre-activate the associated subjective experience, and thus the latter was dampened. ambiguous cues, instead, participants were unable to implement a reliable prediction and coherently pre-activate the associated subjective experience, and thus the latter was dampened. Concerning previous implicit experience (Experiment 2), we did not find any evidence that subjective affective ratings are shaped by (un)certainty of previous implicit learning. Indeed, all participants reported similar subjective experience. This is consistent with some recent evi- dence about repetition learning, providing strong evidence that awareness is a necessary pre- cursor for learning [61], and suggests that affective stimuli require a prompt response primarily driven by a quick bottom-up evaluation of present inputs rather than by top-down predictions based on past information. However, this does not necessarily exclude that an implicit extraction of statistical regularities from task stimuli contingencies occurred in some ways (e.g., at a covert level, measured as patterns of neural/psychophysiological activity). There is a bunch of evidence, indeed, suggesting that implicit learning may potentially occur across all the statistical learning paradigms, and that these two learning processes are associated with the activation of partially overlapping neural networks [63,64]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0297954 February 9, 2024 General discussion This high- lights the importance of taking context into careful account when studying affective processes in complex situations and/or beyond each specific experimental setup. Notwithstanding, some limitations of our research are worth mentioning. First, we did not assess individual differences in cognitive processes that may influence affective predictions (e.g., attention, memory, etc.), so we cannot exclude that these processes may have exerted influencing effects. However, we can exclude that mood and personality traits modulating PLOS ONE \| https://doi.org/10.1371/journal.pone.0297954 February 9, 2024 19 / 24 PLOS ONE Implicit learning, cue ambiguity and affective predictions affective predictions (i.e., intolerance of uncertainty and negative affectivity, that we measured and controlled for in our analyses; see S5–S6, S9 and S10 Tables) exerted confounding effects. Also, there are some constraints on the generality of our findings [67] since our sample only included WEIRD participants. The lack of participants from other cultural groups precludes our ability to ascertain whether our findings have a broader generalizability. Second, our para- digm remains quite artificial with respect to real-life situations, in which people often experi- ence multimodal and dynamic affective stimuli. Third, previous experience is manipulated only in terms of (un)certain probabilistic relationships between stimuli (i.e., S1-S2 congru- ency). However, other characteristics of previous experience, such as the frequency of expo- sure, or the familiarity with the physical environment in which the stimuli are embedded, may be of interest, too. Fourth, we have only manipulated ambiguity with respect to cues, but also ambiguity of targets can have a great impact on subjective affective experience. Last, in our study we only focused on subjective experience as overtly rated by participants (since we were not able to collect covert -e.g., psychophysiological- measures due to COVID-19 pandemic restrictions), but more subtle modulations may be experimentally captured by measuring also covert processing indices. We thus encourage future studies to complement our results, by integrating both subjective and objective measures of affective processing. Supporting information S1 Table. List of NAPS picture names used as S2s in Experiment 1 and 2, sorted by valence (Neg = negative, Neu = neutral). (PDF) S1 Table. List of NAPS picture names used as S2s in Experiment 1 and 2, sorted by valenc (Neg = negative, Neu = neutral). (PDF) S2 Table. Means (M), standard deviations (SD), and results of two-tailed t-tests assuming unequal variance in luminance, contrast, complexity indices (i.e., JPEG size, entropy), an color space indices (i.e., LABL, LABA, LABB), referred to negative (Neg) and neutral (Neu) NAPS pictures employed as S2s in Experiment 1 and 2. (PDF) S3 Table. Pre-registered exploratory models on Block effect in Experiment 1. (PDF) S4 Table. Pre-registered exploratory models on S2 Congruency effect in Experiment 1. (PDF) S5 Table. Pre-registered exploratory models on Intolerance of Uncertainty Scale (IUS) effect in Experiment 1. (PDF) S6 Table. Pre-registered exploratory models on Depression, Anxiety and Stress Scale (DASS-21) effect in Experiment 1. (PDF) S7 Table. Pre-registered exploratory models on Block effect in Experiment 2. (PDF) S8 Table. Pre-registered exploratory models on S2 Congruency effect in Experiment 2. (PDF) S9 Table. Pre-registered exploratory models on Intolerance of Uncertainty Scale (IUS) effect in Experiment 2. (PDF) S2 Table. Means (M), standard deviations (SD), and results of two-tailed t-tests assuming unequal variance in luminance, contrast, complexity indices (i.e., JPEG size, entropy), and color space indices (i.e., LABL, LABA, LABB), referred to negative (Neg) and neutral (Neu) NAPS pictures employed as S2s in Experiment 1 and 2. (PDF) Acknowledgments We kindly thank Filippo Carnovalini for his valuable help on the experiment programming. Author Contributions Author Contributions Conceptualization: Fiorella Del Popolo Cristaldi, Giulia Buodo, Suzanne Oosterwijk, Gio- vanni Mento. Conceptualization: Fiorella Del Popolo Cristaldi, Giulia Buodo, Suzanne Oosterwijk, Gio- vanni Mento. Data curation: Fiorella Del Popolo Cristaldi, Filippo Gambarota. Formal analysis: Fiorella Del Popolo Cristaldi, Filippo Gambarota. Funding acquisition: Fiorella Del Popolo Cristaldi. Investigation: Fiorella Del Popolo Cristaldi. Methodology: Fiorella Del Popolo Cristaldi, Suzanne Oosterwijk, Giovanni Mento. Project administration: Fiorella Del Popolo Cristaldi, Suzanne Oosterwijk. Resources: Fiorella Del Popolo Cristaldi. Software: Fiorella Del Popolo Cristaldi. Software: Fiorella Del Popolo Cristaldi. Supervision: Suzanne Oosterwijk. Visualization: Fiorella Del Popolo Cristaldi, Filippo Gambarota. Writing – original draft: Fiorella Del Popolo Cristaldi, Filippo Gambarota. Writing – review & editing: Fiorella Del Popolo Cristaldi, Giulia Buodo, Filippo Gambarota, Suzanne Oosterwijk, Giovanni Mento. S4 Table. Pre-registered exploratory models on S2 Congruency effect in Experiment 1. (PDF) S5 Table. Pre-registered exploratory models on Intolerance of Uncertainty Scale (IUS) effect in Experiment 1. (PDF) S6 Table. Pre-registered exploratory models on Depression, Anxiety and Stress Scale (DASS-21) effect in Experiment 1. (PDF) S7 Table. Pre-registered exploratory models on Block effect in Experiment 2. (PDF) S8 Table. Pre-registered exploratory models on S2 Congruency effect in Experiment 2. (PDF) S9 Table. Pre-registered exploratory models on Intolerance of Uncertainty Scale (IUS) effect in Experiment 2. (PDF) 20 / 24 PLOS ONE \| https://doi.org/10.1371/journal.pone.0297954 February 9, 2024 PLOS ONE Implicit learning, cue ambiguity and affective predictions S10 Table. Pre-registered exploratory models on Depression, Anxiety and Stress Scale (DASS-21) effect in Experiment 2. (PDF) S10 Table. Pre-registered exploratory models on Depression, Anxiety and Stress Scale (DASS-21) effect in Experiment 2. (PDF) References Current Opinion in Behavioral Sciences. 2018 Feb 1; 19:67–72. 16. Barrett LF, Wilson-Mendenhall CD, Barsalou LW. The conceptual act theory: A roadmap. In: Barrett LF, Russell JA, editors. The psychological construction of emotion. New York, NY: Guilford Press; 2014. p. 83–110. 17. Seth AK. Interoceptive inference, emotion, and the embodied self. Trends in Cognitive Sciences. 2013; 17(11):565–73. https://doi.org/10.1016/j.tics.2013.09.007 PMID: 24126130 18. Del Popolo Cristaldi F, Gambarota F, Oosterwijk S. Does your past define you? The role of previous visual experience in subjective reactions to new affective pictures and sounds. Emotion. 2023 Aug; 23 (5):1317–33. https://doi.org/10.1037/emo0001168 PMID: 36074619 19. Mercado F, Hinojosa JA, Peñacoba C, Carretie´ L. The emotional S1-S2 paradigm for exploring brain mechanisms underlying affective modulation of expectancy. In: Brain Mapping Research Develop- ments. Hauppauge, NY: Nova Science Publishers; 2008. p. 197–209. 20. Del Popolo Cristaldi F, Mento G, Buodo G, Sarlo M. What’s next? Neural correlates of emotional predic- tions: A high-density EEG investigation. Brain and Cognition. 2021; 150:105708. https://doi.org/10. 1016/j.bandc.2021.105708 PMID: 33714004 21. Dieterich R, Endrass T, Kathmann N. Uncertainty is associated with increased selective attention and sustained stimulus processing. Cognitive, Affective, & Behavioral Neuroscience. 2016; 16(3):447–56. https://doi.org/10.3758/s13415-016-0405-8 PMID: 26810702 22. Grupe DW, Nitschke JB. Uncertainty is associated with biased expectancies and heightened responses to aversion. Emotion. 2011; 11(2):413–24. https://doi.org/10.1037/a0022583 PMID: 21500909 23. Lin H, Liang J, Jin H, Zhao D. Differential effects of uncertainty on LPP responses to emotional events during explicit and implicit anticipation. International Journal of Psychophysiology. 2018; 129:41–51. https://doi.org/10.1016/j.ijpsycho.2018.04.012 PMID: 29704580 24. Qiao Z, Geng H, Wang Y, Li X. Anticipation of uncertain threat modulates subsequent affective responses and covariation bias. Frontiers in Psychology. 2018; 9:2547. https://doi.org/10.3389/fpsyg. 2018.02547 PMID: 30618968 25. Sarinopoulos I, Grupe DW, Mackiewicz KL, Herrington JD, Lor M, Steege EE, et al. Uncertainty during anticipation modulates neural responses to aversion in human insula and amygdala. Cerebral Cortex. 2010; 20(4):929–40. https://doi.org/10.1093/cercor/bhp155 PMID: 19679543 26. Chen JTH, Lovibond PF. Intolerance of Uncertainty Is Associated With Increased Threat Appraisal and Negative Affect Under Ambiguity but Not Uncertainty. Behavior Therapy. 2016; 47(1):42–53. https://doi. org/10.1016/j.beth.2015.09.004 PMID: 26763496 27. Bermpohl F, Pascual-Leone A, Amedi A, Merabet LB, Fregni F, Gaab N, et al. Attentional modulation of emotional stimulus processing: An fMRI study using emotional expectancy. Human Brain Mapping. 2006; 27(8):662–77. https://doi.org/10.1002/hbm.20209 PMID: 16317710 28. Greenberg T, Carlson JM, Rubin D, Cha J, Mujica-Parodi L. References 1. Friston K. The free-energy principle: A unified brain theory? Nature Reviews Neuroscience. 2010; 11 (2):127–38. https://doi.org/10.1038/nrn2787 PMID: 20068583 2. Shipp S. Neural elements for predictive coding. Frontiers in Psychology. 2016; 7:1792. https://doi.org/ 10.3389/fpsyg.2016.01792 PMID: 27917138 3. Barrett LF. The theory of constructed emotion: an active inference account of interoception and catego- rization. Social Cognitive and Affective Neuroscience. 2017; 12(1):1–23. https://doi.org/10.1093/scan/ nsw154 PMID: 27798257 4. Seth AK, Friston KJ. Active interoceptive inference and the emotional brain. Philosophical Transactions of the Royal Society B: Biological Sciences. 2016; 371(1708):20160007. https://doi.org/10.1098/rstb. 2016.0007 PMID: 28080966 5. Clark A. Whatever next? Predictive brains, situated agents, and the future of cognitive science. Behav- ioral and Brain Sciences. 2013; 36(3):181–204. https://doi.org/10.1017/S0140525X12000477 PMID: 23663408 6. Bar M. The proactive brain: memory for predictions. Philosophical Transactions of the Royal Society B: Biological Sciences. 2009; 364(1521):1235–43. https://doi.org/10.1098/rstb.2008.0310 PMID: 19528004 7. Pezzulo G, Zorzi M, Corbetta M. The secret life of predictive brains: what’s spontaneous activity for? Trends in Cognitive Sciences [Internet]. 2021 Jun 16 [cited 2021 Jul 6];0(0). Available from: https:// www.cell.com/trends/cognitive-sciences/abstract/S1364-6613(21)00128-5 PMID: 34144895 8. Sterling P. Allostasis: A model of predictive regulation. Physiology & Behavior. 2012 Apr 12; 106(1):5– 15. 21 / 24 PLOS ONE \| https://doi.org/10.1371/journal.pone.0297954 February 9, 2024 PLOS ONE Implicit learning, cue ambiguity and affective predictions 9. Sterling P, Laughlin S. Principles of neural design. MIT press; 2015. 9. Sterling P, Laughlin S. Principles of neural design. MIT press; 2015. 10. Alink A, Schwiedrzik CM, Kohler A, Singer W, Muckli L. Stimulus predictability reduces responses in pri- mary visual cortex. Journal of Neuroscience. 2010; 30(8):2960–6. https://doi.org/10.1523/ JNEUROSCI.3730-10.2010 PMID: 20181593 11. Kok P, Rahnev D, Jehee JFM, Lau HC, de Lange FP. Attention reverses the effect of prediction in silencing sensory signals. Cerebral cortex. 2019; 22(9):2197–206. 12. Stefanics G, Heinzle J, Horva´th AA, Stephan KE. Visual mismatch and predictive coding: A computa- tional single-trial ERP study. Journal of Neuroscience. 2018; 38(16):4020–30. https://doi.org/10.1523/ JNEUROSCI.3365-17.2018 PMID: 29581379 13. Duma GM, Granziol U, Mento G. Should I stay or should I go? How local-global implicit temporal expec- tancy shapes proactive motor control: An hdEEG study. NeuroImage. 2020; 220:117071. https://doi. org/10.1016/j.neuroimage.2020.117071 PMID: 32574807 14. Mento G, Vallesi A. Spatiotemporally dissociable neural signatures for generating and updating expec- tation over time in children: A High Density-ERP study. Developmental Cognitive Neuroscience. 2016; 19:98–106. https://doi.org/10.1016/j.dcn.2016.02.008 PMID: 26946428 15. LeDoux JE, Hofmann SG. The subjective experience of emotion: a fearful view. PLOS ONE \| https://doi.org/10.1371/journal.pone.0297954 February 9, 2024 References Anticipation of high arousal aversive and positive movie clips engages common and distinct neural substrates. Social Cognitive and Affective Neuroscience. 2015; 10(4):605–11. https://doi.org/10.1093/scan/nsu091 PMID: 24984958 29. Johnen AK, Harrison NR. The effects of valid and invalid expectations about stimulus valence on beha- vioural and electrophysiological responses to emotional pictures. International Journal of Psychophysi- ology. 2019; 144:47–55. https://doi.org/10.1016/j.ijpsycho.2019.08.002 PMID: 31398378 22 / 24 PLOS ONE \| https://doi.org/10.1371/journal.pone.0297954 February 9, 2024 PLOS ONE Implicit learning, cue ambiguity and affective predictions 30. Lin H, Liang J, Liu T, Liang Z, Jin H. Cue Valence Influences the Effects of Cue Uncertainty on ERP Responses to Emotional Events. Frontiers in Human Neuroscience. 2020; 14:140. https://doi.org/10. 3389/fnhum.2020.00140 PMID: 32351374 31. Lin H, Jin H, Liang J, Yin R, Liu T, Wang Y. Effects of Uncertainty on ERPs to Emotional Pictures Depend on Emotional Valence. Frontiers in Psychology. 2015; 6(1927). https://doi.org/10.3389/fpsyg. 2015.01927 PMID: 26733916 32. Lin H, Xiang J, Li S, Liang J, Jin H. Anticipation of negative pictures enhances the P2 and P3 in their later recognition. Frontiers in Human Neuroscience. 2015; 9:646. https://doi.org/10.3389/fnhum.2015. 00646 PMID: 26648860 33. Lin H, Xiang J, Li S, Liang J, Zhao D, Yin D, et al. Cued uncertainty modulates later recognition of emo- tional pictures: An ERP study. International Journal of Psychophysiology. 2017; 116:68–76. https://doi. org/10.1016/j.ijpsycho.2017.03.004 PMID: 28323026 34. Green P, MacLeod CJ. SIMR: an R package for power analysis of generalized linear mixed models by simulation. Methods in Ecology and Evolution. 2016; 7(4):493–8. 35. Marchewka A, \.Zurawski Lukasz, Jednoro´g K, Grabowska A. The Nencki Affective Picture System (NAPS): Introduction to a novel, standardized, wide-range, high-quality, realistic picture database. Behavior Research Methods. 2014; 46(2):596–610. https://doi.org/10.3758/s13428-013-0379-1 PMID: 23996831 36. Mathoˆt S, Schreij D, Theeuwes J. OpenSesame: An open-source, graphical experiment builder for the social sciences. Behavior Research Methods. 2012; 44(2):314–24. https://doi.org/10.3758/s13428- 011-0168-7 PMID: 22083660 37. Lange K, Ku¨hn S, Filevich E. "Just Another Tool for Online Studies” (JATOS): An Easy Solution for Setup and Management of Web Servers Supporting Online Studies. PLOS ONE. 2015 Jun 26; 10(6): e0130834. 38. Carleton RN, Norton MAPJ, Asmundson GJG. Fearing the unknown: A short version of the Intolerance of Uncertainty Scale. Journal of Anxiety Disorders. 2007; 21(1):105–17. https://doi.org/10.1016/j. janxdis.2006.03.014 PMID: 16647833 39. Lovibond PF, Lovibond SH. The structure of negative emotional states: Comparison of the Depression Anxiety Stress Scales (DASS) with the Beck Depression and Anxiety Inventories. Behaviour Research and Therapy. 1995 Mar 1; 33(3):335–43. References https://doi.org/10.1016/0005-7967(94)00075-u PMID: 7726811 40. Del Popolo Cristaldi F, Buodo G, Duma GM, Sarlo M, Mento G. Unbalanced functional connectivity at rest affects the ERP correlates of affective prediction in high intolerance of uncertainty individuals: A high density EEG investigation. International Journal of Psychophysiology. 2022 Aug 1; 178:22–33. https://doi.org/10.1016/j.ijpsycho.2022.06.006 PMID: 35709946 41. Del Popolo Cristaldi F, Mento G, Sarlo M, Buodo G. Dealing with uncertainty: A high-density EEG inves- tigation on how intolerance of uncertainty affects emotional predictions. PLOS ONE. 2021 Jul 1; 16(7): e0254045. https://doi.org/10.1371/journal.pone.0254045 PMID: 34197554 42. Gole M, Scha¨fer A, Schienle A. Event-related potentials during exposure to aversion and its anticipa- tion: The moderating effect of intolerance of uncertainty. Neuroscience Letters. 2012; 507(2):112–7. https://doi.org/10.1016/j.neulet.2011.11.054 PMID: 22172930 43. Buehler R, McFarland C, Spyropoulos V, Lam KCH. Motivated Prediction of Future Feelings: Effects of Negative Mood and Mood Orientation on Affective Forecasts. Pers Soc Psychol Bull. 2007 Sep 1; 33 (9):1265–78. https://doi.org/10.1177/0146167207303014 PMID: 17586732 44. Bru¨hl AB, Viebke MC, Baumgartner T, Kaffenberger T, Herwig U. Neural correlates of personality dimensions and affective measures during the anticipation of emotional stimuli. Brain Imaging and Behavior. 2011; 5:86–96. https://doi.org/10.1007/s11682-011-9114-7 PMID: 21264550 45. Leys C, Delacre M, Mora YL, Lakens D, Ley C. How to Classify, Detect, and Manage Univariate and Multivariate Outliers, With Emphasis on Pre-Registration. International Review of Social Psychology [Internet]. 2019 [cited 2021 May 11]; 32(1). Available from: http://www.rips-irsp.com/articles/10.5334/ irsp.289/ 46. Bates D, Ma¨chler M, Bolker BM, Walker SC. Fitting linear mixed-effects models using lme4. Journal of Statistical Software. 2015; 67(1). 47. Kuznetsova A, Brockhoff PB, Christensen RHB. lmerTest Package: Tests in Linear Mixed Effects Mod- els. Journal of Statistical Software. 2017; 82(13):1–26. 48. Nakagawa S, Johnson PCD, Schielzeth H. The coefficient of determination R2 and intra-class correla- tion coefficient from generalized linear mixed-effects models revisited and expanded. Journal of The Royal Society Interface. 2017 Sep 30; 14(134):20170213. https://doi.org/10.1098/rsif.2017.0213 PMID: 28904005 23 / 24 PLOS ONE \| https://doi.org/10.1371/journal.pone.0297954 February 9, 2024 PLOS ONE Implicit learning, cue ambiguity and affective predictions 49. Barrett LF. Context reconsidered: Complex signal ensembles, relational meaning, and population think- ing in psychological science. American Psychologist. 2022; 77(8):894. https://doi.org/10.1037/ amp0001054 PMID: 36409120 50. Barrett LF, Satpute AB. Historical pitfalls and new directions in the neuroscience of emotion. Neurosci- ence Letters. 2019; 693:9–18. https://doi.org/10.1016/j.neulet.2017.07.045 PMID: 28756189 51. Oosterwijk S, Mackey S, Wilson-Mendenhall C, Winkielman P, Paulus MP. References Concepts in context: Pro- cessing mental state concepts with internal or external focus involves different neural systems. Social Neuroscience. 2015; 10(3):294–307. https://doi.org/10.1080/17470919.2014.998840 PMID: 25748274 52. Kirschner H, Hilbert K, Hoyer J, Lueken U, Beesdo-Baum K. Psychophsyiological reactivity during uncertainty and ambiguity processing in high and low worriers. Journal of Behavior Therapy and Experi- mental Psychiatry. 2016; 50:97–105. https://doi.org/10.1016/j.jbtep.2015.06.001 PMID: 26143445 53. Grenier S, Barrette AM, Ladouceur R. Intolerance of Uncertainty and Intolerance of Ambiguity: Similari- ties and differences. Personality and Individual Differences. 2005 Aug 1; 39(3):593–600. 54. Carleton RN. The intolerance of uncertainty construct in the context of anxiety disorders: theoretical and practical perspectives. Expert Review of Neurotherapeutics. 2012 Aug 1; 12(8):937–47. https://doi.org/ 10.1586/ern.12.82 PMID: 23002938 55. Herwig U, Kaffenberger T, Baumgartner T, Ja¨ncke L. Neural correlates of a “pessimistic” attitude when anticipating events of unknown emotional valence. NeuroImage. 2007 Jan; 34(2):848–58. https://doi. org/10.1016/j.neuroimage.2006.09.035 PMID: 17112750 56. Schumacher S, Herwig U, Baur V, Mueller-Pfeiffer C, Martin-Soelch C, Rufer M, et al. Psychophysiolog- ical responses during the anticipation of emotional pictures. Journal of Psychophysiology. 2015; 29 (1):13–9. 57. Ratcliff R. Methods for dealing with reaction time outliers. Psychological bulletin. 1993; 114(3):510. https://doi.org/10.1037/0033-2909.114.3.510 PMID: 8272468 58. Vandierendonck A. A comparison of methods to combine speed and accuracy measures of perfor- mance: A rejoinder on the binning procedure. Behav Res. 2017 Apr 1; 49(2):653–73. https://doi.org/10. 3758/s13428-016-0721-5 PMID: 26944576 59. Wilcox R, Peterson TJ, McNitt-Gray JL. Data Analyses When Sample Sizes Are Small: Modern Advances for Dealing With Outliers, Skewed Distributions, and Heteroscedasticity. Journal of Applied Biomechanics. 2018 Aug 1; 34(4):258–61. https://doi.org/10.1123/jab.2017-0269 PMID: 30045651 60. Fox J, Weisberg S. An R companion to applied regression. Third edition. Thousand Oaks, CA: Sage; 2019. 61. Musfeld P, Souza AS, Oberauer K. Repetition learning is neither a continuous nor an implicit process. Proceedings of the National Academy of Sciences. 2023 Apr 18; 120(16):e2218042120. 62. Clark SC, Benassi VA. Judgment of Contingency: Contrast and Assimilation, Displacement of Judg- ments, and Self-Efficacy. Social Behavior & Personality: an international journal. 1997 May; 25(2):183. 63. Batterink LJ, Paller KA, Reber PJ. Understanding the Neural Bases of Implicit and Statistical Learning. Topics in Cognitive Science. 2019; 11(3):482–503. https://doi.org/10.1111/tops.12420 PMID: 30942536 64. Kim R, Seitz A, Feenstra H, Shams L. Testing assumptions of statistical learning: Is it long-term and implicit? Neuroscience Letters. 2009 Sep 11; 461(2):145–9. https://doi.org/10.1016/j.neulet.2009.06. 030 PMID: 19539701 65. PLOS ONE \| https://doi.org/10.1371/journal.pone.0297954 February 9, 2024 References Chennu S, Noreika V, Gueorguiev D, Blenkmann A, Kochen S, Iba´ñez A, et al. Expectation and atten- tion in hierarchical auditory prediction. Journal of Neuroscience. 2013; 33(27):11194–205. https://doi. org/10.1523/JNEUROSCI.0114-13.2013 PMID: 23825422 66. Barrett LF, Mesquita B, Gendron M. Context in Emotion Perception. Current Directions in Psychological Science. 2011; 20(5):286–90. 67. Simons DJ, Shoda Y, Lindsay DS. Constraints on Generality (COG): A Proposed Addition to All Empiri- cal Papers. Perspect Psychol Sci. 2017 Nov 1; 12(6):1123–8. https://doi.org/10.1177/ 1745691617708630 PMID: 28853993 24 / 24
https://openalex.org/W4319160799	https://www.frontiersin.org/articles/10.3389/fnint.2023.898215/pdf	English	null	Neuropsychiatric feature-based subgrouping reveals neural sensory processing spectrum in female FMR1 premutation carriers: A pilot study	Frontiers in integrative neuroscience	2,023	cc-by	10,544	OPEN ACCESS OPEN ACCESS EDITED BY Elena V. Mnatsakanian, Institute of Higher Nervous Activity and Neurophysiology (RAS), Russia REVIEWED BY Carol Lee Wilkinson, Boston Children’s Hospital and Harvard Medical School, United States Emily G. Allen, Emory University, United States CORRESPONDENCE Lauren E. Ethridge ethri@ou.edu EDITED BY Elena V. Mnatsakanian, Institute of Higher Nervous Activity and Neurophysiology (RAS), Russia REVIEWED BY Carol Lee Wilkinson, Boston Children’s Hospital and Harvard Medical School, United States Emily G. Allen, Emory University, United States CORRESPONDENCE Lauren E. Ethridge ethri@ou.edu Jordan E. Norris1, Lauren M. Schmitt2,3, Lisa A. De Stefano2, Ernest V. Pedapati4,5,6, Craig A. Erickson4,6, John A. Sweeney6 and Lauren E. Ethridge1,7* Jordan E. Norris1, Lauren M. Schmitt2,3, Lisa A. De Stefano2, Ernest V. Pedapati4,5,6, Craig A. Erickson4,6, John A. Sweeney6 and Lauren E. Ethridge1,7* RECEIVED 17 March 2022 ACCEPTED 18 January 2023 PUBLISHED 03 February 2023 CITATION Norris JE, Schmitt LM, De Stefano LA, Pedapati EV, Erickson CA, Sweeney JA and Ethridge LE (2023) Neuropsychiatric feature-based subgrouping reveals neural sensory processing spectrum in female FMR1 premutation carriers: A pilot study. Front. Integr. Neurosci. 17:898215. doi: 10.3389/fnint.2023.898215 1Department of Psychology, The University of Oklahoma, Norman, OK, United States, 2Behavioral Medicine and Clinical Psychology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, United States, 3Department of Pediatrics, University of Cincinnati, Cincinnati, OH, United States, 4Division of Child and Adolescent Psychiatry, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, United States, 5Division of Child Neurology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, United States, 6Department of Psychiatry and Behavioral Neuroscience, University of Cincinnati, Cincinnati, OH, United States, 7Department of Pediatrics, Section on Developmental and Behavioral Pediatrics, The University of Oklahoma Health Sciences Center, Oklahoma City, OK, United States CITATION Norris JE, Schmitt LM, De Stefano LA, Pedapati EV, Erickson CA, Sweeney JA and Ethridge LE (2023) Neuropsychiatric feature-based subgrouping reveals neural sensory processing spectrum in female FMR1 premutation carriers: A pilot study. Front. Integr. Neurosci. 17:898215. doi: 10.3389/fnint.2023.898215 Introduction: Fragile X Syndrome (FXS) is rare genetic condition characterized by a repeat expansion (CGG) in the Fragile X messenger ribonucleoprotein 1 (FMR1) gene where individuals with greater than 200 repeats are deﬁned as full mutation. FXS clinical presentation often includes intellectual disability, and autism- like symptoms, including anxiety and sensory hypersensitivities. TYPE Original Research PUBLISHED 03 February 2023 DOI 10.3389/fnint.2023.898215 TYPE Original Research PUBLISHED 03 February 2023 DOI 10.3389/fnint.2023.898215 TYPE Original Research PUBLISHED 03 February 2023 DOI 10.3389/fnint.2023.898215 Neuropsychiatric feature-based subgrouping reveals neural sensory processing spectrum in female FMR1 premutation carriers: A pilot study OPEN ACCESS EDITED BY Elena V. Mnatsakanian, Institute of Higher Nervous Activity and Neurophysiology (RAS), Russia REVIEWED BY Carol Lee Wilkinson, Boston Children’s Hospital and Harvard Medical School, United States Emily G. Allen, Emory University, United States CORRESPONDENCE Lauren E. Ethridge ethri@ou.edu RECEIVED 17 March 2022 ACCEPTED 18 January 2023 PUBLISHED 03 February 2023 CITATION Norris JE, Schmitt LM, De Stefano LA, Pedapati EV, Erickson CA, Sweeney JA and Ethridge LE (2023) Neuropsychiatric feature-based subgrouping reveals neural sensory processing spectrum in female FMR1 premutation carriers: A pilot study. Front. Integr. Neurosci. 17:898215. doi: 10.3389/fnint.2023.898215 COPYRIGHT 1. Background neuropsychiatric risk (Schmitt et al., 2022). Executive dysfunction and social processing diﬃculties were previously noted in child PMCs, in addition to the developmental diﬀerences between PMCs and TD infants centered around abnormal sensory experiences and non-verbal communication (Wheeler et al., 2016). Fragile X Syndrome (FXS) is a trinucleotide repeat disorder aﬀecting the X chromosome characterized by a cytosine-guanine- guanine (CGG) expansion of the FMR1 gene (Verkerk et al., 1991; Yu et al., 1991; Ciaccio et al., 2017). FXS is the most common monogenic cause of intellectual disability and autism. Repeat expansions > 200 CGG repeats are referred to as full mutations. These expansions result in extensive methylation of the gene, large reductions in Fragile X protein (FXP or Fragile X Messenger Ribonucleoprotein (FMRP), and symptoms of FXS. Repeat expansions falling between a typical CGG repeat count and 200 repeats (i.e., 55 – 200 CGG repeats) on the FMR1 gene result in a premutation, which is associated with increased FMR1 mRNA accompanied by minor reduction in protein levels, but which is not associated with the full cognitive and behavioral presentation of FXS (Fu et al., 1991; Wheeler et al., 2014). While individuals with FXS typically presents with varying levels of sensory processing diﬃculties (i.e., sensory hypersensitivities), individuals with the FMR1 premutation are less aﬀected and demonstrate a diﬀerent pattern of behavioral symptoms than the full mutation (Schmitt et al., 2019). Abnormal sensory processing is a common phenotypical feature of the FXS full mutation manifesting typically as sensory hyperreactivity (Ethridge et al., 2019). Few studies have addressed sensory sensitivity in PMCs and also fail to utilize measures of neural responses such as electroencephalography (EEG) to sensory stimulation (Hunter et al., 2008; Allen et al., 2020; Schmitt et al., 2022). Most prior evaluations focused on child PMCs from a developmental perspective, limiting commentary on how moderate FMR1 expansions impact sensory processing capacity later in the lifespan. Previous studies in child PMCs have documented co- occurring sensory hyper- and hypo-responsivity that vary based on age (Wheeler et al., 2016; Raspa et al., 2018). Most studies of sensory processing abnormalities in adult PMCs focused on their relation to mood and anxiety (Allen et al., 2020; Schmitt et al., 2022). The neural basis of sensory processing alterations remain unclear, as is the mechanism by which alterations at the neural level are related to clinical presentation of neuropsychiatric symptoms in adult PMCs. 1. Background Individuals with the FMR1 premutation (premutation carrier – PMC) exhibit considerable variability in the type and severity of their clinical proﬁles (Budimirovic et al., 2020). PMCs exhibit complex health proﬁles including two known conditions associated with aging: Fragile X Tremor Ataxia Syndrome (FXTAS) and Fragile X Primary Ovarian Insuﬃciency (FXPOI) (Berry-Kravis et al., 2007). Beyond FXTAS and FXPOI, PMCs present with increased rates of psychopathology risk that change across the lifespan. Children with the premutation present with more frequent diagnoses of Attention Deﬁcient Hyperactivity Disorder (ADHD), autism spectrum disorder (ASD), anxiety, and social diﬃculties, whereas adult PMCs primarily present with increased rates of anxiety and depression, particularly in females, who are twice as likely to have a premutation status than males due to the location of the FMR1 gene on the X chromosome (Hunter et al., 2014; Cordeiro et al., 2015; Hagerman et al., 2018). Neuropsychiatric features of the premutation based on patterns of psychiatric, health, and behavioral symptoms were recently classiﬁed as Fragile X Associated Neuropsychiatric Disorder (FXAND) signifying the prevalence of psychiatric symptoms among PMCs. Further, PMCs are at higher risk for exacerbation of underlying psychopathology by comorbidities associated with the premutation and external experiences (e.g., close relative with the full mutation) resulting in greater impact of the premutation on quality of life (Hall et al., 2016; Wheeler et al., 2017; Hagerman et al., 2018; Allen et al., 2020; Klusek et al., 2020). Female adult PMCs may experience impaired executive function and social processing diﬃculties in addition to increased We recently addressed clinical proﬁle variability in the neuropsychiatric, executive function, eye tracking, and resting EEG features of PMCs using cluster-based methods. Clustering successfully categorized PMCs into three distinct subgroups based on aﬀective, cognitive, and behavioral features. A three-cluster solution neatly identiﬁed one group with increased neuropsychiatric features (cluster 1) including increased depression and anxiety, but relatively typical resting EEG features. The second cluster (cluster 2) was characterized by increased executive dysfunction and higher resting gamma power suggesting a group more comparable to full mutation FXS. Finally, the third cluster (cluster 3) was comprised of members who were relatively unaﬀected by psychiatric or cognitive symptoms (Schmitt et al., 2022). However, this initial study did not examine or utilize sensory self-report or sensory EEG (such as sensory evoked potentials) measures or EEG measures directly related to mood and anxiety (e.g., frontal asymmetry) in deriving the cluster solution. OPEN ACCESS Individuals with 55 to <200 CGG repeats are said to have the FMR1 premutation, which is not associated with primary characteristics of the full mutation, but with an increased risk for anxiety, depression, and other affective conditions, as well as and impaired cognitive processing differences that vary in severity. Deﬁning subgroups of premutation carriers based on distinct biological features may identify subgroups with varying levels of psychiatric, cognitive, and behavioral alterations. © 2023 Norris, Schmitt, De Stefano, Pedapati, Erickson, Sweeney and Ethridge. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Methods: The current pilot study utilized 3 cluster subgroupings deﬁned by previous k means cluster analysis on neuropsychiatric, cognitive, and resting EEG variables in order to examine basic sensory auditory chirp task-based EEG parameters from 33 females with the FMR1 premutation (ages 17–78). Results: Based on the predeﬁned, neuropsychiatric three-cluster solution, premutation carriers with increased neuropsychiatric features and higher CGG repeat counts (cluster 1) showed decreased stimulus onset response, similar to previous ERP ﬁndings across a number of psychiatric disorders but opposite to ﬁndings in individuals with full mutation FXS. Premutation carriers with increased executive dysfunction and resting gamma power (cluster 2) exhibited decreased gamma phase locking to a chirp stimulus, similar to individuals with full mutation FXS. Cluster 3 members, who were relatively unaffected by psychiatric or cognitive symptoms, showed the most normative task-based EEG metrics. Discussion: Our ﬁndings suggest a spectrum of sensory processing characteristics present in subgroups of premutation carriers that have been previously understudied due to lack of overall group differences. Our ﬁndings also further validate the pre-deﬁned clinical subgroups by supporting links between Frontiers in Integrative Neuroscience Frontiers in Integrative Neuroscience 01 frontiersin.org Norris et al. 10.3389/fnint.2023.898215 10.3389/fnint.2023.898215 disturbances in well-deﬁned neural pathways and behavioral alterations that may be informative for identifying the mechanisms supporting speciﬁc risk factors and divergent therapeutic needs in individuals with the FMR1 premutation. sensory processing, neural oscillations and entrainment, FMR1 premutation carrier, clustering analysis, EEG Frontiers in Integrative Neuroscience 2.3. EEG recording and data reduction EEG recordings were collected using 128 channel hydrocel nets (Electrical Geodesics, Eugene, Oregon). EEG was recorded continuously and digitized at 1000 Hz, ﬁltered from 0.01 to 200 Hz, referenced to Cz, and ampliﬁed 10,000x. Sensors were placed with landmark sensors according to the International 10/10 system (Chatrian et al., 1985; Luu and Ferree, 2005). Raw data were visually inspected oﬄine. Bad sensors were interpolated using spherical spline interpolation (no more than 5% per subject for a total of 6 electrodes, no more than two adjacent, 96.97% of participants had no sensors interpolated within the 23 channels used to calculate general EEG variables for ﬁnal analyses, 100% did not have F3 or F4 interpolated) implemented in BESA 6.1 (MEGIS Software, Grafelﬁng, Germany). Data were digitally ﬁltered from 0.5 to 120 Hz (12 and 24 db/octave roll-oﬀ, respectively; zero-phase; 60 Hz notch) and data segments with large amounts of artifact were removed prior to individual component removal to facilitate algorithm convergence while running independent component analysis (ICA; Infomax). Lastly, large muscle artifacts (e.g., eye movement, cardiac, and muscle movement artifacts) were removed blind to participant group using ICA implemented in EEGLAB through Matlab 2018b software (Delorme and Makeig, 2004; The Mathworks, Natick, MA, United States). Data were transformed to average reference and epoched into 3,250 ms trials (-500 to 2,750 ms). Chirp task data were averaged across trials. Any trial with post-ICA amplitude exceeding 120 µV was considered residual artifact and removed prior to averaging (i.e., trials containing large quantities of movement artifact post-ICA were rejected). The same 23 electrodes were selected a priori for general level analysis from prior work with the FXS full mutation based on a fronto-central spatial distribution of the scalp best poised to capture auditory cortex activity (Luck, 2014; Ethridge et al., 2019). Intertrial coherence (ITC) and single trial power were calculated with un-baseline-corrected epoched single- trial data according to wavelet methods used previously for Ethridge et al. (2019) and using the same power bands across the entire epoch and ITC time-frequency clusters: low gamma ITC (30-55 Hz) during the chirp, high gamma ITC (65-80 Hz) during the chirp, chirp stimulus onset, and oﬀset theta/alpha ITC (corresponding to time windows encompassing approximately 50-400 ms after chirp stimulus onset and again post- chirp stimulus oﬀset (2,050-2,400 ms in Figure 1), and a 4-13 Hz frequency range from previous work. 1. Background As changes in sensory function measured both by self/caregiver report and sensory EEG are highly prevalent in full mutation FXS (Ethridge et al., 2019), it is of interest to examine these features, their distribution, and relevance to more commonly reported neuropsychiatric features in the FXS premutation. Frontal asymmetry measures have previously been linked to variations in mood and anxiety (Allen et al., 2004; Coan and Allen, 2004; Thibodeau et al., 2006; Stewart et al., 2010; Smith et al., 2017; van der Vinne et al., 2017), relevant to individuals with the FXS premutation. Changes in frontal asymmetry have also been reported in full 02 Frontiers in Integrative Neuroscience frontiersin.org Norris et al. 10.3389/fnint.2023.898215 10.3389/fnint.2023.898215 10.3389/fnint.2023.898215 2.3. EEG recording and data reduction Single trial power evaluates the amplitude of the neural response whereas ITC evaluates consistency of phase of the neural response frequency at any given timepoint across trials (Delorme and Makeig, 2004). 2.2. Procedure mutation FXS, related to sensory processing and adaptive behavior (Norris et al., 2022). Use of predeﬁned clustering may provide insight into the neural basis of sensory, mood, and anxiety processing in adult PMCs speciﬁc to the features that deﬁned each cluster and provide deﬁned neural pathways and behavioral alterations associated with varying clinical proﬁles. In this sense, any diﬀerences in sensory or asymmetry EEG measures between clusters serves as an external validation of cluster results, in that these measures were not used to deﬁne initial clusters but rather support additional sources of variation linked to cluster-deﬁning features of PMCs. Participants underwent dense-array EEG while listening passively to a chirp stimulus. The chirp stimulus was an amplitude modulated white noise burst carrier stimulus increasing in frequency from 0- 100 Hz over the course of 2,000 ms. Chirp stimuli were presented 200 times with each trial separated by an intertrial period that alternated between 1,500 ms and 2,000 ms. Stimuli were delivered via headphones at 65 db SPL while the participants watched a silent movie, consistent with prior studies using individuals with FXS (Ethridge et al., 2016, 2017, 2019; Smith et al., 2021; Pedapati et al., 2022). The current pilot study aimed to extend the work detailed in Schmitt et al. (2022) by evaluating cluster membership in relation to task-based EEG parameters obtained during an auditory task, considering dynamic evaluations of oscillatory frequency bands in order to test the hypothesis that neuropsychiatric features are present in PMC populations supported by similar neural processes observed in full mutation FXS. Although the current study should be considered a pilot study and hypothesis generating due to the relatively small sample size and sparse literature on EEG in PMCs, we opted to also evaluate cluster diﬀerences across clinical measures as they related to variations in task-related frontal hemispheric asymmetry during the auditory task, as frontal asymmetry measures have previously been linked to variations in mood and anxiety hypothesized to map onto similar characteristics in PMCs (Allen et al., 2004; Coan and Allen, 2004; Thibodeau et al., 2006; Stewart et al., 2010; Smith et al., 2017; van der Vinne et al., 2017). Frontiers in Integrative Neuroscience 2.1. Participants Premutation carriers (N = 33) Cluster 1 (N = 8) Cluster 2 (N = 10) Cluster 3 (N = 11) General M SD M SD M SD Age 46.73 17.19 50.47 14.54 51.79 8.92 CGG repeat count 107.33 11.61 93.11 14.63 96.11 20.84 Clinical mood measures ASI 24.38 6.32 18.29 9.88 11.22 7.66 BDI 22.25 5.95 10.56 10.11 5.80 4.16 Demographic variables for PMCs by cluster. (A) Intertrial coherence for Clusters 1, 2, and 3 for the chirp task. (B) Difference plots for cluster 3 minus cluster 1, cluster 2 minus cluster 1, and cluster 3 minus cluster 2. Red boxes on difference maps indicate areas with signiﬁcant group differences. Warmer colors in difference maps indicate higher phase-locking values for the ﬁrst cluster and the cooler colors indicate higher values for the second cluster. 2.4. Frontal asymmetry EEG analysis (4-7 Hz), alpha (8-13 Hz), and low gamma (30-55 Hz) ranges for all recordings. All variables calculated using F3 and F4 are referenced as F3/F4 frontal EEG variables (e.g., F3/F4 frontal hemispheric power) to diﬀerentiate from power measures calculated using the initial 23 electrodes from previous work, referenced as general frontal power. Data were transformed to current source density (CSD)- Laplacian in BESA 6.1 for frontal asymmetry analyses and hemisphere speciﬁc power analyses. CSD computation reduces volume conduction eﬀects (i.e., signal smearing) and improves both temporal and spatial resolution (Stewart et al., 2010; Burle et al., 2015; Smith et al., 2017). Data from one electrode in each hemisphere were used to calculate frontal asymmetry scores according to conventional frontal asymmetry analysis methods (Allen et al., 2004; Stewart et al., 2010). Electrode F3 (left hemisphere) and electrode F4 (right hemisphere) were selected for data extraction and asymmetry was operationalized as (R – L)/(R + L) to compute a normalized diﬀerence score (NDS) that normalizes for overall power. The correlation between NDS and the natural log diﬀerence scores [i.e., ln(R)- ln(L)] was checked to conﬁrm linearity with NDSs and found to correlate highly (rho > 0.9, p < 0.001) with the natural log calculation, as expected (Allen et al., 2004). Single trial power was extracted from each electrode to compute asymmetry in the theta 2.1. Participants Participants were 33 adult females with the FMR1 premutation [Mean (M) age = 48.36, SD = 13.17; age range 19.61 – 78.36] (Table 1). Only female PMCs were recruited to control for biological sex and ensure feasible ascertainment. The PMC sample was a convenience sample recruited from primary relatives (i.e., parent, grandparent, or sibling) of individuals with FXS through the Cincinnati Fragile X Research and Treatment Center, to pilot test for diﬀerences in individuals with the FMR1 premutation on measures that show signiﬁcant diﬀerences in individuals with the FMR1 full mutation. PMC status was conﬁrmed via medical record review or by a previously completed PCR quantiﬁcation of allele speciﬁc CGG repeat length. Eleven PMC were currently taking antidepressant (AD) medications, consistent with reported rates of AD use and recommendations for treating PMCs experiencing mood disorders (Cordeiro et al., 2015; Hagerman et al., 2018). Exclusion criteria included history of seizures and current use of anticonvulsant and benzodiazepine medications due to known EEG eﬀects of both medications. Clinical self-report assessments included the Adolescent/Adult Sensory Proﬁle (Brown et al., 2001), Anxiety Depression and Mood Scale (ADAMS, Esbensen et al., 2003), Becks’ Depression Inventory (BDI, Beck et al., 1961), and the Anxiety Sensitivity Index (ASI, Reiss et al., 1986). All participants provided written informed consent prior to participation, as approved by the Cincinnati Children’s Hospital Institutional Review Board. 03 frontiersin.org 10.3389/fnint.2023.898215 Norris et al. TABLE 1 Participant demographics. Premutation carriers (N = 33) Cluster 1 (N = 8) Cluster 2 (N = 10) Cluster 3 (N = 11) General M SD M SD M SD Age 46.73 17.19 50.47 14.54 51.79 8.92 CGG repeat count 107.33 11.61 93.11 14.63 96.11 20.84 Clinical mood measures ASI 24.38 6.32 18.29 9.88 11.22 7.66 BDI 22.25 5.95 10.56 10.11 5.80 4.16 Demographic variables for PMCs by cluster. FIGURE 1 (A) Intertrial coherence for Clusters 1, 2, and 3 for the chirp task. (B) Difference plots for cluster 3 minus cluster 1, cluster 2 minus cluster 1, and cluster 3 minus cluster 2. Red boxes on difference maps indicate areas with signiﬁcant group differences. Warmer colors in difference maps indicate higher phase-locking values for the ﬁrst cluster and the cooler colors indicate higher values for the second cluster. Frontiers in Integrative Neuroscience 3.3. Correlations across the PMC sample Correlation analyses reﬂected an exploratory evaluation of EEG variables and clinical measures. Signiﬁcant correlations for PMCs between general frontal EEG, F3/F4 frontal EEG, and clinical variables are shown in Table 4. General physiological correlations are presented in Table 5 to provide a broad assessment of the PMC neural phenotype across relationships generated from full mutation EEG ﬁndings. General frontal gamma power was correlated with all F3/F4 EEG measures across the alpha, theta, and low gamma frequency bands demonstrating unilateral use of high and low frequency bands. Power measures at the hemisphere level also demonstrated a pattern suggesting right hemisphere gamma was more strongly correlated with general frontal gamma, but this pattern was not signiﬁcant, z(32) = −0.79, p = 0.21. Correlations between BDI scores and neural responses to the onset of the chirp stimulus adds support to the conclusion that hypo-responsivity shares a signiﬁcant relationship with increased symptoms of depression. Increased general anxiety on the ADAMs also was associated with decreased responses to the onset of the chirp stimulus, but not decreased mood scores. Only one correlation was retained as signiﬁcant between F3/F4 frontal EEG variables and clinical variables (not shown in Table 4): right alpha power was inversely correlated with manic/hyperactive behavior scores on the ADAMs (rho = −0.77, p = 0.015). TABLE 2 Univariate results for EEG data by cluster MANCOVA. TABLE 2 Univariate results for EEG data by cluster MANCOVA. EEG variable F(2,24) P value Effect size (Partial eta squared) ITC stimulus onset 3.61 0.043 0.231 ITC stimulus oﬀset 1.69 0.206 0.123 ITC low gamma 3.76 0.038 0.239 ITC high gamma 2.07 0.148 0.147 Theta power 1.33 0.283 0.100 Gamma power 0.256 0.776 0.021 Theta power asymmetry 1.56 0.231 0.115 Alpha power asymmetry 1.56 0.229 0.116 Gamma power asymmetry 3.01 0.068 0.200 See section “3.1. Cluster diﬀerences on EEG measures” of the text for Bonferroni-corrected individual cluster comparisons for signiﬁcant univariate tests. 2.5. Statistical analysis All statistical analyses were conducted in SPSS 27. Multivariate general linear models were utilized to assess hypothesis driven diﬀerences across clusters on EEG variables and clinical measures assessing sensory processing. Cluster membership was obtained from Schmitt et al. (2022), based on a k-means cluster analysis of the same participants using neuropsychiatric, executive functioning, social attention, eye-tracking measures, and resting EEG features. Eﬀect sizes are reported as partial eta squared. All analyses included age as a covariate, retained where signiﬁcant. All cluster comparisons were hypothesis driven. Clinical correlations were examined with all 04 frontiersin.org Norris et al. 10.3389/fnint.2023.898215 3.2. Cluster differences on clinical measures EEG variables. Exploratory correlations were also evaluated between power in frequency bands of interest (i.e., theta, alpha, and gamma) and hypothesis-driven associations with gamma ITC from FXS ﬁndings (Ethridge et al., 2019). All correlations were bivariate and conducted using Spearman’s rho. Clinical correlations and power band correlations were considered to be exploratory and hypothesis generating due to the current limited scope of neuropsychiatric research with PMC populations, and thus not corrected for multiple comparisons. EEG variables. Exploratory correlations were also evaluated between power in frequency bands of interest (i.e., theta, alpha, and gamma) and hypothesis-driven associations with gamma ITC from FXS ﬁndings (Ethridge et al., 2019). All correlations were bivariate and conducted using Spearman’s rho. Clinical correlations and power band correlations were considered to be exploratory and hypothesis generating due to the current limited scope of neuropsychiatric research with PMC populations, and thus not corrected for multiple comparisons. Results for anxiety and depression scale scores by cluster are reported in Schmitt et al. (2022). A MANCOVA was conducted examining cluster diﬀerences across Adolescent/Adult Sensory Proﬁle sub-scores to evaluate self- reported sensory experiences across clusters with diﬀering clinical proﬁles. A signiﬁcant diﬀerence was found on low registration (F(2,17) = 4.30, p = 0.031, ES = 0.34) and sensation sensitivity scores (F(2,17) = 5.06, p = 0.019, ES = 0.37) with diﬀerences on touch processing trending toward signiﬁcance (F(2,17) = 3.43, p = 0.056, ES = 0.29). After Bonferroni correction, low registration diﬀerences between clusters were trending with Cluster 3 (M = 25.38, SD = 6.46) exhibiting lower scores compared to Cluster 1 (M = 35.00, SD = 2.94) and Cluster 2 (M = 32.56, SD = 6.73). Sensory sensitivity scores were signiﬁcantly diﬀerent between Cluster 2 (M = 37.00, SD = 5.52) and Cluster 3 (M = 27.25, SD = 7.01), with Cluster 2 exhibiting increased scores compared to Cluster 3. Cluster 1 showed intermediate sensory sensitivity scores (M = 34.50, SD = 6.14) and did not diﬀer from either Cluster 2 or 3. Touch processing scores trended toward increases in Cluster 2 (M = 39.22, SD = 7.82) compared to Cluster 3 (M = 31.75, SD = 3.28), which Cluster 1 again showing an intermediate score (M = 33.50, SD = 4.51). (See Table 3 for full univariate results including non-signiﬁcant comparisons, and Figure 3). 3.1.1. ITC and power differences between clusters 3.1.1. ITC and power differences between clusters A MANCOVA was conducted to evaluate both general EEG variables (i.e., ITC to stimulus onset, ITC to stimulus oﬀset, low gamma ITC to chirp, high gamma ITC to chirp, theta power, alpha power, gamma power) and F3/F4 frontal asymmetry variables across frequency bands (i.e., alpha, theta, and gamma) by PMC cluster membership. Age was a signiﬁcant covariate for ITC in the low gamma range, only. ITC to the onset of the chirp stimulus was signiﬁcantly diﬀerent between clusters, F(2, 24) = 3.61, p = 0.043, ES = 0.23. Post hoc Bonferroni corrected comparisons identiﬁed trending diﬀerences on ITC onset between Cluster 3 (M = 0.15, SE = 0.02) and Cluster 1 (M = 0.05, SE = 0.02). ITC in the low gamma range was also signiﬁcantly diﬀerent between clusters with cluster 2 (M = 0.07, SE = 0.05) exhibiting reduced ITC compared to cluster 3 (M = 0.15, SE = 0.07), F(2, 24) = 3.76, p = 0.038, ES = 0.24. Lastly, there was a trending diﬀerence between clusters on frontal low gamma asymmetry with cluster 2 (M = 0.06, SE = 0.09) exhibiting trending increases in frontal low gamma asymmetry compared to cluster 1 (M = –0.03, SE = 0.09) F(2, 24) = 3.01, p = 0.068, ES = 0.20. None of the comparisons between clusters on power variables were signiﬁcant; cluster diﬀerences were primarily in ITC. ITC reﬂects the ability to reliably reproduce neural oscillations across multiple trials, and in the case of the chirp stimulus, to reliably reproduce the oscillatory properties of the stimulus in time. (See Table 2 for univariate results including non-signiﬁcant comparisons, Figure 1 for ITC plots and Figure 2 for box plots with individual data points). See section “3.1. Cluster diﬀerences on EEG measures” of the text for Bonferroni-corrected individual cluster comparisons for signiﬁcant univariate tests. Frontiers in Integrative Neuroscience frontiersin.org 4. Discussion Cluster diﬀerences on clinical measures” of the text for Bonferroni-corrected individual cluster comparisons for signiﬁcant univariate tests. such as withdrawal, lethargy, and disinterest (i.e., low registration). Cluster 1 exhibiting higher low registration scores further supports this conclusion. Sensory hyposensitivity may mirror symptoms of depression or possibly render PMCs vulnerable to heightened self- reﬂection and internalizing behaviors (Kotsiris et al., 2020). Previous ﬁndings in individuals with depression have indicated a complex relationship between depressive symptoms and sensory processing, with individuals endorsing higher levels of sensory hypersensitivity and hyposensitivity (low registration) than controls (Engel-Yeger et al., 2016). Correlations between the neural response to onset and oﬀset of the stimulus and BDI scores is consistent with the interpretation that sensory hypo-responsivity may contribute to risk or onset of aﬀective disturbances (i.e., Cluster 1). Interestingly, individuals with full mutation FXS show increased neural response and hyperresponsivity to sensory stimuli, the opposite of these PMC ﬁndings, highlighting the diﬀerences in underlying pathophysiology between the two conditions (Ethridge et al., 2017, 2019). 4. Discussion The current study aimed to extend the understanding of FMR1 PMC subgroupings established in a recent exploratory, data- driven approach that identiﬁed discrete clinical subgroups of PMC Frontiers in Integrative Neuroscience 05 frontiersin.org Norris et al. 10.3389/fnint.2023.898215 FIGURE 2 Signiﬁcance markers reﬂect non-corrected MANCOVA results. N.S. = not signiﬁcant, ∗p < 0.05. (A) Boxplot depicting group comparison across clusters for ITC to the onset of the chirp stimulus showed overall signiﬁcance (p = 0.04) where cluster 1 showed a trend toward signiﬁcantly lower ITC than cluster 3 (p = 0.05). (B) Boxplot depicting group comparison across clusters for low gamma ITC with signiﬁcant overall (p = 0.04) where cluster 2 showed signiﬁcantly lower ITC than cluster 3 (p = 0.03). FIGURE 2 Signiﬁcance markers reﬂect non-corrected MANCOVA results. N.S. = not signiﬁcant, ∗p < 0.05. (A) Boxplot depicting group comparison across clusters for ITC to the onset of the chirp stimulus showed overall signiﬁcance (p = 0.04) where cluster 1 showed a trend toward signiﬁcantly lower ITC than cluster 3 (p = 0.05). (B) Boxplot depicting group comparison across clusters for low gamma ITC with signiﬁcant overall (p = 0.04) where cluster 2 showed signiﬁcantly lower ITC than cluster 3 (p = 0.03). individuals (Schmitt et al., 2022) that varied in protein levels (FXP), CGG repeat length, environmental factors, and aging-associated outcomes (e.g., increased mRNA toxicity). We found that PMC with increased neuropsychiatric features and higher CGG repeat counts (Cluster (1) showed decreased stimulus onset response, similar to previous ERP ﬁndings across a number of psychiatric disorders (Drake et al., 1991; Ford et al., 2001; Ethridge et al., 2015) but opposite to ﬁndings in individuals with full mutation FXS (Ethridge et al., 2016, 2019). Premutation carriers with increased executive dysfunction and resting gamma power (Cluster 2) exhibited decreased gamma phase locking to a chirp stimulus, similar to individuals with full mutation FXS. Cluster 3 members, who were relatively unaﬀected by psychiatric or cognitive symptoms, showed the most normative task-based EEG metrics. Sensory variable F(2,17) P value Effect size (Partial eta squared) Auditory processing 2.02 0.163 0.192 Low registration 4.30 0.031 0.336 Sensation seeking 0.352 0.708 0.040 Sensation sensitivity 5.06 0.019 0.373 Sensation avoiding 1.562 0.238 0.155 Touch processing 3.43 0.056 0.287 See section “3.2. Cluster diﬀerences on clinical measures” of the text for Bonferroni-corrected individual cluster comparisons for signiﬁcant univariate tests. See section “3.2. Frontiers in Integrative Neuroscience 4.1. Neurophysiological differences by cluster Diﬀerences among PMC subgroups suggest relevant sensory processing outcomes. Female PMCs in Cluster 1 (the “psychiatric” subgroup) showed the greatest reduction in neural response to stimulus onset relative to other clusters. The simplest interpretation of reduced stimulus onset response is sensory hyposensitivity, which may be linked to general neural hypo-responsivity and/or perceptual disturbances (Donkers et al., 2015). Clinically, Cluster 1 is comprised of individuals with aﬀective disturbances (i.e., elevated depression and anxiety symptoms or the presence of symptoms related to aﬀective disorders like depression or anxiety), suggesting that sensory hypo-responsivity may contribute to and/or co-occur with neuropsychiatric features for these individuals. This is a clinically relevant ﬁnding in that sensory systems present a novel target for therapy in PMC that may also have broader-ranging eﬀects on mood and anxiety. Signiﬁcant correlations between neural response to stimulus onset and clinical variables indicate PMCs may have increased sensory detection thresholds, which may lead to symptoms Cluster 2 (the “executive dysfunction and atypical resting electrophysiology” subgroup) also showed reduced neural response to stimulus onset, and additionally showed reduced ITC to the chirp stimulus in the low gamma range. Cluster 2 also exhibited increased sensory sensitivity compared to other subgroups suggesting 06 frontiersin.org Norris et al. 10.3389/fnint.2023.898215 FIGURE 3 Signiﬁcance markers reﬂect non-corrected MANCOVA results. N.S. = not signiﬁcant, ∗p < 0.05. (A) Box plots depicting group comparison across clusters for ASP sensory sensitivity with overall signiﬁcance (p = 0.02) where cluster 2 showed signiﬁcantly higher sensitivity scores than cluster 3 (p = 0.02). (B) Box plots depicting group comparison across clusters for ASP low registration with overall signiﬁcance (p = 0.03) where cluster 1 showed a trend toward signiﬁcantly higher low registration scores than cluster 3 (p = 0.07). Signiﬁcance markers reﬂect non-corrected MANCOVA results. N.S. = not signiﬁcant, ∗p < 0.05. (A) Box plots depicting group comparison across clusters for ASP sensory sensitivity with overall signiﬁcance (p = 0.02) where cluster 2 showed signiﬁcantly higher sensitivity scores than cluster 3 (p = 0.02). (B) Box plots depicting group comparison across clusters for ASP low registration with overall signiﬁcance (p = 0.03) where cluster 1 showed a trend toward signiﬁcantly higher low registration scores than cluster 3 (p = 0.07). full mutation FXS (Norris et al., 2022). 4.1. Neurophysiological differences by cluster Cluster 2 may exhibit reduced ability to mobilize compensatory activity in the theta range (Wang et al., 2017; Schmitt et al., 2022), which in this case may exacerbate sensory sensitivity symptoms. Indeed, the combination of reduced response to the onset of the chirp but comparably typical low gamma ITC in Cluster 1 suggests recovery in subsequent sustained high frequency sensory processing capacity, which is notably absent in Cluster 2, and which may diﬀerentiate neural pathways contributing to neural compensation and resilience in PMC. Individuals with FXS also show reductions in low gamma ITC to the chirp, which has been consistently correlated with increased autistic features (Ethridge et al., 2017, 2019), consistent with its potential contribution to alterations in sensory processing, and social and executive function deﬁcits. By subdividing the heterogenous PMC group into more homogenous clusters, we hoped to recover a more direct relationship between frontal asymmetry abnormalities and depression and anxiety symptoms, particularly in Cluster 1 individuals who show increased BDI and ASI scores. We did not however ﬁnd any signiﬁcant diﬀerences among clusters for theta and alpha frontal asymmetry, and only a trending diﬀerence between clusters for gamma asymmetry. We also did not ﬁnd any correlations between frontal asymmetry measures and BDI or ASI scores across clusters, suggesting that these relationships identiﬁed in other patient populations with aﬀective illness may not hold or be less robust in PMCs. The lack of relationship between frontal asymmetry and measures of mood dysregulation in PMCs may even reﬂect diﬀerential contributions of pathophysiology and environment to depression risk in PMC, who are typically identiﬁed for research studies due to their relationship to individuals in their household with FXS and who may experience additional stressors related to caring for a relative with a complex neurodevelopmental disorder (e.g., maternal stress; Hunter et al., 2019). Coupled with recent ﬁndings linking FMR1 CGG repeat count to abnormal cortisol responses to stress in female PMC who were also mothers to a child with FXS (Hong et al., 2021), the absence of frontal Consistent with our previous study (Schmitt et al., 2022), Cluster 3 consisted of individuals who were largely unaﬀected with the exception of notable resting state diﬀerences. Resting EEG diﬀerences in this subgroup may represent compensatory responses that reduce risk for psychiatric, executive function, and sensory processing deﬁcits, although further research will be necessary to conﬁrm a causative role. 4.1. Neurophysiological differences by cluster Frontal alpha asymmetry has shown high internal consistency and moderate test-retest reliability in populations with depression, particularly greater right hemisphere frontal activity (Allen et al., 2004; Coan and Allen, 2004; Thibodeau et al., 2006; Stewart et al., 2010; van der Vinne et al., 2017). The relationship between frontal asymmetry and anxiety is similarly characterized by greater right hemisphere frontal activity (Thibodeau et al., 2006; Mathersul et al., 2008). However, individual diﬀerences in frontal alpha asymmetry may be related to underlying pathophysiology contributing to depression symptoms (Smith et al., 2017; van der Vinne et al., 2017). Rather than reﬂecting stable physiological alteration, measures of asymmetry may index more dynamic utilization of oscillatory frequency bands between hemispheres uniquely impacted by the FMR1 premutation, which appear to be related to aﬀective disturbances (Schmitt et al., 2022). heightened sensory detection. Based on previous clustering results (Schmitt et al., 2022), Cluster 2 was comprised of individuals with symptoms of executive dysfunction and some resting EEG abnormalities. In particular, Cluster 2 showed reduced theta band resting power compared to typically developing controls, and theta power within Cluster 2 was associated with social processing in the opposite direction to the less aﬀected Cluster 3, who showed increased theta power. Cluster 2 may exhibit reduced ability to mobilize compensatory activity in the theta range (Wang et al., 2017; Schmitt et al., 2022), which in this case may exacerbate sensory sensitivity symptoms. Indeed, the combination of reduced response to the onset of the chirp but comparably typical low gamma ITC in Cluster 1 suggests recovery in subsequent sustained high frequency sensory processing capacity, which is notably absent in Cluster 2, and which may diﬀerentiate neural pathways contributing to neural compensation and resilience in PMC. Individuals with FXS also show reductions in low gamma ITC to the chirp, which has been consistently correlated with increased autistic features (Ethridge et al., 2017, 2019), consistent with its potential contribution to alterations in sensory processing, and social and executive function deﬁcits. heightened sensory detection. Based on previous clustering results (Schmitt et al., 2022), Cluster 2 was comprised of individuals with symptoms of executive dysfunction and some resting EEG abnormalities. In particular, Cluster 2 showed reduced theta band resting power compared to typically developing controls, and theta power within Cluster 2 was associated with social processing in the opposite direction to the less aﬀected Cluster 3, who showed increased theta power. Frontiers in Integrative Neuroscience 4.2. Links between frontal asymmetry, depression, and anxiety Previous evidence in the psychiatric literature links changes in frontal asymmetry to anxiety and depression (Smith et al., 2017), and frontal asymmetry diﬀerences have been described in 07 frontiersin.org Norris et al. 10.3389/fnint.2023.898215 TABLE 4 Correlations between general frontal EEG variables, F3/F4 frontal EEG variables, and clinical variables for PMCs. BLE 4 Correlations between general frontal EEG variables, F3/F4 frontal EEG variables, and clinical variables for PMCs. General frontal EEG variables F3/F4 frontal EEG variables (N = 32) Gamma power Theta power ITC stimulus onset ITC stimulus offset ITC low gamma ITC high gamma Left alpha power 0.41 0.62 −0.22 −0.15 0.03 −0.24 Right alpha power 0.62 0.63** −0.13 −0.15 −0.05 −0.38* Left theta power 0.41* 0.59 −0.25 −0.09 0.04 −0.21 Right theta power 0.57 0.50 −0.09 −0.08 −0.04 −0.31 Left low gamma power 0.67 0.41* 0.03 0.18 −0.05 −0.22 Right low gamma power 0.75** 0.21 0.06 −0.01 −0.08 −0.31 Frontal alpha power asymmetry 0.05 −0.10 0.09 0.03 −0.12 −0.15 Frontal theta power asymmetry 0.004 −0.09 0.17 0.08 0.01 −0.05 Frontal low gamma power asymmetry 0.06 −0.12 0.23 0.03 −0.01 −0.005 Clinical variables BDI (N = 30) −0.08 0.16 −0.46* −0.41* −0.19 −0.09 ASI (N = 27) −0.05 0.15 −0.26 −0.21 −0.21 −0.19 ADAMs (N = 9) Manic/Hyperactive behavior −0.10 −0.92** −0.31 −0.35 −0.44 0.08 Depressed mood 0.19 −0.68* −0.39 −0.18 −0.35 −0.30 Social avoidance −0.07 −0.60 −0.24 −0.26 −0.24 0.18 General anxiety 0.08 −0.90** −0.44 −0.31 −0.42 −0.11 Obsessive/Compulsive behavior 0.24 −0.67* −0.71* −0.44 −0.84** −0.30 ASP (N = 27) Auditory processing −0.03 −0.27 0.14 0.03 0.04 0.19 Taste/Smell processing 0.01 0.03 0.11 0.39 0.29 −0.02 Low registration −0.33 −0.07 −0.37 (p = 0.058) −0.25 −0.02 −0.03 Movement processing 0.20 −0.16 0.02 −0.25 −0.46* −0.39 Visual processing −0.05 0.11 −0.25 0.21 −0.04 −0.02 Touch processing −0.03 −0.18 0.23 0.14 −0.15 0.11 Activity level 0.35 −0.11 0.08 0.35 −0.05 −0.07 Sensation seeking −0.31 −0.19 −0.12 0.03 0.13 0.11 Sensation sensitivity −0.24 −0.19 −0.02 0.19 −0.05 −0.11 Sensation avoiding 0.09 −0.08 −0.06 0.22 −0.19 −0.33 All l ti S ’ h d ﬂ t ll PMC l t N t i k N S * 0 05 ** 0 01 TABLE 5 Correlations between General EEG Variables for PMCs. All correlations are Spearman’s rho and reﬂect all PMC across clusters. No asterisk = N.S. p < 0.05, p < 0.01. 4.2. Links between frontal asymmetry, depression, and anxiety Conclusions of reduced theta reﬂecting an underlying mechanism for depression and anxiety speciﬁc to PMCs is complicated by non-signiﬁcant correlations between EEG measures and both BDI and ASI scores suggesting that reduced theta may reﬂect more lifetime risk than current depression symptoms. Current use of mood stabilizing medication may also explain EEG outcomes with BDI and ASI scores, as PMCs that reported current use of antidepressant medications (N = 11) likely exhibited reduced symptoms of mood dysregulation at the time of evaluation (Iosifescu, 2011). Our data does suggest however that the FMR1 premutation, coupled with potential diﬀerences in life stressors, may create unique contributions to depression and anxiety outcomes; further evaluation of underlying mechanisms is necessary to provide more speciﬁc therapeutic interventions (Hagerman et al., 2018; Maltman et al., 2021; Schmitt et al., 2022). y In contrast to our frontal asymmetry data, we did ﬁnd signiﬁcant correlations between decreased theta/alpha ITC to stimulus onset and increased scores on the BDI across the entire PMC group, suggesting that for PMC there is a relationship between sensory processing hypo-responsivity and mood. Reduced ERP amplitude and reduced ITC to sensory stimuli have been found in other psychiatric and neurodevelopmental groups, namely individuals with bipolar disorder (Isomura et al., 2016), autism (De Stefano et al., 2019; Hornung et al., 2019; Seymour et al., 2020; Roberts et al., 2021), and psychosis (Krishnan et al., 2009; Sugiyama et al., 2021), consistent with the psychiatric implications of these speciﬁc ERP features in PMCs. Reductions in theta power also were associated with increased symptoms of anxiety and depression in individuals who completed the ADAMS, including obsessive compulsive symptoms, suggesting a potential broader contribution for theta power to limbic regulation. Although these ﬁndings must be veriﬁed with a larger sample, the strong correlations do suggest a clinically meaningful association between reduced theta power, a universal feature of the PMC group rather than unique to one cluster, and overall risk for mood symptoms. Indeed, individuals in Cluster 3, who showed the smallest reductions in task-related theta power, and enhanced theta power at rest (Schmitt et al., 2022), also showed the fewest psychiatric and behavioral symptoms. While theta power typically decreases with healthy cognitive aging, age was not a signiﬁcant correlate in any analyses regarding theta power, suggesting decreases in theta may reﬂect a typical neural presentation of the FMR1 premutation (Cummins and Finnigan, 2007; Cellier et al., 2021). 4.2. Links between frontal asymmetry, depression, and anxiety Theta power ﬁndings may be related to known anatomical shifts to limbic circuitry mediated by FMRP/elevated FMR1 mRNA thought to heighten risk for psychopathology (Cordeiro et al., 2015). In conjunction with clinical correlations, these ﬁndings raise questions about the mechanistic underpinnings of psychiatric symptoms. We interpret the correlation results between clinical measures of mood dysregulation and EEG measures as reﬂective of underlying neural processes related to depression and anxiety but acknowledge the inability to attribute causality. Correlations between theta and mood dysregulation are potentially reﬂective of (1) the eﬀect of depression on EEG measures, or (2) antecedent/causal of depression. Theta power may therefore be a clinically relevant measure for evaluating mood and anxiety in PMC, as well as a target for assessing eﬀect of therapeutics in advance of clinically meaningful behavioral changes. The PMC sample was limited due to (1) the nature of the FMR1 premutation, and (2). the convenience nature of the sample. Primarily, PMCs were asked to participate if their child, sibling, or grandchild was participating in on-going FXS-speciﬁc research or were at the clinic for regular clinical care. Limited participant numbers per cluster impacted the capacity to attribute correlations strictly to individual clusters and correlations may reﬂect population parameters confounded by heterogenous premutation presentation. More research is required to parse the extent that EEG measures of decreased theta reﬂect a unique mechanism of depressed mood in PMC. This study was initially conducted as a pilot to determine whether task-based EEG features related to clinical variation could be identiﬁed in PMCs. The preliminary ﬁndings suggest that future studies are warranted, particularly in delineating whether these EEG features that diﬀered between clusters simply reﬂect a spectrum of variation that, while potentially informative regarding mechanism, still entirely falls within normative values, or whether, for example, the reductions in stimulus onset and chirp processing responses in Clusters 1 and 2 are signiﬁcantly decreased relative to non-PMC controls. Future studies with larger samples, enabling medication use and psychiatric comorbidities to be better disentangled, are also necessary to conﬁrm these ﬁndings. Lastly, due to the focus on mood and anxiety, the sample was limited to female PMCs and lacks applicability to males with the premutation. Future work should aim to evaluate males only or include equal numbers to assess clinical proﬁles. 4.5. Limitations and future research Twelve participants reported current AD use (36.4%) suggesting a clinically relevant level of mood disturbance within the sample, though many clinical reports did not report a diagnostic history of depression. Use of AD medication was consistent across all clusters indicating successful treatment potentially impacted cluster membership (Schmitt et al., 2022). Standard measures of depression and anxiety, including the BDI and ASI, are self- report measures that potentially prompt participants to respond according to their current mood state (Beck et al., 1961). The ADAMS is another self-report measure of broad spectrum psychiatric conditions, including depressed mood and anxiety, that also potentially prompted current mood states rather than lifetime Frontiers in Integrative Neuroscience 4.2. Links between frontal asymmetry, depression, and anxiety General EEG variables EEG variables Gamma power Theta power ITC Stimulus onset ITC Stimulus offset ITC low gamma ITC high gamma Gamma Power − 0.48* −0.18 −0.07 −0.17 −0.49 Theta Power − −0.12 0.001 0.19 −0.10 ITC Stimulus Onset − 0.52 0.49 0.47 ITC Stimulus Oﬀset − 0.27 0.24 ITC Low Gamma − 0.64** ITC High Gamma − All correlations are Spearman’s rho and reﬂect all PMCs across clusters. No asterisk = N.S. p < 0.05, *p < 0.01. 08 Frontiers in Integrative Neuroscience frontiersin.org Norris et al. 10.3389/fnint.2023.898215 10.3389/fnint.2023.898215 asymmetry ﬁndings may indicate diﬀerential pathways to depression and anxiety risk in PMCs. symptom experiences. Further, the ADAMs has not been used in PMC samples, and was only completed by a subset of the sample (n = 9), thus additional caution was taken with data interpretation. PMCs with a medical history of current AD use may also report fewer symptoms on the BDI due to response items on the BDI prompting reports of current mood disturbance (as reﬂective of their AD use) compared to their life history reports (Marques et al., 2013). Conclusions of reduced theta reﬂecting an underlying mechanism for depression and anxiety speciﬁc to PMCs is complicated by non-signiﬁcant correlations between EEG measures and both BDI and ASI scores suggesting that reduced theta may reﬂect more lifetime risk than current depression symptoms. Current use of mood stabilizing medication may also explain EEG outcomes with BDI and ASI scores, as PMCs that reported current use of antidepressant medications (N = 11) likely exhibited reduced symptoms of mood dysregulation at the time of evaluation (Iosifescu, 2011). Our data does suggest however that the FMR1 premutation, coupled with potential diﬀerences in life stressors, may create unique contributions to depression and anxiety outcomes; further evaluation of underlying mechanisms is necessary to provide more speciﬁc therapeutic interventions (Hagerman et al., 2018; Maltman et al., 2021; Schmitt et al., 2022). symptom experiences. Further, the ADAMs has not been used in PMC samples, and was only completed by a subset of the sample (n = 9), thus additional caution was taken with data interpretation. PMCs with a medical history of current AD use may also report fewer symptoms on the BDI due to response items on the BDI prompting reports of current mood disturbance (as reﬂective of their AD use) compared to their life history reports (Marques et al., 2013). frontiersin.org References Allen, E. G., Charen, K., Hipp, H. S., Shubeck, L., Amin, A., He, W., et al. (2020). Clustering of comorbid conditions among women who carry an FMR1 premutation. Genet. Med. 22, 758–766. doi: 10.1038/s41436-019-0733-5 Chatrian, G. E., Lettich, E., and Nelson, P. L. (1985). Ten percent electrode system for topographic studies of spontaneous and evoked EEG activities. Am. J. EEG Technol. 25, 83–92. doi: 10.1080/00029238.1985.11080163 Ciaccio, C., Fontana, L., Milani, D., Tabano, S., Miozzo, M., and Esposito, S. (2017). Fragile X syndrome: A review of clinical and molecular diagnoses. Ital. J. Pediatr. 43:39. doi: 10.1186/s13052-017-0355-y Allen, J. J., Urry, H. L., Hitt, S. K., and Coan, J. A. (2004). The stability of resting frontal electroencephalographic asymmetry in depression. Psychophysiology 41, 269–280. doi: 10.1111/j.1469-8986.2003.00149.x Coan, J. A., and Allen, J. J. (2004). Frontal EEG asymmetry as a moderator and mediator of emotion. Biol. Psychol. 67, 7–49. doi: 10.1016/j.biopsycho.2004.03.002 Beck, A. T., Ward, C. H., Mendelson, M., Mock, J., and Erbaugh, J. (1961). An inventory for measuring depression. Arch. Gen. Psychiatry 4, 561–571. doi: 10.1001/ archpsyc.1961.01710120031004 Cordeiro, L., Abucayan, F., Hagerman, R., Tassone, F., and Hessl, D. (2015). Anxiety disorders in fragile X premutation carriers: Preliminary characterization of probands and non-probands. Intractable Rare Dis. Res. 4, 123–130. doi: 10.5582/irdr.2015.01029 Berry-Kravis, E., Goetz, C. G., Leehey, M. A., Hagerman, R. J., Zhang, L., Li, L., et al. (2007). Neuropathic features in fragile X premutation carriers. Am. J. Med. Genet. A 143A, 19–26. doi: 10.1002/ajmg.a.31559 Cummins, T. D., and Finnigan, S. (2007). Theta power is reduced in healthy cognitive aging. Int. J. Psychophysiol. 66, 10–17. doi: 10.1016/j.ijpsycho.2007.0 5.008 Brown, C., Tollefson, N., Dunn, W., Cromwell, R., and Filion, D. (2001). The adult sensory proﬁle: Measuring patterns of sensory processing. Am. J. Occup. Ther. 55, 75–82. doi: 10.5014/ajot.55.1.75 Delorme, A., and Makeig, S. (2004). EEGLAB: An open source toolbox for analysis of single-trial EEG dynamics including independent component analysis. J. Neurosci. Methods 134, 9–21. doi: 10.1016/j.jneumeth.2003.10.009 Budimirovic, D. B., Schlageter, A., Filipovic-Sadic, S., Protic, D. D., Bram, E., Mahone, E. M., et al. (2020). A genotype-phenotype study of high-resolution FMR1 nucleic acid and protein analyses in fragile X patients with neurobehavioral assessments. Brain Sci. 10:694. doi: 10.3390/brainsci10100694 De Stefano, L. A., Schmitt, L. M., White, S. P., Mosconi, M. W., Sweeney, J. A., and Ethridge, L. E. (2019). Developmental eﬀects on auditory neural oscillatory synchronization abnormalities in autism spectrum disorder. Front. Integr. Neurosci. 13:34. Data availability statement We would like to acknowledge the families of individuals with FXS and the typically developing controls who participated in this study. We also want to thank the clinical research coordinators for their assistance with data collection and data entry. The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. Funding symptom presentations reﬂect underlying spectrum characteristics forming unique subgroupings. Importantly, these neuropsychiatric groupings also reﬂect diﬀerences in basic neural sensory processing characteristics, suggesting a tie between basic synaptic function, sensory systems, and risk for speciﬁc neuropsychiatric phenotypes within individuals with the FMR1 premutation. This research study was supported by the U54 Fragile X Center (U54HD082008, JS and U54HD104461, CE) and the University of Oklahoma Libraries’ Open Access Fund. Publisher’s note JN led all EEG data preprocessing, management, data analyses, and wrote the full manuscript. LS led all clinical data collection and analyses. LD consulted on data preprocessing and analyses. EP led all EEG data collection. CE and JS supervised all aspects of study design, funding, and participant recruitment. LE supervised all aspects of EEG experimental design, data analysis, and manuscript preparation. All authors contributed signiﬁcantly to manuscript preparation. All claims expressed in this article are solely those of the authors and do not necessarily represent those of their aﬃliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. All claims expressed in this article are solely those of the authors and do not necessarily represent those of their aﬃliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Conﬂict of interest The studies involving human participants were reviewed and approved by Cincinnati Children’s Hospital Medical Center. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin. The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. The reviewer EA declared a past co-authorship with the authors CE and JS to the handling editor at the time of review. 5. Conclusion Overall, PMCs exhibited relatively typical electrophysiological measures at the group level. However, congruent with Schmitt et al. (2022), we ﬁnd diﬀerences notable by cluster membership supporting initial cluster characterizations and conclusions that heterogenous 09 frontiersin.org Norris et al. 10.3389/fnint.2023.898215 References doi: 10.3389/fnint.2019.00034 Burle, B., Spieser, L., Roger, C., Casini, L., Hasbroucq, T., and Vidal, F. (2015). Spatial and temporal resolutions of EEG: Is it really black and white? A scalp current density view. Int. J. Psychophysiol. 97, 210–220. doi: 10.1016/j.ijpsycho.2015.05.004 Donkers, F. C., Schipul, S. E., Baranek, G. T., Cleary, K. M., Willoughby, M. T., Evans, A. M., et al. (2015). Attenuated auditory event-related potentials and associations with atypical sensory response patterns in children with autism. J. Autism Dev. Disord. 45, 506–523. doi: 10.1007/s10803-013-1948-y Cellier, D., Riddle, J., Petersen, I., and Hwang, K. (2021). The development of theta and alpha neural oscillations from ages 3 to 24 years. Dev. Cogn. Neurosci. 50:100969. doi: 10.1016/j.dcn.2021.100969 Frontiers in Integrative Neuroscience 10 frontiersin.org Norris et al. Norris et al. 10.3389/fnint.2023.898215 Drake, M. E. Jr., Pakalnis, A., Phillips, B., Padamadan, H., and Hietter, S. A. (1991). Auditory evoked potentials in anxiety disorder. Clin. Electroencephalogr. 22, 97–101. doi: 10.1177/155005949102200209 Luu, P., and Ferree, T. (2005). Determination of the hydrocel geodesic sensory nets’ average electrode positiions and their 10-10 international equivalents. Technical document. Eugene, OR: Electrical Geodesics, Inc. Maltman, N., Guilfoyle, J., Nayar, K., Martin, G. E., Winston, M., Lau, J. C. Y., et al. (2021). The phenotypic proﬁle associated with the FMR1 premutation in women: An investigation of clinical-behavioral, social-cognitive, and executive abilities. Front. Psychiatry 12:718485. doi: 10.3389/fpsyt.2021.718485 Engel-Yeger, B., Muzio, C., Rinosi, G., Solano, P., Geoﬀroy, P. A., Pompili, M., et al. (2016). Extreme sensory processing patterns and their relation with clinical conditions among individuals with major aﬀective disorders. Psychiatry Res. 236, 112–118. doi: 10.1016/j.psychres.2015.12.022 Esbensen, A. J., Rojahn, J., Aman, M. G., and Ruedrich, S. (2003). Reliability and validity of an assessment instrument for anxiety, depression, and mood among individuals with mental retardation. J. Autism Dev. Disord. 33, 617–629. doi: 10.1023/b: jadd.0000005999.27178.55 Marques, L. A., Galduroz, J. C., Fernandes, M. R., Oliveira, C. C., Beijo, L. A., and Noto, A. R. (2013). Assessment of the eﬀectiveness of pharmacotherapy follow-up in patients treated for depression. J. Manag. Care Pharm. 19, 218–227. doi: 10.18553/jmcp.2013.19. 3.218 Mathersul, D., Williams, L. M., Hopkinson, P. J., and Kemp, A. H. (2008). Investigating models of aﬀect: Relationships among EEG alpha asymmetry, depression, and anxiety. Emotion 8, 560–572. doi: 10.1037/a0012811 Ethridge, L. E., Hamm, J. P., Pearlson, G. D., Tamminga, C. A., Sweeney, J. A., Keshavan, M. S., et al. (2015). References Event-related potential and time-frequency endophenotypes for schizophrenia and psychotic bipolar disorder. Biol. Psychiatry 77, 127–136. doi: 10.1016/j.biopsych.2014.03.032 Norris, J. E., De Stefano, L. A., Schmitt, L. M., Pedapati, E. V., Erickson, C. A., Sweeney, J. A., et al. (2022). Hemisphreic utilization of alpha oscillatory dynamics as a unique biomarker of neural compensation in females with Fragile X Syndrome. ACS Chem. Neurosci. 13, 3389–3402. doi: 10.1021/acschemneuro.2c 00404 Ethridge, L. E., De Stefano, L. A., Schmitt, L. M., Woodruﬀ, N. E., Brown, K. L., Tran, M., et al. (2019). Auditory EEG biomarkers in fragile X syndrome: Clinical relevance. Front. Integr. Neurosci. 13:60. doi: 10.3389/fnint.2019.00060 Ethridge, L. E., White, S. P., Mosconi, M. W., Wang, J., Byerly, M. J., and Sweeney, J. A. (2016). Reduced habituation of auditory evoked potentials indicate cortical hyper- excitability in fragile X syndrome. Transl. Psychiatry 6:e787. doi: 10.1038/tp.2016.48 Pedapati, E. V., Schmitt, L. M., Ethridge, L. E., Miyakoshi, M., Sweeney, J. A., Liu, R., et al. (2022). Neocortical localization and thalamocortical modulation of neuronal hyperexcitability contribute to Fragile X Syndrome. Commun. Biol. 5:442. doi: 10.1038/ s42003-022-03395-9 Ethridge, L. E., White, S. P., Mosconi, M. W., Wang, J., Pedapati, E. V., Erickson, C. A., et al. (2017). Neural synchronization deﬁcits linked to cortical hyper-excitability and auditory hypersensitivity in fragile X syndrome. Mol. Autism 8:22. doi: 10.1186/s13229- 017-0140-1 Raspa, M., Wylie, A., Wheeler, A. C., Kolacz, J., Edwards, A., Heilman, K., et al. (2018). Sensory diﬃculties in children with an FMR1 premutation. Front. Genet. 9:351. doi: 10.3389/fgene.2018.00351 Ford, J. M., Mathalon, D. H., Kalba, S., Marsh, L., and Pfeﬀerbaum, A. (2001). N1 and P300 abnormalities in patients with schizophrenia, epilepsy, and epilepsy with schizophrenialike features. Biol. Psychiatry 49, 848–860. doi: 10.1016/s0006-3223(00) 01051-9 Reiss, S., Peterson, R. A., Gursky, D. M., and McNally, R. J. (1986). Anxiety sensitivity, anxiety frequency and the prediction of fearfulness. Behav. Res. Ther. 24, 1–8. doi: 10.1016/0005-7967(86)901 Fu, Y. H., Kuhl, D. P., Pizzuti, A., Pieretti, M., Sutcliﬀe, J. S., Richards, S., et al. (1991). Variation of the CGG repeat at the fragile X site results in genetic instability: Resolution of the sherman paradox. Cell 67, 1047–1058. doi: 10.1016/0092-8674(91)9 0283-5 Roberts, T. P. L., Bloy, L., Liu, S., Ku, M., Blaskey, L., and Jackel, C. (2021). Magnetoencephalography studies of the envelope following response during amplitude- modulated sweeps: Diminished phase synchrony in autism spectrum disorder. Front. Hum. Neurosci. 15:787229. doi: 10.3389/fnhum.2021.787229 Hagerman, R. References J., Protic, D., Rajaratnam, A., Salcedo-Arellano, M. J., Aydin, E. Y., and Schneider, A. (2018). Fragile X-associated neuropsychiatric disorders (FXAND). Front. Psychiatry 9:564. doi: 10.3389/fpsyt.2018.00564 Schmitt, L. M., Shaﬀer, R. C., Hessl, D., and Erickson, C. (2019). Executive function in fragile X syndrome: A systematic review. Brain Sci. 9:15. doi: 10.3390/brainsci9010015 Schmitt, L. M., Dominick, K. C., Liu, R., Pedapati, E. V., Ethridge, L. E., Smith, E., et al. (2022). Evidence for three subgroups of female FMR1 premutation carriers deﬁned by distinct neuropsychiatric features: A pilot study. Front. Int. Neuro. 15:48. doi: 10.3389/fnint.2021.797546 Hall, D. A., Robertson-Dick, E. E., O’Keefe, J. A., Hadd, A. G., Zhou, L., and Berry- Kravis, E. (2016). X-inactivation in the clinical phenotype of fragile X premutation carrier sisters. Neurol. Genet. 2:e45. doi: 10.1212/NXG.0000000000000045 Seymour, R. A., Rippon, G., Gooding-Williams, G., Sowman, P. F., and Kessler, K. (2020). Reduced auditory steady state responses in autism spectrum disorder. Mol. Autism 11:56. doi: 10.1186/s13229-020-00357-y Hong, J., Kapoor, A., DaWalt, L. S., Maltman, N., Kim, B., Berry-Kravis, E. M., et al. (2021). Stress and genetics inﬂuence hair cortisol in FMR1 premutation carrier mothers of children with fragile X syndrome. Psychoneuroendocrinology 129:105266. doi: 10.1016/j.psyneuen.2021.105266 Smith, E. G., Pedapati, E. V., Liu, R., Schmitt, L. M., Dominick, K. C., Shaﬀer, R. C., et al. (2021). Sex diﬀerences in resting EEG power in fragile X syndrome. J. Psychiatr. Res. 138, 89–95. doi: 10.1016/j.jpsychires.2021.03.057 Hornung, T., Chan, W. H., Muller, R. A., Townsend, J., and Keehn, B. (2019). Dopaminergic hypo-activity and reduced theta-band power in autism spectrum disorder: A resting-state EEG study. Int. J. Psychophysiol. 146, 101–106. doi: 10.1016/j.ijpsycho. 2019.08.012 Smith, E. E., Reznik, S. J., Stewart, J. L., and Allen, J. J. (2017). Assessing and conceptualizing frontal EEG asymmetry: An updated primer on recording, processing, analyzing, and interpreting frontal alpha asymmetry. Int. J. Psychophysiol. 111, 98–114. doi: 10.1016/j.ijpsycho.2016.11.005 Hunter, J. E., Jenkins, C. L., Grim, V., Leung, S., Charen, K. H., Hamilton, D. R., et al. (2019). Feasibility of an app-based mindfulness intervention among women with an FMR1 premutation experiencing maternal stress. Res. Dev. Disabil. 89, 76–82. doi: 10.1016/j.ridd.2019.03.008 Stewart, J. L., Bismark, A. W., Towers, D. N., Coan, J. A., and Allen, J. J. (2010). Resting frontal EEG asymmetry as an endophenotype for depression risk: Sex-speciﬁc patterns of frontal brain asymmetry. J. Abnorm. Psychol. 119, 502–512. References doi: 10.1037/a00 19196 Hunter, J., Rivero-Arias, O., Angelov, A., Kim, E., Fotheringham, I., and Leal, J. (2014). Epidemiology of fragile X syndrome: A systematic review and meta-analysis. Am. J. Med. Genet. A 164A, 1648–1658. doi: 10.1002/ajmg.a.36511 Sugiyama, S., Ohi, K., Kuramitsu, A., Takai, K., Muto, Y., Taniguchi, T., et al. (2021). The auditory steady-state response: Electrophysiological index for sensory processing dysfunction in psychiatric disorders. Front. Psychiatry. 12:644541. doi: 10.3389/fpsyt. 2021.644541 Hunter, J. E., Allen, E. G., Abramowitz, A., Rusin, M., Leslie, M., Novak, G., et al. (2008). No evidence for a diﬀerence in neuropsychological proﬁle among carriers and noncarriers of the FMR1 premutation in adults under the age of 50. Am. J. Hum. Genet. 83, 692–702. doi: 10.1016/j.ajhg.2008.10.021 Thibodeau, R., Jorgensen, R. S., and Kim, S. (2006). Depression, anxiety, and resting frontal EEG asymmetry: A meta-analytic review. J. Abnorm. Psychol. 115, 715–729. doi: 10.1037/0021-843X.115.4.715 Iosifescu, D. V. (2011). Electroencephalography-derived biomarkers of antidepressant response. Harv. Rev. Psychiatry 19, 144–154. doi: 10.3109/10673229.2011.586549 van der Vinne, N., Vollebregt, M. A., van Putten, M., and Arns, M. (2017). Frontal alpha asymmetry as a diagnostic marker in depression: Fact or ﬁction? A meta-analysis. Neuroimage Clin. 16, 79–87. doi: 10.1016/j.nicl.2017.07.006 Isomura, S., Onitsuka, T., Tsuchimoto, R., Nakamura, I., Hirano, S., Oda, Y., et al. (2016). Diﬀerentiation between major depressive disorder and bipolar disorder by auditory steady-state responses. J. Aﬀect Disord. 190, 800–806. doi: 10.1016/j.jad.2015. 11.034 Verkerk, A. J., Pieretti, M., Sutcliﬀe, J. S., Fu, Y. H., Kuhl, D. P., Pizzuti, A., et al. (1991). Identiﬁcation of a gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome. Cell 65, 905–914. doi: 10.1016/0092-8674(91)90397-h Klusek, J., Hong, J., Sterling, A., Berry-Kravis, E., and Mailick, M. R. (2020). Inhibition deﬁcits are modulated by age and CGG repeat length in carriers of the FMR1 premutation allele who are mothers of children with fragile X syndrome. Brain Cogn. 139:105511. doi: 10.1016/j.bandc.2019.105511 Wang, J., Ethridge, L. E., Mosconi, M. W., White, S. P., Binder, D. K., Pedapati, E. V., et al. (2017). A resting EEG study of neocortical hyperexcitability and altered functional connectivity in fragile X syndrome. J. Neurodev. Disord. 9:11. doi: 10.1186/s11689-017- 9191-z Kotsiris, K., Westrick, J., and Little, L. (2020). Sensory processing patterns and internalizing behaviors in the pediatric and young adult general population: A scoping review. Open J. Occup. Ther. 8, 1–13. doi: 10.15453/2168-6408.1624 Krishnan, G. P., Hetrick, W. Frontiers in Integrative Neuroscience Yu, S., Pritchard, M., Kremer, E., Lynch, M., Nancarrow, J., Baker, E., et al. (1991). Fragile X genotype characterized by an unstable region of DNA. Science 252, 1179–1181. doi: 10.1126/science.252.5009.1179 Wheeler, A. C., Sideris, J., Hagerman, R., Berry-Kravis, E., Tassone, F., and Bailey, D. B. Jr. (2016). Developmental proﬁles of infants with an FMR1 premutation. J. Neurodev. Disord. 8:40. doi: 10.1186/s11689-016-9171-8 10.3389/fnint.2023.898215 References P., Brenner, C. A., Shekhar, A., Steﬀen, A. N., and ODonnell, B. F. (2009). Steady state and induced auditory gamma deﬁcits in schizophrenia. Neuroimage. 47, 1711–1719. doi: 10.1016/j.neuroimage.2009. 03.085 Wheeler, A., Raspa, M., Hagerman, R., Mailick, M., and Riley, C. (2017). Implications of the FMR1 premutation for children, adolescents, adults, and their families. Pediatrics 139, S172–S182. doi: 10.1542/peds.2016-1159D Wheeler, A. C., Bailey, D. B. Jr., Berry-Kravis, E., Greenberg, J., Losh, M., Mailick, M., et al. (2014). Associated features in females with an FMR1 premutation. J. Neurodev. Disord. 6:30. doi: 10.1186/1866-1955-6-30 Luck, S. J. (2014). An Introduction to the event-related potential technique. Cambridge, MA: The MIT Press. Frontiers in Integrative Neuroscience 11 frontiersin.org Norris et al. 10.3389/fnint.2023.898215 12 frontiersin.org Frontiers in Integrative Neuroscience
https://openalex.org/W2144489030	https://aacr.figshare.com/articles/journal_contribution/Supplementary_Figure_and_Table_Legends_from_ETV6-NTRK3_Mediated_Breast_Epithelial_Cell_Transformation_Is_Blocked_by_Targeting_the_IGF1R_Signaling_Pathway/22389704/1/files/39835223.pdf	English	null	ETV6-NTRK3–Mediated Breast Epithelial Cell Transformation Is Blocked by Targeting the IGF1R Signaling Pathway	Cancer research	2,011	cc-by	452	Blocking IGF1R signaling in ETV6-NTRK3 breast cancer Blocking IGF1R signaling in ETV6-NTRK3 breast cancer SUPPLEMENTARY FIGURE LEGENDS Supplementary Figure S1. Expression of EN in EpH4 cells has no effect on morphology or signaling in monolayer cultures. A. Western blot analysis of serum starved control and EpH4-EN cells grown in monolayer. B. Morphology of control (MSCV) and two independent EN-expressing EpH4 cells lines (EN1 and EN2). Scale: 50μm. Supplementary Figure S2. Movement of EpH4-MSCV and EpH4-EN cells in serum free conditions. Each colored line represents the track or movement of one cell followed by time lapse microscopy over a 24 hr period. Supplementary Figure S3. Insulin stimulation results in IGF1R activation. A. IGF1R positive murine embryo fibroblast (R+) cells engineered to express EN were serum starved overnight and then stimulated with 100ng/ml IGF-1 or 100ng/ml of insulin for 30 minutes. B. Various concentrations of IGF-1 and insulin were used to stimulate serum starved MCF10A cells for 30 minutes. Anti-phosphotyrosine immunoprecipitations followed by IGF1R or INSR immunoblotting was performed. Supplementary Figure S4. siRNA knock down of INSR in MCF10A-EN cells decreases spheroid size and cyclin D1 levels. A. Cells were grown in Matrigel cultures for 10 days. Pictures=Day 7. Size bar: 100μM. B. The effects of 4 independent siRNAs were assessed for their effects on the levels of INSR and cyclin D1 by Western blotting. 1 1 Blocking IGF1R signaling in ETV6-NTRK3 breast cancer Blocking IGF1R signaling in ETV6-NTRK3 breast cancer Supplementary Figure S5. Comparing effects of BMS-536924 and BMS-754807 on 3T3-EN soft agar colony formation. Control and EN expressing NIH 3T3 cells were plated in soft agar in the absence or presence of increasing concentrations of BMS- 536924 and BMS-754807. Average and standard deviation of three independent experiments is depicted in the graph. Supplementary Figure S6. EN requires activation of the PI3K-Akt pathway to transform EpH4 cells in 3D Matrigel cultures. A. Photomicrographs of spheroids formed in 3D Matrigel cultures. Cells were cultured in IGF-1 (100ng/ml)-containing media with either vehicle control (DMSO), PI3K inhibitor LY294002 (25μM), or MEK inhibitor U0126 (25μM). Spheroid morphology (Top panels; Scale:200μm), immunofluorescence microscopy for E-cadherin and Ki67 (Middle panels; Scale: 100μm), and BrdU staining (Bottom panels; Scale: 400μm). B. Western blots of EpH4-EN stimulated with Insulin or IGF-1 +/- the PI3K inhibitor LY294002 (25μM). Supplementary Table S1. Quantification of the effects of LY294002 and U0126 on EN mediated transformation of EpH4 cells grown in 3D culture. Each cell line was grown in 3D Matrigel cultures for 3 days and analyzed by immunofluorescence for BrDU staining. Spheroids were considered positive if they possessed >4 stained nuclei. Diameters were measured by confocal microscopy. Table represents the measurements of 100 random spheroids grown under each condition for each parameter. 2 2
https://openalex.org/W4304146326	https://eurjmedres.biomedcentral.com/counter/pdf/10.1186/s40001-022-00828-3	English	null	Biomarkers of interstitial lung disease associated with primary Sjögren's syndrome	European journal of medical research	2,022	cc-by	8,291	© The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Clinical trial registration: None. Keywords: Interstitial lung disease (ILD), Serum biomarkers, Predictive factor, Primary Sjögren’s syndrome (pSS) Abstract Objectives: The aim of this study was to investigate serum biomarkers linked to primary Sjögren’s syndrome (pSS)- associated interstitial lung disease (ILD). Methods: 69 pSS patients were consecutively enrolled and evaluated via quantitative ILD scoring based on high- resolution computed tomography (HRCT). Biomarkers of interest were assessed by multiplex enzyme-linked immuno- sorbent assays (ELISAs). Results: Among consecutively enrolled patients with pSS, the presence of pSS–ILD was 50% based on the presence of radiographically defined interstitial lung abnormalities (ILA) meeting specified criteria for mild/moderate (ILA 2) or severe (ILA 3) disease. Age, immunoglobulin M (IgM), C-reactive protein (CRP), and serum levels of eotaxin/CCL11, Krebs von den Lungen-6 (KL-6), TNFα, and TGFα were significantly higher in the combined pSS–ILD group (ILA 2 + ILA 3) than in the pSS–no-ILD and pSS–indeterminate ILD groups (ILA 0 and ILA 1, respectively) in unadjusted analyses (p < 0.05 for all variables). A binary logistic regression model revealed that disease duration and KL-6 levels were associ- ated with the presence of pSS–ILD (p < 0.05). Complementary least absolute shrinkage and selection operator (LASSO) modeling showed that age, KL-6, and TNF-α effectively differentiated pSS–ILD (ILA 2 + ILA3) from pSS without ILD (ILA 0 + ILA 1), with an area under the curve (AUC) of 0.883 (p value < 0.0001). Conclusions: Patient age, disease duration, and serum levels of both KL-6 and TNFα were the most discriminating factors associated with the presence of ILD in our pSS patients. Higher levels of CRP, IgM, eotaxin, TGFα, and TNFα should also prompt the search for occult as well as clinically evident lung involvement based on statistically significant univariate associations with pSS–ILD. Clinical trial registration: None. Weng et al. European Journal of Medical Research (2022) 27:199 https://doi.org/10.1186/s40001-022-00828-3 Weng et al. European Journal of Medical Research (2022) 27:199 https://doi.org/10.1186/s40001-022-00828-3 Weng et al. European Journal of Medical Research (2022) 27:199 https://doi.org/10.1186/s40001-022-00828-3 European Journal of Medical Research Correspondence: DASCHER@pitt.edu; juchen@xmu.edu.cn 1 Department of Rheumatology, The First Affiliated Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen 361003, China 9 Division of Rheumatology and Clinical Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA Full list of author information is available at the end of the article Biomarkers of interstitial lung disease associated with primary Sjögren’s syndrome Lin Weng1†, Yaqiong Chen1†, Tao Liang2†, Yihua Lin3, Dehao Liu4, Ciyong Yu1, Yudi Hu5, Wei Lui6, Yongliang Liu6, Xiangfang Chen7, Qiyuan Li8, Shengxiang Ge6, Dana P. Ascherman9 and Juan Chen1* Abstract Objectives: The aim of this study was to investigate serum biomarkers linked to primary Sjögren’s syndrome (pSS)- associated interstitial lung disease (ILD). Methods: 69 pSS patients were consecutively enrolled and evaluated via quantitative ILD scoring based on high- resolution computed tomography (HRCT). Biomarkers of interest were assessed by multiplex enzyme-linked immun sorbent assays (ELISAs). Results: Among consecutively enrolled patients with pSS, the presence of pSS–ILD was 50% based on the presence of radiographically defined interstitial lung abnormalities (ILA) meeting specified criteria for mild/moderate (ILA 2) or severe (ILA 3) disease. Age, immunoglobulin M (IgM), C-reactive protein (CRP), and serum levels of eotaxin/CCL11, Krebs von den Lungen-6 (KL-6), TNFα, and TGFα were significantly higher in the combined pSS–ILD group (ILA 2 + ILA 3) than in the pSS–no-ILD and pSS–indeterminate ILD groups (ILA 0 and ILA 1, respectively) in unadjusted analyses (p < 0.05 for all variables). A binary logistic regression model revealed that disease duration and KL-6 levels were assoc ated with the presence of pSS–ILD (p < 0.05). Complementary least absolute shrinkage and selection operator (LASSO modeling showed that age, KL-6, and TNF-α effectively differentiated pSS–ILD (ILA 2 + ILA3) from pSS without ILD (IL 0 + ILA 1), with an area under the curve (AUC) of 0.883 (p value < 0.0001). Conclusions: Patient age, disease duration, and serum levels of both KL-6 and TNFα were the most discriminating factors associated with the presence of ILD in our pSS patients. Higher levels of CRP, IgM, eotaxin, TGFα, and TNFα should also prompt the search for occult as well as clinically evident lung involvement based on statistically significa univariate associations with pSS–ILD. Clinical trial registration: None. Keywords: Interstitial lung disease (ILD), Serum biomarkers, Predictive factor, Primary Sjögren’s syndrome (pSS) Background Interstitial lung diseases (ILD) consist of a heterogeneous group of parenchymal lung disorders that are character- ized by variable degrees of inflammation and fibrosis [1, 2]. Although different subsets of ILD may share com- mon radiologic, pathologic, and clinical manifestations, they are associated with quite different etiologies and co-morbidities [3]. In primary Sjögren’s syndrome (pSS), the annual incidence of respiratory manifestations is estimated at 10% 1 year after diagnosis and increases to †Lin Weng, Yaqiong Chen, and Tao Liang have contributed equally to this manuscript Correspondence: DASCHER@pitt.edu; juchen@xmu.edu.cn © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Weng et al. European Journal of Medical Research (2022) 27:199 Page 2 of 10 Page 2 of 10 20% after 5 years [4]. In a recent report from China, the prevalence of ILD in pSS among those undergoing HRCT examination is as high as 39.1% [5]. Importantly, patients with pSS and lung involvement have an increased risk of death in comparison with those without lung involve- ment [6], with an estimated 5-year survival rate ranging from 84% [7] to 87.3% [8]. arthritis, systemic lupus erythematosus, mixed connec- tive tissue disease, polymyositis, and dermatomyositis were excluded. Other illnesses producing clinical mani- festations of xerophthalmia and/or xerostomia—such as past history of head and neck irradiation, pre-existing lymphoma, hepatitis B or C infection, acquired immu- nodeficiency, sarcoidosis, or graft vs. host disease—were also excluded. For eligible participants, patient-specific variables, including age, sex, and disease duration, were recorded. HRCT images were obtained from all patients at enrollment (see below for details). Serum samples were obtained at the time of clinical data collection for all of the consecutively enrolled patients and stored at – 80 ℃. While 10–51% of patients develop ILD years before the onset of pSS [9], pSS–ILD begins at the same time as other systemic manifestations in approximately 10% of cases. In the remainder of cases, ILD develops late in the course of disease [10]. Laboratory testing A t i Laboratory testing Autoimmune serology, serum immunoglobulin lev- els (including IgG, IgA and IgM), C3, C4, and C-reac- tive protein (CRP) were assessed in all patients. Antinuclear antibody (ANA) testing was performed using indirect immunofluorescence and qualitatively graded based on the serum dilution yielding positive staining (ANA+ = ANA 1:80 − 1:160; ANA++ = ANA 1:320 − 1:640; ANA+++ = ANA ≥ 1:1280). Anti-SSA/ Ro60, anti-Ro52, and anti-SSB/La antibodies were assessed via immunoblotting. Previous studies have shown that prognostic factors associated with the occurrence of pSS–ILD include older age, male sex, disease duration, smoking, an increase in anti-nuclear antibodies or rheumatoid factor, the pres- ence of anti-SSA/Ro52 antibodies [11], low levels of circulating C3, and increased C-reactive protein levels [12–14]. However, there are few reports on other serum protein biomarkers in pSS–ILD that potentially shed light on underlying disease mechanisms. In this study, we, therefore, focused on clinical risk factors and alterna- tive serum protein biomarkers capable of discriminating pSS–ILD from pSS–no-ILD. High resolution computed tomog- raphy (HRCT) of the chest represents the main imaging tool for evaluating pSS-related pulmonary abnormali- ties, because HRCT is very sensitive in detecting mild pSS-related pulmonary abnormalities, even in asymp- tomatic patients [10]. However, screening pSS patients with HRCT is not currently standard of care, despite the relatively high prevalence of ILD in this disease and the potential for significant morbidity. Therefore, identifying clinical risk factors and other serum protein biomark- ers associated with the development of pSS–ILD will be critically important in facilitating non-invasive detection of lung involvement at earlier, more treatable stages of disease. Ethics approval for the study was obtained from the Ethics Committee of the first affiliated Hospital of Xia- men University, School of Medicine, Xiamen Univer- sity, China (Approval number: KY2017-026). Written informed consent was obtained from each participant before enrollment. HRCT and ILD scoring HRCT (Aquilion 16; Toshiba Medical Systems) of the chest without contrast was performed for all patients during end inspiration using 1–2-mm collimation at 1–2- mm intervals in the supine position. HRCT classification was based on readings from at least two independent pulmonologists/radiologists blinded to the disease char- acteristics of the patients. Although the intra- and inter- observer correlations exceeded 90% for the HRCT image readers in previous studies, discrepancies in the cur- rent study were resolved by consensus agreement with one additional reader. Radiographic features indicative of ILD included ground-glass opacities, septal thicken- ing, reticulation, traction bronchiectasis, and/or honey- combing. Additional characterization utilized a scale of interstitial lung abnormalities (ILAs) ranging from 0 to 3 [17], where ILA 0 = no ILD, ILA 1 = indeterminate ILD (focal or unilateral ground-glass attenuation, focal or unilateral reticulation, or patchy ground-glass abnor- mality involving < 5% of the lung), ILA 2 = mild/moder- ate ILD (changes affecting > 5% of any lobar region with Materials and methods Patients Sixty-nine patients with pSS who met the 2016 Ameri- can College of Rheumatology (ACR)–European League Against Rheumatism (EULAR) classification criteria for primary Sjögren’s syndrome [15, 16] were consecutively enrolled between September 2013 and June 2017 through the Department of Rheumatology at the first affiliated Hospital of Xiamen University, School of Medicine, Xia- men University and Fuqing City Hospital affiliated with Fujian Medical University, China. Recruited patients in this observational study were from different provinces, mainly in the south of China. Patients with Sjogren’s features occurring in the context of other well-defined connective tissue diseases (CTD) such as rheumatoid Weng et al. European Journal of Medical Research (2022) 27:199 Weng et al. European Journal of Medical Research (2022) 27:199 Weng et al. European Journal of Medical Research (2022) 27:199 Weng et al. European Journal of Medical Research (2022) 27:199 Page 3 of 10 Finally, least absolute shrinkage and selection operator (LASSO) modeling was used as a penalized regression tool to develop a clinical prediction algorithm for detect- ing the presence of pSS–ILD. We determined the lowest shrinkage parameter (λ) with which to select final clini- cal risk factors and protein biomarkers (and their coeffi- cients) for predicting the probability of being diagnosed as having pSS–ILD. ROC curves were then generated to assess the ability of algorithm-based predictions to dis- criminate between the presence vs. absence of pSS–ILD (as measured by AUC). non-dependent ground-glass or reticular abnormalities, diffuse centrilobular nodularity, non-emphysematous cysts, honeycombing, or traction bronchiectasis), ILA 3 = advanced ILD (bilateral fibrosis in multiple lobes associated with honeycombing and traction bronchiec- tasis in a subpleural distribution) [18, 19]. In this study, ILA scores ≥ 2 identified patients with pSS–ILD, while ILA scores of 0 and 1 encompassed pSS patients without definite ILD. Detection of pSS–ILD‑related serum biomarkers by multiplex ELISA All statistical analyses were performed using IBM SPSS Statistics, version 20.0. Two-sided p values ≤ 0.05 were considered statistically significant. To adjust for multiple comparisons, p values were corrected by the false discov- ery rate (FDR). Multiplex enzyme-linked immunosorbent assays (Mul- tiplex ELISAs) were performed using Luminex xMAP technology according to the manufacturer’s instructions (EMD Millipore Corporation, Billerica, MA, USA) [20]. A combined 42-plex assay was used to determine serum levels of a range of cytokines, chemokines and MMPs (listed in Additional file 1) potentially related to the mechanism of pSS–ILD. Detection of KL‑6 by CLEIAh The concentration of Krebs von den Lungen-6 (KL-6) was measured using a commercial chemiluminescent enzyme immunoassay (CLEIA) according to the experimental procedure specified by the manufacturer (Fujirebio Inc., Tokyo, Japan). Patient‑specific variables and pSS–ILD Demographic characteristics of our pSS cohort are sum- marized in Table 1. Most patients were female (84%, n = 58), and the mean age at the time of diagnosis of pSS was 55.04 ± 12.90 years (Table 1). Among the 69 patients with pSS, 19 (28%) had advanced pSS–ILD (ILA score 3), 15 (22%) had mild/moderate pSS–ILD (ILA score 2), 25 (36%) had indeterminate pSS–ILD (ILA score 1), and 10 (14%) had pSS–no ILD (ILA score 0) based on HRCT criteria (Table 1). After combining individuals with ILA score 2 + 3, the prevalence of pSS–ILD in our cohort was 50%. Statistical analysis Age was significantly different between mild/moder- ate ILD (ILA score 2) and no-ILD (ILA score 0) (p < 0.05), and between advanced ILD (ILA score 3) and pSS no-ILD (ILA score 0) (p < 0.001) (Table 1). Overall, the mean age at diagnosis in the combined pSS–ILD group (ILA score 2 + 3) was significantly higher than in individuals who did not meet criteria for pSS–ILD (ILA score 0 + 1) (61 vs. 52, p = 0.003) (Table 2). Area under the curve (AUC) derived from ROC analysis was 0.706 (95% confidence interval: 0.582–0.830, p = 0.003) for age of pSS diagnosis as a discriminating factor for pSS–ILD (Table 3). In con- trast to age at diagnosis, there were no statistically signif- icant differences in gender or disease duration between those meeting criteria for pSS–ILD and those who did not (p > 0.05) (Table 2). All continuous variables were evaluated for normality to ensure uniformity in concentration and dispersion. Median and interquartile range (IQR) were presented for variables that did not follow a normal distribution. Bivariate analyses were conducted using Chi-square or Fisher’s exact test for categorical variables and outcomes (pSS–ILD and pSS no/indeterminate-ILD). Alternatively, Mann–Whitney U tests were used for non-parametric continuous variables (e.g., serum biomarker levels, risk scores) and outcomes. To determine correlations among continuous variables and identify potential confound- ing factors, Spearman correlation coefficients were calculated. Additional analyses were conducted to determine which demographic variables, clinical risk factors, and/ or serum proteins were associated with pSS–ILD. These variables were included in the final logistic regression model as candidate covariates to assess associations with pSS–ILD. Risk scores were derived for each patient based on the final logistic regression model. We further evalu- ated the performance of our model using receiver operat- ing characteristic (ROC) analysis to calculate area under the curve (AUC) values. Laboratory findings (%) 7 (70) 16 (64) 9 (60) 12 (60) Table 1 Clinical, demographic, and laboratory characteristics of pSS patients with different stages of ILD Table 1 Clinical, demographic, and laboratory characteristics of pSS patients with different stages of ILD pSS primary Sjogren’s syndrome, ILA interstitial lung abnormalities, IQR interquartile range, ANA Antinuclear antibody, IgA IgM, IgG Immunoglobulin AM and G, C3 and C4 complement 3 and complement 4, CRP C-reactive protein. Mann–Whitney U test and Fisher’s exact test were used to determine p values p < 0.05, p < 0.01, p < 0.001 when comparing different subcategories of pSS-ILD with pSS-no ILD remodeling proteins (MMPs) previously examined in IPF and other cohorts of CTD–ILD. We then assessed the relationship between the serum levels of these biomark- ers and pSS–ILD disease severity based on HRCT. This analysis revealed that eotaxin/CCL11, KL-6, TGFα and TNFα showed significant associations with ILD severity in selected subgroups of pSS–ILD (Tables 2, 3), whereas no statistically significant differences were found for the other biomarkers. of 0.653 (95% confidence interval: 0.521–0.785, p = 0.029) (Table 3).if CRP was also significantly different between severe ILD and no-ILD in pSS patients (p < 0.001) (Table 1). Further- more, higher levels of CRP distinguished the combined pSS–ILD group (ILA score 2 + 3) from those without definite ILD (p = 0.002) (Table 2), yielding an AUC of 0.713 (95% confidence interval: 0.590–0.836, p = 0.002 (Table 3). Conversely, there were no statistically signifi- cant differences in circulating levels of IgG, IgA, C3, or C4 between the two groups (Table 2). Autoantibodiesh Significant differences were found between the pSS mod- erate/advanced-ILD group (ILA score 2 + 3) and the pSS no-/indeterminate-ILD group (ILA score 0 + 1) based on age, IgM, CRP, eotaxin, KL-6, TGFα and TNFα levels (p < 0.05, Table 2 and Fig. 1). Other variables did not show statistically significant differences in univariate analyses. Corrected by FDR, age, CRP, eotaxin, and KL-6 remained statistically significant (p = 0.017, 0.017, 0.021, < 0.0001, respectively) (Table 2). In ROC analyses, serum levels of eotaxin, KL-6 and TNFα distinguished combined sub- groups of pSS–ILD (ILA score 2 + 3) from subgroups without definite ILD, with respective AUCs of 0.695 (95% confidence interval: 0.571–0.818, p = 0.006), 0.883 (95% confidence interval: 0.798–0.969, p < 0.0001), and The percentage of pSS patients with anti-SSA/Ro60 anti- bodies was significantly higher in the pSS-advanced ILD patients (ILA score 3) than in the pSS no-ILD subgroup (ILA score 0) (p < 0.01) (Table 1). However, the percent- ages of anti-Ro52, anti-SSB/La, and ANA antibody posi- tivity were not significantly different between different pSS subgroups (p > 0.05) (Table 2). Laboratory findings As shown in Table 1, IgM was significantly different between advanced ILD (ILA score 3) and no-ILD (ILA score 0) in pSS patients (p < 0.05). The combined pSS– ILD group (ILA 2 + ILA 3) had higher circulating levels of IgM than the group consisting of pSS–indeterminate and pSS–no ILD (p = 0.029) (Table 2), yielding an AUC Weng et al. European Journal of Medical Research (2022) 27:199 Page 4 of 10 Table 1 Clinical, demographic, and laboratory characteristics of pSS patients with different stages of ILD pSS primary Sjogren’s syndrome, ILA interstitial lung abnormalities, IQR interquartile range, ANA Antinuclear antibody, IgA IgM, IgG Immunoglobulin AM and G, C3 and C4 complement 3 and complement 4, CRP C-reactive protein. Mann–Whitney U test and Fisher’s exact test were used to determine p values p < 0.05, p < 0.01, p < 0.001 when comparing different subcategories of pSS-ILD with pSS-no ILD pSS-no ILD pSS-indeterminate ILD pSS-mild/moderate ILD pSS-advanced ILD (ILA score 0) (ILA score 1) (ILA score 2) (ILA score 3) Demographic parameters Number, no. (%) 10 (14) 25 (36) 15 (22) 19 (28) Female, no. (%) 9 (90) 21 (84) 13 (87) 15 (79) Male, no. (%) 1 (10) 4 (16) 2 (13) 4 (21) Age at diagnosis of pSS, median (IQR), years 49.00 (33.25–53.25) 53.00 (46.50–61.50) 60.00 (48.00–64.00) 62.00 (55.00– 70.00)*** Disease duration, median (IQR), years 1.00 (0.25–5.00) 1.00 (0.75–4.00) 3.00 (1.00–10.00) 2.00 (0.50–5.00) Laboratory findings IgG, g/l 16.85 (13.75–19.23) 18.30 (14.25–21.50) 21.20 (13.50–26.10) 14.00 (10.04–19.20) IgM, g/l 1.16 (0.77–1.47) 1.00 (0.62–2.39) 1.37 (1.06–2.44) 1.58 (1.38–2.98)* IgA, g/l 2.75 (2.19–3.92) 3.36 (2.67–4.60) 2.88 (1.67–4.50) 2.60 (2.49–3.82) C3, g/l 1.01 (0.80–1.25) 0.86 (0.67–1.08) 1.02 (0.98–1.11) 0.93 (0.92–1.12) C4, g/l 0.22 (0.17–0.31) 0.18 (0.11–0.22) 0.15 (0.12–0.22) 0.28 (0.20–0.31) CRP, mg/l 0.66 (0.33–5.45) 2.20 (0.40–6.76) 4.9 (1.41–30.00) 9.95 (4.80–22.60)* ANA positive, no. (%) 7 (70) 20 (80) 12 (80) 10 (50) Anti-SSA/Ro60 positive, no. (%) 6 (60) 14 (56) 8 (53) 4 (20) Anti-SSB/La positive, no. (%) 3 (30) 10 (40) 3 (20) 3 (15) Anti-Ro-52 positive, no. Clinical prediction model 0.656 (95% confidence interval: 0.527–0.785, p = 0.086) (Table 3). To develop complementary clinical prediction tools capable of distinguishing pSS–ILD from pSS–no ILD, we applied LASSO modeling—a machine learning- based, penalized regression method designed to mini- mize data complexity and maximize precision. This analysis demonstrated that the clinical and serum bio- marker signature consisting of age, KL-6, and TNFα effectively differentiated pSS–ILD from pSS–no ILD with high sensitivity and specificity, yielding an AUC of 0.883 (95% CI = 0.7987–0.9677, p value < 0.0001) (Fig. 3). We next used the Spearman coefficient to assess corre- lations between continuous variables, and then selected only one member of each pair of correlated variables (r > 0.3, p < 0.05) to avoid multicollinearity in prediction models. Based on these criteria, disease duration and KL-6 levels were the two variables retained in the final logistic regression model to assess their association with the occurrence of pSS–ILD (p < 0.0001, Table 4). After KL-6 and disease duration were fit into this logistic regression model, we calculated risk scores based on the equation: risk for pSS– ILD = 3.824 + 0.006KL-6 + 0.150disease duration. This analysis demonstrated that risk scores were significantly different between different ILA subcategories, as p values for comparisons of ILA score 0 vs. ILA score 1, ILA score 0 vs. ILA score 2 and ILA score 0 vs. ILA score 3 were 0.4558, 0.004 and < 0.0001, respectively (Fig. 2). Overall, these data indicated that the model incorporating disease duration and KL-6 levels effectively distinguished pSS– ILD from pSS no-ILD. Serum protein biomarkers To address whether serum protein biomarkers are asso- ciated with the presence of pSS–ILD, we measured the serum levels of KL-6 as well as 42 proteins that encompass cytokines, chemokines, growth factors, and Weng et al. European Journal of Medical Research (2022) 27:199 Page 5 of 10 Table 2 Clinical, demographic, and laboratory characteristics of pSS patients segregated by ILD subcategory Eotaxin/CCL11 C–C motif chemokine ligand 11, KL-6 Krebs vonden Lungen-6, TGFa growth factor alpha, TNFa tumor necrosis factor-alpha The continuous variables were presented as median (interquartile range). The significance of differences in demographic variables, clinical features, and serum biomarkers were determined by univariate analyses using Fisher’s exact test for categorical variables and Mann–Whitney U test for continuous variables. IQR interquartile range * p < 0.05, p < 0.01, p < 0.001 when the pSS moderate/advanced-ILD group (ILA score 2 + 3) was compared with the pSS no/indeterminate-ILD group (ILA score 0 + 1) 1. The lower limit of detection for TGF-α was 1.07 pg/ml Bold values represent the statistic significances p<0 05 Variable no/indeterminate ILD moderate/advanced ILD p value (ILA score 0 + 1) (ILA score 2 + 3) (n = 35) (n = 34) Female, no. (%) 30 (86) 28 (82) 0.703 Age at diagnosis of pSS, years 52.00 (44.00–58.00) 61.00 (52.50–66.50) 0.003 Disease duration, years 1.00 (0.50–4.00) 2.50 (1.00–5.00) 0.229 IgG, g/l 17.60 (14.10–21.50) 18.50 (10.09–21.58) 0.792 IgM, g/l 1.09 (0.62–1.86) 1.44 (1.19–2.88) 0.029 IgA, g/l 3.21 (2.53–4.37) 2.74 (2.26–3.83) 0.203 C3, g/l 0.89 (0.72–1.19) 0.98 (0.92–1.11) 0.138 C4, g/l 0.20 (0.14–0.24) 0.21 (0.15–0.30) 0.201 CRP, mg/l 2.09 (0.40–6.23) 7.97 (1.85–23.20) 0.002 ANA positive, no. (%) 31 (89) 28 (82) 0.695 Anti-SSA/Ro60 positive, no. (%) 23 (66) 15 (44) 0.071 Anti-SSB/La positive, no. (%) 13 (37) 6 (18) 0.07 Anti-Ro-52 positive, no. (%) 26 (74) 28 (82) 0.417 eotaxin/CCL11, pg/ml 278.00 (167.00–391.00) 1130.00 (549.25–1538.25) 0.005 KL-6, U/ml 146.00 (119.00–208.00) 202.00 (164.25–300.75) < 0.0001 TGF-α, pg/ml 1.07 (1.07–1.07)1 1.07 (1.07–4.19) 0.044 TNF-α, pg/ml 7.17 (1.83–12.07) 11.60 (5.88–26.86) 0.026 Discussion Based on HRCT criteria, the prevalence of pSS–ILD (ILA 2 + ILA 3) in this cohort of consecutively enrolled pSS patents from our center was 50%. Because patients were enrolled consecutively without respect to underly- ing lung disease, this statistic likely reflects the true fre- quency of this complication that may go undetected in unscreened populations (note that in another study of 527 patients with pSS, 206 (39.1%) had evidence of ILD Weng et al. European Journal of Medical Research (2022) 27:199 Page 6 of 10 Table 3 Factors discriminating pSS–moderate/advanced ILD from pSS–no/indeterminate ILD AUC area under the curve p < 0.05, p < 0.01, p < 0.001 when the pSS moderate/advanced-ILD group (ILA score 2 + 3) was compared with the pSS no/indeterminate-ILD group (ILA score 0 + 1) Bold values represent the statistic significances p < 0.05 Variable Mild/moderate and severe ILD Disease (ILA score 2 + 3) AUC 95% CI p value Age at diagnosis of pSS 0.706 0.582–0.830 0.003* Disease duration 0.584 0.448–0.719 0.232 IgG 0.518 0.377–0.660 0.792 IgM 0.653 0.521–0.785 0.029* IgA 0.589 0.453–0.725 0.203 C3 0.604 0.467–0.741 0.138 C4 0.590 0.453–0.726 0.201 CRP 0.713 0.590–0.836 0.002 ANA positive 0.531 0.394–0.668 0.657 Anti-SSA/RO-60 positive 0.608 0.474–0.742 0.123 Anti-SSB/La positive 0.597 0.463–0.732 0.164 Anti-Ro-52 positive 0.540 0.404–0.677 0.565 eotaxin/CCL11 0.695 0.571–0.818 0.006 KL-6 0.883 0.798–0.969 < 0.0001*** TGF-α 0.618 0.484–0.753 0.091 TNF-a 0.656 0.527–0.785 0.026* Table 3 Factors discriminating pSS–moderate/advanced ILD from pSS–no/indeterminate ILD [5]). Subgroup comparisons indicated that the occur- rence of pSS–ILD was associated with older age and higher serum levels of CRP, immunoglobulin M, eotaxin, KL-6, TGFα and TNFα. Our final binary logistic regres- sion model revealed that KL-6 level and disease duration were key parameters associated with pulmonary involve- ment in Sjögren’s syndrome patients. LASSO modeling complemented these findings, demonstrating that age, KL-6, and TNFα effectively differentiated pSS–ILD from pSS–no ILD with high sensitivity and specificity. [5]). Subgroup comparisons indicated that the occur- rence of pSS–ILD was associated with older age and higher serum levels of CRP, immunoglobulin M, eotaxin, KL-6, TGFα and TNFα. Our final binary logistic regres- sion model revealed that KL-6 level and disease duration were key parameters associated with pulmonary involve- ment in Sjögren’s syndrome patients. LASSO modeling complemented these findings, demonstrating that age, KL-6, and TNFα effectively differentiated pSS–ILD from pSS–no ILD with high sensitivity and specificity. Discussion ILA score 2 + 3) by HRCT in pSS. Panels a–c demonstrate the relationship between the natural log of serum a) KL-6, b eotaxin/CCL11, and c TNFα levels and ILD severity (p = 0.0002, 0006, and 0.0182, respectively). Each symbol represents an individual patient; horizontal lines show the mean value (natural log) of serum levels for specified cytokines. P values were determined by Mann–Whitney U test Fig. 1 Relationship between serum levels of cytokines and the severity of ILD (ILA score 0 vs. ILA score 2 + 3) by HRCT in pSS. Panels a–c demonstrate the relationship between the natural log of serum a) KL-6, b eotaxin/CCL11, and c TNFα levels and ILD severity (p = 0.0002, 0006, and 0.0182, respectively). Each symbol represents an individual patient; horizontal lines show the mean value (natural log) of serum levels for specified cytokines. P values were determined by Mann–Whitney U test Fig. 1 Relationship between serum levels of cytokines and the severity of ILD (ILA score 0 vs. ILA score 2 + 3) by HRCT in pSS. Panels a–c demonstrate the relationship between the natural log of serum a) KL-6, b eotaxin/CCL11, and c TNFα levels and ILD severity (p = 0.0002, 0006, and 0.0182, respectively). Each symbol represents an individual patient; horizontal lines show the mean value (natural log) of serum levels for specified cytokines. P values were determined by Mann–Whitney U test epithelial cell damage or is playing a direct role in aber- rant signaling pathways remains unclear, however. Table 4 Relationship between selected risk factors and pSS-ILD Relationship between selected risk factors and pSS–ILD Descriptive analyses showed differences in risk of pSS–ILD for seventeen indices mentioned above. We conducted Spearman correlation analyses for continuous variables and selected one member of each pair of correlated variables (r > 0.3 and p < 0.05) to include into the logistic regression model to avoid multicollinearity. With those steps, disease duration and KL-6 were selected to include in the final model. Beta coefficient p value Disease duration 0.150 0.032 KL-6 0.006 0.000 Constant − 3.824 0.000 Our study does have several limitations. First, even though we enrolled patients from different provinces Fig. 2 Distribution of risk scores between different ILA subcategories. KL-6 and disease duration were fit into a logistic regression model, yielding an equation for calculation of a combined risk score = − 3.824 + 0.006KL-6 + 0.150disease duration. Discussion SSB positivity as well as high levels of IgG and IgA were shown in a separate study to be independently associated with lung involvement in pSS [23]. Unlike these studies, however, we did not find statistically significant associa- tions between anti-Ro52/La or anti-La/SSB antibodies and pSS–ILD. Serum levels of eotaxin were higher in the pSS–ILD subgroup of our cohort, distinguishing these pSS patients from those without definite ILD. Eotaxin may be involved in the pathogenesis of ILD in pSS—possibly due to acti- vation of fibroblasts, eosinophils, and neutrophils, each of which bears receptors for this chemokine. Although there have been no reports on eotaxin in the context of pSS–ILD, other studies have shown that eotaxin directly affects lung fibroblasts by upregulating procollagen type I gene expression and collagen protein production in lung fibroblasts—suggesting a potential contribution of this CC chemokine to deposition of extracellular matrix (ECM) and the early phase of tissue remodeling in the lung [24]. Interestingly, a recent RA-ILD study found significant associations between eotaxin and the sever- ity of RA-ILD [25], again consistent with a role for this chemokine in lung fibrosis [24–26].h i Advancing age was also strongly associated with the development of pSS–ILD in other studies. A recent sys- tematic review of 6157 pSS patients showed that older age, male sex, and higher CRP levels were risk factors for pSS–ILD [13, 14]. In addition, late age of onset and long duration of disease have also been linked to pSS–ILD [21], consistent with our logistic regression model show- ing that disease duration of pSS plays a critical role in the development of pSS–ILD. Regarding relationships between the presence of autoantibodies and the occurrence of pSS–ILD, the per- centage of anti-SSA/Ro60 antibody in pSS-advanced ILD patients (ILA score 3) was significantly higher than in pSS–no ILD patients in our study as well as in oth- ers [21]. Other studies have also shown that anti-Ro52/ La antibodies are an independent risk factor associated with the occurrence of ILD in pSS [11, 21, 22]. Anti-La/ i The present study showed that the serum level of KL-6 was the most discriminating biomarker for pSS–ILD in our cohort. Indeed, levels of KL-6 significantly correlated Weng et al. European Journal of Medical Research (2022) 27:199 Page 7 of 10 Fig. 1 Relationship between serum levels of cytokines and the severity of ILD (ILA score 0 vs. Discussion P values for ILA score 0 vs. ILA score 1, ILA score 0 vs. ILA score 2 and ILA score 0 vs. ILA score 3 were 0.4558, 0.004 and < 0.0001, respectively. The presented p values were determined by the non-parametric Mann– Whitney U test with the severity of ILD measured by HRCT to a greater degree than other biomarkers associated with pSS–ILD in this study (eotaxin, TGFα and TNFα), as shown in Fig. 1. KL-6 has been classified as a human mucin-like glycoprotein (in the MUC1 family) secreted predomi- nantly by type II pneumocytes in the affected lungs of patients with ILD [27, 28]. Although previous studies have investigated the relationship between serum KL-6 levels and disease activity/severity as well as prognosis in SSc–ILD [29–31], RA-ILD [30, 32, 33], and PM/DM- ILD [30, 32], we found that KL-6 also related to sever- ity of pSS–ILD. Consistent with our findings, Kamiya et al. showed that pSS–ILD patients with higher levels of serum KL-6 (> 800U/mL) had a higher pulmonary mor- tality rate compared to those without elevated serum KL-6 levels [34]. Overall, the ability of serum levels of KL-6 to distinguish CTD–ILD from CTD–no ILD in multiple subsets of early as well as established disease groups demonstrates its value as a biomarker for these disorders. Whether KL-6 is simply serving as a marker of with the severity of ILD measured by HRCT to a greater degree than other biomarkers associated with pSS–ILD in this study (eotaxin, TGFα and TNFα), as shown in Fig. 1. KL-6 has been classified as a human mucin-like glycoprotein (in the MUC1 family) secreted predomi- nantly by type II pneumocytes in the affected lungs of patients with ILD [27, 28]. Although previous studies have investigated the relationship between serum KL-6 levels and disease activity/severity as well as prognosis in SSc–ILD [29–31], RA-ILD [30, 32, 33], and PM/DM- ILD [30, 32], we found that KL-6 also related to sever- ity of pSS–ILD. Consistent with our findings, Kamiya et al. showed that pSS–ILD patients with higher levels of serum KL-6 (> 800U/mL) had a higher pulmonary mor- tality rate compared to those without elevated serum KL-6 levels [34]. Overall, the ability of serum levels of KL-6 to distinguish CTD–ILD from CTD–no ILD in multiple subsets of early as well as established disease groups demonstrates its value as a biomarker for these disorders. pSS: Sjögren’s syndrome; ILD: Interstitial lung disease; HRCT: High-resolution computed tomography; ILA: Interstitial lung abnormalities; CTD: Connective tissue diseases; CRP: C-reactive protein; ANA: Antinuclear antibody; KL-6: Krebs von den Lungen-6; ROC: Receiver operating characteristic; AUC: Area under the curve; FDR: False discovery rate; LASSO: Least absolute shrinkage and selection operator modeling. Conclusions and different hospitals of China, there were small num- bers of patients in each subgroup of pSS–ILD, limit- ing study power. Second, pulmonary function was only tested in 20 patients in this study (data not shown) and, therefore, could not be incorporated into any of our analyses. Of note, however, our findings support the associations between pSS–ILD and serum protein biomarkers, such as eotaxin, KL-6, TGFα, and TNFα. While future cross-sectional studies in larger cohorts will be critical in validating findings from this study, prospective studies will also be important to inves- tigate a wider range of biomarkers and their ability to correlate with disease activity over time. In turn, these corroborative studies will provide greater insight to the underlying pathogenesis of pSS–ILD and elucidate therapeutically targetable pathways. Patient age, disease duration, and serum levels of both KL-6 and TNFα were the most discriminating factors associated with the presence of ILD in our cohort of pSS patients. In addition to these parameters, higher levels of CRP, IgM, eotaxin, TGFα, and TNFα should also prompt the search for occult as well as clinically evident lung involvement based on statistically signifi- cant univariate associations with pSS–ILD in this study. Discussion The corresponding ROC curve reflects performance characteristics of this model, as indicated by area under the curve (AUC). The accompanying table shows regression coefficients for clinical risk factors and specific serum proteins and different hospitals of China, there were small num- bers of patients in each subgroup of pSS–ILD, limit- ing study power. Second, pulmonary function was only tested in 20 patients in this study (data not shown) and, therefore, could not be incorporated into any of our analyses. Of note, however, our findings support the associations between pSS–ILD and serum protein biomarkers, such as eotaxin, KL-6, TGFα, and TNFα. While future cross-sectional studies in larger cohorts will be critical in validating findings from this study, prospective studies will also be important to inves- tigate a wider range of biomarkers and their ability to correlate with disease activity over time. In turn, these corroborative studies will provide greater insight to the underlying pathogenesis of pSS–ILD and elucidate therapeutically targetable pathways. Discussion Whether KL-6 is simply serving as a marker of Fig. 2 Distribution of risk scores between different ILA subcategories. KL-6 and disease duration were fit into a logistic regression model, yielding an equation for calculation of a combined risk score = − 3.824 + 0.006KL-6 + 0.150disease duration. P values for ILA score 0 vs. ILA score 1, ILA score 0 vs. ILA score 2 and ILA score 0 vs. ILA score 3 were 0.4558, 0.004 and < 0.0001, respectively. The presented p values were determined by the non-parametric Mann– Whitney U test Weng et al. European Journal of Medical Research (2022) 27:199 Page 8 of 10 Fig. 3 Least absolute shrinkage and selection operator (LASSO) modeling in the identification of pSS–ILD. Application of LASSO regression modeling that is based on machine learning revealed clinical risk factors and serum protein biomarkers capable of distinguishing pSS–ILD patients with moderate/advanced-ILD from those with no-/indeterminate-ILD. The corresponding ROC curve reflects performance characteristics of this model, as indicated by area under the curve (AUC). The accompanying table shows regression coefficients for clinical risk factors and specific serum proteins Fig. 3 Least absolute shrinkage and selection operator (LASSO) modeling in the identification of pSS–ILD. Application of LASSO regression modeling that is based on machine learning revealed clinical risk factors and serum protein biomarkers capable of distinguishing pSS–ILD patients with moderate/advanced-ILD from those with no-/indeterminate-ILD. The corresponding ROC curve reflects performance characteristics of this model, as indicated by area under the curve (AUC). The accompanying table shows regression coefficients for clinical risk factors and specific serum proteins Fig. 3 Least absolute shrinkage and selection operator (LASSO) modeling in the identification of pSS–ILD. Application of LASSO regression modeling that is based on machine learning revealed clinical risk factors and serum protein biomarkers capable of distinguishing pSS–ILD patients with moderate/advanced-ILD from those with no-/indeterminate-ILD. The corresponding ROC curve reflects performance characteristics of this model, as indicated by area under the curve (AUC). The accompanying table shows regression coefficients for clinical risk factors and specific serum proteins Fig. 3 Least absolute shrinkage and selection operator (LASSO) modeling in the identification of pSS–ILD. Application of LASSO regression modeling that is based on machine learning revealed clinical risk factors and serum protein biomarkers capable of distinguishing pSS–ILD patients with moderate/advanced-ILD from those with no-/indeterminate-ILD. Consent for publication Consent for publication Not applicable. Not applicable. 15. Shiboski CH, Shiboski SC, Seror R, et al. 2016 American college of rheu- matology/European league against rheumatism classification criteria for primary Sjögren’s syndrome. Ann Rheum Dis. 2017;76:9–16. Funding 9. Palm O, Garen T, Berge Enger T, et al. Clinical pulmonary involvement in primary Sjogren’s syndrome: prevalence, quality of life and mortality–a retrospective study based on registry data. Rheumatology (Oxford). 2013;52:173–9. This study was funded by the National Natural Science Foundation of China (Grant No. 81771751) and Fujian Provincial Department of Science and Tech- nology (Grant No. 2018J01383). 10. Luppi F, Sebastiani M, Silva M, et al. Interstitial lung disease in Sjogren’s syndrome: a clinical review. Clin Exp Rheumatol. 2020;38(Suppl 126):291–300. 10. Luppi F, Sebastiani M, Silva M, et al. Interstitial lung disease in Sjogren’s syndrome: a clinical review. Clin Exp Rheumatol. 2020;38(Suppl 126):291–300. Declarations 12. Flament T, Bigot A, Chaigne B, Henique H, Diot E, Marchand-Adam S. Pulmonary manifestations of Sjögren’s syndrome. Eur Respir Rev. 2016;25(140):110–23. Availability of data and materials y The outputs are available from the authors upon request. 11. Buvry C, Cassagnes L, Tekath M, et al. Anti-Ro52 antibodies are a risk fac- tor for interstitial lung disease in primary Sjögren syndrome. Respir Med. 2020;163: 105895. The authors declare no conflicts of interest. 16. Shiboski CH, Shiboski SC, Seror R, et al. 2016 American College of Rheu- matology/European League Against Rheumatism Classification Criteria for Primary Sjögren’s Syndrome: a consensus and data-driven methodol- ogy involving three international patient cohorts. Arthritis Rheumatol (Hoboken, NJ). 2017;69:35–45. Author details 1 1 Department of Rheumatology, The First Affiliated Hospital of Xiamen Univer- sity, School of Medicine, Xiamen University, Xiamen 361003, China. 2 School of Life Sciences, Xiamen University, Xiamen, China. 3 Department of Respira- tory Medicine, The First Affiliated Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, China. 4 Department of Radiology, The First Affiliated Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, China. 5 School of Medicine, Xiamen University, Xiamen, China. 6 State Key Laboratory of Molecular Vaccinology and Molecular Diag- nostics, School of Public Health, Xiamen University, Xiamen, China. 7 Fuqing City Hospital affiliated to Fujian Medical University, Fuzhou, China. 8 Depart- ment of Pediatrics, School of Medicine, The First Affiliated Hospital of Xiamen University National Institute of Data Science in Health and Medicine, Xiamen University, Xiamen, China. 9 Division of Rheumatology and Clinical Immunol- ogy, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA. 17. Chen J, Doyle TJ, Liu Y, et al. Biomarkers of rheumatoid arthritis- associated interstitial lung disease. Arthritis Rheumatol (Hoboken, NJ). 2015;67:28–38. 18. Washko GR, Hunninghake GM, Fernandez IE, et al. Lung volumes and emphysema in smokers with interstitial lung abnormalities. N Engl J Med. 2011;364:897–906. 19. Washko GR, Lynch DA, Matsuoka S, et al. Identification of early interstitial lung disease in smokers from the COPDGene Study. Acad Radiol. 2010;17:48–53. 20. Gorelik E, Landsittel DP, Marrangoni AM, et al. Multiplexed immunobead- based cytokine profiling for early detection of ovarian cancer. Cancer Epidemiol Biomarkers Prev. 2005;14:981–7. 21. Buvry C, Cassagnes L, Tekath M, et al. Anti-Ro52 antibodies are a risk factor for interstitial lung disease in primary Sjogren € syndrome. Respir Med. 2020;2020(163):105895. Received: 4 July 2022 Accepted: 17 September 2022 Received: 4 July 2022 Accepted: 17 September 2022 22. Dong X, Zhou JF, Guo XJ, et al. A retrospective analysis of distinguishing features of chest HRCT and clinical manifestation in primary Sjögren’s syndrome-related interstitial lung disease in a Chinese population. Clin Rheumatol. 2018;37(2981–2988):2018. Acknowledgements Not applicable 6. Parambil JG, Myers JL, Lindell RM, Matteson EL, Ryu JH. Interstitial lung disease in primary Sjögren syndrome. Chest. 2006;130:1489–95. 6. Parambil JG, Myers JL, Lindell RM, Matteson EL, Ryu JH. Interstitial lung disease in primary Sjögren syndrome. Chest. 2006;130:1489–95. Author contributions 7. Ito I, Nagai S, Kitaichi M, et al. Pulmonary manifestations of primary Sjogren’s syndrome: a clinical, radiologic, and pathologic study. Am J Respir Crit Care Med. 2005;171:632–8. 7. Ito I, Nagai S, Kitaichi M, et al. Pulmonary manifestations of primary Sjogren’s syndrome: a clinical, radiologic, and pathologic study. Am J Respir Crit Care Med. 2005;171:632–8. Study conception or design: JC and DA; acquisition of the data: YHL, DHL, CYY, YLL, LW, WL, and SXG.; analysis and interpretation of the data: QYL, LW, YQC, DA, YDH, and TL; drafting and revising the article: JC and DA. All authors read and approved the final manuscript. p 8. Schildgen O, Enomoto Y, Takemura T, et al. Prognostic factors in interstitial lung disease associated with primary Sjögren’s syndrome: a retrospective analysis of 33 pathologically–proven cases. PLoS ONE. 2013;8: e73774. 8. Schildgen O, Enomoto Y, Takemura T, et al. Prognostic factors in interstitial lung disease associated with primary Sjögren’s syndrome: a retrospective analysis of 33 pathologically–proven cases. PLoS ONE. 2013;8: e73774. Received: 4 July 2022 Accepted: 17 September 2022 2. Castelino FV, Varga J. Interstitial lung disease in connective tissue dis- eases: evolving concepts of pathogenesis and management. Arthritis Res Ther. 2010;12:213. 1. Rubio-Rivas M, Royo C, Simeón CP, Corbella X, Fonollosa V. Mortality and survival in systemic sclerosis: systematic review and meta-analysis. Semin Arthritis Rheum. 2014;44:208–19. Abbreviations SS Sjö ’ Page 9 of 10 Weng et al. European Journal of Medical Research (2022) 27:199 Supplementary Information 3. Bagnato G, Harari S. Cellular interactions in the pathogenesis of interstitial lung diseases. Eur Respir Rev. 2015;24:102–14. Competing interests The authors declare no conflicts of interest. Ethics approval and consent to participate Ethical approval for the study was obtained by the Ethical Committee of The first affiliated Hospital of Xiamen University, School of Medicine, Xiamen University, China (Approval number KY2017-026). Written informed consents were obtained from all participants. 13. Wang Y, Hou Z, Qiu M, Ye Q. Risk factors for primary Sjogren syndrome- associated interstitial lung disease. J Thorac Dis. 2018;10:2108–17. 14. He C, Chen Z, Liu S, Chen H, Zhang F. Prevalence and risk factors of interstitial lung disease in patients with primary Sjogren’s syndrome: a systematic review and meta-analysis. Int J Rheum Dis. 2020;23:1009–18. Consent for publication Not applicable. 1. Rubio-Rivas M, Royo C, Simeón CP, Corbella X, Fonollosa V. Mortality and survival in systemic sclerosis: systematic review and meta-analysis. Semin Arthritis Rheum. 2014;44:208–19. 2. Castelino FV, Varga J. Interstitial lung disease in connective tissue dis- eases: evolving concepts of pathogenesis and management. Arthritis Res Ther. 2010;12:213. Supplementary Information 4. Nannini C, Jebakumar AJ, Crowson CS, Ryu JH, Matteson EL. Primary Sjögren’s syndrome 1976–2005 and associated interstitial lung disease: a population-based study of incidence and mortality. BMJ Open. 2013;3: e003569. Additional file 1: Table. CTD–ILD-related biomarkers assessed by Multi- plex ELISA. 5. Dong X, Zhou J, Guo X, et al. A retrospective analysis of distinguishing features of chest HRCT and clinical manifestation in primary Sjögren’s syndrome-related interstitial lung disease in a Chinese population. Clin Rheumatol. 2018;37:2981–8. References Sarcoidosis Vasc Diffuse Lung Dis. 2016;33:216–23. f 34. Kamiya Y, Fujisawa T, Kono M, et al. Prognostic factors for primary Sjogren’s syndrome-associated interstitial lung diseases. Respir Med. 2019;159: 105811. 34. Kamiya Y, Fujisawa T, Kono M, et al. Prognostic factors for primary Sjogren’s syndrome-associated interstitial lung diseases. Respir Med. 2019;159: 105811. References 1. Rubio-Rivas M, Royo C, Simeón CP, Corbella X, Fonollosa V. Mortality and survival in systemic sclerosis: systematic review and meta-analysis. Semin Arthritis Rheum. 2014;44:208–19. 23. Zhao Y, Li Y, Wang L, et al. Primary Sjogren syndrome in Han Chinese: clinical and immunological characteristics of 483 patients. Medicine (Baltimore). 2015;94: e667. 24. Puxeddu I, Bader R, Piliponsky AM, Reich R, Levi-Schaffer F, Berkman N. The CC chemokine eotaxin/CCL11 has a selective profibrogenic effect on human lung fibroblasts. J Allergy Clin Immunol. 2006;117:103–10. 25. Kass DJ, Nouraie M, Glassberg MK, et al. Comparative profiling of serum protein biomarkers in rheumatoid arthritis-associated interstitial lung Page 10 of 10 Weng et al. European Journal of Medical Research (2022) 27:199 disease and idiopathic pulmonary fibrosis. Arthritis Rheumatol (Hoboken, NJ). 2020;72:409–19. disease and idiopathic pulmonary fibrosis. Arthritis Rheumatol (Hoboken, NJ). 2020;72:409–19. 26. Huaux F, Gharaee-Kermani M, Liu T, et al. Role of Eotaxin-1 (CCL11) and CC chemokine receptor 3 (CCR3) in bleomycin-induced lung injury and fibrosis. Am J Pathol. 2005;167:1485–96. i 27. Ishikawa N, Hattori N, Yokoyama A, Kohno N. Utility of KL-6/MUC1 in the clinical management of interstitial lung diseases. Respir Investig. 2012;50:3–13. 28. Xu L, Yang D, Zhu S, et al. Bleomycin-induced pulmonary fibrosis is attenuated by an antibody against KL-6. Exp Lung Res. 2013;39:241–8. 29. Kuwana M, Shirai Y, Takeuchi T. Elevated Serum Krebs von den lungen-6 in early disease predicts subsequent deterioration of pulmonary function in patients with systemic sclerosis and interstitial lung disease. J Rheumatol. 2016;43:1825–31. 30. Oguz EO, Kucuksahin O, Turgay M, et al. Association of serum KL-6 levels with interstitial lung disease in patients with connective tissue disease: a cross-sectional study. Clin Rheumatol. 2016;35:663–6. 30. Oguz EO, Kucuksahin O, Turgay M, et al. Association of serum KL-6 levels with interstitial lung disease in patients with connective tissue disease: a cross-sectional study. Clin Rheumatol. 2016;35:663–6. 31. Yamakawa H, Hagiwara E, Kitamura H, et al. Serum KL-6 and surfactant protein-D as monitoring and predictive markers of interstitial lung disease in patients with systemic sclerosis and mixed connective tissue disease. J Thorac Dis. 2017;9:362–71. 32. Lee JS, Lee EY, Ha Y-J, Kang EH, Lee YJ, Song YW. Serum KL-6 levels reflect the severity of interstitial lung disease associated with connective tissue disease. Arthritis Res Ther. 2019;21:58. 33. Lee YS, Kim HC, Lee BY, et al. The value of biomarkers as predictors of outcome in patients with rheumatoid arthritis-associated usual interstitial pneumonia. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. 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https://openalex.org/W4321480207	https://www.researchsquare.com/article/rs-1242933/latest.pdf	English	null	Ageing threatens sustainability of smallholder farming in China	Nature	2,023	cc-by	17,654	Aging threatens sustainability of smallholder farming in China Physical Sciences - Article Keywords: Posted Date: January 17th, 2022 Posted Date: January 17th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-1242933/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Aging threatens sustainability of smallholder farming in China 1 2 2 Chenchen Ren a,b, Xinyue Zhou c, Chen Wang b,d, Yaolin Guo d, Yu Diao d, Sisi Shen d, 3 Stefan Reis e,f, Wanyue Li c, Jianming Xu d, Baojing Gu d,g,* 4 5 a Department of Land Management, Zhejiang University, Hangzhou 310058, China; 6 b Policy Simulation Laboratory, Zhejiang University, Hangzhou, 310058, China 7 c School of Management, Zhejiang University, Hangzhou 310058, China; 8 d College of Environmental & Resource Sciences, Zhejiang University, Hangzhou 9 310058, China 10 e UK Centre for Ecology & Hydrology, Penicuik, EH26 0QB, United Kingdom 11 f University of Exeter Medical School, Knowledge Spa, Truro, TR1 3HD, United 12 Kingdom 13 g Zhejiang Provincial Key Laboratory of Agricultural Resources and Environment, 14 Zhejiang University, Hangzhou 310058, China 15 16 Corresponding Author: 17 College of Environmental & Resource Sciences, Zhejiang University, Zijingang 18 Campus, 866 Yuhangtang Road, Hangzhou 310058, PR China. Tel & Fax: +86 571 19 8820 6926. E-mail: bjgu@zju.edu.cn 20 21 Rapidly demographic aging substantially affects socioeconomic development 1-4, 22 presents grand challenges for food security and agricultural sustainability 5-8, 23 which have so far not been well understood. Here, by using over 30,000 households 24 survey across China, we show that rural population aging lowers average 25 education level of farmers by 3% (0-11% across different provinces) and reduces 26 farm size by 4% (2-11%) due to land transfer-out and abandonment in 2019. These 27 changes further led to a reduction of agricultural inputs, including fertilizers and 28 machinery, which decrease agricultural output by 4% and labor productivity by 29 9%. Meanwhile, fertilizer use efficiency is reduced by 3%, while increasing 30 fertilizer-related pollutants emission to the environment. New farming models, 31 such as cooperative farming, tend to have larger farm sizes and being operated by 32 younger farmers, who have a higher average education level, hence reducing total 33 labor requirement. Without policy interventions, agricultural output, labor 34 productivity and fertilizer use efficiency would decrease by 3-16% as a 35 consequence of population aging by 2100, compared to 2019 levels. With policy 36 measures, such as new farming models, this decrease could be reversed and in fact 37 an increase by approximately one-third achieved in the same time period. Aging threatens sustainability of smallholder farming in China 1 2 Our 38 analyses suggest that population aging effects on agriculture could be effectively 39 addressed through labor saving in large-scale new farming models, further 40 contributing to a widespread transformation of smallholder farming to 41 sustainable agriculture in China. 42 43 As life expectancy increases and population fertility rates decline, populations are aging 44 at an accelerating rate globally 9. Population aging brings grand challenges on multiple 45 global sustainable development goals (SDGs), mainly in relation to no poverty, zero 46 hunger, education, gender equality, decent work and economic growth, responsible 47 consumption and production, etc 1-4, 10, 11. Labor shortage and innovation constraints 48 may become severe in a society with an aging population, especially in labor-intense 49 economic sectors 3. Policy interventions to increase birth rates seems to be largely 50 2 Chenchen Ren a,b, Xinyue Zhou c, Chen Wang b,d, Yaolin Guo d, Yu Diao d, Sisi Shen d, 3 Stefan Reis e,f, Wanyue Li c, Jianming Xu d, Baojing Gu d,g, 4 a Department of Land Management, Zhejiang University, Hangzhou 310058, China; 6 b Polic Sim lation Laborator Zhejiang Uni ersit Hang ho 310058 China 7 a Department of Land Management, Zhejiang University, Hangzhou 310058, China; b Policy Simulation Laboratory, Zhejiang University, Hangzhou, 310058, China School of Management, Zhejiang University, Hangzhou 310058, China; College of Environmental & Resource Sciences, Zhejiang University, Hangzhou 10058, China Zhejiang Provincial Key Laboratory of Agricultural Resources and Environment, Zhejiang University, Hangzhou 310058, China Rapidly demographic aging substantially affects socioeconomic development 1-4, Rapidly demographic aging substantially affects socioeconomic development 1-4, 22 presents grand challenges for food security and agricultural sustainability 5-8, 23 which have so far not been well understood. Here, by using over 30,000 households 24 survey across China, we show that rural population aging lowers average 25 education level of farmers by 3% (0-11% across different provinces) and reduces 26 farm size by 4% (2-11%) due to land transfer-out and abandonment in 2019. These 27 changes further led to a reduction of agricultural inputs, including fertilizers and 28 machinery, which decrease agricultural output by 4% and labor productivity by 29 9%. Meanwhile, fertilizer use efficiency is reduced by 3%, while increasing 30 fertilizer-related pollutants emission to the environment. Aging threatens sustainability of smallholder farming in China 1 2 New farming models, 31 such as cooperative farming, tend to have larger farm sizes and being operated by 32 younger farmers, who have a higher average education level, hence reducing total 33 labor requirement. Without policy interventions, agricultural output, labor 34 productivity and fertilizer use efficiency would decrease by 3-16% as a 35 consequence of population aging by 2100, compared to 2019 levels. With policy 36 measures, such as new farming models, this decrease could be reversed and in fact 37 an increase by approximately one-third achieved in the same time period. Our 38 analyses suggest that population aging effects on agriculture could be effectively 39 addressed through labor saving in large-scale new farming models, further 40 contributing to a widespread transformation of smallholder farming to 41 sustainable agriculture in China. 42 As life expectancy increases and population fertility rates decline, populations are aging 44 at an accelerating rate globally 9. Population aging brings grand challenges on multiple 45 global sustainable development goals (SDGs), mainly in relation to no poverty, zero 46 hunger, education, gender equality, decent work and economic growth, responsible 47 consumption and production, etc 1-4, 10, 11. Labor shortage and innovation constraints 48 may become severe in a society with an aging population, especially in labor-intense 49 economic sectors 3. Policy interventions to increase birth rates seems to be largely 50 1 ineffective in many countries where aging issues have been considered serious 12, 13. 51 Alternative strategies urgently need to be developed to achieve and maintain sustainable 52 development paths for human society. Agriculture as a typical labor-intensive industry 53 could be one of the sectors substantially affected by population aging, especially in 54 countries where smallholder farming is prevalent 8, 14. However, how aging affects 55 agriculture and rural livelihoods has not been well researched or understood to date. 56 This sector is facing substantial challenges in squaring the circle between maintaining 57 food security and improving environmental protection through sustainable 58 intensification at many levels. However, it is indispensable to identify integrated, 59 comprehensive measures to ensure global food security, while protecting the 60 environment and achieving rural revitalization to achieve common prosperity. 61 62 Older age groups of farmers typically have a lower education levels, are less likely to 63 keep their agricultural skills up-to-date and embrace novel, cutting-edge methods 14-16. Aging threatens sustainability of smallholder farming in China 1 2 64 Due to a consistent labor supply shortage, they can only operate small-scale farms, 65 which leads to an increase in cropland abandonment as rural populations age 17, 18. 66 Meanwhile, the typical agricultural input mix and crop types (labor-intensive crops or 67 not) are likely to shift with rural aging, reducing agricultural productivity and efficiency 68 5-7, 19. These effects are usually worse in smallholder-farming-dominated countries such 69 as China 8. An aging society is defined as one in which more than 7% of the population 70 is over 65 years old 20, and this ratio has doubled in rural China (Fig. 1a) in the period 71 from 2000 to 2019. In the coming decades, China's population aging rate is predicted 72 to accelerate 20, 21. Thus, it is urgent and timely to address the threats of aging on 73 agricultural production in China and also other countries and regions globally, which 74 are facing similar issues. 75 76 Here, we explored the relationship between rural population aging (the proportion of 77 individuals over 65 years old at household-level) and agricultural sustainability based 78 on a panel model, using survey data from more than 30,000 rural households in China. 79 Nine indicators overall were selected to illustrate agricultural sustainability, including 80 farm size, farmer’s education, labor productivity, agricultural total input and output, 81 profit-cost ratio, machine input, fertilizer input and fertilizer use efficiency (FUE). To 82 understand the full range of effects of aging on agriculture, other variables such as 83 transferred and abandoned cropland, pesticide and manure inputs, and technology 84 adoption were also incorporated into the analysis. In addition, the analysis quantified 85 the potential of new farming models, such as industrial, family and cooperative farming 86 for contributing to addressing the threats posed by aging on a predominantly traditional 87 smallholder farming sector. Finally, Shared Socioeconomic Pathways (SSP) scenarios 88 of future development were used to explore options for achieving sustainable 89 intensification of agriculture under conditions of aging rural populations. 90 91 Results and discussion 92 How aging threatens agriculture? 93 Population aging trends have been observed since the 1990s, in both urban and rural 94 areas in China (Fig. 1). However, rural aging is more severe and happening at a more 95 rapidly accelerating rate than that that of urban areas (Fig. 1a). Aging threatens sustainability of smallholder farming in China 1 2 75 ineffective in many countries where aging issues have been considered serious 12, 13. 51 Alternative strategies urgently need to be developed to achieve and maintain sustainable 52 development paths for human society. Agriculture as a typical labor-intensive industry 53 could be one of the sectors substantially affected by population aging, especially in 54 countries where smallholder farming is prevalent 8, 14. However, how aging affects 55 agriculture and rural livelihoods has not been well researched or understood to date. 56 This sector is facing substantial challenges in squaring the circle between maintaining 57 food security and improving environmental protection through sustainable 58 intensification at many levels. However, it is indispensable to identify integrated, 59 comprehensive measures to ensure global food security, while protecting the 60 environment and achieving rural revitalization to achieve common prosperity. 61 62 Older age groups of farmers typically have a lower education levels, are less likely to 63 keep their agricultural skills up-to-date and embrace novel, cutting-edge methods 14-16. 64 Due to a consistent labor supply shortage, they can only operate small-scale farms, 65 which leads to an increase in cropland abandonment as rural populations age 17, 18. 66 Meanwhile, the typical agricultural input mix and crop types (labor-intensive crops or 67 not) are likely to shift with rural aging, reducing agricultural productivity and efficiency 68 5-7, 19. These effects are usually worse in smallholder-farming-dominated countries such 69 as China 8. An aging society is defined as one in which more than 7% of the population 70 is over 65 years old 20, and this ratio has doubled in rural China (Fig. 1a) in the period 71 from 2000 to 2019. In the coming decades, China's population aging rate is predicted 72 to accelerate 20, 21. Thus, it is urgent and timely to address the threats of aging on 73 agricultural production in China and also other countries and regions globally, which 74 are facing similar issues. 75 76 Here, we explored the relationship between rural population aging (the proportion of 77 individuals over 65 years old at household-level) and agricultural sustainability based 78 on a panel model, using survey data from more than 30,000 rural households in China. 79 Nine indicators overall were selected to illustrate agricultural sustainability, including 80 farm size, farmer’s education, labor productivity, agricultural total input and output, 81 profit-cost ratio, machine input, fertilizer input and fertilizer use efficiency (FUE). Aging threatens sustainability of smallholder farming in China 1 2 To 82 understand the full range of effects of aging on agriculture, other variables such as 83 transferred and abandoned cropland, pesticide and manure inputs, and technology 84 adoption were also incorporated into the analysis. In addition, the analysis quantified 85 the potential of new farming models, such as industrial, family and cooperative farming 86 for contributing to addressing the threats posed by aging on a predominantly traditional 87 smallholder farming sector. Finally, Shared Socioeconomic Pathways (SSP) scenarios 88 of future development were used to explore options for achieving sustainable 89 intensification of agriculture under conditions of aging rural populations. 90 Aging threatens sustainability of smallholder farming in China 1 2 The urban rate of aging 96 increased from 6% to 11% during the period between 2000 and 2019, rural aging from 97 7% to 15% over the same period. The rapid aging in rural area has resulted in a 98 substantial decline in the labor force available in the agricultural sector (Fig. 1b). Young 99 rural laborers are attracted to non agricultural jobs in urban areas resulting in an 100 ineffective in many countries where aging issues have been considered serious 12, 13. 51 Alternative strategies urgently need to be developed to achieve and maintain sustainable 52 development paths for human society. Agriculture as a typical labor-intensive industry 53 could be one of the sectors substantially affected by population aging, especially in 54 countries where smallholder farming is prevalent 8, 14. However, how aging affects 55 agriculture and rural livelihoods has not been well researched or understood to date. 56 This sector is facing substantial challenges in squaring the circle between maintaining 57 food security and improving environmental protection through sustainable 58 intensification at many levels. However, it is indispensable to identify integrated, 59 comprehensive measures to ensure global food security, while protecting the 60 environment and achieving rural revitalization to achieve common prosperity. 61 62 Older age groups of farmers typically have a lower education levels, are less likely to 63 keep their agricultural skills up-to-date and embrace novel, cutting-edge methods 14-16. 64 Due to a consistent labor supply shortage, they can only operate small-scale farms, 65 which leads to an increase in cropland abandonment as rural populations age 17, 18. 66 Meanwhile, the typical agricultural input mix and crop types (labor-intensive crops or 67 not) are likely to shift with rural aging, reducing agricultural productivity and efficiency 68 5-7, 19. These effects are usually worse in smallholder-farming-dominated countries such 69 as China 8. An aging society is defined as one in which more than 7% of the population 70 is over 65 years old 20, and this ratio has doubled in rural China (Fig. 1a) in the period 71 from 2000 to 2019. In the coming decades, China's population aging rate is predicted 72 to accelerate 20, 21. Thus, it is urgent and timely to address the threats of aging on 73 agricultural production in China and also other countries and regions globally, which 74 are facing similar issues. Results and discussion Population aging trends have been observed since the 1990s, in both urban and rural 94 areas in China (Fig. 1). However, rural aging is more severe and happening at a more 95 rapidly accelerating rate than that that of urban areas (Fig. 1a). The urban rate of aging 96 increased from 6% to 11% during the period between 2000 and 2019, rural aging from 97 7% to 15% over the same period. The rapid aging in rural area has resulted in a 98 substantial decline in the labor force available in the agricultural sector (Fig. 1b). Young 99 rural laborers are attracted to non-agricultural jobs in urban areas, resulting in an 100 2 imbalance of about 10% more workers in the age group from 15 to 64 being available 101 in urban compared to rural areas. Agriculture has been the sector primarily affected by 102 migration to urban centers as well as rural aging, with young workers becoming an 103 increasingly scarce commodity in rural areas 6. 104 105 Using a fixed effect panel model, we found that for every one unit increase in the 106 proportion of rural aging ratio, there is on average a 1.5-year reduction in education 107 level of farmers (Table 1). In China, the effect of lower education levels is amplified by 108 the rapid socioeconomic development, with more and younger people typically 109 obtaining substantially higher education levels. Thus, regions with above-average 110 numbers of aging households are affected by overall lower education levels compared 111 to other regions. Meanwhile, we observe a correlation between aging and reduction in 112 farm size with a coefficient of -0.27, demonstrating that aging farmers tend to only 113 operate small-scale farms, and croplands are more likely to be abandoned or 114 transferred-out to others (Table 1). Smallholder farming is currently most prevalent in 115 regions of Central and South China, where rural aging is seen to be substantial (Fig. 1c, 116 d). 117 118 Aging is correlated with a decrease in agricultural inputs, mainly including chemicals 119 such as fertilizers and pesticides, and machinery and technology adoption (Table 1 and 120 S1). This typically reduces mechanization and intensification of agriculture. Moreover, 121 aging substantially lowers FUE by -0.13% for every one unit increase in aging. A lower 122 FUE indicates a higher rate of fertilizer-related losses to the environment and, 123 consequently, higher pollution. Impacts on agricultural sustainability 151 Total agricultural inputs 179 are reduced by about 3% (1-9%). The decrease in agricultural inputs reduces 180 agricultural output per area and labor productivity by 4% (2-10%) and 9% (4-22%), 181 respectively. Even though the correlation between aging and profit-cost ratio is not 182 significant, the reduction in farm size and education levels due to aging ultimately 183 reduces profit-cost ratio by about 2% (1-4%). The reduction in fertilizer use did not 184 contribute to an improvement in FUE, with aging reducing FUE by 3% (1-7%) and 185 leading to a 3% (1-12%) increase in fertilizer-related losses to the environment (Fig. 186 S3). 187 188 Aging in China’s rural areas is likely to accelerate in the coming decades, especially in 189 the context of urbanization due to the migration of young, working-age populations 190 from rural to urban areas 20, 21. This will further impact on the viability of agricultural 191 production in areas subjected to accelerated aging 27. The negative effects on 192 agricultural outputs could threaten the food security of 1.4 billion people in China, and 193 the loss in labor productivity would result in a decline in rural per capita income, 194 compromising the attainment of the SDGs focusing on ‘no poverty’ and ‘zero hunger’. 195 The decline in FUE and the increase in fertilizer-related losses to the environment have 196 the potential to further exacerbate environmental pollution and present a threat to both 197 environmental and human health. China is already under pressure to ensure food 198 security while also protecting the environment, so it is critical to address the negative 199 effects of aging on agriculture. 200 p g y We quantified the impact of aging on agricultural sustainability in 2019 at provincial 152 level in China to account for the large spatial variability of aging and other related 153 factors 24, 25. Average education levels of farmers are lower by around 3% in rural areas 154 (0-11% across different provinces) as a result of rural different rates of aging in 155 predominantly urban vs. rural areas (Fig. 3 and S2). The Chongqing-Sichuan area, 156 where aging has been accelerating since the 1990s, is the one of the most affected 157 regions, with an education level decline of 9% (0.6 years) (Fig. 3 and S2). Impacts on agricultural sustainability 151 Impacts on agricultural sustainability 151 We quantified the impact of aging on agricultural sustainability in 2019 at provincial 152 level in China to account for the large spatial variability of aging and other related 153 factors 24, 25. Average education levels of farmers are lower by around 3% in rural areas 154 (0-11% across different provinces) as a result of rural different rates of aging in 155 predominantly urban vs. rural areas (Fig. 3 and S2). The Chongqing-Sichuan area, 156 where aging has been accelerating since the 1990s, is the one of the most affected 157 regions, with an education level decline of 9% (0.6 years) (Fig. 3 and S2). Provinces 158 such as Zhejiang, Anhui, and Fujian are also affected, with a level of education 6% 159 lower (0.4 years) on average. Lower levels in farmers' education is associated with less 160 knowledge and adoption of technologies and scientific methods in agricultural practice, 161 inhibiting agricultural performance and modernization. Due to a lack of knowledge and 162 skills, aging farmers typically operate small-scale farms with outdated farming methods. 163 As a result, aging populations are associated with average farm size reduction by 4% 164 (2-11%) in 2019 nationally. Aging farmers tend to reduce farm size by cropland 165 abandonment or transferring increasing amounts of farmland out to others. 166 Approximately an additional 1 million hectares of cropland area would be expected to 167 be abandoned in 2019 as a result of aging (Fig. S3, Table S3). The abandonment ratio 168 is higher in hilly areas and comparatively lower in plain areas as a result of aging 18, 169 however, the abandonment increasing rate is markedly higher in plain areas such as 170 Shandong and Jiangsu provinces (Fig. S3). This poses a severe threat to the preservation 171 of croplands as a key contributor to safeguarding food security. 172 173 The decline in farm size further leads to a reduction in the use of agricultural fixed 174 inputs such as machinery 26. We found that machinery inputs decreased by about 5% on 175 average with aging, and the largest decrease was estimated at 25% found in Chongqing. 176 In addition, aging also leads to a reduction in the use of non-fixed inputs such as 177 chemical fertilizers, pesticide, and manure fertilizer (Table 1 and S1). Chemical 178 fertilizer use is reduced by an average of 2% (0-8%) (Fig. 3). Results and discussion In addition, aging reduces the amount of manure input 124 (Table S1), inhibiting the recycling of manure and aggravating environmental pollution. 125 Consequently, agricultural output per area and labor productivity decrease substantially 126 with aging, despite the fact that the profit-cost ratio does not vary significantly (Fig. 127 S1). This decline in labor productivity reduces farmers’ per capita income, further 128 discouraging young people from engaging in agriculture, and in turn further exacerbates 129 aging effects in the sector. 130 131 Aging affects agriculture both directly and indirectly as shown by a structural equation 132 model (Fig. 2). The standardized path coefficients for the direct effects of aging on labor 133 productivity and yield are both -0.05. This means that each standard deviation increase 134 in aging would lead to 0.05 standard deviations of decrease in labor productivity and 135 yield. Similarly, the direct effects of aging on fertilizer use and the FUE are -0.08 and - 136 0.02, respectively. These direct effects are only a small share of the total effect of aging 137 on agriculture. 138 139 More effects are factored in indirectly through farm size and education. For example, 140 the direct effect of aging on labor productivity has a standardized path coefficient of - 141 0.05, but the indirect effect through farm size and education is -0.08 and -0.02, 142 respectively. It results in a net effect of -0.15 of aging on labor productivity, of which 143 the direct effect accounts for one-third, while the indirect effect through farm size and 144 education accounts for two-thirds (Table S2). Similarly, 29%, 33%, and 67% of the net 145 effect of yield, fertilizer, and the FUE stem from indirect effects, respectively. This 146 implies that multiple initiatives are needed to mitigate the consequences of rural aging, 147 such as attracting young people to consider work in the agriculture sector, better 148 imbalance of about 10% more workers in the age group from 15 to 64 being available 101 in urban compared to rural areas. Agriculture has been the sector primarily affected by 102 migration to urban centers as well as rural aging, with young workers becoming an 103 increasingly scarce commodity in rural areas 6. 104 3 Impacts on agricultural sustainability 151 231 232 The reason why new farming models have better agricultural performance than 233 traditional smallholder farming is mainly because it has younger farmers with higher 234 education level. These younger farmers also have the opportunities to realize higher 235 incomes by working in non-agricultural sectors in cities, which means they face 236 substantial opportunity costs if they decide to engage in farming. To offset the 237 opportunity cost, these farmers have to increase their income by increasing farm size 238 and improving farm management. In contrast, older farmers in traditional farming do 239 not have such opportunities and are thus not prone to respond to incentives. Thus, we 240 can see a shift in agricultural input mix as well as an increase in output and labor 241 productivity under new farming models. Furthermore, as these new farming farmers are 242 younger and well educated, they are more likely to have scientific knowledge and 243 embrace new technologies 15, resulting in increased mechanization and FUE, 244 contributing to agricultural modernization and long-term sustainability. 245 246 Mitigation pathways 247 To quantitatively assess future trends of aging and its impacts we accounted for 248 New farming models matter 202 Chinese agriculture is currently dominated by smallholder farms 28. Traditional 203 smallholder farming emerged in the 1980s as a consequence of the introduction of the 204 Household Contract Responsibility System (HCRS) in China 23. The HCRS allocated 205 cropland equally to each rural household based on family size and the quality of 206 croplands. This resulted in small farm sizes as a status-quo across China, with a high 207 degree of cropland fragmentation 23. New agricultural farming models have been 208 encouraged by Chinese government and increasingly emerging since 2010s, mainly 209 including family, cooperative and industrial farming, to improve overall agricultural 210 performance 29, 30. Family farming is still by rural households, but with larger farm size, 211 compared to traditional smallholders’ farming. Cooperative farming is characterized by 212 shared agricultural equipment, such as machinery, across several families with much 213 larger farm size. Industrial farms are large-scale agricultural enterprises targeting 214 marketing and sales with large scales and professional production to maximize profits. 215 Also, young farmers with higher education levels are drawn to these new farming 216 models. Here, we considered these three models together as new farming models. Impacts on agricultural sustainability 151 Provinces 158 such as Zhejiang, Anhui, and Fujian are also affected, with a level of education 6% 159 lower (0.4 years) on average. Lower levels in farmers' education is associated with less 160 knowledge and adoption of technologies and scientific methods in agricultural practice, 161 inhibiting agricultural performance and modernization. Due to a lack of knowledge and 162 skills, aging farmers typically operate small-scale farms with outdated farming methods. 163 As a result, aging populations are associated with average farm size reduction by 4% 164 (2-11%) in 2019 nationally. Aging farmers tend to reduce farm size by cropland 165 abandonment or transferring increasing amounts of farmland out to others. 166 Approximately an additional 1 million hectares of cropland area would be expected to 167 be abandoned in 2019 as a result of aging (Fig. S3, Table S3). The abandonment ratio 168 is higher in hilly areas and comparatively lower in plain areas as a result of aging 18, 169 however, the abandonment increasing rate is markedly higher in plain areas such as 170 Shandong and Jiangsu provinces (Fig. S3). This poses a severe threat to the preservation 171 of croplands as a key contributor to safeguarding food security. 172 The decline in farm size further leads to a reduction in the use of agricultural fixed 174 inputs such as machinery 26. We found that machinery inputs decreased by about 5% on 175 average with aging, and the largest decrease was estimated at 25% found in Chongqing. 176 In addition, aging also leads to a reduction in the use of non-fixed inputs such as 177 chemical fertilizers, pesticide, and manure fertilizer (Table 1 and S1). Chemical 178 fertilizer use is reduced by an average of 2% (0-8%) (Fig. 3). Total agricultural inputs 179 are reduced by about 3% (1-9%). The decrease in agricultural inputs reduces 180 agricultural output per area and labor productivity by 4% (2-10%) and 9% (4-22%), 181 respectively. Even though the correlation between aging and profit-cost ratio is not 182 significant, the reduction in farm size and education levels due to aging ultimately 183 reduces profit-cost ratio by about 2% (1-4%). The reduction in fertilizer use did not 184 contribute to an improvement in FUE, with aging reducing FUE by 3% (1-7%) and 185 leading to a 3% (1-12%) increase in fertilizer-related losses to the environment (Fig. 186 S3). Impacts on agricultural sustainability 151 187 4 201 New farming models matter 202 Chinese agriculture is currently dominated by smallholder farms 28. Traditional 203 smallholder farming emerged in the 1980s as a consequence of the introduction of the 204 Household Contract Responsibility System (HCRS) in China 23. The HCRS allocated 205 cropland equally to each rural household based on family size and the quality of 206 croplands. This resulted in small farm sizes as a status-quo across China, with a high 207 degree of cropland fragmentation 23. New agricultural farming models have been 208 encouraged by Chinese government and increasingly emerging since 2010s, mainly 209 including family, cooperative and industrial farming, to improve overall agricultural 210 performance 29, 30. Family farming is still by rural households, but with larger farm size, 211 compared to traditional smallholders’ farming. Cooperative farming is characterized by 212 shared agricultural equipment, such as machinery, across several families with much 213 larger farm size. Industrial farms are large-scale agricultural enterprises targeting 214 marketing and sales with large scales and professional production to maximize profits. 215 Also, young farmers with higher education levels are drawn to these new farming 216 models. Here, we considered these three models together as new farming models. 217 218 New farming models was observed to have better performance than that of traditional 219 smallholder farming in several aspects in 2019 (Fig. 4). Compared to traditional 220 farming, aging ratio in new farming was 33% lower, with 64% higher in education level 221 and 20% larger in farm size. In other words, new farming models attract younger 222 farmers to participate in agricultural practices who have a higher education level and 223 capability to operate large-scale farming. The total input and machinery input were 41% 224 and 68% higher in the new farming, respectively, therefore, the output per area and 225 labor productivity increased by 24% and 29% in the new farming, respectively, 226 although the profit-cost ratio was slightly changed. Furthermore, despite fertilizer input 227 was slightly higher in new farming, there is no statistically significant difference 228 compared to the traditional smallholder farming (Fig. 4, Table S4). On the contrary, 229 new farming had a 4% higher FUE than smallholder farmers in 2019, suggesting less 230 environmental pollution in new farming. Impacts on agricultural sustainability 151 217 New farming models was observed to have better performance than that of traditional 219 smallholder farming in several aspects in 2019 (Fig. 4). Compared to traditional 220 farming, aging ratio in new farming was 33% lower, with 64% higher in education level 221 and 20% larger in farm size. In other words, new farming models attract younger 222 farmers to participate in agricultural practices who have a higher education level and 223 capability to operate large-scale farming. The total input and machinery input were 41% 224 and 68% higher in the new farming, respectively, therefore, the output per area and 225 labor productivity increased by 24% and 29% in the new farming, respectively, 226 although the profit-cost ratio was slightly changed. Furthermore, despite fertilizer input 227 was slightly higher in new farming, there is no statistically significant difference 228 compared to the traditional smallholder farming (Fig. 4, Table S4). On the contrary, 229 new farming had a 4% higher FUE than smallholder farmers in 2019, suggesting less 230 environmental pollution in new farming. 231 The reason why new farming models have better agricultural performance than 233 traditional smallholder farming is mainly because it has younger farmers with higher 234 education level. These younger farmers also have the opportunities to realize higher 235 incomes by working in non-agricultural sectors in cities, which means they face 236 substantial opportunity costs if they decide to engage in farming. To offset the 237 opportunity cost, these farmers have to increase their income by increasing farm size 238 and improving farm management. In contrast, older farmers in traditional farming do 239 not have such opportunities and are thus not prone to respond to incentives. Thus, we 240 can see a shift in agricultural input mix as well as an increase in output and labor 241 productivity under new farming models. Furthermore, as these new farming farmers are 242 younger and well educated, they are more likely to have scientific knowledge and 243 embrace new technologies 15, resulting in increased mechanization and FUE, 244 contributing to agricultural modernization and long-term sustainability. 245 g p y To quantitatively assess future trends of aging and its impacts, we accounted for 248 demographic change in Shared Socioeconomic Pathways (SSPs) scenarios 31. SSP1 249 (Taking the green road), SSP2 (Middle of Road), SSP3 (A rocky road) and SSP4 (A 250 5 road divided) are included in this analysis 32. Impacts on agricultural sustainability 151 SSP5 (Taking the highway) is excluded 251 due to having a similar population structure to SSP1. Among these scenarios, SSP1 252 includes the world's most pronounced population aging, with more than 60% of the 253 world's population over the age of 65 by 2100 (Fig. S4). Total population first increases 254 in the coming decades and then starts to decrease by the 2060s. Even with high 255 urbanization rates and economic growth under the green development path, high levels 256 of aging would exert substantial negative impacts on socioeconomic development and 257 agricultural production. SSP4 comprises the smallest rate of aging, with only 24% of 258 over 65s in 2100. However, in this scenario, the global population rises to 12.7 billion 259 by 2100, with an urbanization rate of under 60% and a severe slowdown in economic 260 growth. 261 262 Despite the different assumptions for future development paths, the aging trend 263 continues to rise in all scenarios (Fig. S4). We found that for China, the abandonment 264 ratio of croplands would increase from 5% in 2020 to 15-47% by 2100 nationally due 265 to aging, while average farm size would decrease by 5-16% (0.04-0.11 ha), and 266 education levels would fall by 8-28% (0.6-2 years) (Fig. 5, S5 and S6). Meanwhile, 267 agricultural performance would decline, with machinery and fertilizer inputs decreasing 268 by 3-5% and 1-5% by 2100, respectively. The overall input per area would reduce by 269 1-2% as well, leading to a decrease in output and profit-cost ratio by 3-5% and 3-13%, 270 respectively. Labor productivity is projected to decrease by about 7-14%, from an $7.2 271 per labor hour in 2020 to $5.2-6.6 in 2100. In addition, FUE would decrease by 5-16%, 272 and fertilizer loss increase by 3-5%. Without interventions, food security challenges 273 and environmental degradation would pose substantial threats to ecosystem and human 274 health and well-being. 275 276 Fortunately, the decline in agricultural performance caused by aging can be reversed by 277 promoting an increasing uptake of new farming models. With new farming models, 278 average farm size would increase by 11-22% from 2020 to 2100, accompanied by an 279 improvement in the average education level. This would lead to a 14-15% increase in 280 total inputs, a 13-46% increase in mechanization and a 4-5% decrease in fertilizer use. Impacts on agricultural sustainability 151 281 As a result, output and labor productivity would increase by 12-14% and 57-72%, 282 respectively, FUE improve by 12-25% and fertilizer loss be reduced by 4-7%. New 283 farming models could mitigate impacts of aging on agriculture, resulting in a substantial 284 improvement in agricultural sustainability for China. In addition, new farming models 285 would accelerate the transformation of the agricultural sector from traditional 286 smallholder farming to large-scale sustainable agriculture. 287 288 Social co-benefits 289 The implementation of new farming may present a cost-effective solution for China, 290 especially considering the dual challenges of maintaining food security and protecting 291 the environment 24. This strategy would also lead to additional social co-benefits, not 292 limited to agriculture. 293 294 The proportion of the population engaged in agricultural production has continued to 295 fall over time, while the proportion of people employed in non-agricultural sectors has 296 continued to rise (Fig. S7). In China, young people tend to move to cities, leaving older 297 people in rural areas maintaining an involvement in agriculture. But most rural young 298 people do not settle in city, continue to support rural families and eventually return to 299 the countryside. As a result, agriculture is no longer the primary source of income for 300 road divided) are included in this analysis 32. SSP5 (Taking the highway) is excluded 251 due to having a similar population structure to SSP1. Among these scenarios, SSP1 252 includes the world's most pronounced population aging, with more than 60% of the 253 world's population over the age of 65 by 2100 (Fig. S4). Total population first increases 254 in the coming decades and then starts to decrease by the 2060s. Even with high 255 urbanization rates and economic growth under the green development path, high levels 256 of aging would exert substantial negative impacts on socioeconomic development and 257 agricultural production. SSP4 comprises the smallest rate of aging, with only 24% of 258 over 65s in 2100. However, in this scenario, the global population rises to 12.7 billion 259 by 2100, with an urbanization rate of under 60% and a severe slowdown in economic 260 growth. 261 Despite the different assumptions for future development paths, the aging trend 263 continues to rise in all scenarios (Fig. S4). Impacts on agricultural sustainability 151 We found that for China, the abandonment 264 ratio of croplands would increase from 5% in 2020 to 15-47% by 2100 nationally due 265 to aging, while average farm size would decrease by 5-16% (0.04-0.11 ha), and 266 education levels would fall by 8-28% (0.6-2 years) (Fig. 5, S5 and S6). Meanwhile, 267 agricultural performance would decline, with machinery and fertilizer inputs decreasing 268 by 3-5% and 1-5% by 2100, respectively. The overall input per area would reduce by 269 1-2% as well, leading to a decrease in output and profit-cost ratio by 3-5% and 3-13%, 270 respectively. Labor productivity is projected to decrease by about 7-14%, from an $7.2 271 per labor hour in 2020 to $5.2-6.6 in 2100. In addition, FUE would decrease by 5-16%, 272 and fertilizer loss increase by 3-5%. Without interventions, food security challenges 273 and environmental degradation would pose substantial threats to ecosystem and human 274 health and well-being. 275 Fortunately, the decline in agricultural performance caused by aging can be reversed by 277 promoting an increasing uptake of new farming models. With new farming models, 278 average farm size would increase by 11-22% from 2020 to 2100, accompanied by an 279 improvement in the average education level. This would lead to a 14-15% increase in 280 total inputs, a 13-46% increase in mechanization and a 4-5% decrease in fertilizer use. 281 As a result, output and labor productivity would increase by 12-14% and 57-72%, 282 respectively, FUE improve by 12-25% and fertilizer loss be reduced by 4-7%. New 283 farming models could mitigate impacts of aging on agriculture, resulting in a substantial 284 improvement in agricultural sustainability for China. In addition, new farming models 285 would accelerate the transformation of the agricultural sector from traditional 286 smallholder farming to large-scale sustainable agriculture. 287 6 7 rural households. In 2019, income derived from agricultural activities accounted for 301 just 23% of rural household income (Fig. S7). This essentially implies that farmers do 302 not rely on agriculture any more and agricultural vitality is diminishing. 303 304 The increasing number of young people leaving to seek employment in cities may have 305 additional socio-economic impacts, e.g. contributing to a rising mortality rate of rural 306 elderlies and children in recent years 33, 34. Impacts on agricultural sustainability 151 The mortality rate of the elderly over 65 307 years old in rural areas due to accidents, mental illness and suicide is much higher than 308 that in urban areas (Fig. S8). That is mainly because elderly who are left behind in rural 309 areas face additional risks of living alone 33. Furthermore, the mortality rate of children 310 less than 14 years old in rural areas are often greater than for urban locations (Fig. S9). 311 Aside from variations in medical care provision, the greater prevalence of accidental 312 mortality among rural compared to urban children can also be attributed to the neglect 313 of children left behind due to their young parents moving to cities 35, 36. 314 315 However, new farming models can reinvigorate agriculture and improve rural income 316 levels, promoting rural revitalization and common prosperity. Meanwhile, it contributes 317 to the return of young people to their rural villages and hometowns, resolving the issue 318 of elderly and children being left behind on their own and sustaining rural communities. 319 These additional benefits are intertwined with agricultural sustainability, contributing 320 to attaining multiple SDGs. 321 322 Policy changes and feasibility 323 Adverse impacts of population aging would – without policy interventions - be 324 unavoidable in the future, and in particular the impacts on agriculture and rural 325 communities require urgent attention 37. Mitigating these adverse impacts is a great 326 challenge, but also presents a tremendous opportunity, which requires multiple 327 stakeholders’ efforts. Increasing fertility policy is an important measure with long-term 328 benefits to address aging issues despite it may bring challenges on feeding more people. 329 It can directly stimulate childbirth, reduce demographic aging and mitigate its adverse 330 impacts. Under the family planning strategy in China, a couple was restricted to have 331 only one child on average before 2016 13. In recent years, the government has 332 authorized the second and third children to each couple to increase birthrate. Meanwhile, 333 social security measures including maternity subsidies, insurance and maternity leave 334 have been improved gradually. However, changes of fertility policy may not 335 substantially reverse the population aging according to the experiences in other 336 developed countries/regions such as Japan and European Union. Other policies and 337 measures are still required to complement the interventions on aging. Impacts on agricultural sustainability 151 338 339 Policy changes on Hukou and land tenure systems would make the implementation of 340 new farming models feasible, benefiting the controls on adverse impacts of population 341 aging. The smallholder aging issues are mainly due to the unique Chinese household 342 registration system that divides the Chinese population into two categories: rural and 343 urban, allocating lands and other public resources based on the Hukou status of 344 residents 28. The growing urbanization in recent years has resulted in young rural people 345 migrating to cities to engage in non-agricultural sectors, leaving the elderly in rural 346 areas. Rural elders are unable to move to cities because they are unable to access urban 347 social services such as pension and medical care under the restriction of Hukou system. 348 The large rural aging population inhibits the extension of new farming models. Thus, 349 new policies are needed to move rural aging population to urban areas and exchange 350 rural households. In 2019, income derived from agricultural activities accounted for 301 just 23% of rural household income (Fig. S7). This essentially implies that farmers do 302 not rely on agriculture any more and agricultural vitality is diminishing. 303 rural households. In 2019, income derived from agricultural activities accounted for 301 just 23% of rural household income (Fig. S7). This essentially implies that farmers do 302 not rely on agriculture any more and agricultural vitality is diminishing. 303 y g y Adverse impacts of population aging would – without policy interventions - be 324 unavoidable in the future, and in particular the impacts on agriculture and rural 325 communities require urgent attention 37. Mitigating these adverse impacts is a great 326 challenge, but also presents a tremendous opportunity, which requires multiple 327 stakeholders’ efforts. Increasing fertility policy is an important measure with long-term 328 benefits to address aging issues despite it may bring challenges on feeding more people. 329 It can directly stimulate childbirth, reduce demographic aging and mitigate its adverse 330 impacts. Under the family planning strategy in China, a couple was restricted to have 331 only one child on average before 2016 13. In recent years, the government has 332 authorized the second and third children to each couple to increase birthrate. Meanwhile, 333 social security measures including maternity subsidies, insurance and maternity leave 334 have been improved gradually. Impacts on agricultural sustainability 151 However, changes of fertility policy may not 335 substantially reverse the population aging according to the experiences in other 336 developed countries/regions such as Japan and European Union. Other policies and 337 measures are still required to complement the interventions on aging. 338 7 their rural resources (mainly croplands and homesteads) for urban social services, 351 especially the elderly whose children have moved to urban areas. It has been reported 352 that the Jiangsu governments have compensated farmers who give up croplands and 353 migrate to cities with a cash transfer equal to five times their annual income as social 354 security 29. This effectively encourages rural-to-urban migration of aging people. 355 Meanwhile, croplands of these migrating aging farmers can be integrated for large-scale 356 farming and their homesteads can also be reclaimed for farming 38. It is estimated that 357 the reclaimed lands due to rural-to-urban migration under urbanization could increase 358 the cropland area by 4.1% by 2050 nationally, approximately 5.8 million hectares 39. 359 And average farm size can increase from less 0.6 ha to over 16 ha through land 360 consolidation in some regions 40, benefiting the implementation of new farming models. 361 Reforming Hukou and land tenure systems to achieve such changes is an effective 362 pathway for the new farming models 30. 363 their rural resources (mainly croplands and homesteads) for urban social services, 351 especially the elderly whose children have moved to urban areas. It has been reported 352 that the Jiangsu governments have compensated farmers who give up croplands and 353 migrate to cities with a cash transfer equal to five times their annual income as social 354 security 29. This effectively encourages rural-to-urban migration of aging people. 355 Meanwhile, croplands of these migrating aging farmers can be integrated for large-scale 356 farming and their homesteads can also be reclaimed for farming 38. It is estimated that 357 the reclaimed lands due to rural-to-urban migration under urbanization could increase 358 the cropland area by 4.1% by 2050 nationally, approximately 5.8 million hectares 39. 359 And average farm size can increase from less 0.6 ha to over 16 ha through land 360 consolidation in some regions 40, benefiting the implementation of new farming models. 361 Reforming Hukou and land tenure systems to achieve such changes is an effective 362 pathway for the new farming models 30. Impacts on agricultural sustainability 151 363 364 Given the experiences in Jiangsu Providence, it should be feasible for the reform of 365 rural communities through reallocation of lands and other public resources to increase 366 their use efficiencies. Over 40,000 hectares of homesteads have been reclaimed during 367 past decade, suggests that it is not only feasible but also happening in practice 39. These 368 released croplands could be integrated for large-scale new farming, which normally 369 have much higher cost-efficiencies than that in smallholders (Fig. 4). Similarly, urban 370 areas have higher efficiency on the supply of non-agricultural services such as health 371 care and education, and the rural-to-urban migration would benefit the increase of labor 372 productivity through training and better health care. Therefore, it should be cost- 373 effective to compensate rural-to-urban migrated farmers 29. This strategy would bring 374 in multiple social benefits that far outweigh its cost. Increasing cropland area through 375 both homesteads reclamation and decreasing the abandonment benefit food security 376 that is the top priority of national strategy. Improving rural income under new farming 377 models encourages return of young people, promoting rural revitalization and 378 sustainable rural communities. The cost of land consolidation can also be recovered in 379 the following decades, despite a large number of one-time inputs 40. When the 380 environmental and human health benefits are considered, the reforming cost should be 381 much lower than the benefits. Chinese central government has already issued a policy 382 of giving 'Priority to Development of Agriculture, Rural Areas and Farmers' in early 383 2019 to accelerate the construction of large-scale high-standard croplands. These 384 ongoing changes suggest that reforming Chinese agriculture to address the threats of 385 rural population aging on the sustainability of smallholder farming is not only important 386 but also feasible. 387 388 Methods 389 Given the experiences in Jiangsu Providence, it should be feasible for the reform of 365 rural communities through reallocation of lands and other public resources to increase 366 their use efficiencies. Over 40,000 hectares of homesteads have been reclaimed during 367 past decade, suggests that it is not only feasible but also happening in practice 39. These 368 released croplands could be integrated for large-scale new farming, which normally 369 have much higher cost-efficiencies than that in smallholders (Fig. 4). Impacts on agricultural sustainability 151 Similarly, urban 370 areas have higher efficiency on the supply of non-agricultural services such as health 371 care and education, and the rural-to-urban migration would benefit the increase of labor 372 productivity through training and better health care. Therefore, it should be cost- 373 effective to compensate rural-to-urban migrated farmers 29. This strategy would bring 374 in multiple social benefits that far outweigh its cost. Increasing cropland area through 375 both homesteads reclamation and decreasing the abandonment benefit food security 376 that is the top priority of national strategy. Improving rural income under new farming 377 models encourages return of young people, promoting rural revitalization and 378 sustainable rural communities. The cost of land consolidation can also be recovered in 379 the following decades, despite a large number of one-time inputs 40. When the 380 environmental and human health benefits are considered, the reforming cost should be 381 much lower than the benefits. Chinese central government has already issued a policy 382 of giving 'Priority to Development of Agriculture, Rural Areas and Farmers' in early 383 2019 to accelerate the construction of large-scale high-standard croplands. These 384 ongoing changes suggest that reforming Chinese agriculture to address the threats of 385 rural population aging on the sustainability of smallholder farming is not only important 386 but also feasible. 387 The province and country-level population aging and rural aging data was derived ovince and country-level population aging and rural aging data was derived 8 from the China Population & Employment Statistics Yearbook (available at 401 https://data.cnki.net/yearbook/Single/N2021020056). County-level population aging 402 data in 2020 shown in Fig.1c is taken from the Sixth National Census in 2010 403 (available at http://www.stats.gov.cn/tjsj/pcsj/rkpc/6rp/indexch.htm) and weighted by 404 province-level aging data for the year 2020 from the Seventh National Census 405 (available at http://www.stats.gov.cn/tjsj/tjgb/rkpcgb/qgrkpcgb/). County-level farm 406 size data in Fig.1d is from the statistics of the Second National Pollution Census (non- 407 public database), which is averaged by investigated household farm size. 408 409 from the China Population & Employment Statistics Yearbook (available at 401 https://data.cnki.net/yearbook/Single/N2021020056). County-level population aging 402 data in 2020 shown in Fig.1c is taken from the Sixth National Census in 2010 403 (available at http://www.stats.gov.cn/tjsj/pcsj/rkpc/6rp/indexch.htm) and weighted by 404 province-level aging data for the year 2020 from the Seventh National Census 405 (available at http://www.stats.gov.cn/tjsj/tjgb/rkpcgb/qgrkpcgb/). Impacts on agricultural sustainability 151 County-level farm 406 size data in Fig.1d is from the statistics of the Second National Pollution Census (non- 407 public database), which is averaged by investigated household farm size. 408 409 409 We obtained future population data across all age groups based on the Shared 410 Socioeconomic Pathways (SSP) database hosted by the IIASA Energy Program 411 (https://tntcat.iiasa.ac.at/SspDb). In this paper, scenario SSP5 was not considered as 412 its population change is in line with that of scenario SSP1. 413 409 We obtained future population data across all age groups based on the Shared 410 Socioeconomic Pathways (SSP) database hosted by the IIASA Energy Program 411 (https://tntcat.iiasa.ac.at/SspDb). In this paper, scenario SSP5 was not considered as 412 its population change is in line with that of scenario SSP1. 413 We obtained future population data across all age groups based on the Shared 410 Socioeconomic Pathways (SSP) database hosted by the IIASA Energy Program 411 (https://tntcat.iiasa.ac.at/SspDb). In this paper, scenario SSP5 was not considered as 412 its population change is in line with that of scenario SSP1. 413 Statistical analysis. CRHPS data allowed estimating the relationships between 415 population aging and agricultural performance. Aging ratio, the proportion of people 416 65 years and older in rural households, was adopted as an indicator to demonstrate 417 population aging at household-level. Adult farmer’s ratio, defined as the proportion of 418 people over 15 but less 64 years old in rural households, was also considered to 419 demonstrate age groups. Nine different indicators were determined to illustrate 420 agricultural sustainability, including farm size (ha), education (yr), labor productivity 421 ($ hr-1), total agricultural input ($ ha-1), output ($ ha-1), profit-cost ratio, machine input 422 ($ ha-1), fertilizer input ($ ha-1) and fertilizer use efficiency (FUE). Other variables 423 concerning transferred-out cropland, abandoned cropland, pesticide, pesticide use 424 efficiency, manure and technology adoption were also incorporated to demonstrate 425 agricultural performance changes under population aging. The detailed interpretation 426 of these indicators is documented in Supplementary Information. 427 428 To explore the relationships between population aging and agricultural sustainability, 429 we first used a fixed effect panel (FEP) regression model to conduct the longitudinal 430 analysis, while controlling for confounding factors such as crop type, plot numbers, 431 regional and year effect. Impacts on agricultural sustainability 151 We estimated the following equation using data on 432 households from 2015, 2017 and 2019: 433 𝐴𝑔𝑟𝑖𝑐𝑢𝑙𝑡𝑢𝑟𝑒𝑖𝑡= 𝛼+ 𝛽∙𝐴𝑔𝑖𝑛𝑔𝑖𝑡+ 𝛾∙𝐸𝑑𝑢𝑐𝑎𝑡𝑖𝑜𝑛𝑖𝑡+ 𝛿∙𝐹𝑎𝑟𝑚𝑠𝑖𝑧𝑒𝑖𝑡+ 434 ∑ 𝜃𝑚∙𝑝𝑚𝑖𝑡 𝑚 + 𝜀𝑖𝑡+ 𝜎𝑘 (1) 435 𝐴𝑔𝑟𝑖𝑐𝑢𝑙𝑡𝑢𝑟𝑒𝑖𝑡= 𝛼+ 𝛽∙𝐴𝑔𝑖𝑛𝑔𝑖𝑡+ 𝛾∙𝐸𝑑𝑢𝑐𝑎𝑡𝑖𝑜𝑛𝑖𝑡+ 𝛿∙𝐹𝑎𝑟𝑚𝑠𝑖𝑧𝑒𝑖𝑡+ 434 ∑ 𝜃𝑚∙𝑝𝑚𝑖𝑡 𝑚 + 𝜀𝑖𝑡+ 𝜎𝑘 (1) 435 𝑖𝑡+ (1) ∑ 𝜃𝑚∙𝑝𝑚𝑖𝑡 𝑚 + 𝜀𝑖𝑡+ 𝜎𝑘 where subscript i, k and t denote household, county and time, respectively. 436 𝐴𝑔𝑟𝑖𝑐𝑢𝑙𝑡𝑢𝑟𝑒𝑖𝑡 refers to the agricultural sustainability indicators and other inputs and 437 outputs on household level. 𝐴𝑔𝑖𝑛𝑔 refers to the proportion of people ≥ 65 years in 438 rural households. 𝐸𝑑𝑢𝑐𝑎𝑡𝑖𝑜𝑛 refers to average years of education of family 439 members aged 15 and over. 𝐹𝑎𝑟𝑚𝑠𝑖𝑧𝑒 is the logarithm of total cultivated area 440 covering all crops. 𝑝𝑚 is control variable including adult farmer’s ratio, income ratio 441 from non-agricultural sectors, crop type, plot numbers, county-level regional effect 442 and year effect. 𝛼 is a constant, 𝜀𝑖𝑡 and 𝜎𝑘 are error items. 𝛽, 𝛾, 𝛿 𝑎𝑛𝑑 𝜃 are 443 coefficients that need to be estimated. The detailed results are listed in Table 1 and S1. 444 445 Given too many zeros in variables of variables of transfer-out cropland, abandoned 446 cropland, machine input and owned, and manure fertilizer input, we also used a Tobit 447 regression model to validate the relationship between these variables and aging. We 448 estimated the following equation using data on households in 2015, 2017 and 2019: 449 9 𝑦𝑖𝑡 ∗= 𝛼+ 𝛽∙𝐴𝑔𝑖𝑛𝑔𝑖𝑡+ 𝛾∙𝐸𝑑𝑢𝑐𝑎𝑡𝑖𝑜𝑛𝑖𝑡+ 𝛿∙𝐹𝑎𝑟𝑚𝑠𝑖𝑧𝑒𝑖𝑡+ ∑𝜑𝑛𝑞𝑛,𝑖𝑡 𝑛 + 𝜀𝑖𝑡 450 (2) 451 where subscript i and t denote household and time, respectively. 𝑦𝑗𝑡 ∗ refers to latent 452 variables including transfer-out cropland, abandoned cropland, machine input and 453 owned, and manure fertilizer input in each household. Latent variables can be 454 observed when over 0, truncated at 0 when the value is equal to 0 or less 0. 𝐴𝑔𝑖𝑛𝑔 455 refers to the proportion of people over 65 years old in rural households. 𝐸𝑑𝑢𝑐𝑎𝑡𝑖𝑜𝑛 456 refers to average years of education of family members aged 15 and over. 𝐹𝑎𝑟𝑚𝑠𝑖𝑧𝑒 457 is the logarithm of total cultivated area covering all crops. 𝑞𝑛 is control variable 458 including adult farmer’s ratio, income ratio from non-agricultural sectors, crop type, 459 plot numbers, city-level regional effect and year effect. 𝛼 is a constant and 𝜀𝑖𝑡 is the 460 error item. 𝛽, 𝛾, 𝛿 𝑎𝑛𝑑 𝜑 are coefficients that need to be estimated. Impacts on agricultural sustainability 151 Farm size was 461 removed in explaining variables in the equation with explained variable of transfer- 462 out and abandoned cropland considering multi-collinearity. The results are listed in 463 Table 1 and S1. 464 The latent variable is expressed in terms of the observed variable 𝑦𝑖𝑡 ∗ in the following 465 form: 466 {𝑦𝑖𝑡= 𝑦𝑖𝑡 ∗ 𝑖𝑓 𝑦𝑖𝑡 ∗> 0 𝑦𝑖𝑡= 0 𝑖𝑓 𝑦𝑖𝑡 ∗≤0 (3) 467 468 Besides, a Logit regression model was adopted as technology adoption (Tech) is a 469 binary variable. This model is formulated as follows based on household-level data 470 from 2015, 2017 and 2019: 471 𝐿𝑜𝑔𝑖𝑡{𝑃𝑖𝑡(Tech𝑖𝑡= 1)} = 𝛼+ 𝛽1 ∙𝐴𝑔𝑒𝑖𝑡+ 𝛽2 ∙𝐴𝑔𝑒𝑖𝑡 2 + 𝛾∙𝐸𝑑𝑢𝑐𝑎𝑡𝑖𝑜𝑛𝑖𝑡+ 𝛿∙ 472 𝐹𝑎𝑟𝑚𝑠𝑖𝑧𝑒𝑖𝑡+ ∑𝜑𝑛𝑞𝑛,𝑖𝑡 𝑛 + 𝜀𝑖𝑡 (4) 473 where subscript i and t denotes household and time, respectively. 𝑃𝑖𝑡 is the probability 474 of the occurrence of Tech (Tech𝑖𝑡=1: event occurs; Tech𝑖𝑡=0: event does not occur). 475 𝐴𝑔𝑒 refers to the log-transformed age of the head of the investigated household. 476 𝐸𝑑𝑢𝑐𝑎𝑡𝑖𝑜𝑛 refers to education years of the head of the investigated household. 477 𝐹𝑎𝑟𝑚𝑠𝑖𝑧𝑒 is the logarithm of total cultivated area covering all crops. 𝑞𝑛 is control 478 variable including income ratio from non-agricultural sectors, crop type, plot 479 numbers, city-level regional effect and year effect. 𝛼 is a constant and 𝜀𝑖𝑡 is the 480 error item. 𝛽, 𝛾, 𝛿 𝑎𝑛𝑑 𝜑 are coefficients that need to be estimated. The results are 481 listed in Table S1. 482 483 To test the difference of agricultural performance of traditional and new farming 484 model under aging, a binary variable indicating the occurrence of new farming (NF𝑖𝑡) 485 (NF𝑖𝑡=1: event occurs; NF=0: event does not occur) was introduced based on Eq. (1) 486 and (2). The equations are expressed in the following forms: 487 𝐴𝑔𝑟𝑖𝑐𝑢𝑙𝑡𝑢𝑟𝑒𝑖𝑡= 𝛼+ 𝜔∙NF𝑖𝑡+ 𝛽∙𝐴𝑔𝑖𝑛𝑔𝑖𝑡+ 𝛾∙𝐸𝑑𝑢𝑐𝑎𝑡𝑖𝑜𝑛𝑖𝑡+ 𝛿∙𝐹𝑎𝑟𝑚𝑠𝑖𝑧𝑒𝑖𝑡+ 488 ∑ 𝜃𝑚∙𝑝𝑚𝑖𝑡 𝑚 + 𝜀𝑖𝑡 + 𝜎𝑘 (5) 489 𝑦𝑖𝑡 ∗= 𝛼+ 𝜔∙NF𝑖𝑡+ 𝛽∙𝐴𝑔𝑖𝑛𝑔𝑖𝑡+ 𝛾∙𝐸𝑑𝑢𝑐𝑎𝑡𝑖𝑜𝑛𝑖𝑡+ 𝛿∙𝐹𝑎𝑟𝑚𝑠𝑖𝑧𝑒𝑖𝑡+ 490 ∑𝜑𝑛𝑞𝑛,𝑖𝑡 𝑛 + 𝜀𝑖𝑡 (6) 491 𝜔, 𝛽, 𝛾, 𝛿, 𝜃 𝑎𝑛𝑑 𝜑 are coefficients that need to be estimated. Related results are listed 492 in Table S4. Note that as new farming is not widely prompted in China, new farming 493 data is limited. Thus, we consider that when the variable NF with p<0.05 in Table S4, 494 there is a significant difference. Impacts on agricultural sustainability 151 495 496 𝑦𝑖𝑡 ∗= 𝛼+ 𝛽∙𝐴𝑔𝑖𝑛𝑔𝑖𝑡+ 𝛾∙𝐸𝑑𝑢𝑐𝑎𝑡𝑖𝑜𝑛𝑖𝑡+ 𝛿∙𝐹𝑎𝑟𝑚𝑠𝑖𝑧𝑒𝑖𝑡+ ∑𝜑𝑛𝑞𝑛,𝑖𝑡 𝑛 + 𝜀𝑖𝑡 450 (2) 451 𝑦𝑖𝑡 ∗= 𝛼+ 𝛽∙𝐴𝑔𝑖𝑛𝑔𝑖𝑡+ 𝛾∙𝐸𝑑𝑢𝑐𝑎𝑡𝑖𝑜𝑛𝑖𝑡+ 𝛿∙𝐹𝑎𝑟𝑚𝑠𝑖𝑧𝑒𝑖𝑡+ ∑𝜑𝑛𝑞𝑛,𝑖𝑡 𝑛 + 𝜀𝑖𝑡 (2) (2) (2) 451 where subscript i and t denote household and time, respectively. 𝑦𝑗𝑡 ∗ refers to latent 452 variables including transfer-out cropland, abandoned cropland, machine input and 453 owned, and manure fertilizer input in each household. Latent variables can be 454 observed when over 0, truncated at 0 when the value is equal to 0 or less 0. 𝐴𝑔𝑖𝑛𝑔 455 refers to the proportion of people over 65 years old in rural households. 𝐸𝑑𝑢𝑐𝑎𝑡𝑖𝑜𝑛 456 refers to average years of education of family members aged 15 and over. 𝐹𝑎𝑟𝑚𝑠𝑖𝑧𝑒 457 is the logarithm of total cultivated area covering all crops. 𝑞𝑛 is control variable 458 including adult farmer’s ratio, income ratio from non-agricultural sectors, crop type, 459 plot numbers, city-level regional effect and year effect. 𝛼 is a constant and 𝜀𝑖𝑡 is the 460 error item. 𝛽, 𝛾, 𝛿 𝑎𝑛𝑑 𝜑 are coefficients that need to be estimated. Farm size was 461 removed in explaining variables in the equation with explained variable of transfer- 462 out and abandoned cropland considering multi-collinearity. The results are listed in 463 Table 1 and S1. 464 The latent variable is expressed in terms of the observed variable 𝑦𝑖𝑡 ∗ in the following 465 form: 466 {𝑦𝑖𝑡= 𝑦𝑖𝑡 ∗ 𝑖𝑓 𝑦𝑖𝑡 ∗> 0 𝑦𝑖𝑡= 0 𝑖𝑓 𝑦𝑖𝑡 ∗≤0 (3) {𝑦𝑖𝑡= 𝑦𝑖𝑡 ∗ 𝑖𝑓 𝑦𝑖𝑡 ∗> 0 𝑦𝑖𝑡= 0 𝑖𝑓 𝑦𝑖𝑡 ∗≤0 (3) (3) Besides, a Logit regression model was adopted as technology adoption (Tech) is a 469 binary variable. This model is formulated as follows based on household-level data 470 from 2015, 2017 and 2019: 471 𝐿𝑜𝑔𝑖𝑡{𝑃𝑖𝑡(Tech𝑖𝑡= 1)} = 𝛼+ 𝛽1 ∙𝐴𝑔𝑒𝑖𝑡+ 𝛽2 ∙𝐴𝑔𝑒𝑖𝑡 2 + 𝛾∙𝐸𝑑𝑢𝑐𝑎𝑡𝑖𝑜𝑛𝑖𝑡+ 𝛿∙ 𝐹𝑎𝑟𝑚𝑠𝑖𝑧𝑒𝑖𝑡+ ∑𝜑𝑛𝑞𝑛,𝑖𝑡 𝑛 + 𝜀𝑖𝑡 (4) 𝑖𝑡 𝐹𝑎𝑟𝑚𝑠𝑖𝑧𝑒𝑖𝑡+ ∑𝜑𝑛𝑞𝑛,𝑖𝑡 𝑛 + 𝜀𝑖𝑡 where subscript i and t denotes household and time, respectively. 𝑃𝑖𝑡 is the probability p , p y 𝑖𝑡 p y of the occurrence of Tech (Tech𝑖𝑡=1: event occurs; Tech𝑖𝑡=0: event does not occur). q p g 𝑢𝑟𝑒𝑖𝑡= 𝛼+ 𝜔∙NF𝑖𝑡+ 𝛽∙𝐴𝑔𝑖𝑛𝑔𝑖𝑡+ 𝛾∙𝐸𝑑𝑢𝑐𝑎𝑡𝑖𝑜𝑛𝑖𝑡+ 𝛿∙𝐹𝑎𝑟𝑚𝑠𝑖𝑧𝑒𝑖𝑡+ ∑ 𝜃𝑚∙𝑝𝑚𝑖𝑡 𝑚 + 𝜀𝑖𝑡 + 𝜎𝑘 (5) 𝑦𝑖𝑡 ∗= 𝛼+ 𝜔∙NF𝑖𝑡+ 𝛽∙𝐴𝑔𝑖𝑛𝑔𝑖𝑡+ 𝛾∙𝐸𝑑𝑢𝑐𝑎𝑡𝑖𝑜𝑛𝑖𝑡+ 𝛿∙𝐹𝑎𝑟𝑚𝑠𝑖𝑧𝑒𝑖𝑡+ ∑𝜑𝑛𝑞𝑛,𝑖𝑡 𝑛 + 𝜀𝑖𝑡 (6) 𝜔, 𝛽, 𝛾, 𝛿, 𝜃 𝑎𝑛𝑑 𝜑 are coefficients that need to be estimated. Related results are listed 492 in Table S4. Impacts on agricultural sustainability 151 Note that as new farming is not widely prompted in China, new farming 493 data is limited. Thus, we consider that when the variable NF with p<0.05 in Table S4, 494 there is a significant difference. 495 10 Meanwhile, we replaced aging ratio with the age of the head of the investigated 497 household and average education years with the head’s education for a robustness 498 check. The results of this robustness check are detailed in Table S1 and S5. 499 500 We then used a structural equation model (SEM) to understand the direct and 501 indirect effects of aging on agriculture 41. Considering the multi-collinearity of 502 agricultural indicators, such as output with labor productivity, fertilizer input with 503 total input, only some of typical indicators were selected. Also, we excluded 504 variables with nonlinear relationships with aging (e.g. machine input) to deduce 505 comparable standardized path coefficients. Income ratio from non-agricultural 506 sectors and the ratio of cash crops area to total cultivated area were controlled when 507 doing SEM analysis. In this model, the goodness-of-fit statistics are reasonable 508 based on CRHPS data in 2019: non-normed fit index (NNFI) > 0.90, comparative 509 fit index (CFI) > 0.90, root mean square error (RMSE) < 0.05, and standardized 510 root mean square residual (SRMR) < 0.05. Using the results from the SEM, the 511 direct and indirect effects of aging with standardized path coefficients are shown in 512 Fig.2. 513 514 Summary statistics for main variables used in statistical regression analysis are 515 listed in Table S7. We did all statistical analysis in Stata12.0 software. 516 517 Impact of aging on agricultural sustainability. We assume that the population age 518 distribution remains at the 1990 level to represent no aging. Province-level population 519 data for 1990 from the China Population & Employment Statistics Yearbook was 520 introduced after weighting by the difference between this data and CRHPS to reduce 521 the error caused by variations in data stemming from different databases. Then 522 province-level population data was downscaled to household-level proportionally. 523 Finally, aging and adult farmer’s ratio without aging are deduced. We assume that other 524 variables remain the same and calculated the predicted value of agriculture without 525 aging on each household based on coefficients estimated in Eq. (1). Impacts on agricultural sustainability 151 For household i, 526 the predicted without aging is calculated as follows: 527 𝑌𝑖𝑗𝑡 𝑃𝑟𝑒𝑑𝑖𝑐𝑡𝑒𝑑 𝑊𝑖𝑡ℎ𝑜𝑢𝑡 𝐴𝑔𝑖𝑛𝑔= exp(𝐿𝑛 𝑌𝑖𝑗𝑡 𝑂𝑏𝑠𝑒𝑟𝑣𝑒𝑑 + 𝐿𝑛 𝑌𝑖𝑗𝑡 𝑊𝑖𝑡ℎ𝑜𝑢𝑡 𝐴𝑔𝑖𝑛𝑔−𝐿𝑛 𝑌𝑖𝑗𝑡 𝑊𝑖𝑡ℎ 𝐴𝑔𝑖𝑛𝑔) 528 (7) 529 where the subscript i, j and t denote household, explained variable and year, respectively. 530 𝑌𝑗 includes farm size, output, input, labor productivity, fertilization and 531 FUE. 𝑌𝑖𝑗𝑡 𝑂𝑏𝑠𝑒𝑟𝑣𝑒𝑑 refers to the observed value of each household. 𝑌𝑖𝑗𝑡 𝑊𝑖𝑡ℎ𝑜𝑢𝑡 𝐴𝑔𝑖𝑛𝑔 is the 532 deduced value based on estimated coefficients in Eq. (1) using the aging and adult 533 farmer’s ratio data in 1990. 𝑌𝑖𝑗𝑡 𝑊𝑖𝑡ℎ 𝐴𝑔𝑖𝑛𝑔 is the deduced value using the observed aging 534 and adult farmer’s ratio (age over 15 but less than 64) data from CRHPS. For explained 535 variables without log-transformed such as education and profit-cost ratio, the predicted 536 is calculated as follows: 537 𝑌𝑖𝑗𝑡 𝑃𝑟𝑒𝑑𝑖𝑐𝑡𝑒𝑑 𝑊𝑖𝑡ℎ𝑜𝑢𝑡 𝐴𝑔𝑖𝑛𝑔= 𝑌𝑖𝑗𝑡 𝑂𝑏𝑠𝑒𝑟𝑣𝑒𝑑+ 𝑌𝑖𝑗𝑡 𝑊𝑖𝑡ℎ𝑜𝑢𝑡 𝐴𝑔𝑖𝑛𝑔−𝑌𝑖𝑗𝑡 𝑊𝑖𝑡ℎ 𝐴𝑔𝑖𝑛𝑔 538 (8) 539 𝑌𝑗 includes education and profit-cost ratio. 540 For some variables with nonlinear relationship with aging, the following equation was 541 adopted to calculate the impact of aging based on Eq. (2): 542 Meanwhile, we replaced aging ratio with the age of the head of the investigated 497 household and average education years with the head’s education for a robustness 498 check. The results of this robustness check are detailed in Table S1 and S5. 499 11 g y check. The results of this robustness check are detailed in Table S1 and S5. 499 500 We then used a structural equation model (SEM) to understand the direct and 501 indirect effects of aging on agriculture 41. Considering the multi-collinearity of 502 agricultural indicators, such as output with labor productivity, fertilizer input with 503 total input, only some of typical indicators were selected. Also, we excluded 504 variables with nonlinear relationships with aging (e.g. machine input) to deduce 505 comparable standardized path coefficients. Income ratio from non-agricultural 506 sectors and the ratio of cash crops area to total cultivated area were controlled when 507 doing SEM analysis. In this model, the goodness-of-fit statistics are reasonable 508 based on CRHPS data in 2019: non-normed fit index (NNFI) > 0.90, comparative 509 fit index (CFI) > 0.90, root mean square error (RMSE) < 0.05, and standardized 510 root mean square residual (SRMR) < 0.05. Impacts on agricultural sustainability 151 Using the results from the SEM, the 511 direct and indirect effects of aging with standardized path coefficients are shown in 512 Fig.2. 513 514 Summary statistics for main variables used in statistical regression analysis are 515 listed in Table S7. We did all statistical analysis in Stata12.0 software. 516 517 Impact of aging on agricultural sustainability. We assume that the population age 518 distribution remains at the 1990 level to represent no aging. Province-level population 519 data for 1990 from the China Population & Employment Statistics Yearbook was 520 introduced after weighting by the difference between this data and CRHPS to reduce 521 the error caused by variations in data stemming from different databases. Then 522 province-level population data was downscaled to household-level proportionally. 523 Finally, aging and adult farmer’s ratio without aging are deduced. We assume that other 524 variables remain the same and calculated the predicted value of agriculture without 525 aging on each household based on coefficients estimated in Eq. (1). For household i, 526 the predicted without aging is calculated as follows: 527 𝑌𝑖𝑗𝑡 𝑃𝑟𝑒𝑑𝑖𝑐𝑡𝑒𝑑 𝑊𝑖𝑡ℎ𝑜𝑢𝑡 𝐴𝑔𝑖𝑛𝑔= exp(𝐿𝑛 𝑌𝑖𝑗𝑡 𝑂𝑏𝑠𝑒𝑟𝑣𝑒𝑑 + 𝐿𝑛 𝑌𝑖𝑗𝑡 𝑊𝑖𝑡ℎ𝑜𝑢𝑡 𝐴𝑔𝑖𝑛𝑔−𝐿𝑛 𝑌𝑖𝑗𝑡 𝑊𝑖𝑡ℎ 𝐴𝑔𝑖𝑛𝑔) 528 (7) 529 where the subscript i, j and t denote household, explained variable and year, respectively. 530 𝑌𝑗 includes farm size, output, input, labor productivity, fertilization and 531 FUE. 𝑌𝑖𝑗𝑡 𝑂𝑏𝑠𝑒𝑟𝑣𝑒𝑑 refers to the observed value of each household. 𝑌𝑖𝑗𝑡 𝑊𝑖𝑡ℎ𝑜𝑢𝑡 𝐴𝑔𝑖𝑛𝑔 is the 532 deduced value based on estimated coefficients in Eq. (1) using the aging and adult 533 farmer’s ratio data in 1990. 𝑌𝑖𝑗𝑡 𝑊𝑖𝑡ℎ 𝐴𝑔𝑖𝑛𝑔 is the deduced value using the observed aging 534 and adult farmer’s ratio (age over 15 but less than 64) data from CRHPS. For explained 535 variables without log-transformed such as education and profit-cost ratio, the predicted 536 is calculated as follows: 537 𝑌𝑖𝑗𝑡 𝑃𝑟𝑒𝑑𝑖𝑐𝑡𝑒𝑑 𝑊𝑖𝑡ℎ𝑜𝑢𝑡 𝐴𝑔𝑖𝑛𝑔= 𝑌𝑖𝑗𝑡 𝑂𝑏𝑠𝑒𝑟𝑣𝑒𝑑+ 𝑌𝑖𝑗𝑡 𝑊𝑖𝑡ℎ𝑜𝑢𝑡 𝐴𝑔𝑖𝑛𝑔−𝑌𝑖𝑗𝑡 𝑊𝑖𝑡ℎ 𝐴𝑔𝑖𝑛𝑔 538 (8) 539 𝑌𝑗 includes education and profit-cost ratio. 540 For some variables with nonlinear relationship with aging, the following equation was 541 adopted to calculate the impact of aging based on Eq. (2): 542 𝐼𝑚𝑝𝑎𝑐𝑡𝑖𝑗𝑡= 𝛽∙∆𝐴𝑔𝑖𝑛𝑔𝑖𝑗𝑡+ 𝛾∙∆𝐸𝑑𝑢𝑐𝑎𝑡𝑖𝑜𝑛𝑖𝑗𝑡+ 𝛿∙∆𝐹𝑎𝑟𝑚𝑠𝑖𝑧𝑒𝑖𝑗𝑡+ 𝜑1 ∙ 543 ∆𝐴𝑑𝑢𝑙𝑡𝑖𝑗𝑡 (9) 544 We then used a structural equation model (SEM) to understand the direct and 501 indirect effects of aging on agriculture 41. Impacts on agricultural sustainability 151 Considering the multi-collinearity of 502 agricultural indicators, such as output with labor productivity, fertilizer input with 503 total input, only some of typical indicators were selected. Also, we excluded 504 variables with nonlinear relationships with aging (e.g. machine input) to deduce 505 comparable standardized path coefficients. Income ratio from non-agricultural 506 sectors and the ratio of cash crops area to total cultivated area were controlled when 507 doing SEM analysis. In this model, the goodness-of-fit statistics are reasonable 508 based on CRHPS data in 2019: non-normed fit index (NNFI) > 0.90, comparative 509 fit index (CFI) > 0.90, root mean square error (RMSE) < 0.05, and standardized 510 root mean square residual (SRMR) < 0.05. Using the results from the SEM, the 511 direct and indirect effects of aging with standardized path coefficients are shown in 512 Fig.2. 513 Summary statistics for main variables used in statistical regression analysis are listed in Table S7. We did all statistical analysis in Stata12.0 software. g g 𝑜𝑢𝑡 𝐴𝑔𝑖𝑛𝑔= exp(𝐿𝑛 𝑌𝑖𝑗𝑡 𝑂𝑏𝑠𝑒𝑟𝑣𝑒𝑑 + 𝐿𝑛 𝑌𝑖𝑗𝑡 𝑊𝑖𝑡ℎ𝑜𝑢𝑡 𝐴𝑔𝑖𝑛𝑔−𝐿𝑛 𝑌𝑖𝑗𝑡 𝑊𝑖𝑡ℎ 𝐴𝑔𝑖𝑛𝑔) (7) (8) (9) 11 where the subscript i, j and t denote household, explained variable and year, respectively. 545 ∆ refers to the value difference with and without aging. 𝑌𝑗 includes abandoned 546 cropland and machine input. 𝐴𝑑𝑢𝑙𝑡𝑖𝑗𝑡 refers to adult farmer’s ratio in rural household. 547 Then the predicted value on each household without aging was calculated: 548 𝑌𝑖𝑗𝑡 𝑃𝑟𝑒𝑑𝑖𝑐𝑡𝑒𝑑 𝑊𝑖𝑡ℎ𝑜𝑢𝑡 𝐴𝑔𝑖𝑛𝑔= 𝑌𝑖𝑗𝑡 𝑂𝑏𝑠𝑒𝑟𝑣𝑒𝑑−𝐼𝑚𝑝𝑎𝑐𝑡𝑖𝑗𝑡 (10) 549 Note that we first derived the predicted education without aging. Then abandoned 550 cropland area was derived based on predicted education as it is one of the explanatory 551 variables. Farm size was derived based on predicted education and considering 552 abandoned cropland changes. The remaining variables were finally calculated based on 553 predicted education and farm size. 554 555 When the household-level predicted value in 2019 was calculated, the provincial 556 predicted means including input, output, machine and fertilization were averaged after 557 weighted by the total cultivated area on province-level. The rest were arithmetic 558 averages. 559 560 Scenario analysis. We based our analysis of future socio-economic development on 561 the SSP scenarios to determine the impact of population aging on agricultural 562 sustainability. We obtained population age data (2015-2100) from the SSP database. Impacts on agricultural sustainability 151 547 Then the predicted value on each household without aging was calculated: 548 𝑌𝑖𝑗𝑡 𝑃𝑟𝑒𝑑𝑖𝑐𝑡𝑒𝑑 𝑊𝑖𝑡ℎ𝑜𝑢𝑡 𝐴𝑔𝑖𝑛𝑔= 𝑌𝑖𝑗𝑡 𝑂𝑏𝑠𝑒𝑟𝑣𝑒𝑑−𝐼𝑚𝑝𝑎𝑐𝑡𝑖𝑗𝑡 (10) 549 g g 𝑌𝑖𝑗𝑡 𝑃𝑟𝑒𝑑𝑖𝑐𝑡𝑒𝑑 𝑊𝑖𝑡ℎ𝑜𝑢𝑡 𝐴𝑔𝑖𝑛𝑔= 𝑌𝑖𝑗𝑡 𝑂𝑏𝑠𝑒𝑟𝑣𝑒𝑑−𝐼𝑚𝑝𝑎𝑐𝑡𝑖𝑗𝑡 (10) (10) Note that we first derived the predicted education without aging. Then abandoned 550 cropland area was derived based on predicted education as it is one of the explanatory 551 variables. Farm size was derived based on predicted education and considering 552 abandoned cropland changes. The remaining variables were finally calculated based on 553 predicted education and farm size. 554 When the household-level predicted value in 2019 was calculated, the provincial 556 predicted means including input, output, machine and fertilization were averaged after 557 weighted by the total cultivated area on province-level. The rest were arithmetic 558 averages. 559 Scenario analysis. We based our analysis of future socio-economic development on 561 the SSP scenarios to determine the impact of population aging on agricultural 562 sustainability. We obtained population age data (2015-2100) from the SSP database. We 563 undertook a comparison between the population age data for 2015 in the SSP and 564 CRHPS databases, and weighted aging and adult farmer’s ratio by their difference to 565 reduce the error. Then country-level population data was downscaled to household level 566 proportionally. Then the aging and adult farmer’s ratio data of the SSP scenarios for the 567 period between 2020 and 2100 were calculated for each household. Finally, the 568 predicted values (𝑌𝑖𝑗𝑡 𝑃𝑟𝑒𝑑𝑖𝑐𝑡𝑒𝑑 𝑆𝑆𝑃 ) of nine agricultural sustainability indicators and 569 abandoned cropland under future aging were calculated based on Eqns. (7), (8), (9) and 570 (10) under the hypothesis that other variables remain stable at 2019 levels. The 571 equations are expressed in the following forms: 572 q p g 𝑌𝑖𝑗𝑡 𝑃𝑟𝑒𝑑𝑖𝑐𝑡𝑒𝑑 𝑆𝑆𝑃= exp(𝐿𝑛 𝑌𝑖𝑗,2019 𝑂𝑏𝑠𝑒𝑟𝑣𝑒𝑑 + 𝐿𝑛 𝑌𝑖𝑗𝑡 𝑆𝑆𝑃−𝐿𝑛 𝑌𝑖𝑗,2019 𝑊𝑖𝑡ℎ 𝐴𝑔𝑖𝑛𝑔) (11) 573 𝑌𝑖𝑗𝑡 𝑃𝑟𝑒𝑑𝑖𝑐𝑡𝑒𝑑 𝑆𝑆𝑃= 𝑌𝑖𝑗,2019 𝑂𝑏𝑠𝑒𝑟𝑣𝑒𝑑+ 𝑌𝑖𝑗𝑡 𝑆𝑆𝑃−𝑌𝑖𝑗,2019 𝑊𝑖𝑡ℎ 𝐴𝑔𝑖𝑛𝑔 (12) 574 𝐼𝑚𝑝𝑎𝑐𝑡𝑖𝑗𝑡= 𝛽∙∆𝐴𝑔𝑖𝑛𝑔𝑖𝑗𝑡+ 𝛾∙∆𝐸𝑑𝑢𝑐𝑎𝑡𝑖𝑜𝑛𝑖𝑗𝑡+ 𝛿∙∆𝐹𝑎𝑟𝑚𝑠𝑖𝑧𝑒𝑖𝑗𝑡+ 𝜑1 ∙ 575 ∆𝐴𝑑𝑢𝑙𝑡𝑖𝑗𝑡 (13) 576 𝑌𝑖𝑗𝑡 𝑃𝑟𝑒𝑑𝑖𝑐𝑡𝑒𝑑 𝑆𝑆𝑃= 𝑌𝑖𝑗𝑡 𝑂𝑏𝑠𝑒𝑟𝑣𝑒𝑑−𝐼𝑚𝑝𝑎𝑐𝑡𝑖𝑗𝑡 (14) 577 ∆ in Eq. (13) refers to the value difference between year 2019 and year t. 578 (11) (11) (12) (12) (14) To improve agricultural performance and estimate the effectiveness of mitigating 580 negative impacts of future population aging, we assume that new farming models will 581 be promoted as a key element of future agricultural sector development. Impacts on agricultural sustainability 151 We 563 undertook a comparison between the population age data for 2015 in the SSP and 564 CRHPS databases, and weighted aging and adult farmer’s ratio by their difference to 565 reduce the error. Then country-level population data was downscaled to household level 566 proportionally. Then the aging and adult farmer’s ratio data of the SSP scenarios for the 567 period between 2020 and 2100 were calculated for each household. Finally, the 568 predicted values (𝑌𝑖𝑗𝑡 𝑃𝑟𝑒𝑑𝑖𝑐𝑡𝑒𝑑 𝑆𝑆𝑃 ) of nine agricultural sustainability indicators and 569 abandoned cropland under future aging were calculated based on Eqns. (7), (8), (9) and 570 (10) under the hypothesis that other variables remain stable at 2019 levels. The 571 equations are expressed in the following forms: 572 𝑌𝑖𝑗𝑡 𝑃𝑟𝑒𝑑𝑖𝑐𝑡𝑒𝑑 𝑆𝑆𝑃= exp(𝐿𝑛 𝑌𝑖𝑗,2019 𝑂𝑏𝑠𝑒𝑟𝑣𝑒𝑑 + 𝐿𝑛 𝑌𝑖𝑗𝑡 𝑆𝑆𝑃−𝐿𝑛 𝑌𝑖𝑗,2019 𝑊𝑖𝑡ℎ 𝐴𝑔𝑖𝑛𝑔) (11) 573 𝑌𝑖𝑗𝑡 𝑃𝑟𝑒𝑑𝑖𝑐𝑡𝑒𝑑 𝑆𝑆𝑃= 𝑌𝑖𝑗,2019 𝑂𝑏𝑠𝑒𝑟𝑣𝑒𝑑+ 𝑌𝑖𝑗𝑡 𝑆𝑆𝑃−𝑌𝑖𝑗,2019 𝑊𝑖𝑡ℎ 𝐴𝑔𝑖𝑛𝑔 (12) 574 𝐼𝑚𝑝𝑎𝑐𝑡𝑖𝑗𝑡= 𝛽∙∆𝐴𝑔𝑖𝑛𝑔𝑖𝑗𝑡+ 𝛾∙∆𝐸𝑑𝑢𝑐𝑎𝑡𝑖𝑜𝑛𝑖𝑗𝑡+ 𝛿∙∆𝐹𝑎𝑟𝑚𝑠𝑖𝑧𝑒𝑖𝑗𝑡+ 𝜑1 ∙ 575 ∆𝐴𝑑𝑢𝑙𝑡𝑖𝑗𝑡 (13) 576 𝑌𝑖𝑗𝑡 𝑃𝑟𝑒𝑑𝑖𝑐𝑡𝑒𝑑 𝑆𝑆𝑃= 𝑌𝑖𝑗𝑡 𝑂𝑏𝑠𝑒𝑟𝑣𝑒𝑑−𝐼𝑚𝑝𝑎𝑐𝑡𝑖𝑗𝑡 (14) 577 ∆ in Eq. (13) refers to the value difference between year 2019 and year t. 578 579 To improve agricultural performance and estimate the effectiveness of mitigating 580 negative impacts of future population aging, we assume that new farming models will 581 be promoted as a key element of future agricultural sector development. Based on the 582 relative sector share of the new farming models, which is 2% in 2019, we hypothesize 583 that its coverage will increase by about 1.2% annually, reaching 100% by 2100. For 584 household i, the predicted value of nine agricultural sustainability indicators and 585 abandoned cropland under new farming is calculated as follows: 586 𝑌𝑖𝑗𝑡 𝑃𝑟𝑒𝑑𝑖𝑐𝑡𝑒𝑑 𝑁𝐹= exp(𝐿𝑛 𝑌𝑖𝑗𝑡 𝑃𝑟𝑒𝑑𝑖𝑐𝑡𝑒𝑑 𝑆𝑆𝑃+ 𝜌𝑗∙𝐶𝑜𝑣𝑒𝑟𝑎𝑔𝑒𝑡) 587 (15) 588 where the subscript i, j and t denote household, explained variable and year, respectively. 589 𝑌 i l d f i t t i t l b d ti it f tili ti d FUE 590 where the subscript i, j and t denote household, explained variable and year, respectively. 545 ∆ refers to the value difference with and without aging. 𝑌𝑗 includes abandoned 546 cropland and machine input. 𝐴𝑑𝑢𝑙𝑡𝑖𝑗𝑡 refers to adult farmer’s ratio in rural household. Impacts on agricultural sustainability 151 Based on the 582 relative sector share of the new farming models, which is 2% in 2019, we hypothesize 583 that its coverage will increase by about 1.2% annually, reaching 100% by 2100. For 584 household i, the predicted value of nine agricultural sustainability indicators and 585 abandoned cropland under new farming is calculated as follows: 586 (15) ( ) where the subscript i, j and t denote household, explained variable and year, respectively. 589 𝑌𝑗 includes farm size, output, input, labor productivity, fertilization and FUE. 590 𝑌𝑖𝑗𝑡 𝑃𝑟𝑒𝑑𝑖𝑐𝑡𝑒𝑑 𝑁𝐹 is the predicted value new farming (NF) models. 𝑌𝑖𝑗𝑡 𝑃𝑟𝑒𝑑𝑖𝑐𝑡𝑒𝑑 𝑆𝑆𝑃 is the 591 12 predicted value under SSP scenarios. 𝜌𝑗 refers to estimated coefficients of new 592 farming responding to explained variable j. The results of these estimated coefficients 593 are detailed in Table S4. 𝐶𝑜𝑣𝑒𝑟𝑎𝑔𝑒𝑡 means the assuming coverage of new farming in 594 year t. 595 predicted value under SSP scenarios. 𝜌𝑗 refers to estimated coefficients of new 592 farming responding to explained variable j. The results of these estimated coefficients 593 are detailed in Table S4. 𝐶𝑜𝑣𝑒𝑟𝑎𝑔𝑒𝑡 means the assuming coverage of new farming in 594 year t. 595 596 (16) Country-level mean values of each year including input, output, machine and 608 fertilization were averaged after weighting by the total cultivated area across the 609 whole country. The rest were arithmetic averages. 610 References: 612 1. Harper, S. Economic and social implications of aging societies. Science 346, 587- 595 (2014). 1. Harper, S. Economic and social implications of aging societies. Science 346, 587- 595 (2014). 2. Chen, C., Goldman, D.P., Zissimopoulos, J. & Rowe, J.W. Multidimensional comparison of countries’ adaptation to societal aging. Proc. Natl. Acad. Sci. U. S. A. 115, 9169-9174 (2018). 2. Chen, C., Goldman, D.P., Zissimopoulos, J. & Rowe, J.W. Multidimensional comparison of countries’ adaptation to societal aging. Proc. Natl. Acad. Sci. U. S. A. 115, 9169-9174 (2018). 3. Bloom, D.E., et al. Macroeconomic implications of population ageing and selected policy responses. Lancet 385, 649-657 (2015). 3. Bloom, D.E., et al. Macroeconomic implications of population ageing and selected policy responses. Lancet 385, 649-657 (2015). 4. Yu, B., Wei, Y., Kei, G. & Matsuoka, Y. Future scenarios for energy consumption and carbon emissions due to demographic transitions in Chinese households. Nat. Energy 3, 109-118 (2018). 5. Li, M. & Sicular, T. Aging of the labor force and technical efficiency in crop production: Evidence from Liaoning province, China. China Agric. Econ. Rev. 5, 342-359 (2013). 6. Ji, Y., Hu, X., Zhu, J. & Zhong, F. Demographic change and its impact on farmers' field production decisions. China Econ. Rev. 43, 64-71 (2017). 7. Apipoonyanon, C., et al. Socio-economic and environmental barriers to increased agricultural production: New evidence from central Thailand. Outlook Agric. 50, 178-187 (2021). 8. Rigg, J., Phongsiri, M., Promphakping, B., Salamanca, A. & Sripun, M. Who will tend the farm? Interrogating the ageing Asian farmer. The Journal of peasant studies 47, 306-325 (2020). 9. Lutz, W., Sanderson, W. & Scherbov, S. The coming acceleration of global population ageing. Nature 451, 716-719 (2008). 10. Yang, J., et al. Projecting heat-related excess mortality under climate change scenarios in China. Nat. Commun. 12, 1039 (2021). 11. Chen, X., Huang, B. & Li, S. Population ageing and inequality: Evidence from 638 China. World Econ. 41, 1976-2000 (2018). 639 12. Zhang, X., Guo, F. & Zhai, Z. China's Demographic Future Under the New Two- 640 13 Child Policy. Popul. Res. Policy Rev. 38, 537-563 (2019). 641 y 13. Zeng, Y. & Hesketh, T. The effects of China's universal two-child policy. Lancet 642 388, 1930-1938 (2016). 643 14. Satola, L. Problems of the aging of the farmers' population in small farms in 644 Poland. Scientific Papers Series Management, Economic Engineering in 645 Agriculture and Rural Development 19, 305-310 (2019). References: 612 646 15. Cutler, J., et al. Ageing is associated with disrupted reinforcement learning whilst 647 learning to help others is preserved. Nat. Commun. 12, 4440 (2021). 648 16. Huang, X., Lu, Q., Wang, L., Cui, M. & Yang, F. Does aging and off-farm 649 employment hinder farmers' adoption behavior of soil and water conservation 650 technology in the Loess Plateau? Int. J. Clim. Chang. Strateg. Manag. 12, 92-107 651 (2020). 652 17. Min, S., Waibel, H. & Huang, J. Smallholder participation in the land rental market 653 in a mountainous region of Southern China: Impact of population aging, land 654 tenure security and ethnicity. Land Use Pol. 68, 625-637 (2017). 655 18. Li, S., et al. An estimation of the extent of cropland abandonment in mountainous regions of China. Land Degrad. Dev. 29, 1327-1342 (2018). 19. Jansuwan, P. & Zander, K.K. What to do with the farmland? Coping with ageing in rural Thailand. J. Rural Stud. 81, 37-46 (2021). 20. Peng, X. China's demographic history and future challenges. Science 333, 581- 660 587 (2011). 661 21. Chen, R., Xu, P., Song, P., Wang, M. & He, J. China has faster pace than Japan in 662 population aging in next 25 years. Biosci Trends 13, 287-291 (2019). 663 22. Usman, M., Sawaya, A., Igarashi, M., Gayman, J.J. & Dixit, R. Strained 664 agricultural farming under the stress of youths’ career selection tendencies: a case 665 study from Hokkaido (Japan). Hum. Soc. Sci. Commun. 8, 19 (2021). 666 23. Ren, C., et al. The impact of farm size on agricultural sustainability. J. Clean Prod 220, 357-367 (2019). 24. Liao, L., Long, H., Gao, X. & Ma, E. Effects of land use transitions and rural aging on agricultural production in China's farming area: A perspective from changing labor employing quantity in the planting industry. Land Use Pol. 88, 104152 (2019). 25. Wu, Song & Yu. Spatial Differences in China’s Population Aging and Influencing Factors: The Perspectives of Spatial Dependence and Spatial Heterogeneity. Sustainability (Basel, Switzerland) 11, 5959 (2019). y 26. Ren, C., et al. Fertilizer overuse in Chinese smallholders due to lack of fixed inputs. J. Environ. Manage. 293, 112913 (2021). 27. Liu, X., et al. The impact of urbanization and aging on food security in developing 678 countries: The view from Northwest China. J. Clean Prod. 292, 126067 (2021). 679 28. Wu, Y., et al. References: 612 pathways describing world futures in the 21st century. Glob. Environ. Change 42, 691 169-180 (2017). 692 33. Zhou, L., Wang, G., Jia, C. & Ma, Z. Being left-behind, mental disorder, and 693 elderly suicide in rural China: a case–control psychological autopsy study. Psychol. 694 Med. 49, 458-464 (2019). 695 34. Steptoe, A., Shankar, A., Demakakos, P. & Wardle, J. Social isolation, loneliness, 696 and all-cause mortality in older men and women. Proc. Natl. Acad. Sci. U. S. A. 697 110, 5797-5801 (2013). 698 35. Zhao, F. & Yu, G. Parental Migration and Rural Left-Behind Children’s Mental 699 Health in China: A Meta-Analysis Based on Mental Health Test. J. Child Fam. 700 Stud. 25, 3462-3472 (2016). 701 36. Li, Q., Liu, G. & Zang, W. The health of left-behind children in rural China. China 702 Econ. Rev 36, 367-376 (2015). 703 37. O'Meara, P. The ageing farming workforce and the health and sustainability of 704 agricultural communities: A narrative review. Aust. J. Rural Health 27, 281-289 705 (2019). 706 38. Gu, B., Zhang, X., Bai, X., Fu, B. & Chen, D. Four steps to food security for 707 swelling cities. Nature 566, 31-33 (2019). 708 39. Wang, S., et al. Urbanization can benefit agricultural production with large-scale 709 farming in China. Nat. Food 2, 183-191 (2021). 710 40. Duan, J., et al. Consolidation of agricultural land can contribute to agricultural 711 sustainability in China. Nat. Food (2021). 712 41. Garland, G., et al. Crop cover is more important than rotational diversity for soil 713 multifunctionality and cereal yields in European cropping systems. Nat. Food 2, 714 28-37 (2021). 715 716 717 Data availability 718 Data supporting the findings of this study are available within the article and its 719 supplementary information files. 720 721 Acknowledgements 722 This study was supported by the National Natural Science Foundation of China 723 (42061124001, 41822701 and 41773068). 724 725 Author contributions 726 B.G. designed the study. C.R. conducted the research. B.G. and C.R. wrote the first 727 draft of the paper, S.R. revised the paper. C.W., Y.G., Y.D., S.S., and W.L. processed 728 the raw data. X.Z. and J.X. contributed to the discussion of the paper. 729 730 Declaration of competing interest 731 All authors have no conflicts of interest to report. 732 33. Zhou, L., Wang, G., Jia, C. & Ma, Z. References: 612 Policy distortions, farm size, and the overuse of agricultural 680 chemicals in China. Proc. Natl. Acad. Sci. U. S. A. 115, 7010-7015 (2018). 681 29. Yu, Y., Hu, Y., Gu, B., Reis, S. & Yang, L. Reforming smallholder farms to 682 mitigate agricultural pollution. Environ. Sci. Pollut. Res. (2021). 683 30. Fang, D. & Guo, Y. Induced Agricultural Production Organizations under the 684 Transition of Rural Land Market: Evidence from China. Agriculture 11, 881 685 (2021). 686 31. Guo, Ding, Zhong, Cheng & Huang. Predicting the Future Chinese Population 687 using Shared Socioeconomic Pathways, the Sixth National Population Census, and 688 a PDE Model. Sustainability (Basel, Switzerland) 11, 3686 (2019). 689 y ( ) ( ) 32. O Neill, B.C., et al. The roads ahead: Narratives for shared socioeconomic 14 pathways describing world futures in the 21st century. Glob. Environ. Change 42, 691 169-180 (2017). 692 33. Zhou, L., Wang, G., Jia, C. & Ma, Z. Being left-behind, mental disorder, and 693 elderly suicide in rural China: a case–control psychological autopsy study. Psychol. 694 Med. 49, 458-464 (2019). 695 34. Steptoe, A., Shankar, A., Demakakos, P. & Wardle, J. Social isolation, loneliness, 696 and all-cause mortality in older men and women. Proc. Natl. Acad. Sci. U. S. A. 697 110, 5797-5801 (2013). 698 35. Zhao, F. & Yu, G. Parental Migration and Rural Left-Behind Children’s Mental 699 Health in China: A Meta-Analysis Based on Mental Health Test. J. Child Fam. 700 Stud. 25, 3462-3472 (2016). 701 36. Li, Q., Liu, G. & Zang, W. The health of left-behind children in rural China. China 702 Econ. Rev 36, 367-376 (2015). 703 37. O'Meara, P. The ageing farming workforce and the health and sustainability of 704 agricultural communities: A narrative review. Aust. J. Rural Health 27, 281-289 705 (2019). 706 38. Gu, B., Zhang, X., Bai, X., Fu, B. & Chen, D. Four steps to food security for 707 swelling cities. Nature 566, 31-33 (2019). 708 39. Wang, S., et al. Urbanization can benefit agricultural production with large-scale 709 farming in China. Nat. Food 2, 183-191 (2021). 710 40. Duan, J., et al. Consolidation of agricultural land can contribute to agricultural 711 sustainability in China. Nat. Food (2021). 712 41. Garland, G., et al. Crop cover is more important than rotational diversity for soil 713 multifunctionality and cereal yields in European cropping systems. Nat. Food 2, 714 28-37 (2021). References: 612 715 716 717 Data availability 718 Data supporting the findings of this study are available within the article and its 719 supplementary information files. 720 721 Acknowledgements 722 This study was supported by the National Natural Science Foundation of China 723 (42061124001, 41822701 and 41773068). 724 725 Author contributions 726 B.G. designed the study. C.R. conducted the research. B.G. and C.R. wrote the first 727 draft of the paper, S.R. revised the paper. C.W., Y.G., Y.D., S.S., and W.L. processed 728 the raw data. X.Z. and J.X. contributed to the discussion of the paper. 729 730 Declaration of competing interest 731 All authors have no conflicts of interest to report. 732 pathways describing world futures in the 2 691 169-180 (2017). 692 33. Zhou, L., Wang, G., Jia, C. & Ma, Z. B 693 elderly suicide in rural China: a case–contr 694 Med. 49, 458-464 (2019). 695 34. Steptoe, A., Shankar, A., Demakakos, P. & 696 and all-cause mortality in older men and 697 110, 5797-5801 (2013). 698 35. Zhao, F. & Yu, G. Parental Migration an 699 Health in China: A Meta-Analysis Based 700 Stud. 25, 3462-3472 (2016). 701 36. Li, Q., Liu, G. & Zang, W. The health of le 702 Econ. Rev 36, 367-376 (2015). 703 37. O'Meara, P. The ageing farming workfor 704 agricultural communities: A narrative rev 705 (2019). 706 38. Gu, B., Zhang, X., Bai, X., Fu, B. & Ch 707 swelling cities. Nature 566, 31-33 (2019). 708 39. Wang, S., et al. Urbanization can benefit 709 farming in China. Nat. Food 2, 183-191 (2 710 40. Duan, J., et al. Consolidation of agricult 711 sustainability in China. Nat. Food (2021). 712 41. Garland, G., et al. Crop cover is more im 713 multifunctionality and cereal yields in Eu 714 28-37 (2021). 715 716 717 Data availability 718 Data supporting the findings of this study are a 719 supplementary information files. 720 721 Acknowledgements 722 This study was supported by the National 723 (42061124001, 41822701 and 41773068). 724 725 Author contributions 726 B.G. designed the study. C.R. conducted the 727 draft of the paper, S.R. revised the paper. C.W 728 the raw data. X.Z. and J.X. contributed to the d 729 730 Declaration of competing interest 731 All authors have no conflicts of interest to repo 732 pathways describing world futures in the 21st century. Glob. Environ. Change 42, 169-180 (2017). References: 612 Being left-behind, mental disorder, and elderly suicide in rural China: a case–control psychological autopsy study. Psychol. Med. 49, 458-464 (2019). 34. Steptoe, A., Shankar, A., Demakakos, P. & Wardle, J. Social isolation, loneliness, and all-cause mortality in older men and women. Proc. Natl. Acad. Sci. U. S. A. 110, 5797-5801 (2013). 35. Zhao, F. & Yu, G. Parental Migration and Rural Left-Behind Children’s Mental Health in China: A Meta-Analysis Based on Mental Health Test. J. Child Fam. Stud. 25, 3462-3472 (2016). 36. Li, Q., Liu, G. & Zang, W. The health of left-behind children in rural China. China 02 Econ. Rev 36, 367-376 (2015). 03 37. O'Meara, P. The ageing farming workforce and the health and sustainability of 704 agricultural communities: A narrative review. Aust. J. Rural Health 27, 281-289 705 (2019). 706 38. Gu, B., Zhang, X., Bai, X., Fu, B. & Chen, D. Four steps to food security for swelling cities. Nature 566, 31-33 (2019). 39. Wang, S., et al. Urbanization can benefit agricultural production with large-scale farming in China. Nat. Food 2, 183-191 (2021). 40. Duan, J., et al. Consolidation of agricultural land can contribute to agricultura sustainability in China. Nat. Food (2021). 41. Garland, G., et al. Crop cover is more important than rotational diversity for soil 713 multifunctionality and cereal yields in European cropping systems. Nat. Food 2, 714 28-37 (2021). 715 multifunctionality and cereal yields in European cropping systems. Nat. Food 2, 714 28-37 (2021). 715 716 717 Data availability 718 Data supporting the findings of this study are available within the article and its 719 supplementary information files. 720 721 Acknowledgements 722 This study was supported by the National Natural Science Foundation of China 723 (42061124001, 41822701 and 41773068). 724 725 Author contributions 726 B.G. designed the study. C.R. conducted the research. B.G. and C.R. wrote the first 727 draft of the paper, S.R. revised the paper. C.W., Y.G., Y.D., S.S., and W.L. processed 728 the raw data. X.Z. and J.X. contributed to the discussion of the paper. 729 730 Declaration of competing interest 731 All authors have no conflicts of interest to report. 732 Data supporting the findings of this study are available within the article and its 719 supplementary information files. 720 All authors have no conflicts of interest to report. References: 612 732 15 Table 1 Fixed effect panel models determining the effect of aging on agricultural sustainability 33 Farm size and Education Cost and Benefit Fertilizer and Machinery Ln Farm size (ha) Education (yr) Transfer -out Abando ned (ha) Ln Output ($ ha-1) Ln Input ($ ha-1) Profit- cost ratio Ln LP ($ hr-1) Ln Fertilization ($ ha-1) Ln FUE Machine input ($ ha-1) Machine owned ($ ha-1) g -0.27* -1.54* 0.76* 0.19 -0.32* -0.30* -0.03 -0.37* -0.23* -0.13* -112* -919* arm size (ha) -0.14* -0.20* 0.13* 0.61* -0.28* 0.10* -25.2* 464* ation (yr) 0.01* 0.04 -0.01 0.03* 0.01* 0.03* 0.03* -0.003 0.04* 12.8* 55.0*** nty Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes 16767 16767 16137 12304 16767 15602 15460 16247 15447 15292 16542 16593 st R2 0.51 0.29 0.26 0.20 0.08 0.41 0.21 0.13 el FEP FEP Tobit Tobit FEP FEP FEP FEP FEP FEP Tobit Tobit p < 0.05, p < 0.01, and *p< 0.005. LP, Labor productivity. FUE, Fertilizer use efficiency. FEP, Fixed effect panel. Farm size is the total 34 cultivated area covering all crops. Education refers to average years of education of family members aged 15 and over. Transfer-out are the ratio 35 of transferred-out cropland area to the current total cropland area. Abandoned indicates the area of abandoned cropland by farmers. Profit-cost 36 ratio means the ratio of difference between agricultural income and input to the input. Labor productivity (LP) indicates agricultural output per 37 working hour of labor. Fertilizer use efficiency (FUE) is the ratio of agricultural output to fertilizer input, measuring fertilizer contribution and 38 reflecting its loss. Machine input is total machine cost including machinery rent and depreciation to total cultivated area. Machine owned means 39 original value of agricultural machinery the household owned per area. Aging refers to the proportion of people over 65 years old in rural 40 households. County and city mean county-level or city-level regional effect is controlled, respectively. Adult farmer’s ratio, income ratio from 41 non-agricultural sectors, crop type, plot numbers and year effect have been controlled in all regression equations. 42 effect panel models determining the effect of aging on agricultural sustainability Farm size and Education Cost and Benefit Fertilizer and Machinery Model FEP FEP Tobit Tobit FEP FEP FEP FEP FEP FEP Tobit Tob p < 0.05, p < 0.01, and p< 0.005. LP, Labor productivity. FUE, Fertilizer use efficiency. References: 612 FEP, Fixed effect panel. Farm size is the total 734 cultivated area covering all crops. Education refers to average years of education of family members aged 15 and over. Transfer-out are the ratio 735 of transferred-out cropland area to the current total cropland area. Abandoned indicates the area of abandoned cropland by farmers. Profit-cost 736 ratio means the ratio of difference between agricultural income and input to the input. Labor productivity (LP) indicates agricultural output per 737 working hour of labor. Fertilizer use efficiency (FUE) is the ratio of agricultural output to fertilizer input, measuring fertilizer contribution and 738 reflecting its loss. Machine input is total machine cost including machinery rent and depreciation to total cultivated area. Machine owned means 739 original value of agricultural machinery the household owned per area. Aging refers to the proportion of people over 65 years old in rural 740 households. County and city mean county-level or city-level regional effect is controlled, respectively. Adult farmer’s ratio, income ratio from 741 non-agricultural sectors, crop type, plot numbers and year effect have been controlled in all regression equations. 742 cultivated area covering all crops. Education refers to average years of education of family members aged 15 and over. Transfer-out are the ratio 735 of transferred-out cropland area to the current total cropland area. Abandoned indicates the area of abandoned cropland by farmers. Profit-cost 736 ratio means the ratio of difference between agricultural income and input to the input. Labor productivity (LP) indicates agricultural output per 737 working hour of labor. Fertilizer use efficiency (FUE) is the ratio of agricultural output to fertilizer input, measuring fertilizer contribution and 738 reflecting its loss. Machine input is total machine cost including machinery rent and depreciation to total cultivated area. Machine owned means 739 original value of agricultural machinery the household owned per area. Aging refers to the proportion of people over 65 years old in rural 740 households. County and city mean county-level or city-level regional effect is controlled, respectively. Adult farmer’s ratio, income ratio from 741 non-agricultural sectors, crop type, plot numbers and year effect have been controlled in all regression equations. 742 16 743 Fig. 1 Population aging and farm size in China. (a) Aging trend in rural and urban 744 China from 2000 to 2019; (b) Age stages in rural and urban China in 2019; (c) County- 745 level aging in 2020; (d) Farm size in 2017. References: 612 Aging in (a) and (c) refers to the proportion 746 of people over 65 years old on country and county-level, respectively. It is calculated 747 by the number of people over 65 years old to the total population. 748 743 Fig. 1 Population aging and farm size in China. (a) Aging trend in rural and urban 744 China from 2000 to 2019; (b) Age stages in rural and urban China in 2019; (c) County- 745 level aging in 2020; (d) Farm size in 2017. Aging in (a) and (c) refers to the proportion 746 of people over 65 years old on country and county-level, respectively. It is calculated 747 by the number of people over 65 years old to the total population. 748 17 17 749 Fig. 2 Structural equation model of aging impacts on agricultural sustainability. 750 p < 0.05, p < 0.01, and p< 0.005. The numbers adjacent to the lines are the 751 standardized path coefficients, which means how much standard deviation changes in 752 dependent variable if each of the independent variable changes by one standard 753 deviation. The blue and red lines represent positive and negative effects, respectively. 754 Dark and light red indicate the direct and indirect negative effects of aging on 755 agriculture, respectively. The line width is proportional to the strength of the 756 standardized path coefficient. Variables in this figure except aging, abandoned cropland 757 and education have been log-transformed. It is worth noting that the path coefficient 758 between aging and abandoned cropland was not incorporated in this structural equation 759 model considering the nonlinear relationship between these two variables. The 760 coefficient of “0.01” between aging and abandoned cropland means the abandoned 761 cropland area increase when aging ratio increase by one unit averagely. Then the 762 abandoned cropland squarely decreases household-level farm size. 763 764 749 Fig. 2 Structural equation model of aging impacts on agricultural sustainability. 750 p < 0.05, p < 0.01, and p< 0.005. The numbers adjacent to the lines are the 751 standardized path coefficients, which means how much standard deviation changes in 752 dependent variable if each of the independent variable changes by one standard 753 deviation. The blue and red lines represent positive and negative effects, respectively. 754 Dark and light red indicate the direct and indirect negative effects of aging on 755 agriculture, respectively. References: 612 The line width is proportional to the strength of the 756 standardized path coefficient. Variables in this figure except aging, abandoned cropland 757 and education have been log-transformed. It is worth noting that the path coefficient 758 between aging and abandoned cropland was not incorporated in this structural equation 759 model considering the nonlinear relationship between these two variables. The 760 coefficient of “0.01” between aging and abandoned cropland means the abandoned 761 cropland area increase when aging ratio increase by one unit averagely. Then the 762 abandoned cropland squarely decreases household-level farm size. 763 764 749 Fig. 2 Structural equation model of aging impacts on agricultural sustainability. 750 p < 0.05, p < 0.01, and p< 0.005. The numbers adjacent to the lines are the 751 standardized path coefficients, which means how much standard deviation changes in 752 dependent variable if each of the independent variable changes by one standard 753 deviation. The blue and red lines represent positive and negative effects, respectively. 754 Dark and light red indicate the direct and indirect negative effects of aging on 755 agriculture, respectively. The line width is proportional to the strength of the 756 standardized path coefficient. Variables in this figure except aging, abandoned cropland 757 and education have been log-transformed. It is worth noting that the path coefficient 758 between aging and abandoned cropland was not incorporated in this structural equation 759 model considering the nonlinear relationship between these two variables. The 760 coefficient of “0.01” between aging and abandoned cropland means the abandoned 761 cropland area increase when aging ratio increase by one unit averagely. Then the 762 abandoned cropland squarely decreases household-level farm size. 763 764 18 65 Fig. 3 Impact of aging on agricultural sustainability across China’s provinces in 66 2019. (a) Farm size; (b) Education; (c) Labor productivity; (d) Total agricultural input; 67 (e) Agricultural output; (f) Profit-cost ratio; (g) Machine input; (h) Fertilization; (i) 68 Fertilizer use efficiency. It is calculated by determining the difference between the 69 predicted values without aging and the observed values with aging divided by the 70 observed value on province-level, and the result is carried out in percentage terms. 71 Acronyms for 31 provinces, autonomous regions, and municipalities directly under the 72 Central Government are listed in Table S6. Shanghai, Tibet and Xinjiang aren’t depicted 73 due to data limitation. 74 Fig. References: 612 3 Impact of aging on agricultural sustainability across China’s provinces in 765 Fig. 3 Impact of aging on agricultural sustainability across China’s provinces in 766 2019. (a) Farm size; (b) Education; (c) Labor productivity; (d) Total agricultural input; 767 (e) Agricultural output; (f) Profit-cost ratio; (g) Machine input; (h) Fertilization; (i) 768 Fertilizer use efficiency. It is calculated by determining the difference between the 769 predicted values without aging and the observed values with aging divided by the 770 observed value on province-level, and the result is carried out in percentage terms. 771 Acronyms for 31 provinces, autonomous regions, and municipalities directly under the 772 Central Government are listed in Table S6. Shanghai, Tibet and Xinjiang aren’t depicted 773 due to data limitation. 774 19 775 Fig. 4 Comparison of smallholder and new farming models on agricultural 776 sustainability in 2019. (a) Aging farmer’s ratio; (b) Education; (c) Farm size; (d) Labor 777 productivity; (e) Agricultural output; (f) Total agricultural input; (g) Fertilization; (h) 778 Fertilizer use efficiency (FUE); (i) Machine input. p < 0.05, p < 0.01, and p< 0.005, 779 which indicates the significant difference level between smallholder and new farming 780 responding to indicators of agricultural sustainability. Error bars refer to standard errors. 781 The statistical results are in Table S4. 782 783 10% 16% 22% 28% Aging* (a) 0.0 0.6 1.2 1.8 Farm size (ha) (c) 6 7 8 9 10 Education* (yr) (b) 0.0 0.8 1.6 2.4 Input* (103 $ ha-1) (f) 0 2 4 6 Smallholder New farming Fertilization (102 $ ha-1) (g) 0 5 10 15 Smallholder New farming FUE* (h) 1.5 2.0 2.5 3.0 Output* (103 $ ha-1) (e) 0 2 4 6 Smallholder New farming Machine* (102 $ ha-1) (i) 0.3 1.1 1.9 2.7 Labor productivity* ($ hr-1) (d) 15 0 5 10 15 Smallholder New farming FUE* (h) 0 2 4 6 Smallholder New farming Machine* (102 $ ha-1) (i) 775 Fig. 4 Comparison of smallholder and new farming models on agricultural 776 sustainability in 2019. (a) Aging farmer’s ratio; (b) Education; (c) Farm size; (d) Labor 777 productivity; (e) Agricultural output; (f) Total agricultural input; (g) Fertilization; (h) 778 Fertilizer use efficiency (FUE); (i) Machine input. p < 0.05, p < 0.01, and **p< 0.005, 779 which indicates the significant difference level between smallholder and new farming 780 responding to indicators of agricultural sustainability. References: 612 Error bars refer to standard errors. 781 The statistical results are in Table S4. 782 783 20 784 Fig. 5 Future agricultural sustainability changes due to aging under SSP 785 scenarios by 2100. (a) Farm size; (b) Education; (c) Labor productivity; (d) Total 786 agricultural input; (e) Agricultural output; (f) Profit-cost ratio; (g) Machine input; (h) 787 Fertilization; (i) Fertilizer use efficiency. The Baseline assumed no changes in the 788 future. SSP1-4 refers to Shared Socioeconomic Pathways (SSPs) scenarios. NF, New 789 farming. The changes of input, output, machine and fertilization are all weighted with 790 the total cultivated area. 791 1.15 1.25 1.35 1.45 Input (103 $ ha-1) (d) 5.0 5.7 6.4 7.1 7.8 Education (yr) (b) 0.7 0.8 0.9 1.0 1.1 Farm size (ha) (a) 16 17 18 19 20 21 Output (102 $ ha-1) (e) 0.9 1.4 1.9 2.4 Labor productivity ($ hr-1) (c) 2.0 2.4 2.8 3.2 3.6 2020 2040 2060 2080 2100 Machine (102 $ ha-1) Year (g) SSP1 SSP2 SSP3 SSP4 Baseline SSP1 with NF SSP2 with NF SSP3 with NF SSP4 with NF 0.5 0.7 0.9 1.1 Profit/cost (f) 2.9 3.0 3.1 3.2 2020 2040 2060 2080 2100 Fertilization (102 $ ha-1) Year (h) 6 7 8 9 10 2020 2040 2060 2080 2100 Fertilizer use efficiency Year (i) Labor productivity ($ hr-1) 5.0 16 17 18 19 20 21 Output (102 $ ha-1) (e) 0 2.9 3.0 3.1 3.2 2020 2040 2060 2080 2100 Fertilization (102 $ ha-1) Year (h) Fertilizer use efficiency Year Fig. 5 Future agricultural sustainability changes due to aging under SSP 784 Fig. 5 Future agricultural sustainability changes due to aging under SSP 785 scenarios by 2100. (a) Farm size; (b) Education; (c) Labor productivity; (d) Total 786 scenarios by 2100. (a) Farm size; (b) Education; (c) Labor productivity; (d) Total 786 agricultural input; (e) Agricultural output; (f) Profit-cost ratio; (g) Machine input; (h) 787 Fertilization; (i) Fertilizer use efficiency. The Baseline assumed no changes in the 788 future. SSP1-4 refers to Shared Socioeconomic Pathways (SSPs) scenarios. NF, New 789 farming. The changes of input, output, machine and fertilization are all weighted with 790 the total cultivated area. 791 21 Supplementary Files This is a list of supplementary ¦les associated with this preprint. Click to download. SupplementaryInformation.docx
https://openalex.org/W4385618040	https://zenodo.org/records/8220992/files/IJISRT23JUL1538.pdf	English	null	Self-Efficacy and Motivation as Predictors to Mathematics Performance in Online Learning	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	21,737	Volume 8, Issue 7, July – 2023 Volume 8, Issue 7, July – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Self-Efficacy and Motivation as Predictors to Mathematics Performance in Online Learning A thesis Presented to The Faculty of the Graduate school Assumption College of Nabunturan Nabunturan, Davao De Oro In partial fulfillment of the Requirments for the Degree of Master of Arts in Education Major in Mathematics In partial fulfillment of the Requirments for the Degree of Master of Arts in Education Major in Mathematics In partial fulfillment of the Requirments for the Degree of Master of Arts in Education Major in Mathematics 2Romulo G. Doronio Adviser IJISRT23JUL1538 IJISRT23JUL1538 2202 www.ijisrt.com Volume 8, Issue 7, July – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 ISSN No:-2456-2165 Keywords:- Online Learning Education, Motivation, Self-Efficacy, Mathematics Performance, Descriptive-Correlational Study ABSTRACT This study wants to determine if motivation and self-efficacy can be a predictor of students’ Mathematics performance in an online learning context. A descriptive-correlational design was conducted to explore the study. The participants were selected from 4 campuses of Davao De Oro State College. All students who are enrolled in the Mathematics of Investment subject were included in the study using a universal sampling technique. This study used adapted 4 points Likert scale questionnaires for motivation and self-efficacy and self-tailored summative assessment for Mathematics performance. The study concluded that the students had a moderate level of self-efficacy and motivation in an online learning context. The result showed that both motivation and self-efficacy have a negligible relationship and were not statistically significant. The study also found that motivation and self-efficacy were not significant predictors of students’ Mathematics performance. The study recommends that students develop personal strategies to enhance self-efficacy and motivation, while teachers should design and implement effective instructional strategies to cater to varied learning styles. Furthermore, school administrators should develop policies and programs that promote effective online learning practices in Mathematics education. Keywords:- Online Learning Education, Motivation, Self-Efficacy, Mathematics Performance, Descriptive-Correlational Study IJISRT23JUL1538 IJISRT23JUL1538 www.ijisrt.com 2203 Volume 8, Issue 7, July – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Volume 8, Issue 7, July – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 TABLE OF CONTENT TITLE PAGE CHAPTER ONE INTRODUCTION 2205 CHAPTER TWO METHODOLOGY 2212 CHAPTER THREE RESULTS 2215 CHAPTER FOUR DISCUSSION AND CONCLUSION 2219 REFERENCES 2220 APPENDICES 2224 IJISRT23JUL1538 IJISRT23JUL1538 www.ijisrt.com 2204 International Journal of Innovative Science and Research Technology Volume 8, Issue 7, July – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 ISSN No:-2456-2165 A. The Problem and its Background g Mathematics is considered a fundamental subject as it develops students’ logical reasoning. Though the mathematics performance of the students is heavily influenced by the cognitive domain as they mostly use their cognitive faculties, affective factors are equally significant. Affective factors such as motivation and self-efficacy play an important role in order for students to achieve academic success (Sikhwari, 2007; Hiller et. al, 2022). Self-efficacy refers to the level of learners’ confidence in their ability to complete a certain task. On the other hand, motivation refers to a psychological state that drives an individual to engage in an activity. The environment also influences the learning process (Chukwuemeka, 2013). Most countries all over the world such as the USA, Pakistan, India, South Africa, and China transitioned to online learning due to the COVID-19 pandemic. Some educators and learners were caught off-guard by the sudden change which caused some issues in the students’ motivation and self-efficacy. Changes in the environment can interfere with self-efficacy and motivation. Self-efficacy and motivation are different in every context (Hodges, 2008; Brachtl et al., 2023). As a result, students’ self-efficacy and motivation in online learning settings differ from their self-efficacy and motivation in face-to-face learning settings, and it is regarded as a critical component for successful online learning (Shen et al., 2013). Consequently, the changes in students’ level of motivation and self-efficacy significantly influence the students’ performance (Mijares III, 2022). In the Philippines, as the Department of Education (DepEd) and Commission on Higher Education (CHED) implemented an online learning mode of instruction, it brought challenges to educators and especially students as they experienced poor internet connection, outdated devices, lack of resources, and many more (Cabansag, 2020). It resulted in low motivation to continue education amidst the pandemic. In addition, as the nation faces the COVID-19 pandemic, the Philippine Association of State Universities and Colleges (PASUC) (2020) estimates that a total of 44, 069 college students won't enroll for the academic year 2020–2021. Many believe that motivation and self-efficacy are predictors of students’ performance (Sartawi et al., 2012; Ugwuanyi et al., 2020; Kim et al., 2014). Interestingly, the study of Mijares III (2022) was contrary to the previous statement, it showed that motivation and self-efficacy are not significant predictors of students’ mathematics performance. A. The Problem and its Background In Davao De Oro State College – Maragusan Branch, where the researcher is presently teaching, the researcher and his colleagues noticed that some students lack the motivation to participate in any online class. In addition, students who have been recognized as having poor engagement have expressed a lack of confidence in their ability to do the task properly. With the mentioned context, this led the researcher to determine if motivation and self-efficacy can be a predictor of students’ performance in an online learning context. In addition, the researcher aims to determine if motivation and self-efficacy can be predictors of students’ mathematics performance and identify any factors that may influence it. By examining this topic, students can better understand the importance of motivation and self-efficacy in their academic performance. CHAPTER ONE INTRODUCTION A. The Problem and its Background B. Review of Related Literature and Studies Various related literature and related studies are presented to provide direction and a frame of reference for this study. It includes literature as well as studies, which are directly or indirectly related to this study. International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Rifandi (2013) suggests that achieving success in mathematics has a significant impact on one's motivation to succeed. Several findings from the survey imply that factors contributing to the drop in favorable attitudes toward mathematics include a lack of teacher support as well as the environment in which students are taught. Students' motivation can be aided by rationales that induce two effects: students must recognize the importance and personal value of the assignment, and students must sense a high degree of autonomy while working on the task (Jang, 2008). Furthermore, it has been noted in the literature that students' belief in mathematics can be influenced by their views in the following aspects: the nature of mathematics, the method of teaching and three learning, and their beliefs in their instructors (Lazim et al., 2004). In addition, students' perceptions of their own achievement in mathematics learning are substantially correlated with their own motivational attitudes. Teachers' behaviors, attitudes, and the quality of instructional design for mathematics sessions have all had a significant impact on students' motivation (Rifandi, 2013). Paas et al. (2005) asserted that meaningful learning can only occur when it is combined with motivation, which they believe is the case. Using a motivational perspective in the classroom, they claimed, would help discover the task qualities that drove students to put out more mental effort and achieve higher levels of performance. In addition, It is the belief of Keller (2008) that only when the motivating features of students are discovered and, consequently, techniques for addressing their areas of weakness are determined that the learning environment is optimized. Many people believe that mathematics is only necessary for those working in industries such as engineering, science, and finance. This is a frequent mistake. Some people even believe that mathematics is solely concerned with computations. In reality, mathematics is a subject that may be applied to a variety of situations in everyday life. Students learn how to discern between the suitable use of mathematical information for different scenarios through the study of mathematics, which is to say, they learn how to determine which mathematical knowledge is appropriate for which reasons. The value of mathematics can be infused through the process of problem-solving. They can be word problems, non-routine problems, or real-life situations that require a variety of methodologies to be applied in order to be solved (Taplin, 1998, cited by Albay, 2020). International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Student involvement in learning is predicated on their level of motivation to study (Saeed & Zyngier, 2012). According to research, a motivated learner possesses the inner strength necessary for learning and adapting to the demands of the school environment (Ferreira et al., 2011). A motivated learner is more likely to be actively engaged and exhibits improved performance, persistence, and creativity (Schunk et al., 2008). It is not viable to substitute online learning for students who are not sufficiently motivated (Aduayi-Akue et al., 2017), as a lack of motivation is a major cause of student attrition in distant education (Aduayi- Akue et al., 2017; Smith et al., 2005; Visser et al., 2002, cited by Murphy & Rodriguez-Manzanares, 2009). Students' motivation in the classroom has lately been investigated using five frameworks of social–cognitive dimensions, as described by Pintrich (2003). In accordance with the concept of self-beliefs (e.g., self-efficacy), when students believe that they can do well in educational environments, they feel confident, are more likely to try their hardest, persevere longer, and perform better overall (Bandura, 1997). It is anticipated that students who have a stronger sense of personal control over their learning will perform better academically and achieve higher levels of success than those who don't. For a more sophisticated differentiation of extrinsic motivation in higher education, two theories have been developed to complement the traditional distinction between students' intrinsic and extrinsic motivation: students' interest and intrinsic motivation. It has been discovered that pupils' diverse types of interests lead to a variety of different learning outcomes (Ryan & Deci, 2000). There are a number of factors that have a substantial impact on student motivation and achievement as a result of creating effective and efficient learning-teaching processes in educational systems. Among the many psychological, social, and cultural motivational factors are intrinsic and extrinsic directions, parental influence and participation, family history, peer pressure, self- efficacy expectations, effort, value attributed to a relative, anxiety, self-regulation and goal determination, talent perceptions, learning strategies, teaching style, and school environment (Yilmaz et al., 2017). Osborne et al. (2003) also note that one of the elements that influence students' attitudes toward science is the high quality of the teaching process, which encourages interest and involvement in the subject. As a result, this article will provide an explanation of motivation as well as the use of an appropriate environment to boost students' motivation in mathematics learning.  Motivation Motivation is a Psychology term that refers to the desire for something and the fuel that serves as a driving force to continue pursuing that desire (Motevalli et al., 2020). Motivation is an essential component of learning and plays a critical role in assisting students to become involved in academic activities. Motivation is defined as a situation that provides energy to, directs, and sustains behavior. This includes goals and activities that require the objectives to provide motivation to move and act. To operate for an extended period of time, action necessitates effort and persistence. Motivation entails a collection of beliefs, perceptions, values, information, and actions that are all interconnected. Motivation can lead to a variety of behaviors, and it is critical to comprehend the significance of motivation in the educational setting (Suhag et al., 2016). Due to the fact that motivation speeds up cognitive processing, it affects how information is processed. As a result, motivated students are more likely to comprehend and carefully consider the subject at hand as opposed to merely superficially monitoring other people's learning activities (Yilmaz et al., 2017). And in relation to the psychological perspective, motivation refers to an internal process that stimulates individuals' conduct, guides them, and helps them to maintain their behavior through time (Anderman et al., 2013; Pintrich, 2003; Zimmerman & Schunk, 2011. In addition, motivation can be thought of as "the motor that keeps us moving," the desire to accomplish tasks that are important to people in one way or another, and the drive to succeed in these endeavors. (Nyman & Sumpter, 2019). IJISRT23JUL1538 www.ijisrt.com 2205 Volume 8, Issue 7, July – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165  Self Efficacy f ff y Bandura (1997) defined self-efficacy as an individual’s belief in their ability to complete a task in a different situation. Since the covid-19 pandemic, the popularity of online learning dramatically increase. According to him, mastery experience, vicarious experience, verbal persuasion, and physiological affective states are the four main sources of knowledge that influence or develop self-efficacy. Students’ learning might be affected by their self-efficacy (Wang et al., 2017). It has an impact on how students conduct investigations, as seen by their actions, efforts, perseverance, adaptability in differences, and achievement of goals. Strong self- efficacy beliefs improve cognitive processes and performance in various settings, including academic contexts (Zulkosky, 2009). Similar research shows that students who have a high sense of self-efficacy are committed to completing difficult assignments and going the extra mile when facing challenges (Hong et al., 2017). The learning process is intrinsically tied to the learning environment. Changes in the environment can interfere with self- efficacy. Self-efficacy is different in every context (Hodges, 2008). Students' self-efficacy differs from their self-efficacy in face- to-face learning environments as a result, and it is seen to be a crucial element for successful online learning (Shen et al., 2013). Moreover, online learning self-efficacy is further defined by Zimmerman and Kulikowich (2016) as learners' assurance in their capacity to successfully complete the learning criteria for online courses. Bandura (1997) defines self-efficacy as a characteristic that influences how well-aware an individual is of himself or herself and how his or her behavior develops, specifically in terms of the ability to plan the essential actions to successfully carry out a certain performance. Zimmerman and Kulikowich (2016) designed an instrument called Online Learning Self-Efficacy Scale (OLSES) to measure students’ self-efficacy in online learning. In Turkey, Yavuzalp and Bahcivan (2019) tested the validity and reliability of OLSES. The original form of scale is in English and consists of 22 items that have three factors: learning in the online environment, time management, and technology used. After factor analysis, the second item, “find course syllabus online,” was removed from the scale because it scored below 0.40, indicating exclusion from the scale. Gender and school-type variables were not statistically significant, according to the findings of the investigation. International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Students are more appreciative and motivated to master mathematics when they can apply what they have learned in class to their daily lives. As a result, we should provide students with a comprehensive set of abilities that will enable them to meet the challenges of their lives. IJISRT23JUL1538 2206 www.ijisrt.com Volume 8, Issue 7, July – 2023 Volume 8, Issue 7, July – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 ISSN No:-2456-2165 ISSN No:-2456-2165 Teachers serve as role models and a mentor for students who want to pursue higher education. An investigation carried out by Zhang (2014) also revealed that the teacher's enthusiasm was an efficient predictor of students' intrinsic motivation, according to the findings. The teacher's passion acted as a beneficial external catalyst in fostering students' interest, curiosity, and willingness to study by igniting their internal motivation. Effective instructors put out the effort necessary to build meaningful relationships with their pupils. Teachers communicate with their students in an empathic and caring manner, and they make every effort to assist students who are experiencing course-related or personal difficulties. In order for pupils to be motivated to learn, teachers' supportive relationships are essential (Wang, 2014). Teachers are more eager, motivated, and equipped to teach and evaluate when the students have a greater capacity to increase their desire to learn (Williams & Williams, 2011). Moreover, according to Bayraktar (2015), in order to create an effective teaching and learning environment in a classroom, teachers' effective use of methods, techniques, tools, and materials in the relevant teaching field has a significant effect on student motivation. The importance of motivation in mathematics instruction cannot be overstated. Mathematical educators are asked to adapt their instructional practices in order to promote and improve students' motivation to learn Math (Wong & Wong, 2017). An emotion-controlling system is equipped with the ability to direct behavior, and this is what motivates people. This potential can present itself in several ways, including intellect, emotion, and conduct (Hannula, 2004). They are present as part of one's goal structures, as well as one's views about what is essential, and they influence one's decision to engage in a certain endeavor. The importance of good teachers in encouraging student motivation and accomplishment cannot be overemphasized. International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Those dispositions, which are repeating patterns of thoughts, feelings, or actions that result in better levels of performance as a teacher, are described as effective teachers' dispositions (Hutajulu et al., 2019; McCune & Entwistle, 2011). International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 However, there was no significant relationship between Internet self-efficacy and students’ performance on the written test. In a survey of 219 distance learners, Tang and Tseng (2013) discovered that individuals with higher self-efficacy for information searching and the ability to apply information also had better self-efficacy for online learning and had more awareness of online resources. Martin and Tutty (2008) and Martin et al. (2010) found that the self-efficacy of online learners was significantly higher than hybrid learners. However, LMS self-efficacy does not significantly affect course performance for online learners, but it positively influences the course performance for the hybrid learners. More research into the role of self-efficacy in online learning environments is still needed (Alquarashi, 2016). In addition, Tanius et al. (2020) studied the correlation between learners’ motivation, computer anxiety, and social support with self-efficacy on online learning technology due to the COVID-19 pandemic. The result indicated that online learning technology experience, learners’ attitudes, learners’ motivation, computer anxiety, and social support correlate with self-efficacy in online learning technology. Furthermore, the finding revealed that male and female respondents and different ages have similar opinions on the factors that contribute to online learning technology. This study’s most significant finding reveals that students have self-efficacy on online learning technology, even though this is the first time they use it as mandatory learning. The study also highlighted that social support in online learning. Yantraprakorn and colleagues (2018) conducted a study on why some highly efficacious learners failed in an online foreign language course based on Bandura’s theory of self-efficacy. The learning performance of six very effective distant language learners was examined at a reputable English language tutoring school in Bangkok, Thailand. Goal setting, attribution shifts, and insufficient feedback were found to be factors that may reduce online learners’ efficacy and influence their decision to drop out of a program. The implications of this study provide recommendations on support to help online learners succeed. In Mathematics, it is not enough to know how to perform the given task; self-efficacy in the appropriateness of its concepts and procedures is also required (Garfield & Ben-Zvi, 2009). For example, when doing calculations manually or by using a calculator, the element of self-efficacy is in it (Negara, et al., 2021). When people have low self-efficacy for a task, they are less likely to put forth an effort and finish it. International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 ISSN No:-2456-2165 Meanwhile, according to Alquarashi (2016), three categories of self-efficacy in online learning environments were discovered between 1997 and 2015: computer self-efficacy, internet and information-seeking self-efficacy, and LMS (Learning Management System) self-efficacy. The study of self-efficacy and computers is primarily concerned with learners’ confidence in their ability to use computers and other types of technology (Alquarashi, 2016). Additionally, the findings of Jan's (2015) study revealed a positive and significant relationship between computer self-efficacy and prior online learning experience, academic self-efficacy and prior online learning experience, and academic self-efficacy and student satisfaction. Lim (2001) discovered that computer self-efficacy is a statistically significant predictor of student satisfaction and that there is a positive relationship between student satisfaction and future intention to take online courses. Lim (2001) aims to create a model that predicts adult learners’ satisfaction with a Web-based distance education course and their intention to engage in future Web-based distance education courses. Computer self-efficacy, academic self-concept, age, gender, academic position, years of computer use, frequency of computer use, computer training, Internet experience in a class, and participation in a Web-based course workshop were among the variables investigated. The only statistically significant predictor variable was computer self- efficacy. Lee and Hwang (2007) proposed a model for measuring online learning effectiveness, claiming that computer self-efficacy is critical in student satisfaction with e-learning. Research on self-efficacy and the Internet, learners’ confidence in their ability to use the Internet to seek information is related; this is similar to information-seeking self-efficacy in online learning (Alquarashi, 2016). In web-based instruction, Joo et al. (2000) looked into the relationship between Internet self-efficacy and students’ success on written and search assessments. In addition, the study of Kou et al. (2020) also shows that student performance was significantly associated with Internet self-efficacy. Adult students’ Internet self-efficacy, self-regulation, and performance in online learning environments were investigated. The effect of student characteristics on Internet self-efficacy and self-regulation was determined using a quantitative approach to find the correlations between the variables. The participants of this study were African American students from a university in the United States. They participated in two web-based research courses offered in summer. An online survey was used to gather information. Student achievement was connected with Internet self-efficacy but not with self-regulation. Gender and age differences had no significant effect on Internet self-efficacy and self-regulation. Their Internet self-efficacy predicted students’ performance on the search exam.  Self Efficacy Moreover, Bates and Khasawneh (2007) discovered that four factors influence self-efficacy in the context of online learning: (1) previous success with online learning, (2) pre-course training, (3) instructor feedback, and (4) online learning technology anxiety. Though in a different context, we can see that it is closely related to or derived from Bandura’s self-efficacy theory. Their research proposed a mediated model in which a group of antecedent factors influenced students' self-efficacy for online learning, which in turn affected their expectations for their learning outcomes, their perceptions of their mastery, and the number of hours they allocated each week to using online learning technology to complete their university course assignments. The results are consistent with a partially mediated model in which the block of antecedents had an indirect effect on the outcomes via their influence on self-efficacy, a direct impact on the outcome measures, and a direct impact on the outcome measures. The data suggest that the relationships between self-efficacy, its precursors, and a number of outcomes of online learning are more nuanced than has previously been thought in studies. www.ijisrt.com IJISRT23JUL1538 2207 Volume 8, Issue 7, July – 2023 Volume 8, Issue 7, July – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Understanding self-efficacy in online learning is essential to improving online education, which can be a key component of academic success in distance education (Hodges, 2008). Negara et al., (2021) investigated the mathematics performance score on trigonometry and the Mathematics self-efficacy score of 75 students from one of Mataram’s institutions. The findings revealed that the majority of respondents had a high level of mathematical self-efficacy when it came to online learning. Further research reveals a link between mathematics self-efficacy and math performance. IJISRT23JUL1538 2208 www.ijisrt.com Volume 8, Issue 7, July – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 ISSN No:-2456-2165 The previously unfamiliar new normal has become standard at almost all schools and universities where classes are held entirely online, requiring us to learn essential abilities and attitudes that will enable us to adapt and succeed in an uncertain future. Understanding the contributing factors of the student’s learning process is a necessary part of improving their skills and academic performance. In determining the level of self-efficacy and academic performance of 183 out of 366 students, the study of Flores (2020) in Batangas City, Philippines, shows that increased self-efficacy also increases academic performance. According to the literature, studies in the field show that self-efficacy has a significant and positive impact on math achievement (Aksu & Guzeller, 2016; Duran & Bekdemir, 2013; Ozcal, 2019; Özcan & Kültür, 2021). Moreover, Dullas (2010) implied that self-efficacy is a good predictor of academic performance in Mathematics. Also, Hasan et al. (2014), who found a positive relationship between self-efficacy and academic performance. Kuo et al. (2014) present a case study on using a web-based videoconferencing tool (Interwise) for synchronous learning sessions in an industrial technology course offered by a university in northern Taiwan. Undergraduate students from two semesters of the same course were among those who participated in the study. They examined into students’ perceptions of interactions with the instructor and other students, their confidence in using the Internet (Internet self-efficacy), and their overall satisfaction with Interwise’s learning process. They also looked at how interactions and Internet self-efficacy affected student satisfaction. Overall, learners thought Interwise was a reasonably easy tool to use for synchronous learning, according to the findings. Interwise features like emotion icons, conversation, and raising a hand were preferred by students when interacting with their instructors.  Mathematics Performance Collins and O’Brien (2011) define mathematics performance as a student’s level of achievement in mathematics or on examinations meant to measure mastery of a subject matter. This refers to how well a person has completed specific educational goals in a school, college, or university. Examinations are frequently used to define and measure it (Cambridge University Reporter, 2003). It is critical to identify the elements that influence student performance because institutions and instructors must develop ways to improve student performance and motivate students to do better (Uyan & Gungormus, 2011). As the usage of online education grows, there is a pressing need to understand better how students learn in this new environment (Picciano, 2002). In the traditional classroom, research has shown the relevance of learning methods, but it has yet to look into what tactics are relevant and beneficial in the online learning classroom (Weinstein et al., 2000). Research shows a significant relationship between learning strategies, motivation, self-efficacy, and student’s performance in an online context (Wadsworth, L et al., 2007). The study of Uyan and Gungormus (2011) on determining the impact of eight variables, namely: gender, score in the university entrance examination, high school GPA, age, GPA, Math grade, Accounting Grade, and Attendance to students’ mathematics performance in an accounting course, shows that gender and score in mathematics university examination are not significantly correlated. Additionally, the student’s age has a statistically negative significant impact on their performance. Students’ performance in the financial accounting course is influenced by their high school grade point average, prior understanding of accounting, grade point average, attendance, and math grade. In a public national high school in Cebu, Philippines, Peteros et al. (2019) conducted descriptive correlational research between gender and self-concept on their students’ mathematics performance. Results revealed that they had a moderate level of self-concept toward learning Mathematics. There was no gender difference in the respondents’ self-concept. However, there was a strong association between self-concept and academic achievement in Mathematics. As a result, adopting and evaluating a Math performance improvement plan is strongly advised. A cluster analysis approach in Zambia by Mulinga and Marban (2020) investigated the prospective online learning mathematics activities in the age of the COVID-19 pandemic. Cluster analysis results revealed that online learning mathematics activities have significant mean differences in clustering. International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Learners expressed high confidence in gathering data and obtaining assistance via the Internet, but low confidence in resolving Internet-related issues. Learner-learner and learner-instructor interactions were both significant predictors of student satisfaction, although Internet self-efficacy had little bearing on satisfaction. According to Bandura’s four sources of self-efficacy information, Huang and Mayer (2018) investigated the effectiveness of adding four features of self-efficacy to an online statistics course. Results of analyses of variance showed that the treatment group performed better on practice (d = 0.36), retention (d = 0.39), and transfer (d = 0.42) tests as well as reporting higher self- efficacy (d = 0.44) and lower task anxiety (d = −0.45). In addition, mediation analyses revealed that task anxiety and self-efficacy fully mediated the effect of the treatment group on their performance. The findings justify the inclusion of self-efficacy features in online mathematics sessions when the goal is to improve learning outcomes by lowering anxiety and improving self-efficacy. International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 ISSN No:-2456-2165 ISSN No:-2456-2165 ISSN No:-2456-2165 Obtaining information on a student’s self-efficacy can offer the educator with more information about the student’s subsequent performance. Nuruddin et al. (2020) investigated the link between self-efficacy and mathematics performance among university students studying Applied Science. The sample consisted of first-year Applied Science university students with matriculation and diploma backgrounds. Students were given two sets of survey questionnaires created by the researchers to assess their self-efficacy and competence to solve integral calculus problems. The questions were divided into four domains: the concept of calculus, translation from concept to formula, integration techniques, and recognition of functions. This study’s findings did not provide sufficient evidence to support the view that positive self-efficacy beliefs in mathematics improve mathematics performance because there was no significant difference between what students perceived and what they actually scored. The study also showed that self–efficacy is correlated with all of the aspects of performance. C. Theoretical Framework This study draws on Social Cognitive Theory (SCT) to provide a comprehensive understanding of the relationship between students’ self-efficacy, motivation and Mathematics performance in online learning. This theory emphasizes the role of self- efficacy in predicting behavior (Bandura, 1997). SCT suggests that learners’ motivation and performance are influenced by their beliefs about their ability to perform well (self-efficacy) and the support they receive from their peers and instructors (Bandura, 1986). This theory will be used to investigate the level of students’ self-efficacy and its relationship with mathematics performance. In the context of online learning, where technology and digital resources have transformed educational practices, SCT provides a relevant framework for understanding the factors influencing students’ Mathematics performance. SCT acknowledges the interplay between cognitive, behavioral, and environmental factors in shaping behavior. The role of self-efficacy in students’ mathematics performance is very important, it reflects their confidence in understanding and solving mathematical problems, significantly impacting their engagement and persistence. It means that having greater confidence in your skills will lead to a good mathematics performance. Motivation plays a crucial role in driving students’ active participation and investment in Mathematics learning online. Moreover, observational learning and social support mechanisms are particularly relevant in the online context, where the students can learn from the success and experience of their peers and instructors. This study aims to explore the interplay between self- efficacy, motivation and Mathematics performance in this educational setting. The conceptual framework of the study in figure 1 presents the interplay of the variables in the study. It shows that motivation and self-efficacy would be measured and used to determine if those are significant predictors to students Mathematics performance in online learning mode. Fig 1 Conceptual Framework Fig 1 Conceptual Framework  Mathematics Performance Cluster 2 recorded the best performance, implying that students in this cluster exhibited excellent online learning skills for mathematics in technology-rich environments in which they will be forced to study and work in the future. www.ijisrt.com IJISRT23JUL1538 2209 Volume 8, Issue 7, July – 2023 Volume 8, Issue 7, July – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 D. Statement of the Problem f The purpose of this study is to determine the relationship between students’ mathematics performance and self-efficacy and motivation in the online learning mode of the first-year mathematics major students at Davao De Oro State College, and it would seek to answer the following questions:  What is the level of student’s self-efficacy?  What is the level of student’s motivation?  what is the level of mathematics performance of the students?  is there a significant relationship between:  What is the level of student’s self-efficacy?  What is the level of student’s motivation?  4.1. self-efficacy and mathematics performance?   4.2. motivation and mathematics performance?  5. Can motivation and self-efficacy predict the mathematics performance of the student? 2210 www.ijisrt.com IJISRT23JUL1538 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Volume 8, Issue 7, July – 2023 E. Null Hypotheses  𝐻𝑜: There is no significant relationship between motivation and self-efficacy and the mathematics performance of the students.  𝐻𝑜: There is no significant relationship between motivation and self-efficacy and the mathematics performance of the students.  𝐻𝑜: There is no significant influence between motivation and self-efficacy on the mathematics performance of the students.  𝐻𝑜: There is no significant influence between motivation and self-efficacy on the mathematics performance of the students.  Teachers The study results would inform the teachers about the design and implementation of effective instructional strategies that promote student motivation and self-efficacy in mathematics, especially in the context of online learning. Teachers can use the findings of this study to create learning materials that cater to different learning styles and preferences of their students, to incorporate motivational and self-efficacy enhancing techniques into their teaching practices.  School Administrators The study results would inform the development of policies and programs that promote effective online learning practices in mathematics education. The findings of this study may be used to evaluate and improve existing online learning programs and to design new ones that are tailored to the needs of students and teachers.  Students The finding of this study would help the students to better understand the importance of motivation and self-efficacy in mathematics learning, and how these factors can affect their performance in the subject. By knowing their own level of motivation and self-efficacy, students can develop strategies to improve their learning and achieve better outcomes in their mathematics courses. F. Scope and Delimitation of the Study This study focused on determining the relationship between motivation, self-efficacy, and mathematics performance of the selected students using universal sampling technique. It includes 104 first-year mathematics major students in all campuses of Davao De Oro State College who are currently enrolled in Mathematics of Investment subject for the first semester of 2022-2023. Moreover, the researcher wants to determine if motivation and self-efficacy can predict students’ mathematics performance, specifically in the context of online learning. G. Significance of the study The findings of the study will benefit the following; The findings of the study will benefit the following;  Students  Future Researchers. The study would serve as a basis for further investigations into the relationship between motivation, self-efficacy, and mathematics performance in different contexts and populations. The findings of this study may also provide insights into the factors that contribute to successful online learning experiences in mathematics education, which can be further explored and developed. H. Definition of terms For the purpose of understanding how they are used in the study, the following words are defined operationally of terms urpose of understanding how they are used in the study, the following words are defined operationally;  Motivation – It refers to a psychological state that drives an individual to engage in a particular behavior or activity.  Motivation – It refers to a psychological state that drives an individual to engage in a particular behavior or activity.  Self-Efficacy –  Self-Efficacy – It refers to the level of learners’ confidence in their ability to complete the learning requirements for online courses successfully.  Self Efficacy It refers to the level of learners’ confidence in their ability to complete the learning requirements for online courses successfully.  Mathematics Performance – It refers to the summative test scores obtained by the students. 2211 IJISRT23JUL1538 www.ijisrt.com Volume 8, Issue 7, July – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 A. Research Design. A. Research Design. This is a quantitative study. The research design employed was a descriptive correlational desi gathers information to assess whether there is a relationship or difference between or among specific v This study used a descriptive correlational research design since the researcher seeks to determine the student’s level of motivation, self-efficacy, and performance. Also, to determine if motivation and self-efficacy have a significant relationship and will be able to predict students’ mathematics performance in online learning. CHAPTER TWO METHODOLOGY This chapter presents the research design, locale, population and respondents of the study, research instruments, validation of the research instruments, data gathering procedure, scoring quantification of data, and the statistical treatment of the data. B. Research Locale This study was conducted in Davao De Oro where the four campuses of Davao de Oro State College (DDOSC) are located. Davao De Oro historacally known as Compostela Valley is located in the Davao region. It comprises 11 municipalities with stunning natural features such as rivers, lakes, waterfalls, caves, hot and cold springs, and mountain ranges. Davao De Oro State College (DDOSC) formerly known as Compostela Valley State College (CVSC) by RA 11575. DDOSC is a public institution of higher learning located in the southern part of the Philippines. The college has four campuses, namely: Compostela (Main), New Bataan, Montevista, and Maragusan. All of these are offering Bachelor of Secondary Education major in Mathematics program where the researcher had gathered his data from. The Compostela campus is located in the Municipality of Compostela, where the main administration building and the majority of the college’s academic program are located, it is situated in a town that boasts of its natural attractions such as the famous Aliwagwag Falls, one of the highest waterfalls in the country. The New Bataan Campus is located in the Municipality of New Bataan is described as an ecological paradise, and is home to diverse flora and fauna species. The town is also famous for having Mount Pandadagsaan which is the 3rd highest peak in the Davao region and one of the best hiking destinations of Davo de Oro. The Montevista Campus is situated in the Municipality of Montevista that features a one-stop adventure destination which is Jorge Town. A person can enjoy various activities such as a mini zoo, swan boat, zip line, and many more in just one destination. The Maragusan campus is located in the Municipality of Maragusan and is often referred to as the “Summer Capital of Davao” due to its cool climate and natural attractions such as the Tagbibinta Falls, Marangig Falls, and the Mainit hot Spring. The town is also known for its eco-tourism sites, including the Mount Candalaga Range, which is a popular hiking destination, and the many sea of clouds observation decks that bring breathtaking sceneries. Figure 2 presents the location of the study: Compostela (Main), New Bataan, Montevista, and Maragusan campus. 2212 Figure 2 presents the location of the study: Compostela (Main), New Bataan, Montevista, and Maragusan campus. IJISRT23JUL1538 www.ijisrt.com Volume 8, Issue 7, July – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 olume 8, Issue 7, July – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Fig 2 The Map of Davao De Oro Showing Location of Four Municipalities Namely Compostela, New Bataan, Montivista, and Maragusan where Each of the Campuses Of Davao De Oro State College are Located. ISSN No:-2456-2165 ISSN No:-2456-2165 ao De Oro Showing Location of Four Municipalities Namely Compostela, New Bataan, Montivista, and aragusan where Each of the Campuses Of Davao De Oro State College are Located. Fig 2 The Map of Davao De Oro Showing Location of Four Municipalities Namely Compostela, New B Maragusan where Each of the Campuses Of Davao De Oro State College are Locate C. Research Respondents The respondents of this study were the 1st year Mathematics major students who are currently taking the subject Mathematics of Investment in the 2nd semester of the Academic Year 2022-2023 in the selected school Davao De Oro State College. There are a total of 104 enrolled students of the target respondents. The samples are all the 1st year Mathematics major students using the universal sampling technique. Table 1 Subjects of the Study Campus TOTAL N % Davao De Oro State College-Compostela 30 29 Davao De Oro State College-New Bataan 26 25 Davao De Oro State College-Montevista 21 20 Davao De Oro State College-Maragusan 27 26 TOTAL 104 100 F. Research Procedures The researcher will write a formal request letter to the School research office outlining the study's purpose, objectives, and methodology. The letter will be accompanied by a copy of the research proposal, content forms, and any relevant documents required by the school. The researcher will seek ethical clearance from the school’s Research Ethics Committee. The committee will review the proposal and ensure that the study complies with ethical guidelines for research involving human participants. The research will address any concerns raised by the committee and obtain the necessary approvals before proceeding. Once permission has been granted, the researcher will set a schedule for data collection. The researcher will inform the selected schools and obtain permission from the school administrators and teachers to conduct the study. A brief orientation will be conducted with the class advisers on the nature and scope of the study, emphasizing the importance of maintaining confidentiality and the voluntary participation of the students. The participants will be informed that their participation is completely voluntary and that they may withdraw from the study at any time without consequence. The participants will also be assured that their anonymity and confidentiality will be protected throughout the study. The data collection activity will take place on the scheduled date. The class advisers will administer the research questionnaire to the students via Google Forms. The respondents will answer the research questionnaire within 1 hr and 30 minutes. After administering the said activity the researcher will ensure the privacy of the participants is protected. The data will be tabulated and analyzed statistically. The researcher will ensure that the data is anonymous and that the privacy and confidentiality of the participants are maintained throughout the data analysis and reporting stage. Finally, the researcher will prepare a report detailing the findings of the study and present it to the School Research Office. The researcher will acknowledge the contributions of the school and the participants in the study. E. Validation of the Instrument The research instrument such as Motivation, Self-efficacy, and Summative tests were presented to the researcher’s validators and research adviser for comments and suggestions. After validation, the researcher revised the instruments based on the given comments and suggestions. The summative test was syllabus based and with a Table of Specifications (TOS). The summative test was meticulously crafted and organized, it included the presentation of a TOS to ensure that the test items were distributed properly. Pilot testing of the said test questionnaire to the 2nd year mathematics students was conducted to test the reliability of the questionnaire. The sample respondents are selected who are not connected but with the same characteristics as the identified respondents. Using a suitable statistical tool, the result showed a reliability index of 0.710. D. Research Instruments The instruments used in this study were a researcher-made test and an adapted instrument. The researcher used the Academic Motivation Scale (AMS) adapted from Valerland et al. (1993) questionnaire consisting of 16 items. Each item is measured using a Likert scale of four points, ranging from strongly disagree (1) to strongly agree (4). The researcher also used the Online www.ijisrt.com 2213 IJISRT23JUL1538 2213 www.ijisrt.com Volume 8, Issue 7, July – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 International Journal of Innovative Science and Research Technology Learning Self-Efficacy Scale (OLSES) adopted from Zimmerman and Kulikowich’s (2016) questionnaire consisting of 17 items. Each item is measured using a Likert scale consisting of four points, ranging from strongly disagree (1) to strongly agree (4). The researcher-made summative test was used consisting of 45 items.  Mean  Mean This statistical tool will determine the level of students’ self-efficacy, motivation, and Mathematics Performance.  Mean This statistical tool will determine the level of students’ self-efficacy, motivation, and Mathematics Performance.  Standard deviation This will be used to measure how dispersed the data is in relation to the mean.  Standard deviation This will be used to measure how dispersed the data is in relation to the mean. G. Statistical Treatment of Data After collecting quantifiable information, the researcher will use the mean, standard deviation, and frequency distribution table to analyze the levels of measurement of motivation, self-efficacy, and mathematics performance of the students. The Pearson product-moment correlation will be employed to determine the correlation between motivation, self-efficacy, and mathematics performance. Multiple regression analysis will be applied to determine if motivation and self-efficacy can predict mathematics performance. The significance level will be set at p<0.05. All statistical analysis will be performed using the Statistical Package for the Social Sciences (SPSS).  Correlation  Correlation This statistical tool will determine the significant relationship between self-efficacy and Mathematics performance, and motivation and Mathematics performance.  Multiple Regression This is used to analyze the predictive power of self-efficacy and motivation to the students’ Mathematics performance.  Multiple Regression This is used to analyze the predictive power of self-efficacy and motivation to the students’ Mathematics performance.  Multiple Regression This is used to analyze the predictive power of self-efficacy and motivation to the students’ Mathematics performance. IJISRT23JUL1538 CHAPTER THREE RESULTS The data that were acquired using the study’s research instruments are presented in this chapter. It also provides the statistical analysis and the interpretation of data to answer the problems stated in the problems of the study. A. The Level of Students’ Self-efficacy The table below shows the level of self-efficacy of the students. A. The Level of Students Self efficacy The table below shows the level of self-efficacy of the students. Table 2 Level of Students’ Self-efficacy Items Weighted Mean Descriptive Rating I am confident in my ability to navigate online course materials efficiently 2.64 Agree I believe that I can communicate effectively with technical support via e-mail, telephone, or live online chat. 2.75 Agree I am confident that I can submit assignments to an online Dropbox. (i.e., google drive) 2.94 Agree I am confident in my ability to overcome technical difficulties on my own. 2.71 Agree I believe that I can navigate the online grade book. (i.e., scores or grades in google forms quizzes) 2.66 Agree I am confident that I can manage time effectively. 2.68 Agree I believe that I can complete all assignments on time. 2.86 Agree I am confident in my ability to learn to use a new type of technology efficiently 2.86 Agree I believe that I can learn without being in the same room as the instructor 2.55 Agree 0. I am confident that I can learn without being in the same room as other students. 2.63 Agree 1. I believe that I can search the Internet to find the answer to a course-related question. 2.77 Agree 2. I am confident that I can meet deadlines with very few reminders. 2.72 Agree 3. I believe that I can complete a group project entirely online. 2.58 Agree 4. I am confident that I can use synchronous technology to communicate with others (i.e., messenger, google meet, zoom). 2.86 Agree 5. I believe that I can focus on schoolwork when faced with distractions. 2.47 Agree 6. I am confident that I can develop and follow a plan for completing all required work on time. 2.72 Agree 7. I believe that I can promptly ask questions in the appropriate forum (e-mail, google classroom, discussion board, etc.) when a problem arises. 2.65 Agree Overall Weighted Responses 2.71 Agree Items Table 2 shows the level of students’ self-efficacy in online learning. www.ijisrt.com 2214 Volume 8, Issue 7, July – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 CHAPTER THREE RESULTS “I am confident that I can submit assignments to an online Dropbox. (i.e., google drive)” has the highest mean of 2.94 and has a descriptive rating of “Agree”. This means that the students are familiar with and have sufficient knowledge of using this platform when submitting tasks online. On the other hand, the “I believe that I can focus on schoolwork when faced with distractions” has the lowest mean of 2.47 and has a descriptive rating of “Agree”. This shows that only some students are confident that they can focus on their school works online due to the many distractions that online learning may bring such as social media, online games, and many more. The overall mean is 2.71 which has a descriptive rating of “Agree”. This mean score suggests that the students have a moderate level of self-efficacy in online learning. Based on the result, the majority of the students have a moderate level of self- efficacy. It means that they have some confidence in their abilities related to the various situations and feel moderately capable in online learning tasks. In addition, It implies that they require occasional support and guidance. Also, it entails that there is a need to increase their level of self-efficacy. Similar results from the study on the level of self-efficacy in online learning have been reported by Ali (2021). In the study, it is said that online learners had a moderate level of self-efficacy and suggested that to improve students’ self-efficacy a course in ICT may be offered to all online learners as a prerequisite for any online course. As online learning is student-centered and self-directed, it is necessary for students to improve their self-efficacy to perform well in online learning. There are studies for measuring self-efficacy in online learning. The study of Lai et al (2021) shows that students with a higher level of self-efficacy are more likely to experience learning satisfaction. IJISRT23JUL1538 2215 www.ijisrt.com International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Volume 8, Issue 7, July – 2023 Volume 8, Issue 7, July – 2023 B. The Level of Students’ Motivation Data on the level of students’ motivation are presented in the Table below. Table 3 Level of Students’ Motivation Items Weighted Mean Descriptive Rating . CHAPTER THREE RESULTS I really feel that I am wasting my time in school 3.54 Strongly Disagree . I once had good reasons for going to school; however, now I wonder whether I should continue 3.17 Agree . I cannot see why I go to school and frankly, I could not care less. 3.38 Agree . I cannot understand what I am doing in school 3.46 Agree . I believe that without good grades in Mathematics, I will not be able to find a high-paying job. 2.71 Agree . In order to obtain a more prestigious job later on. 2.73 Agree . I want to have “the good life” later on. 3.40 Agree . I want to prove to myself that I am capable of completing my college degree. 3.39 Agree . I feel important when I succeed in school. 3.29 Agree 0. It is to show myself that I am an intelligent person. 2.66 Agree 1. I want to show myself that I can succeed in my studies. 3.35 Agree 2. This will help me make a better choice regarding my career orientation. 3.13 Agree 3. Eventually, it will enable me to enter the job market in a field that I like. 3.02 Agree 4. I believe that my college education will improve my competence as a worker. 3.28 Agree 5. I experience pleasure and satisfaction while learning new things. 3.13 Agree 6. I experience pleasure and satisfaction in broadening my knowledge about Mathematics. 3.10 Agree Overall Weighted Responses 3.17 Agree As presented in Table 3, “I really feel that I am wasting my time in school” has the highest mean of 3.54 which has descriptive of “Strong Disagree”. It implies that students think that attending school is a worthwhile and beneficial thing to do. The statement “I believe that without good grades in Mathematics, I will not be able to find a high-paying job” has the lowest mean and has a descriptive rating of “Agree”. It means that some of the students believe that it is necessary to have good grades to find high-paying jobs in the future. The responses gave an overall mean of 3.17 which has a descriptive rating of “Agree”. This suggests that the students possess a moderate level of motivation in their academic pursuits. In addition, this means that the students generally display an average level of interest and engagement. CHAPTER THREE RESULTS Almost the same result was found by Basar et al. (2021) that students motivation in online learning was low. This shows that students require additional support or strategies to enhance their motivation. According to Ozer & Badem (2022) who exclaimed that students who have high motivation tend to have higher grades. This is because they are more engaged in the learning process, especially in an online platform. Hence, they were also able to grasp the content of the subject matter, regardless of the challenges of online learning. Moreover, the study of Aduayi-Akue et al., (2017) also supports this claim stating that motivation has a significant effect on students’ performance in online learning. Motivation plays a significant role in education, especially in increasing the performance of the learners. C. The Level of Students’ Mathematics Performance The table below shows the level of students’ Mathematics performance in a summative test. Table 4 Level of Students’ Mathematics Performance Mean Standard Deviation Proficiency Descriptive rating Summative test 24.43 10.51 54.29% Did Not Meet Expectations C. The Level of Students’ Mathematics Performance The table below shows the level of students’ Mathematics performance in a summative test. C. The Level of Students’ Mathematics Performance The table below shows the level of students’ Mat Table 4 Level of Students’ Mathematics Performance Mean Standard Deviation Proficiency Descriptive rating Summative test 24.43 10.51 54.29% Did Not Meet Expectations Table 4 describes the students’ average score of 24.3 and proficiency level of 54.29% this implies that, on average, the students attained half of the total possible score. It suggests that the students did not meet the expected level of proficiency in the Mathematics subject. This shows that students may have faced various challenges in learning mathematics in online learning. Bringula and colleagues (2021) found that internet connectivity and power outages are the most problematic aspects of online learning. Furthermore, Mamolo (2022) said that students are struggling to learn in this online learning context because they lack the motivation to do self-study and have a lot of household activities and chores. These issues make it tough for students to learn. IJISRT23JUL1538 2216 www.ijisrt.com Volume 8, Issue 7, July – 2023 International Journal of Innovative Science and Research Technology International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Nevertheless, Gürsul and Keser (2009) found that students perform better in online learning when compared to face-to-face. CHAPTER THREE RESULTS As online learning will be permanently integrated into the education system it is necessary to address the challenges to help improve the teaching-learning process in this type of learning modality. D. The Significant Relationship between Motivation and Mathematics Performance  The Significant Relationship between Self-Efficacy and Mathematics Performance Th bl b l h h l i l i f S lf Effi d M h i P  The Significant Relationship between Self-Efficacy and Mathematics Performance The table below shows the correlation analysis of Self-Efficacy and Mathematics Performance. The table below shows the correlation analysis of Self-Efficacy and Mathematics Performance. Table 5 Significant Relationship between Self-Efficacy and Mathematics Performance Variables Correlation Coefficient P-value Remarks Self-efficacy .020 .838 Not Significant Mathematics Performance Table 5 revealed a negligible positive relationship between self-efficacy and Mathematics performance (r = .020, p =.838 ). The magnitude of the r suggests a negligible relationship between these variables. Furthermore, the p-value of .838 indicates that the observed correlation is not statistically significant. Contrary to the initial hypothesis, the results of the correlation analysis did not support a significant relationship between self-efficacy and Mathematics performance. The weak and non-significant correlation suggests that the level of self-efficacy among the students’ had minimal influence on their Mathematics performance in online learning. These findings align with some previous studies that reported weak or inconsistent associations between self-efficacy and Mathematics performance. According to Mijares III (2022), students’ mathematics performance is not significantly affected by self-efficacy. The same result with the correlation between motivation and mathematics performance, it is fascinating since it contradicts the vast majority of academic research that asserts motivation has a significant relationship on students’ mathematics performance (Wang et al., 2017; Kou et al., 2020; Flores, 2020; Aksu & Guzeller, 2016; Delioglu, 2017; Duran & Bekdemir, 2013). Whatever the outcome, it is also necessary to improve students’ self-efficacy during online learning, as supported by much literary evidence. According to Watsons (2015), students with higher self-efficacy are more successful in mathematics because they can perform better cognitively, have more motivation to continue in the face of difficulties, have less math anxiety, and are more likely to study mathematics. The table below shows the correlation analysis of Motivation and Mathematics Performance. The table below shows the correlation analysis of Motivation and Mathematics Performance. CHAPTER THREE RESULTS Table 6 Significant Relationship between Motivation and Mathematics Performance Variables Correlation Coefficient P-value Remarks Motivation .074 .454 Not Significant Mathematics Performance Table 6 revealed a negligible positive relationship between motivation and Mathematics performance (r = .074, p=.454). The magnitude of the r suggests a negligible relationship between these variables. Furthermore, the p-value of .454 indicates that the observed correlation is not statistically significant. In contrast to the first hypothesis, the results of the correlation analysis did not support a significant relationship between motivation and mathematics performance. The weak and non-significant correlation suggests that the level of motivation among the students had minimal influence on their Mathematics performance in online learning. These findings align with some previous studies that reported weak or inconsistent associations between motivation and mathematics performance. Mijares III (2022) asserts that motivation does not significantly affect students’ mathematics performance. It is intriguing since it goes against the great majority of academic studies that claim motivation has a big influence on students’ performance (Schunk et al., 2008; Paas et al., 2005; Ugwuanyi et al., 2020). Regardless of the result, it is still necessary to improve learners’ motivation to help students engage in the online classroom. Also, the importance of good teachers in encouraging student motivation is one of the key factors to having a successful academic performance. E. The Self-efficacy and Motivation as Predictors of Students’ Mathematics Performance E. The Self-efficacy and Motivation as Predictors of Students’ Mathematics Performanc f ff y f f The table below shows the Multiple regression analysis results to determine the predictive power of self-efficacy and motivation to the students’ Mathematics performance. The table below shows the Multiple regression analysis results to determine the predictive pow motivation to the students’ Mathematics performance. IJISRT23JUL1538 www.ijisrt.com 2217 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Volume 8, Issue 7, July – 2023 Table 7 Self-Efficacy and Motivation as Predictors of Students’ Mathematics Performance Model R R Square Adjusted R square F Sig. 1 .080 .006 -.013 .329 .720 Note: 𝛼= 0.05 The above table revealed a negligible positive correlation (R=.080) between self-efficacy, motivation, and Mathematics performance. The coefficient of determination (R Square) indicated that .6% of the variance in Mathematics performance could be explained by the predictors. However, after adjusting for the number of predictors, the variance in Mathematics performance was not meaningful. Furthermore, the F-test results showed that the model as a whole was not statistically significant (F=.329, p=.720) at the alpha level of .05. le below was the result of multiple regression analysis it provides information about the individual nd their relationship with the dependent variable. The coefficient table below was the result of multiple regression analysis it provides information about the individual predictors in the model and their relationship with the dependent variable. Table 8 Self-Efficacy and Motivation as Predictors of Students’ Mathematics Performance Coefficient Table Model Unstandardized Coefficients Standardized Coefficients t Sig. B Std. Error Beta 1 constant 19.547 7.791 2.509 .014 Motivation 2.308 2.938 .098 .786 .434 Self-efficacy -.900 2.873 -.039 -.313 .755 Dependent Variable: Mathematics Performance Regarding the predictor variables presented in the table above, it showed that motivation had a non-significant relationship with Mathematics performance (B=2.308, t=.786, p=.434). Similarly, self-efficacy also exhibited a non-significant relationship with Mathematics performance (B=-.900, t=-.313, p=.755). The result shows that while self-efficacy and motivation have a negligible relationship with Mathematics performance, they may not be strong predictors in online learning. The low adjusted R square value indicates the addition of self-efficacy and motivation to the model did not significantly contribute to explaining the variance in Mathematics performance. However, this still shows that self-efficacy and motivation contribute to online learning lowly. E. The Self-efficacy and Motivation as Predictors of Students’ Mathematics Performanc Özcan and Gümüş (2019) show that mathematics motivation predicts mathematics self-efficacy which in turn predicts retrospective metacognitive experience i.e,., students' narrative of their metacognitive activities after solving a mathematics task. Understanding self-efficacy in online learning is essential to improving online education, which can be a key component of academic success in distance education (Hodges, 2008). 2218 IJISRT23JUL1538 www.ijisrt.com Volume 8, Issue 7, July – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 CHAPTER FOUR DISCUSSION AND CONCLUSION This chapter presents the summary of findings of the study, conclusions and recommendations of the conducted study. A. Summary of Findings The results of the data analysis and interpretation were as follows: A. Summary of Findings The results of the data analysis and interpretation were as follows:  The students’ self-efficacy was at a moderate level.  The students’ self-efficacy was at a moderate level.  The students’ motivation was at a moderate level.  The students’ mathematics performance did not meet the level of expected proficiency level.  The students’ mathematics performance did not meet the level of expected proficiency level.  There is a negligible relationship between the level of students’ self-efficacy and mathematics performance.  There is a negligible relationship between the level of students’ self-efficacy and mathematics performance.  There is a negligible relationship between the level of students’ self-efficacy and mathematics perform  There is a negligible relationship between the level of students’ motivation and mathematics performan  The level of students’ self-efficacy and motivation were not significant predictors of students’ mathematics performance  The level of students’ self-efficacy and motivation were not significant predictors of students’ mathematics performance.  The level of students’ self-efficacy and motivation were not significant predictors of students’ mathematics performance. C. Recommendations C. Recommendations C. Recommendations Based on the conclusions derived from the study, the following recommendations are hereby presented: Based on the conclusions derived from the study, the following recommendations are hereby presented:  There is a need to improve students’ self-efficacy and motivation. Students should develop their own personal strategies t improve self-efficacy and motivation in learning Mathematics in an online learning mode.  There is a need to improve students’ self-efficacy and motivation. Students should develop their own personal strategies to improve self-efficacy and motivation in learning Mathematics in an online learning mode.  Teachers should design and implement an effective strategy that can improve students’ motivation and self-efficacy in Mathematics, especially in the context of online learning. Teachers should create learning materials that can cater to different learning styles and preferences of the students, to incorporate motivational and self-efficacy-enhancing techniques into their teaching practices.  Teachers should design and implement an effective strategy that can improve students’ motivation and self-efficacy in Mathematics, especially in the context of online learning. Teachers should create learning materials that can cater to different learning styles and preferences of the students, to incorporate motivational and self-efficacy-enhancing techniques into their teaching practices.  The school administrator should develop policies and programs that promote effective online learning practices in Mathematics education. Based on the results, they should revisit the existing online learning programs to cater the needs of students and teachers.  The school administrator should develop policies and programs that promote effective online learning practices in Mathematics education. Based on the results, they should revisit the existing online learning programs to cater the needs of students and teachers.  This study is recommended for future researchers as a basis to investigate further the relationship between motivation and self- efficacy toward Mathematics performance in different contexts and populations. This study also provides insights into the factors that contribute to successful online learning experiences in Mathematics, which can be further explored and developed.  This study is recommended for future researchers as a basis to investigate further the relationship between motivation and self- efficacy toward Mathematics performance in different contexts and populations. This study also provides insights into the factors that contribute to successful online learning experiences in Mathematics, which can be further explored and developed. 2219 B. Conclusion World Health Organization chief declares the end to Covid-19 as a global health emergency, this virus changes the education system globally. To continue education one of the learning modes in education is online learning during the time of pandemic. Though in the present time, most schools transitioned back to face-to-face classes, still online learning is the new alternative way of delivering instructions. In fact, some of the schools also remained in a full online learning mode. Thus, the findings of this study are very important. This will be an essential help for the school administrators to revisit their curriculum and course syllabi in adjusting to the new normal. Through this, students may be able to adapt whenever there is an urgent need to switch back to online learning vis-à-vis. In summary, the student’s level of self-efficacy and motivation was at a moderate level. Students had moderate levels of self- efficacy and motivation to perform academic tasks in an online learning environment. The study concluded that both motivation and self-efficacy have a negligible relationship and are not statistically significant. Also, motivation and self-efficacy were not significant predictors of students’ Mathematics performance. C. Recommendations Based on the conclusions derived from the study, the following recommendations are hereby presented: IJISRT23JUL1538 www.ijisrt.com International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Volume 8, Issue 7, July – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 REFERENCES Bringula, R., Reguyal, J., Tan, D., & Ulfa, S. (2021). Mathematics self-concept and challenges of learners in an online learning environment during COVID-19 pandemic. Smart Learning Environments. [14]. Cabansag, P. D., Cabansag, V. C., & Soriano, R. B. (2020). Students' Motivation toward Onlin Making. Asian Society of Teachers for Research, Inc. [15]. Çalışkan, H., & Kılınç, G. (2002). The Relationship Between the Learning Styles of Students a Social Studies Course. Elsevier Ltd. Selection. doi: 10.1016/j.sbspro.2012.09.476 . j p [16]. Capinding, A. T. (2022). Impact of Modular Distance Learning on High School Students Mathematics Motivation, I t t/Attit d A i t d A hi t d i th COVID 19 P d i E J l f Ed ti R h j p [16]. Capinding, A. T. (2022). Impact of Modular Distance Learning on High School Students Mathematics Motivation, Interest/Attitude, Anxiety and Achievement during the COVID-19 Pandemic. European Journal of Education Research. [17]. Cepada, C. M., & Grepon, B. G. (2020). ABSENTEEISM AND PARENTAL INVOLVEMENT IN HOME AND SCHOOL AMONG MIDDLE SCHOOL STUDENTS OF PUBLIC SCHOOLIN NORTHERN MINDANAO, PHILIPPINES: BASIS FOR INTERVENTION. Asian Journal of Education. https://doi.org/10.5281/zenodo.4459797. p g [18]. Chukwuemeka, O. (2013). Environmental Influence on Academic Performance of Secondary School Students in Port Harcourt Local Government Area of Rivers State. Journal of Economics and Sustainable Development. p [19]. Collins III, J. W., & O'Brien, N. P. (2003). The Greenwood Dictionary of Education. Santa Barbara, California: Greenwood Press. [20]. Dullas, A. (2010). Academic Performance and Self-Efficacy of Filipino Science High School Students on Mathematics and English Subjects. Available at SSRN: https://ssrn.com/abstract=2152791. [21]. Duran, M., & Bekdemir, M. (2013). Evaluation of visual math literacy self efficacy perception with visual mathematics accomplishment. Pegem Eğitim ve Öğretim Dergisi. 5). In the mind of the actor: The structure of adolescents’ achievement task values and expectancy-related y and Social Psychology Bulletin, 260–267. [22]. Eccles, J. &. (1995). In the mind of the actor: The structure of adolescents’ achievement task value beliefs. Personality and Social Psychology Bulletin, 260–267. y y gy , [23]. Elliot, Α. H. (2005). Achievement goals, performance contingencies, and performance attainment: An experimental test. Journal of Educal Psychology, 630–640. y gy [24]. Ercan Yilmaz, M. S. (2017). Variables Affecting Student Motivation Based on Academic. Journal of Education and Practice , 112-120. [25]. Ferreira, M., Cardoso, A. P., & Abrantes, J. L. (2011). REFERENCES [1]. Aduayi-Akue, J., Lotchi, K., Parveen, S., Onatsu, T., & Pehkonen-Elmi, T. (2017). Motivation of Online Learners. Pedagogy in practice. g gy p [2]. Aksu, G., & Güzeller, C. O. (2016). Classification of PISA 2012 mathematical literacy scores using Decision-Tree Method: Turkey sampling. Egitim ve Bilim. y p g g [3]. Albay, E. M. (2020). Towards a 21st Century Mathematics Classroom: Investigating the Effects of the Problem-Solving Approach Among Tertiary Education Students. Asia-Pacific Social Science Review. Albay, E. M. (2020). Towards a 21st Century Mathematics Classroom: Investigating the Effects of the A h A T i Ed i S d A i P ifi S i l S i R i E. M. (2020). Towards a 21st Century Mathematics Classroom: Investigating the Effects of the Proble ch Among Tertiary Education Students Asia-Pacific Social Science Review Albay, E. M. (2020). Towards a 21st Century Mathematics Classroom: Investigating the Effects of the Approach Among Tertiary Education Students. Asia-Pacific Social Science Review. [4]. Ali, S. (2021). E-Learners’ Self-efficacy for Online Courses: Self-efficacy for IT Use as a Predictor for Academic Self- efficacy. Pakistan Journal of Distance & Online Learning . [5]. Alquarashi, E. (2016). Self-Efficacy In Online Learning Environments: A Literature Review. USA: Contemporary Issues in Education Research. [6]. Anderman, E. G. (2013). Motivation and classroom learning. Handbook of psychology, 99–116. [7]. Bandura, A. (1997). Self-efficacy: The exercise of control. New York: Freeman. [8]. Basar, Z. M., Mansor, A. N., Jamaludin, K. A., & Alias, B. S. (2021). The Effectiveness and Challenges of Online Learning for Secondary School Students - A Case Study. Asian Journal of University Education. [9]. Bates, R., & Khasawneh, S. (2007). Self-efficacy and college students' perceptions and use of online learning systems. United Kingdom: Elsevier Science, Oxford. http://dx.doi.org/10.1016/j.chb.2004.04.004. [10]. Bayraktar, H. V. (2015). Student Motivatıon In Classroom Management And Factors That Affect Motivation. Turkish Studies International Periodical For The Languages, Literature and History of Turkish or Turkic, 1079-1100. [11]. Brachtl, S., Ipser, C., Aschenberger, F., Oppl, S., Oppl, S., Pakoy, E., & Radinger, G. (2023). Physical home-learning environments of traditional and non-traditional students during the COVID pandemic: exploring the impact of learning space on students’ motivation, stress and well-being. Smart Learning Environments. p , g g [12]. Brennan. (2019). Diversity of Online Behaviours Associated with Physical Attendance in Lectures. Journal of Learning Analytics. http://learning-analytics.info/journals/index.php/JLA/. y p g y j p p [13]. REFERENCES Kuo, Y.-C., Tseng, H., & Kuo, Y.-T. (2020). Internet Self-Efficacy, Self-Regulation, and Student Performance: African- American Adult Students in Online Learning. Waynesville, NC USA: Association for the Advancement of Computing in Education (AACE). https://www.learntechlib.org/primary/p/181355/. [44]. Kuo, Y.-C., Tseng, H., & Kuo, Y.-T. (2020). Internet Self-Efficacy, Self-Regulation, and Student Performance: African- American Adult Students in Online Learning. Waynesville, NC USA: Association for the Advancement of Computing in Education (AACE). https://www.learntechlib.org/primary/p/181355/. [45]. Kuo, Y.-C., Walker, A. E., Belland, B. R., Schroder, K. E., & Kuo, Y.-T. (2014). A Case Study of Integrating Interwise: Interaction, Internet Self-Efficacy, and Satisfaction in Synchronous Online Learning Environments. Taiwan: THE INTERNATIONAL REVIEW OF RESEARCH IN OPEN AND DISTANCE LEARNING. http://www.irrodl.org/index.php/irrodl/article/view/1664. p g p p [46]. Lai, C. S., Au, K. M., & Low, C. S. (2021). Beyond conventional classroom learning: Linking emotions and self-efficacy to academic achievement and satisfaction with online learning during the COVID-19 pandemic. Journal of Education and e-Learning Research. g [47]. Lazim, M. A. (2004). The Statistical Efidence in Describing the Students' Beliefs about Mathematics. Int. J. Math. Teach. Learn. [48]. Lee, J.-K., & Hwang, C.-Y. (2007). The effects of computer self-efficacy and learning management system quality on e- Learner's satisfaction. Proceedings of the 2007 European LAMS Conference: designing the future of learning. [49]. Lim, C. K. (2001). Computer self-efficacy, academic self-concept, and other predictors of satisfaction and future participation of adult distance learners. American Journal of Distance Education. . doi:10.1080/08923640109527083. [50]. Mamolo, L. A. (2022). Online Learning and Students' Mathematics Motivation, Self-Efficacy, and Anxiety in the "new Normal". Education Research International. [51]. Martin, F., & Tutty, J. I. (2008). Learning Management System Self-Efficacy of online and hybrid learners: Using LMSES Scale. Paper presented at the Proceeding of the UNC Teaching and Learning with Technology Conference, Raleigh, NC. [52]. Martin, F., Tutty, J. I., & Su, Y. (2010). Influence of Learning Management Systems Self-Efficacy on E-Learning Performance. Journal on School Educational Technology. [53]. McCune, V. &. (2011). Cultivating the disposition to understand in 21st century university education. Learning and Individual Differences, 303-310. 5 ij [54]. Mijares III, B. (2022). SCHOOL HEADS' INSTRUCTIONAL SUPERVISIONS AND ITS IMPACT ON TEACHERS' JOB SATISFACTION View project Research Ouput View project. Phsychology and Education: A Multidisciplinary Journal. [55]. Motevalli, S. P. (2020). Motivating Students to Learn: An Overview of Literature in Educational Psychology. International Journal of Academic Research in Progressive Education & Development, 63–74. [56]. REFERENCES Motivation and relationship of the student with the school as factors involved in the perceived learning. Procedia - Social and Behavioral Sciences. [26]. Flores, I. M. (2020). Self-Efficacy and Mathematics Performance of Students’ in the New Normal in Education. Batangas City, Philippines: World Journal of Educational Research: www.scholink.org/ojs/index.php/wjer. [27]. Flores, I. M. (2020). Self-Efficacy and Mathematics Performance of Students’ in the New Normal in Education. World Journal of Educational Research. n-Zvi, D. (2009). Helping Students Develop Statistical Reasoning: Implementing a Statistical Reasoning ment. Teaching Statistics. [28]. Garfield, J., & Ben-Zvi, D. (2009). Helping Students Develop Statistical Reasoning: Implementin Learning Environment. Teaching Statistics. g g [29]. Gürsul, F., & Kesser, H. (2009). The effects of online and face to face problem based learning environments in mathematics education on student's academic achievement. Procedia - Social and Behavioral Sciences. [30]. Hannula, M. S. (2004). Regulation motivation in mathematics. 10th International Congress on Mathematical Education. IJISRT23JUL1538 2220 www.ijisrt.com Volume 8, Issue 7, July – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 International Journal of Innovative Science and Research Technology [31]. Hasan, M. Z., Hasan, B., Tareq, M., Hossain, B., & Islam, M. A. (2014). Factors Affecting Self-Efficacy Towards Academic Performance: A Study on Polytechnic Students in Malaysia. Advances in Environmental Biology. y y y gy [32]. Hiller, S. E., Kitsantas, A., Cheema, J. E., & Poulou, M. (2022). Mathematics anxiety and self-efficacy as predictors of mathematics literacy. International Journal of Mathematical Education in Science and Technology. [32]. Hiller, S. E., Kitsantas, A., Cheema, J. E., & Poulou, M. (2022). Mathematics anxiety and self-efficacy as predictors of mathematics literacy. International Journal of Mathematical Education in Science and Technology. y gy [33]. Hodges, C. B. (2008). Self-efficacy in the context of online learning environments: A review of the literature and directions for research. Performance Improvement Quarterly, 20(3-4), 7-25. doi:10.1002/piq.20001. [33]. Hodges, C. B. (2008). Self-efficacy in the context of online learning environments: A review of the literature and directions for research. Performance Improvement Quarterly, 20(3-4), 7-25. doi:10.1002/piq.20001. [34]. Huang, X., & Mayer, R. E. (2018). Adding Self-Efficacy Features to an Online Statistics Lesson. Journal of Educational Computing Research. https://doi.org/10.1177/0735633118771085. [34]. Huang, X., & Mayer, R. E. (2018). Adding Self-Efficacy Features to an Online Statistics Lesson. Journal of Educational Computing Research. https://doi.org/10.1177/0735633118771085. p g p g [35]. Hutajulu, M. W. (2019). REFERENCES The effect of mathematical disposition and learning motivation on problem solving: an analysis. Infinity Journal, 229-238. y [36]. Aduayi-Akue, K. L.-E. (2017). Motivation of online learners evolving pedagogy. y g p g gy [37]. Jan, S. K. (2015). The Relationships Between Academic Self-Efficacy, Computer Self-Efficacy, Prior Experience, and Satisfaction With Online Learning. American Journal of Distance Education. doi:10.1080/08923647.2015.994366. [38]. Jang, H. (2008). Supporting Students’ Motivation, Engagement, and Learning During an Uninteresting Activity. Journal of Educational Psychology, 798–811. [39]. Joo, Y. J., Bong, M., & Choi, H. J. (2000). Self-efficacy, self-regulation and performance in web-based instruction. Educational Technology Research and Development. doi:10.1007/BF02313398. [39]. Joo, Y. J., Bong, M., & Choi, H. J. (2000). Self-efficacy, self-regulation and performance in web-based instruction. Educational Technology Research and Development. doi:10.1007/BF02313398. [40]. K.C. Williams, C. W. (2011). Five Key Ingredients for Improving Student Motivation. es Journal, 1-23. . Williams, C. W. (2011). Five Key Ingredients for Improving Student Motivation. esearch in High rnal, 1-23. [41]. Keller, J. (2008). An integrative theory of motivation, volition, and performance. Cognition and Learning, 175–185. [41]. Keller, J. (2008). An integrative theory of motivation, volition, and performance. Cognition and Learning, 175–185. [42]. Kim, C., Park, S., & Cozart, J. (2014). Affective and motivational factors of learning in online mathematics courses. British [41]. Keller, J. (2008). An integrative theory of motivation, volition, and performance. Cognition and Learning, 175–185. [42]. Kim, C., Park, S., & Cozart, J. (2014). Affective and motivational factors of learning in online mathematics courses. British J l f Ed i l T h l ( ) g y p g g [42]. Kim, C., Park, S., & Cozart, J. (2014). Affective and motivational factors of learning in online mathematics courses. British Journal of Educational Technology. [43] KOON A N (2016) CASE STUDY FACTORS INFLUENCING STUDENT’S MOTIVATION IN LEARNING MUFY [42]. Kim, C., Park, S., & Cozart, J. (2014 Journal of Educational Technology. [42]. Kim, C., Park, S., & Cozart, J. (2014). Affective and motivational factors of learning in online mathematics courses. British Journal of Educational Technology. [43]. KOON, A. N. (2016). CASE STUDY: FACTORS INFLUENCING STUDENT’S MOTIVATION IN LEARNING MUFY J l f Ed ti d S i l S i 72 82 Journal of Educational Technology. Journal of Educational Technology. [43]. KOON, A. N. (2016). CASE STUDY: FACTORS INFLUENCING STUDENT’S MOTIVATION IN LEARNING MUFY . Journal of Education and Social Sciences, 72-82. [44]. REFERENCES Motevalli, S., Perveen, A., & Tresa Anak Michael, M. (2020). Motivating Students to Learn: An Overview of Literature in Educational Psychology. International Journal of Academic Research in Progressive Education and Development. [57]. Mulenga, E. M., & Marban, J. M. (2020). Prospective Teachers' Online Learning Mathematics Activities in the Age of COVID-19: A Cluster Analysis Approach. EURASIA J Math Sci Tech Ed, 2020 - Volume 16 Issue 9. https://doi.org/10.29333/ejmste/8345. [58]. Mun, Y. S., & Sam, T. L. (2022). ONLINE LEARNING MOTIVATION DURING COVID-19 PANDEMIC: THE ROLE OF LEARNING ENVIRONMENT, STUDENT SELF-EFFICACY AND LEARNER-INSTRUCTOR INTERACTION. Malaysian Journal of Learning and Instruction. IJISRT23JUL1538 2221 www.ijisrt.com Volume 8, Issue 7, July – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 International Journal of Innovative Science and Research Technology [59]. Murphy, E., & Rudriguez-Manzanares, M. (2009). Teachers’ Perspectives on Motivation in High School Distance Education. Journal of Distance Education. [60]. Negara, H. R., Nurlaelah, E., Wahyudin, Herman, T., & Tamur, M. (2021). Mathematics self-efficacy and mathematics performance in online learning. Indonesia: Journal of Physics: Conference Series:Conf. Ser. 1882 012050. [61]. Nieuwoudt, J. E. (2020). Investigating synchronous and asynchronous classattendance as predictors of academic success in online. Australia: Australasian Journal of Educational Technology. https://doi.org/10.14742/ajet.5137. [62]. Nurrudin, M., Noor, C. N., & Abidin, A. W. (2020). Exploring the Relationship between Self-efficacy and Mathematics Performance in Integral Calculus among Applied Science University Students. Shah Alam, Malaysia: Journal of Physics: Conference Ser. 1496 012018. [63]. Nyman, M. S. (2019). The issue of ‘proudliness’: Primary students’ motivation towards mathematics. LUMAT: International Journal on Math, Science and Technology Education, 81-96. [64]. Osborne, J., Simon, S., & Collins, S. (2003). Attitude towards science: a review of the literature and its implications. International Journal Science Education. [65]. Özcan, B., & Kültür, Y. Z. (2021). The Relationship Between Sources of Mathematics Self-Efficacy and Mathematics Test and Course Achievement in High School Seniors. SAGE Open. [66]. Özcan, Z. Ç., & Gümüş, A. E. (2019). A modeling study to explain mathematical problem-solving performance through metacognition, self-efficacy, motivation, and anxiety. Australian Journal of Education. [67]. Ozer, O., & Badem, N. (2022). Student Motivation and Academic Achievement in Online EFL Level. Language Education and Acquisition Research Network . [68]. Ozkal, N. (2019). Relationships between self-efficacy beliefs, engagement and academic performance in math lessons. Cypriot Journal of Educational Sciences. yp [69]. Paas, F. T. (2005). REFERENCES A motivational perspective on the relation between mental effort and performance: Optimizing learner involvement in instruction. ETR&D, 25–34. [70]. Peteros, E., Gamboa, A., Etcuban, J. O., Dianauanao, A., Sitoy, R., & Arcadio, R. (2019). Factors Affecting Mathematics Performance of Junior High School. Cebu City, Philippines: INTERNATIONAL ELECTRONIC JOURNAL OF MATHEMATICS EDUCATION. https://doi.org/10.29333/iejme/5938. p g j [71]. Picciano, A. G. (2002). Beyond Student Perceptions: Issues of Interaction, Presence, and Performance in an Online Course. Journal of Asynchronous Learning Network. [72]. Pintrich, P. (2003). A motivational science perspective on the role of student motivation in learning and teaching contexts. Journal of Educational Psychology, 667–686. y gy [73]. Rapposelli, J. A. (2014). The Correlation between Attendance and Participation with respect to student Achievement in an Online Learning. United States: ProQuest LLC. g [74]. Reporter, C. U. (2013). Indicators of academic performance. p p [75]. Rifandi, R. (2013). Improving Students’ Motivation in Learning Mathematics By Using Contextual Teaching Strategies. 1- 10. [76]. Rodr´ıguez-Manzanares, E. M. (2009). Teachers’ perspectives on motivation in high school distance education. The Journal of Distance Education, 1-24. [77]. Ryan, R. M. (2000). Self-determination theory and the facilitation of intrinsic motivation, social development, and well- being. American Psychologist, 68-78. [77]. Ryan, R. M. (2000). Self-determination theory and the facilitation of intrinsic motivation, social development, and well- being. American Psychologist, 68-78. [78] Saeed S & Zyngier D (2012) How Motivation Influences Student Engagement: A Qualitative Case Study Journal of y g Zyngier, D. (2012). How Motivation Influences Student Engagement: A Qualitative Case Study. Journa Learning. g y g [78]. Saeed, S., & Zyngier, D. (2012). How Motivation Influences Student Engagement: A Qualitative Education and Learning. g [79]. Santibañez, L., & Guarino, C. (2020). The Effects of Absenteeism on Academic and Social-Emotional Outcomes: Lessons for COVID-19. Policy Analysis for California Education. https://doi.org/10.3102%2F0013189X21994488. [80]. Sartawi, A., Alsawaie, O. N., Dodeen, H., Tibi, S., & Alghazo, I. M. (2012). Predicting Mathematics Achievement by Motivation and Self-Efficacy Across Gender and Achievement Levels. Interdisciplinary Journal of Teaching and Learning. [80]. Sartawi, A., Alsawaie, O. N., Dodeen, H., Tibi, S., & Alghazo, I. M. (2012). Predicting Mathematics Achievement by Motivation and Self-Efficacy Across Gender and Achievement Levels. Interdisciplinary Journal of Teaching and Learning. [81]. Schunk, D. H., Pintrich, P. R., & Meece, J. L. (2008). Motivation in education: theory, research, and applications. Upper Saddle River, N.J.: PearsonMerrill Prentice Hall. y p y g g [81]. Schunk, D. REFERENCES H., Pintrich, P. R., & Meece, J. L. (2008). Motivation in education: theory, research, and applications. Upper Saddle River, N.J.: PearsonMerrill Prentice Hall. [82]. Shen, D., Cho, M. H., Tsai, C. L., & Marra, R. (2013). Unpacking online learning experiences: Online learning self- efficacy and learning satisfaction. Internet and Higher Education. [83]. Sikhwari, T. (2007). The relationship between affective factors and the academic achievement at t South African Journal of Higher Edcuation. 7). The relationship between affective factors and the academic achievement at the University of Venda. rnal of Higher Edcuation. g [84]. Smith, R., Clark, T., & Blomeyer, R. L. (2005). A synthesis of New Research on K-12 Online Learning. Learnin Point Associates. [85]. Suhag, A. K. (2016). Student ACademic Motivation Of Secondary Schools Of Khaırpur Mır’s. Academic Research International , 100-109. [86]. Tang, Y., & Tseng, H. W. (2013). Distance learners’ self-efficacy and information literacy skills. Journal of Academic Librarianship. [87]. Tanius, E. (2020). Correlation between Learners' Motivation, Computer Anxiety and Social Support with Self-efficacy on Online Learning Technology due to COVID-19 pandemic. IJIRMPS. http://dx.doi.org/10.37082/IJIRMPS.2020.v08i06.011. 2222 IJISRT23JUL1538 www.ijisrt.com International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Volume 8, Issue 7, July – 2023 [88]. Taplin, M. (1998). Education in human values through mathematics: Mathematics through education in human values. Hong Kong: Sathya Sai Baba Center of Hong Kong. [89]. Ugwuanyi, C. S. (2020). Motivation and Self-efficacy as Predictors of Learners’ Academic Achievement. JOURNAL OF SOCIOLOGY AND SOCIAL ANTHROPOLOGY. [90]. Uyar, A., & Güngörmüş, A. H. (2011). Factors Associated with Student Performance in Financial Accounting Course. Turkey: European Journal of Economic and Political Studies. [91]. Uyar, A., & Güngörmüş, A. H. (2011). Factors Associated with Student Performance in Financial Accounting Course. European Journal of Economic and Political Studies . p [92]. Vallerland, R. J., Pelletier, L. G., Blais, M. R., Brière, N. M., Senécal, C., & Vallières, E. F. (1993). On the assessment of intrinsic, extrinsic, and amotivation in education: Evidence on the concurrent and construct validity of the Academic Motivation Scale. Educational and Psychological Measurement. y g [93]. Visser, L., Plomp, T., Amirault, R., & Kuiper, W. (2002). Motivating students at a distance: The case of an international audience. Education Technology Research and Development. [94]. Wadsworth, L. M., Husman, J., Duggan, M. A., & Pennington, M. N. (2007). Online Mathematics Achievement: Effects of Learning Strategies and Self-Efficacy. Arizona: JOURNAL of DEVELOPMENTAL EDUCATION. [95]. Wang, T. R. REFERENCES Online Learning Self-Efficacy With and Without Online Learning. American Journal of Distance Education. http://dx.doi.org/10.1080/08923647.2016.1193801. p g [105]. Zulkosky, K. (2009). Self-Efficacy: A Concept Analysis. Nursing Forum. ]. Zulkosky, K. (2009). Self-Efficacy: A Concept An [106]. Zyngier, S. S. (2012). How motivation influences student engagement: a qualitative case study. Journal of Education and Learning, 252–267. 2223 REFERENCES (2014). Assessing the effects of instructor enthusiasm on classroom engagement, learning goal orientation, [94]. Wadsworth, L. M., Husman, J., Duggan, M. A., & Pennington, M. N. (2007). Online Mathematics Achievement: Effects of Learning Strategies and Self-Efficacy. Arizona: JOURNAL of DEVELOPMENTAL EDUCATION. Learning Strategies and Self-Efficacy. Arizona: JOURNAL of DEVELOPMENTAL EDUCATION. [95]. Wang, T. R. (2014). Assessing the effects of instructor enthusiasm on classroom engagement, learning goal orientation, and academic self-efficacy. . Communication Teacher, 44-56. g g y [95]. Wang, T. R. (2014). Assessing the effects of instructor enthusiasm on classroom engagement, le and academic self-efficacy. . Communication Teacher, 44-56. [96]. Wang, Y.-L., Liang, J.-C., Lin, C.-Y., & Tsai, C.-C. (2017). Identifying Taiwanese junior-high school students' mathematics learning profiles and their roles in mathematics learning self-efficacy and academic performance. Elsevier Ltd. Selection. https://doi.org/10.1016/j.lindif.2017.01.008. p g j [97]. Watson, K. L. (2015). Examining the Effects of College Algebra on Students' Mathematical Dispositions. (Publication No. 5601) [Master of arts]. Brigham Young University. 17). Motivation towards Mathematics Learning in the Technology-enhanced Environment. Asia-Pacific uters in Education, 109-115. [98]. Wong, S. W. (2017). Motivation towards Mathematics Learning in the Technology-enhanced En Society for Computers in Education, 109-115. y p , [99]. Yantraprakorn, P., Darasawang, P., & Wiriyakarun, P. (2018). Self-efficacy and Online Language Learning: Causes of Failure. Thailand: Academy Publication. http://dx.doi.org/10.17507/jltr.0906.22. y p g j [100]. Yavuzalp, N., & Bahcivan, E. (2020). The Online Learning Self-Efficacy Scale: Its Adaptation into Turkish and Interpretation According to Various Variables. Turkish Online Journal of Distance Education. [101]. Yilmaz, E., Şahin, M., & Turgut, M. (2017). Variables Affecting Student Motivation Based on Academic Publications. Journal of Education and Practice. [102]. Zhang, Q. (2014). Assessing the effects of instructor enthusiasm on classroom engagement, learning goal orientation, and academic self-efficacy. Communication Teacher, 44-56. y [103]. Zhu, L., Liugen, E., Defazio, J., & Hook, S. A. (2019). Impact of the Stringency of Attend Attendance/Participation and Course Grades. Indiana: Journal of the Scholarship of Teaching and L E., Defazio, J., & Hook, S. A. (2019). Impact of the Stringency of Attendance Policies on Class ipation and Course Grades. Indiana: Journal of the Scholarship of Teaching and Learning. Attendance/Participation and Course Grades. Indiana: Journal of the Scholarship of Teaching and Learning. [104]. Zimmerman, & Kulikowich. (2016). Online Learning Self-Efficacy With and Without Online Learning. American Journal of Distance Education http://dx doi org/10 1080/08923647 2016 1193801 p p g g [104]. Zimmerman, & Kulikowich. (2016). IJISRT23JUL1538 www.ijisrt.com Volume 8, Issue 7, July – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 2225 2224 IJISRT23JUL1538 IJISRT23JUL1538 www.ijisrt.com Volume 8, Issue 7, July – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Appendix A Endorsement letter Appendix A Endorsement letter IJISRT23JUL1538 Volume 8, Issue 7, July – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Appendix B Certificate of Approval of Davao De Oro State Research Ethics Committee www.ijisrt.com 2226 www.ijisrt.com Volume 8, Issue 7, July – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 IJISRT23JUL1538 2227 www.ijisrt.com Volume 8, Issue 7, July – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Appendix D: Research Questionnaire Appendix D: Research Questionnaire Research Questionnaire IJISRT23JUL1538 www.ijisrt.com 2228 IJISRT23JUL1538 2228 www.ijisrt.com International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Volume 8, Issue 7, July – 2023 Volume 8, Issue 7, July – 2023 ISSN No:-2456-2165  Motivation Academic Motivation Scale (AMS) adopted from Vallerland et al. (1993) The instrument uses the 4-points Likert Scale with a categorical response; 4-Strongly Agree 3-Agree 2-Disagree 1-Strongly Disagree Table 9 Why do you spend time studying Mathematics? Items Strongly Disagree (1) Disagree (2) Agree (3) Strongly Agree (4) . I really feel that I am wasting my time in school . I once had good reasons for going to school; however, now I wonder whether I should continue . I cannot see why I go to school and frankly, I could not care less. . I cannot understand what I am doing in school . I believe that without good grades in Mathematics, I will not be able to find a high-paying job. . In order to obtain a more prestigious job later on. . I want to have “the good life” later on. . I want to prove to myself that I am capable of completing my college degree. . I feel important when I succeed in school. 0. It is to show myself that I am an intelligent person. 1. I want to show myself that I can succeed in my studies. 2. This will help me make a better choice regarding my career orientation. 3. Eventually, it will enable me to enter the job market in a field that I like. 4. I believe that my college education will improve my competence as a worker. 5. I experience pleasure and satisfaction while learning new things. 6. I experience pleasure and satisfaction in broadening my knowledge about Mathematics. Table 10 Level of Motivation Point Scale Range Description Interpretation 4 3.50 – 4.00 Strongly Agree The respondent strongly agrees that the statement or item accurately represents their academic motivation. They fully identify with and endorse the motivation described. 3 2.50 – 3.49 Agree The respondent agrees to some extent with the statement or item. They identify with  Motivation The instrument uses the 4-points Likert Scale with a categorical response; g y g Table 9 Why do you spend time studying Mathematics? Items Strongly Disagree (1) Disagree (2) Agree (3) Strongly Agree (4) . I really feel that I am wasting my time in school . I once had good reasons for going to school; however, now I wonder whether I should continue . I cannot see why I go to school and frankly, I could not care less. . I cannot understand what I am doing in school . I believe that without good grades in Mathematics, I will not be able to find a high-paying job. . In order to obtain a more prestigious job later on. . I want to have “the good life” later on. . I want to prove to myself that I am capable of completing my college degree. . I feel important when I succeed in school. 0. It is to show myself that I am an intelligent person. 1. I want to show myself that I can succeed in my studies. 2. This will help me make a better choice regarding my career orientation. 3. Eventually, it will enable me to enter the job market in a field that I like. 4. I believe that my college education will improve my competence as a worker. 5. I experience pleasure and satisfaction while learning new things. 6. I experience pleasure and satisfaction in broadening my knowledge about Mathematics. Table 10 Level of Motivation Point Scale Range Description Interpretation 4 3.50 – 4.00 Strongly Agree The respondent strongly agrees that the statement or item accurately represents their academic motivation. They fully identify with and endorse the motivation described. 3 2.50 – 3.49 Agree The respondent agrees to some extent with the statement or item. They identify with and endorse certain aspects of motivation described, but there may be room for improvement or further alignment. 2 1.50 – 2.49 Disagree The respondent disagrees to some extent with the statement or item. While they may acknowledge certain aspects of the motivation described, they do not fully agree with it. 1 1.00 – 1.49 Strongly Disagree The respondent strongly disagrees that the statement or item accurately represents their academic motivation.  Self-Efficacy Online Learning Self-Efficacy Scale (OLSES) adapted from Zimmerman and Kulikowich (2016) The instrument uses the 4-point Likert Scale with a categorical response; nline Learning Self-Efficacy Scale (OLSES) adapted from Zimmerman and Kulikowich (2016) Online Learning Self-Efficacy Scale (OLSES) adapted from Zimmerman and Kulikowich (2016) he instrument uses the 4-point Likert Scale with a categorical response; The instrument uses the 4-point Likert Scale with a categorical response; Table 11 The Instrument uses the 4-Point Likert Scale with a Categorical Response Items Strongly Disagree (1) Disagree (2) Agree (3) Strongly Agree (4) . I am confident in my ability to navigate online course materials efficiently . I believe that I can communicate effectively with technical support via e-mail, telephone, or live online chat. . I am confident that I can submit assignments to an online Dropbox. (i.e., google drive) . I am confident in my ability to overcome technical difficulties on my own. . I believe that I can navigate the online grade book. (i.e., scores or grades in google forms quizzes) . I am confident that I can manage time effectively. . I believe that I can complete all assignments on time. . I am confident in my ability to learn to use a new type of technology efficiently . I believe that I can learn without being in the same room as the instructor 0. I am confident that I can learn without being in the same room as other students. 1. I believe that I can search the Internet to find the answer to a course-related question. 2. I am confident that I can meet deadlines with very few reminders. 3. I believe that I can complete a group project entirely online. 4. I am confident that I can use synchronous technology to communicate with others (i.e., messenger, google meet, zoom). 5. I believe that I can focus on schoolwork when faced with distractions. 6. I am confident that I can develop and follow a plan for completing all required work on time. 7. I believe that I can promptly ask questions in the appropriate forum (e-mail, google classroom, discussion board, etc.) when a problem arises. Table 12 Level of Self-Efficacy Point Scale Range Description Interpretation 4 3.50 – 4.00 Strongly Agree The respondent strongly agrees that the statement or item accurately represents their level of self-efficacy. They fully identify with and endorse the level of self-efficacy described.  Motivation They do not identify with the motivation described in the item Table 10 Level of Motivation Point Scale Range Description Interpretation 4 3.50 – 4.00 Strongly Agree The respondent strongly agrees that the statement or item accurately represents their academic motivation. They fully identify with and endorse the motivation described. 3 2.50 – 3.49 Agree The respondent agrees to some extent with the statement or item. They identify with and endorse certain aspects of motivation described, but there may be room for improvement or further alignment. 2 1.50 – 2.49 Disagree The respondent disagrees to some extent with the statement or item. While they may acknowledge certain aspects of the motivation described, they do not fully agree with it. 1 1.00 – 1.49 Strongly Disagree The respondent strongly disagrees that the statement or item accurately represents their academic motivation. They do not identify with the motivation described in the item 2229 IJISRT23JUL1538 www.ijisrt.com International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Volume 8, Issue 7, July – 2023 Volume 8, Issue 7, July – 2023 ISSN No:-2456-2165  Self-Efficacy Online Learning Self-Efficacy Scale (OLSES) adapted from Zimmerman and Kulikowich (2016) The instrument uses the 4-point Likert Scale with a categorical response; 4-Strongly Agree 3-Agree 2--Disagree 1-Strongly Disagree Table 11 The Instrument uses the 4-Point Likert Scale with a Categorical Response Items Strongly Disagree (1) Disagree (2) Agree (3) Strongly Agree (4) . I am confident in my ability to navigate online course materials efficiently . I believe that I can communicate effectively with technical support via e-mail, telephone, or live online chat. . I am confident that I can submit assignments to an online Dropbox. (i.e., google drive) . I am confident in my ability to overcome technical difficulties on my own. . I believe that I can navigate the online grade book. (i.e., scores or grades in google forms quizzes) . I am confident that I can manage time effectively. . I believe that I can complete all assignments on time. . I am confident in my ability to learn to use a new type of technology efficiently . I believe that I can learn without being in the same room as the instructor 0. I am confident that I can learn without being in the same room as other students. 1.  Motivation I believe that I can search the Internet to find the answer to a course-related question. 2. I am confident that I can meet deadlines with very few reminders. 3. I believe that I can complete a group project entirely online. 4. I am confident that I can use synchronous technology to communicate with others (i.e., messenger, google meet, zoom). 5. I believe that I can focus on schoolwork when faced with distractions. 6. I am confident that I can develop and follow a plan for completing all required work on time. 7. I believe that I can promptly ask questions in the appropriate forum (e-mail, google classroom, discussion board, etc.) when a problem arises. Table 12 Level of Self-Efficacy Point Scale Range Description Interpretation 4 3.50 – 4.00 Strongly Agree The respondent strongly agrees that the statement or item accurately represents their level of self-efficacy. They fully identify with and endorse the level of self-efficacy described. 3 2.50 – 3.49 Agree The respondent agrees to some extent with the statement or item. They identify with and  Self-Efficacy The simple interest on a principal is inversely proportional to the time principal the principal is inv IV. Simple interest does not compound. a. I, II, & IV b. I & IV only II III & IV  Self-Efficacy 3 2.50 – 3.49 Agree The respondent agrees to some extent with the statement or item. They identify with and endorse certain aspects of the level of self-efficacy described, but there may be room for improvement or further alignment. 2 1.50 – 2.49 Disagree The respondent disagrees to some extent with the statement or item. While they may acknowledge certain aspects of the level of self-efficacy described, they do not fully agree with it. 1 1.00 – 1.49 Strongly Disagree The respondent strongly disagrees that the statement or item accurately represents their level of self-efficacy. They do not identify with the level of self-efficacy described in the item. www.ijisrt.com 2230 Volume 8, Issue 7, July – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Volume 8, Issue 7, July – 2023 International Journal of Innovative Science an SUMMATIVE TEST TEST QUESTIONNAIRE I. Multiple Choice GENERAL INSTRUCTION: Read the directions carefully before answering. Direction: Read the following items carefully. Encircle the letter of the correct answer. 1. What is the correct formula for time if we use Banker’s Rule? a. 𝑡𝑜 𝐼𝑜; approximate time divided by ordinary interest b. 𝑡𝑜 𝐼𝑒; approximate time divided by exact interest c. 𝑡𝑒 𝐼𝑜; exact time divided by ordinary interest d. 𝑡𝑒 𝐼𝑒; exact time divided by exact interest 2. It refers to a method in remembering the number of days in every months. a. Banker’s Rule b. Knuckle Mnemonic c. Approximate Time d. Exact Interest 3. What is the number of exact days of the year 2020? a. 365 b. 360 c. 366 d. 361 4. Which of the following simple interest formula gives more Interest (I)? a. 𝐼= Pr( 𝑎𝑝𝑝𝑟𝑜𝑥𝑖𝑚𝑎𝑡𝑒 𝑡𝑖𝑚𝑒 𝑜𝑟𝑑𝑖𝑛𝑎𝑟𝑦 𝑖𝑛𝑡𝑒𝑟𝑒𝑠𝑡) b. 𝐼= Pr( 𝑒𝑥𝑎𝑐𝑡 𝑡𝑖𝑚𝑒 𝑜𝑟𝑑𝑖𝑛𝑎𝑟𝑦 𝑖𝑛𝑡𝑒𝑟𝑒𝑠𝑡) c. 𝐼= Pr( 𝑎𝑝𝑝𝑟𝑜𝑥𝑖𝑚𝑎𝑡𝑒 𝑡𝑖𝑚𝑒 𝑒𝑥𝑎𝑐𝑡 𝑖𝑛𝑡𝑒𝑟𝑒𝑠𝑡) d. 𝐼= Pr( 𝑒𝑥𝑎𝑐𝑡 𝑡𝑖𝑚𝑒 𝑒𝑥𝑎𝑐𝑡 𝑖𝑛𝑡𝑒𝑟𝑒𝑠𝑡) 5. Which of the following value of time is NOT equal with 2 2 3 years? a. 2 8 12 years b. 8 3 years c. 2.666666667 years d. 1.333333333 years 6. Compare the following statement. Which of the following best describes simple interest? I. The simple interest can be found by subtracting the Future value from the Principal value. II. Simple interest is interest calculated on the original principal of a loan or on the amount of an acco III. SUMMATIVE TEST TEST QUESTIONNAIRE What sum will accumulate an interest of ₱ 35,000 in 5 years at 16% simple interest? a. ₱ 630,000.00 b. ₱ 63,000.00 c. ₱ 28,000.00 d. ₱ 19,444.44 10. Sandra needs ₱ 750,000.00 to buy her dream car after 4 years. How much should she invest in a company with a 12% simple interest rate? What is given and asked in the problem? a. Given: Principal amount, interest rate, and maturity date Required: Future amount b. Given: Principal amount, interest rate, and maturity date Required: Interest c. Given: Future amount, interest rate, and maturity date Required: Principal amount d. Given: Principal amount, interest rate, and issue date Required: Future amount 11. What formula should be applied if the given situation is that Racquel wants to buy a computer, so she borrowed Php 20,000.00 at 5% interest rate for 5 years, how much money should she pay back? a. 𝐼= 𝑃𝑟𝑡 b. 𝑃= 𝐹−𝐼 c. 𝑃= 𝐹 1+𝑟𝑡 d. 𝐹= 𝑃(1 + 𝑟𝑡) 7. Find the principal (P) if: F = Php 20,000.00 r = 9 % t = 5 years a. P = Php 434.78 b. P = Php 29,000.00 c. P = Php 13,793.10 d. P = Php 9,000.00 10. Sandra needs ₱ 750,000.00 to buy her dream car after 4 years. How much should she invest in a company with a 12% simple interest rate? What is given and asked in the problem? a. Given: Principal amount, interest rate, and maturity date Required: Future amount b. Given: Principal amount, interest rate, and maturity date Required: Interest c. Given: Future amount, interest rate, and maturity date Required: Principal amount d. Given: Principal amount, interest rate, and issue date Required: Future amount 11. What formula should be applied if the given situation is that Racquel wants to buy a computer, so she borrowed Php 20,000.00 at 5% interest rate for 5 years, how much money should she pay back? a. 𝐼= 𝑃𝑟𝑡 b. 𝑃= 𝐹−𝐼 c. 𝑃= 𝐹 1+𝑟𝑡 d. 𝐹= 𝑃(1 + 𝑟𝑡) 12. Let the accumulated money be ₱ 76,600 when ₱ 50,000 is invested at 11% simple interest. How long should we wait for our invested money to accumulate that much? a. 4.7 𝑦𝑒𝑎𝑟𝑠 b. 52 11 𝑦𝑒𝑎𝑟𝑠 c. 152 11 𝑦𝑒𝑎𝑟𝑠 d. 266 55 𝑦𝑒𝑎𝑟𝑠 a. 4.7 𝑦𝑒𝑎𝑟𝑠 b. 52 11 𝑦𝑒𝑎𝑟𝑠 c. 152 11 𝑦𝑒𝑎𝑟𝑠 d. 266 55 𝑦𝑒𝑎𝑟𝑠 13. SUMMATIVE TEST TEST QUESTIONNAIRE SUMMATIVE TEST TEST QUESTIONNAIRE I. Multiple Choice 4. Which of the following simple interest formula gives more Interest (I)? a. 𝐼= Pr( 𝑎𝑝𝑝𝑟𝑜𝑥𝑖𝑚𝑎𝑡𝑒 𝑡𝑖𝑚𝑒 𝑜𝑟𝑑𝑖𝑛𝑎𝑟𝑦 𝑖𝑛𝑡𝑒𝑟𝑒𝑠𝑡) b. 𝐼= Pr( 𝑒𝑥𝑎𝑐𝑡 𝑡𝑖𝑚𝑒 𝑜𝑟𝑑𝑖𝑛𝑎𝑟𝑦 𝑖𝑛𝑡𝑒𝑟𝑒𝑠𝑡) c. 𝐼= Pr( 𝑎𝑝𝑝𝑟𝑜𝑥𝑖𝑚𝑎𝑡𝑒 𝑡𝑖𝑚𝑒 𝑒𝑥𝑎𝑐𝑡 𝑖𝑛𝑡𝑒𝑟𝑒𝑠𝑡) d. 𝐼= Pr( 𝑒𝑥𝑎𝑐𝑡 𝑡𝑖𝑚𝑒 𝑒𝑥𝑎𝑐𝑡 𝑖𝑛𝑡𝑒𝑟𝑒𝑠𝑡) 5. Which of the following value of time is NOT equal with 2 2 3 years? 5. Which of the following value of time is NOT equal with 2 2 3 years? a. 2 8 12 years b. 8 3 years c. 2.666666667 years d. 1.333333333 years 6. Compare the following statement. Which of the following best describes simple interest? I. The simple interest can be found by subtracting the Future value from the Principal value. II. Simple interest is interest calculated on the original principal of a loan or on the amount of an account. III. The simple interest on a principal is inversely proportional to the time principal the principal is invested or loaned. IV. Simple interest does not compound. a. I, II, & IV b. I & IV only c. II, III & IV d. All of the above 6. Compare the following statement. Which of the following best describes simple interest? I. The simple interest can be found by subtracting the Future value from the Principal value. Si l i i i l l d h i i l i i l f l h f II. Simple interest is interest calculated on the original principal of a loan or on the amount of an account. 2231 IJISRT23JUL1538 www.ijisrt.com Volume 8, Issue 7, July – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Volume 8, Issue 7, July – 2023 International Journal of Innovative Science and Research Technology International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Volume 8, Issue 7, July – 2023 7. Find the principal (P) if: F = Php 20,000.00 r = 9 % t = 5 years a. P = Php 434.78 b. P = Php 29,000.00 c. P = Php 13,793.10 d. P = Php 9,000.00 8. Joana borrowed money from the bank on January 2, 2016, and paid it on April 6, 2016. Find the exact time of the given situation. a. 71 days b. 72 days c. 94 days d. 95 days 9. SUMMATIVE TEST TEST QUESTIONNAIRE Assess which of the following values of r or simple interest rate is correct if F = ₱ 110,000 and P = ₱ 100,000 during 36 months. 1 a. 𝑟= 1 360 b. 𝑟= 1 360 % c. 𝑟= 1 30 d. 𝑟= 0.33 2232 IJISRT23JUL1538 www.ijisrt.com International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Volume 8, Issue 7, July – 2023 14. Evaluate and identify which of the following statement is TRUE. 14. Evaluate and identify which of the following statement is TRUE. 14. Evaluate and identify which of the following statement is TRUE. a. The interest is ₱ 874.27 on ₱ 15,800 at 12 % from January 9, 2017, to June 24 of the same year, using actual time and ordinary interest. b. The interest is ₱ 862.29 on ₱ 15,800 at 12 % from January 9, 2017, to June 24 of the same year, using actual time and ordinary interest. c. The interest is ₱ 900.60 on ₱ 15,800 at 12 % from January 9, 2017, to June 24 of the same year, using actual time and ordinary interest. d. The interest is ₱ 830.00 on ₱ 15,800 at 12 % from January 9, 2017, to June 24 of the same year, using actual time and ordinary interest. a. The interest is ₱ 874.27 on ₱ 15,800 at 12 % from January 9, 2017, to June 24 of the same year, using actual time and ordinary interest. c. The interest is ₱ 900.60 on ₱ 15,800 at 12 % from January 9, 2017, to June 24 of the same year, using actual time and ordinary interest. d. The interest is ₱ 830.00 on ₱ 15,800 at 12 % from January 9, 2017, to June 24 of the same year, using actual time and ordinary interest. 15. Which values of Interest value (𝐼), Principal (𝑃), and time (𝑡) suffice to prove that the value of interest rate (𝑟) is 10%. 𝐼 233 750 𝑃 85 000 𝑑 99 15. Which values of Interest value (𝐼), Principal (𝑃), and time (𝑡) suffice to prove that the value of interest 99 a. 𝐼= 233,750;𝑃= 85,000;𝑎𝑛𝑑 𝑡= 99 360 𝑦𝑒𝑎𝑟𝑠 a. 𝐼= 233,750;𝑃= 85,000;𝑎𝑛𝑑 𝑡= 99 360 𝑦𝑒𝑎𝑟𝑠 a. 𝐼= 233,750;𝑃= 85,000;𝑎𝑛𝑑 𝑡= 360 𝑦𝑒𝑎𝑟𝑠 b. 𝐼= 2,125;𝑃= 85,000;𝑎𝑛𝑑 𝑡= 99 360 𝑦𝑒𝑎𝑟𝑠 c. SUMMATIVE TEST TEST QUESTIONNAIRE 𝐼= 318,750;𝑃= 85,000;𝑎𝑛𝑑 𝑡= 99 360 𝑦𝑒𝑎𝑟𝑠 d. 𝐼= 2,125;𝑃= 85,000;𝑎𝑛𝑑 𝑡= 90 𝑚𝑜𝑛𝑡ℎ𝑠 16. If the interest that is deducted is a discount, what is the actual amount that is given to the borrower? a Principal 17. It is the addition of the discount and proceed. 17. It is the addition of the discount and proceed. 17. It is the addition of the discount and proceed. a. Face value b. Bank discount c. Rate of discount d. Time period 18. What is the correct formula for getting the time in a simple discount? 𝑑 a. t = 𝑑 𝐹𝐷 b. t = I Pr c. t = 𝐷 𝐹𝑑 d. t = r PI 19. Compare the following statements and determine which is NOT TRUE. 19. Compare the following statements and determine which is NOT TRUE. I. For a simple discount, the face value and the maturity are not the same. II. For simple interest, the face value and the maturity are not the same. III. The formula for calculating the discount is similar to the interest formula a. I & II b. I only c. II & III d. All of the statements are not true. 20. Simon decides to continue schooling, but for him to go to school, he needs to have at least a motorcycle worth Php 73,000.00. And to have that amount, he decides to borrow the money at a local savings bank, for 14 months, at a 12% discount rate. How much does he need to apply for? What does this underlined question mean? Th Di t 20. Simon decides to continue schooling, but for him to go to school, he needs to have at least a motorcycle worth Php 73,000.00. And to have that amount, he decides to borrow the money at a local savings bank, for 14 months, at a 12% discount rate. How much does he need to apply for? What does this underlined question mean? Th Di a. The Discount b. The Proceeds c. The Face Value 2233 IJISRT23JUL1538 www.ijisrt.com International Journal of Innovative Science and Research Technology International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Volume 8, Issue 7, July – 2023 21. Douglas signed a Php 20,500 discount note on April 3, 2019. The bank charges an 18% discount rate, and the proceeds are Php 15,500. When will the note mature? SUMMATIVE TEST TEST QUESTIONNAIRE In this problem, what does the underlined phrase means? Th Di t 22. Soledad borrowed Php 18,000 for 8 months from a rural bank so that she could buy an oven for baking. The banker discounts the note at 9%. Find the amount of the discount. 22. Soledad borrowed Php 18,000 for 8 months from a rural bank so that she could buy an oven for baking. The banker discounts the note at 9%. Find the amount of the discount. 23. Sasha decides to go back to school. To get to school, she buys a small car, Php 180,000. She decides to borrow the money at the local savings bank for 14 months at a 12% discount rate. Find the face value of the loan so she will have Php 180,000. a Php 154 800 00 24. Marlon signed a Php 41,000 note at a discount rate of 8%. He was told it would have Php 3,200 interest. Find the length of the loan. a. 0.95 years b. 40 41 years c. 160.16 years d. 5125 32 years 24. Marlon signed a Php 41,000 note at a discount rate of 8%. He was told it would have Php 3,200 interest. Find the length of the loan. a 0 95 years 25. Which could be the correct value of the discount rate (𝑑) if the Php 1,200 note will mature in 90 days and the proceeds were Php 1,179. a. 𝑑= 7 25. Which could be the correct value of the discount rate (𝑑) if the Php 1,200 note will mature in 90 days and the proceeds were Php 1,179. a 𝑑= 7 26. Which of the following shows the correct formula for getting the maturity value in a simple discount? 𝐷 26. Which of the following shows the correct formula for getting the maturity value in a sim 𝐷 26. Which of the following shows the correct formula for getting the maturity value in a simple discount? I. F = 𝐷 𝐹𝑡 II. F = P + D III. F = 𝑃 1−𝑑𝑡 IV. F = P - D a. I,II,& III b. II & IV c. II & III d. I & II 27. Nico received Php 25,756.50 after applying for a loan last Jan 23. He will pay his loan after 2 years. If the bank charges a 13% simple discount, how much did Bistro apply for? SUMMATIVE TEST TEST QUESTIONNAIRE Face value (𝐹) = Php 210,000; discount rate (𝑑) = 9%; and time (𝑡) = 2.5 years ( ) p ( ) ( ) y d. Face value (𝐹) = Php 162,325.11; discount rate (𝑑) = 9%; and time (𝑡) = 2.5 years 30. Assess which of the following values of 𝑑 or discount rate is CORRECT if F = ₱ 13,500, P = ₱ 9,450 and will mature in 2.5 years 30. Assess which of the following values of 𝑑 or discount rate is CORRECT if F = ₱ 13,500, P = ₱ 9,450 and will mature in 2.5 a. 𝑑= 12 b. 𝑑= 0.12 c. 𝑑= − 6 35 d. 𝑑= 0.12% 31. It is the procedure in which interest is periodically calculated and added to the principal. Si l i t t 31. It is the procedure in which interest is periodically calculated and added to the principal. Si l i 32. Which of the following is the stated annual interest rate based on the compound interest calculation? Si l i 34. Virgilio Secundo borrows P47,400 with interest at 18% compounded quarterly. How much should he pay to the creditor after 3 years to pay off his debt? What do these underlined words refer to? 36. Which of the following statement is CORRECT in compounding frequencies and periods? a. The conversion per year of quarterly is 3 because it is every 4 months b. The conversion per year of bimonthly is 2 because it is every 6 months c. The conversion per year of quarterly is 4 because it is every 3 months d. The conversion per year of bimonthly is 6 because it is every 3 months 36. Which of the following statement is CORRECT in compounding frequencies and periods? a. The conversion per year of quarterly is 3 because it is every 4 months b. The conversion per year of bimonthly is 2 because it is every 6 months c. The conversion per year of quarterly is 4 because it is every 3 months d. The conversion per year of bimonthly is 6 because it is every 3 months IJISRT23JUL1538 www.ijisrt.com 2235 SUMMATIVE TEST TEST QUESTIONNAIRE In this problem, what is/are the given and unknown value/s. Gi P d i t t t d t it d t 27. Nico received Php 25,756.50 after applying for a loan last Jan 23. He will pay his loan after 2 years. If the bank charges a 13% simple discount, how much did Bistro apply for? In this problem, what is/are the given and unknown value/s. 27. Nico received Php 25,756.50 after applying for a loan last Jan 23. He will pay his loan after simple discount, how much did Bistro apply for? In this problem, what is/are the given and u a. Given: Proceed, interest rate, and maturity date b. Given: Proceed, discount rate, and maturity date Required: Face Value c. Given: Principal amount, interest rate, and maturity date Required: Face Value 2234 IJISRT23JUL1538 www.ijisrt.com Volume 8, Issue 7, July – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 28. Evaluate and identify which of the following statement is TRUE. 28. Evaluate and identify which of the following statement is TRUE. 28. Evaluate and identify which of the following statement is TRUE. y g a. The face value is Php 28,703.70 if the proceeds is Php 15,500 at 18% discount rate for 3 years. b. The face value is Php 7,130.00 if the proceeds is Php 15,500 at 18% discount rate for 3 years. c. The face value is Php 20,500.00 if the proceeds is Php 15,500 at 18% discount rate for 3 years. d. The face value is Php 33,695.65 if the proceeds is Php 15,500 at 18% discount rate for 3 years. ich values of Face value (𝐹), discount rate (𝑑), and time (𝑡) suffice to prove that the value of Proceeds (𝑃) 29. Which values of Face value (𝐹), discount rate (𝑑), and time (𝑡) suffice to prove that the value of Procee a. Face value (𝐹) = Php 126,131.25; discount rate (𝑑) = 9%; and time (𝑡) = 2.5 years a. Face value (𝐹) = Php 126,131.25; discount rate (𝑑) = 9%; and time (𝑡) = 2.5 years a. Face value (𝐹) = Php 126,131.25; discount rate (𝑑) = 9%; and time (𝑡) = 2.5 years b. Face value (𝐹) = Php 36,618.75; discount rate (𝑑) = 9%; and time (𝑡) = 2.5 years c. www.ijisrt.com IJISRT23JUL1538 Given: Principal amount, equivalent interest rate, maturity date, and number of conversions per year Required: Future amount b. Given: Principal amount, equivalent interest rate, maturity date, and number of conversions per year Required: Face value c. Given: Principal amount, nominal interest rate, and number of conversions per year Required: Future amount d. Given: Principal amount, nominal interest rate, maturity date, and number of conversions per year Required: Future amount 41. How long will it take for Php 11,250.50 to become Php 15,119.73 at 12 % compounded quarterly? a. t = 2.5 b. t = 2 years and 5 months c. t = 2.5 months d. t = 2.5 years 42. The maturity value of an 8 year, Php 60,000 compound-interest investment certificate was Php 80,000. What quarterl compounded rate did the investment certificate earn? a) 3.6 % b) 6.3 % c) 3.3 % d) 3.9 % 43. Evaluate and identify which of the following statement is TRUE. a. The future value of a principal amount of Php 27,000 invested for 2 years at 9% compounded monthly is Php 31,960.00. b. The future value of a principal amount of Php 27,000 invested for 2 years at 9% compounded monthly is Php 32,303.17. c. The future value of a principal amount of Php 27,000 invested for 2 years at 9% compounded monthly is Php 33,203.17. d. The future value of a principal amount of Php 27,000 invested for 2 years at 9% compounded monthly is Php 153,167.60. 44. Which values of Future amount (𝐹), nominal rate (𝑗),number of conversion per year (𝑚), and time (𝑡) suffice to prove that th 40. A note with a maturity value of Php 42,560 is due in 4 years and 9 months. What is the principal amount at 5.6% compounded semiannually? In this problem, what is/are the given and unknown value/s a. Given: Principal amount, equivalent interest rate, maturity date, and number of conversions per year Required: Future amount b. Given: Principal amount, equivalent interest rate, maturity date, and number of conversions per year Required: Face value c. Given: Principal amount, nominal interest rate, and number of conversions per year Required: Future amount d. Given: Principal amount, nominal interest rate, maturity date, and number of conversions per year Required: Future amount 42. The maturity value of an 8 year, Php 60,000 compound-interest investment certificate was Php 80,000. What quarterly compounded rate did the investment certificate earn? IJISRT23JUL1538 www.ijisrt.com Volume 8, Issue 7, July – 2023 ISSN No:-2456-2165 37. If you deposit ₱10,000 into an account paying 2.5% annual interest compounded quarterly, how much money will be in the account after 5 years? a. 𝑃= ₱2,974.55 b. 𝐹= ₱33,618.53 c. 𝑃= ₱8,828.40 d. 𝐹= ₱11,327.08 38. Doggy invested in a company with an interest rate of 6.2% compounded monthly. If, after 10 years, the account balance is ₱ 37,839.45, how much was the beginning investment? a. 𝑃=₱20,150.43 b. 𝑃=₱40,259.11 c. 𝑃=₱20,388.04 d. 𝑃=₱77,228.63 39. Find the interest earned of a Php 100,000.00 at the end of 6 months at 13% compounded semiannually. a. Php 106,500.00 b. Php 212,909.62 c. Php 6,500.00 d. Php 112,909.62 40. A note with a maturity value of Php 42,560 is due in 4 years and 9 months. What is the principal amount at 5.6% compounded semiannually? In this problem, what is/are the given and unknown value/s a. Given: Principal amount, equivalent interest rate, maturity date, and number of conversions per year Required: Future amount b. Given: Principal amount, equivalent interest rate, maturity date, and number of conversions per year Required: Face value c. Given: Principal amount, nominal interest rate, and number of conversions per year Required: Future amount d. Given: Principal amount, nominal interest rate, maturity date, and number of conversions per year Required: Future amount 41 How long will it take for Php 11 250 50 to become Php 15 119 73 at 12 % compounded quarterly? 37. If you deposit ₱10,000 into an account paying 2.5% annual interest compounded quarterly, how much money will be i the account after 5 years? a. 𝑃= ₱2,974.55 b. 𝐹= ₱33,618.53 c. 𝑃= ₱8,828.40 d. 𝐹= ₱11,327.08 38. Doggy invested in a company with an interest rate of 6.2% compounded monthly. If, after 10 years, the account balance i ₱ 37,839.45, how much was the beginning investment? a. 𝑃=₱20,150.43 b. 𝑃=₱40,259.11 c. 𝑃=₱20,388.04 d. 𝑃=₱77,228.63 39. Find the interest earned of a Php 100,000.00 at the end of 6 months at 13% compounded semiannually. a. Php 106,500.00 b. Php 212,909.62 c. Php 6,500.00 d. Php 112,909.62 40. A note with a maturity value of Php 42,560 is due in 4 years and 9 months. What is the principal amount at 5.6% compounded semiannually? In this problem, what is/are the given and unknown value/s a. IJISRT23JUL1538 a) 3 6 % y g a. The future value of a principal amount of Php 27,000 invested for 2 years at 9% compounded monthly is Php 31,960.00. b. The future value of a principal amount of Php 27,000 invested for 2 years at 9% compounded monthly is Php 32,303.17. c. The future value of a principal amount of Php 27,000 invested for 2 years at 9% compounded monthly is Php 33,203.17. d. The future value of a principal amount of Php 27,000 invested for 2 years at 9% compounded monthly is Php 153,167.60. 44. Which values of Future amount (𝐹), nominal rate (𝑗),number of conversion per year (𝑚), and time (𝑡) suffice to prove that the value of Principal amount (𝑃) is Php 73,516.46 a. 𝐹 = Php 63,392.62; 𝑗 = 5%; 𝑚 = 2; and time (𝑡) = 3 years b. 𝐹 = Php 11,740.02; 𝑗 = 5%; 𝑚 = 2; and time (𝑡) = 3 years c. 𝐹 = Php 85,526.55; 𝑗 = 5%; 𝑚 = 2; and time (𝑡) = 3 years d. 𝐹 = Php 85,256.55; 𝑗 = 5%; 𝑚 = 2; and time (𝑡) = 3 years 45. Assess which of the following values of 𝐹 or Future value is CORRECT if Principal (𝑃) = Php 25,324.00 ; nominal rat (𝑗) = 3.5%;number of conversion per year ( 𝑚 )= 4; and time (𝑡) = 5 years 45. Assess which of the following values of 𝐹 or Future value is CORRECT if Principal (𝑃) = Php 25,324.00 ; nominal rat (𝑗) = 3.5%;number of conversion per year ( 𝑚 )= 4; and time (𝑡) = 5 years 45. Assess which of the following values of 𝐹 or Future value is CORRECT if Principal (𝑃) = Php 25,324.00 ; nominal rat (𝑗) = 3.5%;number of conversion per year ( 𝑚 )= 4; and time (𝑡) = 5 years a. 𝐹 = 21,274.60 b. 𝐹 = 30,144.17 c. 𝐹 = 4,820.17 d. 𝐹 = 135,555.65 2236 www.ijisrt.com Volume 8, Issue 7, July – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 ISSN No:-2456-2165 IJISRT23JUL1538 www ijisrt com 2237 Appendix E Summary of Validation Results Name of Researcher: JAYSON KITTS C. IJISRT23JUL1538 POQUITA Degree Enrolled: MAED-Math Title of Research: MOTIVATION AND SELF EFFICACY AS PREDICTORS TO MATHEMATICS PERFORMANCE IN ONLINE LEARNING Motivation Items Expert 1 Expert 2 Expert 3 Expert 4 Expert 5 Descriptive Rating 1 3 3 1 4 4 Good 2 3 4 1 4 1 Fair 3 3 3 1 4 1 Fair 4 3 3 1 4 4 Good 5 3 4 1 4 4 Good 6 3 4 1 4 4 Good 7 3 3 1 4 1 Fair 8 3 3 1 4 1 Fair 9 3 4 1 4 1 Fair 10 3 3 1 4 4 Good 11 3 3 1 4 1 Fair 12 3 4 1 4 4 Good 13 3 4 1 4 1 Fair 14 3 4 1 4 4 Good 15 3 4 1 4 1 Fair 16 3 4 1 4 4 Good 17 3 4 1 4 4 Good 18 3 4 1 4 1 Fair 19 3 3 1 4 4 Good 20 3 4 1 4 1 Fair Self-efficacy Items Expert 1 Expert 2 Expert 3 Expert 4 Expert 5 Descriptive Rating 1 3 4 1 4 4 Good 2 3 3 1 4 1 Fair 3 3 3 1 4 4 Good 4 4 4 1 4 4 Good 5 3 3 1 4 4 Good 6 3 3 1 4 4 Good 7 4 4 1 4 4 Good 8 4 4 1 4 4 Good 9 3 4 1 4 4 Good 10 3 4 1 4 4 Good 11 3 3 1 4 4 Good 12 4 4 1 4 4 Good 13 4 3 1 4 1 Fair 14 3 4 1 4 1 Fair 15 4 3 1 4 4 Good 16 3 3 1 4 4 Good 17 4 4 1 4 4 Good 18 4 3 1 4 4 Good 19 4 4 1 4 4 Good 20 3 3 1 4 1 Fair 21 4 4 1 4 4 Good Appendix E Summary of Validation Results www.ijisrt.com 2237 Volume 8, Issue 7, July – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Panel of Validators: 5 experts rated 3 or 4-Excellent 4 experts rated 3 or 4-Good 3 experts rated 3 or 4-Fair 2 experts rated 3 or 4-Discard International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Volume 8, Issue 7, July – 2023 IJISRT23JUL1538 www.ijisrt.com 2 Panel of Validators: 5 experts rated 3 or 4-Excellent 4 experts rated 3 or 4-Good 3 experts rated 3 or 4-Fair 2 experts rated 3 or 4-Discard 5 experts rated 3 or 4-Excellent 4 experts rated 3 or 4-Good 3 experts rated 3 or 4-Fair 2 experts rated 3 or 4-Discard Panel of Validators: Panel of Validators: 5 experts rated 3 or 4-Excellent 4 experts rated 3 or 4-Good 3 experts rated 3 or 4-Fair 2 experts rated 3 or 4-Discard IJISRT23JUL1538 www.ijisrt.com 2238 Volume 8, Issue 7, July – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 ISSN No:-245 Appendix F Summative Test TOS Appendix F Summative Test TOS 2239 IJISRT23JUL1538 IJISRT23JUL1538 Volume 8, Issue 7, July – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Appendix G Survey and Test Results Survey and Test Results www.ijisrt.com IJISRT23JUL1538 2240 Volume 8, Issue 7, July – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 IJISRT23JUL1538 www.ijisrt.com 2241 Volume 8, Issue 7, July – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 ISSN No:-2456-2165 www.ijisrt.com IJISRT23JUL1538 2242 Volume 8, Issue 7, July – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 IJISRT23JUL1538 2243 www.ijisrt.com Volume 8, Issue 7, July – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Appendix H Photo Documentation International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Volume 8, Issue 7, July – 2023 2244 IJISRT23JUL1538 www.ijisrt.com
https://openalex.org/W3108450240	https://revistavista.pt/index.php/vista/article/download/3049/2945	Portuguese	null	As pessoas nos não-lugares ou as não-pessoas precisam de lugares	Vista	2,019	cc-by	1,062	1 Estas imagens não são profissionais, não as fiz para publicar, a qualidade técnica é reduzida, porque são ficheiros já antigos, cópias dos originais. Todas as fotografias usadas para as montagens são originais meus. Sara Machado da Graça Sou cenógrafa e figurinista. Como tal, gosto de apreciar todas as ricas personagens que se cruzam comigo diariamente no “cenário” de Maputo, cidade em constante bulício colorido, confuso, fluído apesar de caótico e misto de toda a panóplia de emoções. Para quem anda a pé, para quem conduz, para quem anda de chapa, de my love… para cada um há visões diferentes e os ritmos são muitos e muito diversos a coexistir, mas o movimento é constante. É uma cidade que cresceu exponencialmente durante a guerra civil, resultado do êxodo rural, sem ter estrutura para suportar esse excesso demográfico. À cidade “de cimento”, colonial, acrescentaram-se os bairros suburbanos que cresceram desordenadamente, sem plano nem condições sanitárias, onde vive a maioria da população residindo em casas construídas em blocos de cimento e chapa de zinco. Uma grande parte das famílias subsistem através do mercado informal, vendendo os produtos das suas pequenas machambas (hortas) ou itens de toda a espécie, importados da África da Sul. As figuras desumanizadas Quando fotografei estas personagens de Maputo, não sabia que ia depois trabalhá-las, apenas colecionava imagens dos vendedores de rua como atores-marioneta nos diferentes cenários que ia descobrindo. Estas personagens fundem-se com a paisagem, fazem parte dela, mesclam-se numa coreografia cenográfica, urbana e identitária da cidade. 1 Estas imagens não são profissionais, não as fiz para publicar, a qualidade técnica é reduzida, porque são ficheiros já antigos, cópias dos originais. Todas as fotografias usadas para as montagens são originais meus. 229 vista nº 5  2019  Vistas Imperiais: Visualidades coloniais e processos de descolonização  pp. 229-234 Estas pessoas-signo eram uma presença fortíssima nas imagens que fazem parte do meu dia-a-dia - o homem água, o homem almofadas, o homem zips, o homem das tomadas elétricas, o dos óculos, as mulheres carregadas com carga à cabeça... agentes do dumba nengue (mercado informal) geral que circulam, cada um a seu passo, etéreos, estátuas da identidade urbana moçambicana - recolectores, “homem-anúncio”, sucateiros, recicladores dos seus produtos - que tomam formas dignas dos bailarinos triádicos de Oscar Schlemmer, numa espécie de dança sobre um itinerário infinito de ícones pop do espaço africano do consumo. Marc Augé define “não-lugares” como “lugares transitórios que não possuem significado suficiente para serem definidos como um lugar” (1994, p. 10). Segundo o autor, estes espaços são representativos da afirmação da existência da sobremodernidade, participando na antropologia urbana como associados aos viajantes “o espaço do viajante seria assim, o arquétipo do não-lugar”. O movimento acrescenta à coexistência dos mundos e à experiência combinada do lugar antropológico e daquele que não o é mais (pela qual Starobinski define, em suma, a modernidade) a experiência particular de uma forma de solidão, e, em sentido literal, de uma “tomada de posição” – a experiência daquele que, diante da paisagem que é obrigado a contemplar, “toma a posse” e tira da consciência dessa atitude um prazer raro e, às vezes, melancólico. Portanto, não é de espantar que seja, entre os “viajantes” solitários do século passado, (...) viajantes acidentais, que estejam aptos a encontrar a evocação profética de espaço, onde nem a identidade, nem a relação, nem a história fazem realmente sentido, onde a solidão é sentida como superação ou esvaziamento da individualidade, onde só o movimento das imagens deixa entrever, por instantes, àquele que as olha a fugir, a hipótese de um passado e a possibilidade de um futuro” (1994, p.80-81). Sara Machado da Graça  As pessoas nos não-lugares ou as não-pessoas precisam de lugares As não-pessoas precisam de lugares Num segundo momento senti que estas personagens tinham alma, eram pessoas, únicas, solitárias, resilientes e presas ao quotidiano da sobrevivência. Lembravam o Acendedor de Lampiões do Pequeno Príncipe de St Exupéry ou as personagens do universo absurdo de Samuel Beckett, entidades poéticas e simbólicas enredadas num ciclo semelhante aos tormentos de Sísifo. “Pediam” para estar noutro sítio, longe dali, num espaço feito só para eles. Tentei dar-lhes espaços aparentemente transitórios, mas onde a viagem pudesse tornar-se o destino, e que esse fosse o espaço identitário de conforto para que as negativas (não-lugar, não-pessoa) se anulassem e para que pudéssemos ter uma pessoa no seu lugar. vista nº 5  2019  Vistas Imperiais: Visualidades coloniais e processos de descolonização  pp. 229-234 230 Sara Machado da Graça  As pessoas nos não-lugares ou as não-pessoas precisam de lugares Mediterranean; taken and transplanted by early Spanish colonialists from the Canary Islands to the Americas, and distributed by European botanists to and from colonies in Asia, Africa and the Antipodes, and back to Europe, palms have become the symbol of successful uprooted-ness: synonymous with tropical views, pre-lapsarian lands, and exotic holidays. Mediterranean; taken and transplanted by early Spanish colonialists from the Canary Islands to the Americas, and distributed by European botanists to and from colonies in Asia, Africa and the Antipodes, and back to Europe, palms have become the symbol of successful uprooted-ness: synonymous with tropical views, pre-lapsarian lands, and exotic holidays. Mamã das frutas, Sara Machado da Graça, 2019 Mamã das frutas, Sara Machado da Graça, 2019 231 sta nº 5  2019  Vistas Imperiais: Visualidades coloniais e processos de descolonização  pp. 229-234 Man on the moon, Sara Machado da Graça, 2019 Man on the moon, Sara Machado da Graça, 2019 vista nº 5  2019  Vistas Imperiais: Visualidades coloniais e processos de descolonização  pp. 229-234 232 Falta de material, Sara Machado da Graça, 2019 Falta de material, Sara Machado da Graça, 2019 233 sta nº 5  2019  Vistas Imperiais: Visualidades coloniais e processos de descolonização  pp. 229-234 Funâmbulo com balão de almofadas, Sara Machado da Graça, 2019 Funâmbulo com balão de almofadas, Sara Machado da Graça, 2019 Referência bibliográfica Augé, Marc. (1994). Não-lugares, para uma introdução da antropologia da sobremodernidade. Papirus, Campinas. Sara Machado da Graça reside atualmente em Maputo, Moçambique, onde leciona as disciplinas de Cenografia e Figurinos no Curso de Teatro da Escola de Comunicação e Artes da Universidade Eduardo Mondlane, trabalha também como programadora artística da Galeria/Associação Kulungwana. Continua a fazer trabalhos como cenógrafa e figurinista para teatro, cinema, dança e ópera, assim como curadoria para exposições.  sagadexam@gmail.com 234
https://openalex.org/W2996033363	https://digitalcommons.uri.edu/cgi/viewcontent.cgi?article=1192&context=bps_facpubs	English	null	Pectic Oligosaccharides from Cranberry Prevent Quiescence and Persistence in the Uropathogenic Escherichia coli CFT073	Scientific reports	2,019	cc-by	7,054	Follow this and additional works at: https://digitalcommons.uri.edu/bps_facpubs Follow this and additional works at: https://digitalcommons.uri.edu/bps_facpubs Pectic Oligosaccharides from Cranberry Prevent Quiescence and Pectic Oligosaccharides from Cranberry Prevent Quiescence and Persistence in the Uropathogenic Escherichia coli CFT073 Persistence in the Uropathogenic Escherichia coli CFT073 Jiadong Sun Robert W. Deering Zhiyuan Peng Laila Najia Christina Khoo See next page for additional authors University of Rhode Island University of Rhode Island DigitalCommons@URI DigitalCommons@URI University of Rhode Island University of Rhode Island DigitalCommons@URI DigitalCommons@URI Biomedical and Pharmaceutical Sciences Creative Commons License Creative Commons License This work is licensed under a Creative Commons Attribution 4.0 License. This work is licensed under a Creative Commons Attribution 4.0 License. Citation/Publisher Attribution Citation/Publisher Attribution Citation/Publisher Attribution Citation/Publisher Attribution Sun, J., Deering, R.W., Peng, Z. et al. Pectic Oligosaccharides from Cranberry Prevent Quiescence and Persistence in the Uropathogenic Escherichia coli CFT073. Sci Rep 9, 19590 (2019). https://doi.org/ 10.1038/s41598-019-56005-w This Article is brought to you by the University of Rhode Island. It has been accepted for inclusion in Biomedical and Pharmaceutical Sciences Faculty Publications by an authorized administrator of DigitalCommons@URI. For more information, please contact digitalcommons-group@uri.edu. For permission to reuse copyrighted content, contact the author directly. Pectic Oligosaccharides from Cranberry Prevent Quiescence and Persistence in Pectic Oligosaccharides from Cranberry Prevent Quiescence and Persistence in the Uropathogenic Escherichia coli CFT073 the Uropathogenic Escherichia coli CFT073 Creative Commons License Creative Commons License Pectic Oligosaccharides from Cranberry Prevent Quiescence and Persistence in the Uropathogenic Escherichia coli CFT073 EN Jiadong Sun1,2,5, Robert W. Deering1,5, Zhiyuan Peng1, Laila Najia1, Christina Khoo3, Paul S. Cohen4, Navindra P. Seeram1 & David C. Rowley1* Urinary tract infections (UTIs) caused by Escherichia coli create a large burden on healthcare and frequently lead to recurrent infections. Part of the success of E. coli as an uropathogenic bacterium can be attributed to its ability to form quiescent intracellular reservoirs in bladder cells and its persistence after antibiotic treatment. Cranberry juice and related products have been used for the prevention of UTIs with varying degrees of success. In this study, a group of cranberry pectic oligosaccharides (cPOS) were found to both inhibit quiescence and reduce the population of persister cells formed by the uropathogenic strain, CFT073. This is the first report detailing constituents of cranberry with the ability to modulate these important physiological aspects of uropathogenic E. coli. Further studies investigating cranberry should be keen to include oligosaccharides as part of the ‘active’ cocktail of chemical compounds. Bacterial urinary tract infections (UTIs) affect up to 150 million people each year and are among the most com- mon infections worldwide1. It is estimated that the total societal cost burden of UTIs is $3.5 billion each year in the US alone2. Over 50% of women will experience a UTI, with an estimated recurrence rate of about 25%3. While UTIs are typically curable, recurrent infections can require continuous antibiotic prophylaxis for prevention. Frequent use of antibiotics, however, can lead to adverse events such as colitis, the development of antibiotic resistant UTIs, or the development of complicated UTIs2–4. p p Uropathogenic Escherichia coli (UPEC) is responsible for 65–90% of UTI cases2,4–6. For recurrent UTIs, the original infecting UPEC strain is responsible 77% of the time7. While the mechanism of recurrence is not fully understood, it is thought that intracellular, biofilm-like communities (IBCs) and quiescent intracellular reservoirs (QIRs) are contributing factors8–10. IBCs form when UPEC from urine invade and replicate inside of superficial bladder cells6,10. As the infection progresses, subsequent UPEC invasion of urothelial transitional cells can lead to establishment of QIRs. Cells residing in QIRs can remain viable for months6,8,9. Upon exfoliation of bladder epithelial cells, UPEC from QIRs would be able to re-colonize urine and cause a recurrent infection6. Since antibi- otics generally target actively dividing bacteria, quiescent UPEC cells are unlikely to be eradicated by antibiotics, thus contributing to treatment failures and recurrence8,10. Authors Authors Jiadong Sun, Robert W. Deering, Zhiyuan Peng, Laila Najia, Christina Khoo, Paul S. Cohen, Navindra P. Seeram, and David C. Rowley This article is available at DigitalCommons@URI: https://digitalcommons.uri.edu/bps_facpubs/190 www.nature.com/scientificreports Results h The UPEC strain CFT073 demonstrates in vitro quiescence on glucose minimal media agar plates and resumes growth in response to cranberry constituents. We previously reported that the UPEC strain CFT073 grows normally in liquid glucose minimal media but exhibits in vitro quiescence on glucose minimal media agar plates when plated at low density (≤106 CFU)16. It was found that cells exhibiting quiescence under these conditions could be stimulated to grow by transferring a colony of E. coli MG1655 onto the plate or by applying an amino acid cocktail comprising various combinations of lysine, methionine, and tyrosine. Following addition of the stimulus to the agar surface and 24 hour incubation at 37 °C, growing CFT073 cells appear as visible colonies radiating out from the site of the stimulus (see Fig. 1 as an example). The diameter of the zone of growing CFT073 continues to expand with additional incubation time. This experiment is a qualitative test for stimuli that reverse in vitro quiescence of UPEC strains. q Because cranberry has been traditionally used for preventing UTI, we hypothesized that certain cranberry constituents might reverse UPEC quiescence. The above quiescence assay was used to test cranberry phytochem- icals and guide the separation of the active components (Fig. S1). A pectinase degraded cranberry hull extract was separated using C18 reversed phase chromatography to afford an oligosaccharide-enriched fraction, Cranf1b, that induced bacteria colony formation when added (100 µg in 10 µL water) to the surface of an agar plate contain- ing quiescent CFT073 (data not shown). Cranf1b was further separated using porous graphitized carbon (PGC) cartridges. Only a sub-fraction that eluted with 10% ACN/H2O/0.1%TFA showed reversal of E. coli CFT073 qui- escence (Fig. 1). This sub-fraction (cPOS) was further separated using HPLC chromatography and characterized using a combination of NMR, HR-MS and LC-MS/MS. Chemical characterization of cranberry constituents that reverse quiescence. The 1H and HSQC NMR spectra of the active cranberry fraction revealed proton and carbon resonances with chemical shifts charac- teristic for poly-uronic acids. Additional structural features included a methoxy group at δ 3.70/52.8 and a vinylic methine at δ 6.00/111.8 (Figs. S2 and S3). Monosaccharide composition analysis of cPOS determined that the oligosaccharides were comprised of galacturonic acid units (Fig. S4).h g g g The active fraction was further analyzed by hydrophilic interaction liquid chromatography (HILIC) HPLC (Fig. 2). Pectic Oligosaccharides from Cranberry Prevent Quiescence and Persistence in the Uropathogenic Escherichia coli CFT073 EN g Persistence is a physiologic state similar to quiescence for UPEC11–14. Persister cells are a small subpopulation of bacteria that exist in a dormant, non-dividing state characterized by high antibiotic tolerance11,12,14. Persister cells can be isolated and regrown following initial antibiotic treatment, and these populations retain sensitivity to the initial antibiotic and can consistently form another persister cell fraction15. Similar to QIR cells, persister cells are strongly implicated in the pathogenesis of chronic infections, including recurrent UTIs16–20. While proven to be metabolically distinct, the quiescent and persistent physiological states of UPEC are both likely to cause 1Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of Rhode Island, Kingston, RI, 02881, USA. 2Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, 20814, USA. 3Ocean Spray Cranberries, Inc., One Ocean Spray Drive, Lakeville-Middleboro, MA, 02349, USA. 4Department of Cell and Molecular Biology, University of Rhode Island, Kingston, RI, 02881, USA. 5These authors contributed equally: Jiadong Sun and Robert W. Deering. email: drowley@uri.edu www.nature.com/scientificreports/ Figure 1. CFT073 in vitro quiescence assay. The location of spotting a sample is designated by the white ‘x’. (A)- tyrosine, lysine, and methionine positive control (Top) and M9 medium negative control (Bottom), (B)- uG3m2, (C)- Clockwise from bottom: cPOS, uG4m3 and HDP-cPOS. Figure 1. CFT073 in vitro quiescence assay. The location of spotting a sample is designated by the white ‘x’. (A)- tyrosine, lysine, and methionine positive control (Top) and M9 medium negative control (Bottom), (B)- uG3m2, (C)- Clockwise from bottom: cPOS, uG4m3 and HDP-cPOS. recurrent UTIs and treatment failures16. Therapies aimed at preventing or reversing quiescence and persistence could significantly reduce the burden of UPEC recurrent UTIs. C b (V i i p ) d t h b t i l h d f th i bilit t t gi y Cranberry (Vaccinium macrocarpon) products have been extensively researched for their ability to prevent UTIs, especially those caused by UPEC21–26. Past studies have focused on cranberry proanthocyanidins (PACs) with A-type linkages24,27 and fructose28 since these have been shown to interfere with UPEC adherence to bladder cells. Organic acids present in cranberry, including quinic, shikimic, malic and citric acids, were recently demon- strated to help reduce E. coli populations in an experimental mouse model of UTI29. Other recent studies have suggested that cranberry complex carbohydrates might also contribute to the prevention of UTIs. Pectic Oligosaccharides from Cranberry Prevent Quiescence and Persistence in the Uropathogenic Escherichia coli CFT073 EN For example, xyloglucan oligosaccharides from cranberry have been shown to interfere with cell adhesion and biofilm forma- tion by uropathogenic E. coli30–32. We hypothesized that certain cranberry constituents might interfere with quies- cent and persister cells since these are implicated in UTI recurrence. Herein, we report the isolation and chemical characterization of cranberry pectic oligosaccharides (cPOS) that prevent quiescence in the prototypical UPEC strain CFT073 and significantly reduce the population of UPEC persister cells in the presence of ampicillin. Scientific Reports \| (2019) 9:19590 \| https://doi.org/10.1038/s41598-019-56005-w Results h HILIC LC-MS data showed that the predominate ions were consistent with poly-galacturonic acid methyl esters with various degrees of polymerization (DP) (Fig. 3). Each oligosaccharide was present as a pair of α- and β- anomers in equilibrium. These results were in agreement with HR-ESI-MS and HR-ESI-MS/MS data obtained for collected fractions (Figs. S5 and S6). The most abundant ions indicated the presence of fragments with one or two free carboxylic acids and one additional degree of unsaturation. UV absorption maxima at 235 nm was consistent with the presence of an α, β-unsaturated carboxylic acid, which is in agreement of the vinylic methine observed in NMR, and accounting for the additional unsaturation. The major components were therefore deter- mined to be unsaturated pectic oligosaccharides, referred to as uGXmY (uG: unsaturated poly-Galacturonic acid; X: degree of polymerization; Y: number of methyl esters). Saturated poly-galacturonic acid methyl esters were unexpectedly found to be present only trace amounts in cPOS (data not shown). Scientific Reports \| (2019) 9:19590 \| https://doi.org/10.1038/s41598-019-56005-w www.nature.com/scientificreports/ Figure 2. HILIC HPLC chromatogram of cPOS (Top) and cPOS-t (Bottom) at 235 nm. 6,7-dihydromonotropein (a), deacetylasperulsidic acid (b) and monotropein (c). Figure 2. HILIC HPLC chromatogram of cPOS (Top) and cPOS-t (Bottom) at 235 nm. 6,7-dihydromonotropein (a), deacetylasperulsidic acid (b) and monotropein (c). We next isolated two unsaturated poly-galacturonic acid methyl esters, uG3m2 (HRMS (m/z): [M−H]−, C20H27O18, 555.1205, calcd. 555.1197) and uG4m3 (1, HRMS (m/z): [M−H]−, C27H37O24, 745.1692, calcd.745.1674) from the cPOS mixture. Compounds eluting after 1 were pooled into a single fraction containing cPOS with higher DPs (HDP-cPOS). Purified 1 was characterized using 1D and 2D NMR spectra (Figs. S7–S12) and compared to previously reported poly-galacturonic acids33–36. Superimposition of HSQC spectra for both 1 and cPOS showed that both contained similar correlation signals (Fig. S3), confirming 1 as a representative constituent of cPOS. The 1H and 13C NMR chemical shifts of 1 are listed in Table S1. Briefly, five spin systems were identified and were assigned to the four subunits (A-α/A-β, B, C, D) of 1 (Fig. 4). Key HMBC and NOE correlations are shown in Fig. 4. HMBC correlations between the methoxy protons and the C-6 carbonyls were observed for subunits A, B and D, indicating that the free carboxylic acid was located on subunit C. The C-6 of subunit C also has a higher NMR chemical shift (δ 175.0 ppm), consistent with a carboxylic acid at this position. Results h The MS/MS fragmentation patterns indicated sequential neutral losses of 190 Da for 1 (Fig. 5), further supporting the structure. Three previously reported iridoid glucosides were isolated from the cPOS fraction as minor impurities and identified as 6,7-dihydromonotropein, deacetylasperulsidic acid and monotropein37–40. These iridoid glucosides were inactive in the in vitro quiescence inhibition assay (data not shown). We found that these could be removed from the cPOS fraction by exploiting their differential solubility in ethanol. Cranf1b was triturated with 95% aqueous ethanol prior to PGC separation, producing a cPOS fraction (cPOS-t) devoid of iridoid glycosides as determined by HPLC analysis (Fig. 2) and LC-MS ion extraction (data not shown). Purified oligosaccharides uG3m2 and uG4m3 inhibit E. coli CFT073 quiescence. The isolated oli- gosaccharides uG4m3 and uG3m2 were tested alongside cPOS and HDP-cPOS for reversal of E. coli CFT073 qui- escence (Fig. 1). All four samples, applied as 100 μg/10μL aqueous solutions to the agar surface, stimulated the growth of quiescent bacteria, indicating an inhibition of quiescence. The quiescence assay was repeated using glucose-free plates to test if bacteria growth was simply due to bacteria using the added oligosaccharides as a carbon source. The addition of uG3m2 (100 μg/10μL) onto glucose-free plates containing quiescent CFT073 did not result in visible growth of bacteria colonies (Fig. 6). However, co-spotting 100 μg uG3m2 and 2 mg glucose onto agar containing quiescent CFT073 stimulated bacteria growth. This result is consistent with the cranberry oligosaccharides stimulating the quiescent UPEC to grow on glucose. Scientific Reports \| (2019) 9:19590 \| https://doi.org/10.1038/s41598-019-56005-w www.nature.com/scientificreports/ p p Figure 3. Extracted ion chromatograms of cPOS. Select ions of unsaturated poly-galacturonic acid methyl esters were color coded for DP 3–8 with one (Top) and two (Bottom) free carboxylic acids. Figure 3. Extracted ion chromatograms of cPOS. Select ions of unsaturated poly-galacturonic acid methyl esters were color coded for DP 3–8 with one (Top) and two (Bottom) free carboxylic acids. Figure 4. Structure of 1 with labeled subunits (A–D). Key HMBC correlations are designated with single- headed arrows and key NOE correlations are designated by double-headed, dotted arrows. Figure 4. Structure of 1 with labeled subunits (A–D). Key HMBC correlations are designated with single- headed arrows and key NOE correlations are designated by double-headed, dotted arrows. E. coli CFT073 generates a low level of persister cells in liquid glucose M9 minimal medium containing cranberry pectic oligosaccharides. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 5. ESI-TOF-MS/MS of 1. Structure fragments are designated with the corresponding fragment ions. Figure 5. ESI-TOF-MS/MS of 1. Structure fragments are designated with the corresponding fragment ions. Figure 6. CFT073 quiescence assay using glucose-free M9 plates. The location of spotting each sample is designated by the white ‘x’. (A)- glucose control (Top), M9 media control (Bottom). (B)- uG3m2 spotted alone. (C)- uG3m2 co-spot with glucose. Figure 6. CFT073 quiescence assay using glucose-free M9 plates. The location of spotting each sample is designated by the white ‘x’. (A)- glucose control (Top), M9 media control (Bottom). (B)- uG3m2 spotted alone. (C)- uG3m2 co-spot with glucose. Figure 7. E. coli CFT073 persistence in the presence of cPOS. Cultures were grown in 0.4% glucose M9 minimal medium as described in Methods and diluted 20-fold into 0.2% glucose M9 minimal medium either containing or lacking 1 mg/mL cPOS, and containing or lacking ampicillin (100 µg/mL). At 4 h and 24 h, the differences between E. coli CFT073 persisters in the presence of cPOS + ampicillin is significantly different than with ampicillin only (P ≤ 0.01 and P ≤ 0.001, respectively). CFT073 cell viability with and without cPOS treatment were evaluated as controls. Figure 7. E. coli CFT073 persistence in the presence of cPOS. Cultures were grown in 0.4% glucose M9 minimal medium as described in Methods and diluted 20-fold into 0.2% glucose M9 minimal medium either containing or lacking 1 mg/mL cPOS, and containing or lacking ampicillin (100 µg/mL). At 4 h and 24 h, the differences between E. coli CFT073 persisters in the presence of cPOS + ampicillin is significantly different than with ampicillin only (P ≤ 0.01 and ***P ≤ 0.001, respectively). CFT073 cell viability with and without cPOS treatment were evaluated as controls. previously observed16, the viable cell counts in the E. coli CFT073 cultures decreased slightly during the first 4 h of incubation in both the presence and absence of ampicillin. By 24 h, E. coli CFT073 had grown to just over 109 CFU/mL in the absence of ampicillin (Fig. 7). In contrast, the presence of ampicillin reduced the viable counts an additional ~1000-fold over the next 20 h, to about 106 CFU/mL. As shown by the results in Fig. 7, in the presence of 1 mg/mL cPOS and absence of ampicillin, E. coli CFT073 viable counts increased rapidly, reaching stationary phase within 4 h. Results h Normal microbial populations contain persister cells as small subpopulations (10−3 to 10−4%) of dormant cells that are tolerant to antibiotics. Upon regrowth in the absence of the drug, persister cells produce cultures that regain full sensitivity to the antibiotic. Persister cells likely play a role in chronic infections13. We previously found that E. coli CFT073 generates high levels of persister cells in liquid glucose M9 minimal medium in the presence of ampicillin (100 µg/mL), and the addition of a mixture of the 20 standard L-amino acids (100 µg/mL each) reduced persister cell populations by 100-fold during ampicillin treatment16. p Since cPOS reversed E. coli CFT073 quiescence on glucose plates, we were interested in determining whether cPOS addition to cultures of E. coli CFT073 grown in glucose M9 minimal medium in the presence of ampicillin would generate fewer persister cells. Overnight cultures of E. coli CFT073 grown on 0.4% glucose M9 minimal medium were diluted 20-fold into fresh 0.2% glucose M9 minimal medium (A600 of 0.1, ~108 CFU/mL) contain- ing or lacking ampicillin (100 µg/mL), and viable counts were measured after 4 h and 24 h at 37 °C (Fig. 7). As Scientific Reports \| (2019) 9:19590 \| https://doi.org/10.1038/s41598-019-56005-w Discussion Th l h The oligosaccharides discussed in this manuscript suggest previously undiscovered benefits of cranberry con- stituents for mitigating UPEC infections. Other than specific amino acid cocktails, cPOS represents the first molecules shown to reverse a quiescent phenotype and reduce a persister cell fraction in the UPEC strain CFT073. Inhibition of UPEC quiescence is intriguing since this phenotype has been linked with recurrent UTIs6. Persistence and quiescence are both phenotypes involving dormant bacteria cells; however, it remains to be deter- mined if the physiologic change(s) induced in UPEC by cPOS is shared between the two cell types. Prevention of UPEC dormancy may allow host defense mechanisms and common treatment approaches to be more effective. y y ppf CFT073 is a prototypic UPEC strain belonging to the phylogenetic group B2 multilocus sequence type 73 strains (ST73). ST73 is a major UPEC lineage, accounting for 11% and 16.6% of UPEC isolated from patients in recent studies41,42. It was previously shown that CFT073 enters a quiescent state when plated onto M9 minimal medium agar plates containing glucose at concentrations ≤106 CFU16. A recent study found that 30 of 38 ST73 strains (78.9%) entered the quiescent state on M9 glucose plates, demonstrating that quiescence is a common phenotype for this UPEC lineage. UPEC strains belonging to other sequence types (ST141, ST104, ST394, and ST998) have also demonstrated the same in vitro quiescent phenotype16. Additional studies are underway to determine if cPOS reverses quiescence in UPEC strains other than CFT073. We have shown that cranberry pectic oligosaccharides reduce a persister cell fraction of the UPEC strain CFT073 formed in the presence of ampicillin. Ampicillin belongs to the beta-lactam class of antibiotics and inhib- its enzymes critical for cell wall biosynthesis. Further studies are needed to determine if cPOS similarly reduces persister cell fractions when used with other antibacterial agents, especially those that inhibit bacteria via other mechanisms of action such as the fluoroquinolones.h l The dehydration on subunit D is likely due to the enzymatic treatment used to break down insoluble pectic polysaccharides during cranberry juice manufacturing33. Some pectinases can selectively hydrolyze glycosidic bonds by eliminative cleavage and cause oxygen-aglycone bond breakage43–45. www.nature.com/scientificreports/ Importantly, in the presence of both the cPOS and ampicillin, E. coli CFT073 viable Scientific Reports \| (2019) 9:19590 \| https://doi.org/10.1038/s41598-019-56005-w www.nature.com/scientificreports/ counts decreased rapidly within 4 h, and reached a level of about 3 × 102 CFU/mL after 24 h (Fig. 7). Therefore, treating CFT073 with both cPOS and ampicillin resulted in >1000-fold fewer persister cells versus treatment with ampicillin alone. Similar reduction of persisters was obtained when conducting the assay with cPOS-t (Fig. S13). counts decreased rapidly within 4 h, and reached a level of about 3 × 102 CFU/mL after 24 h (Fig. 7). Therefore, treating CFT073 with both cPOS and ampicillin resulted in >1000-fold fewer persister cells versus treatment with ampicillin alone. Similar reduction of persisters was obtained when conducting the assay with cPOS-t (Fig. S13). Discussion Th l h For example, a mechanistic study on a pectin lyase A (PLA) derived from Aspergillus niger, of the same origin as the Klerzyme 150 in this study, showed specific cleavage of a glycosidic bond adjacent to a methyl-esterified galacturonic acid residue, leaving a 4,5-unsaturated galacturonic acid methyl ester at the non-reducing end46. Interestingly, it was also pointed out that free carboxylic acids generally occurred second from the non-reducing end, which is in agreement with our assignment of 146.i Previous studies of cranberry oligosaccharides have focused on xyloglucans and their antibiofilm and antiad- hesion properties30–32. A recent report showed that xyloglucans can be found in pig urine following oral adminis- tration31. In the study presented here, xyloglucans were separated from cPOS by PGC chromatography and were inactive when tested in the quiescence and persister cells assays. Hence, there may be distinct health benefits from the structurally different oligosaccharide fractions found in cranberry.h f The results presented here encourage further investigation of cPOS as agents to combat recurrent E. coli UTIs and support current drug treatment options. Our findings complement those concerning other cranberry con- stituents (e.g. PACs, organic acids and xyloglucans) and their possible benefits in preventing UTI infections. It is rational to speculate that the full benefit of cranberry may not be realized with one chemical class, but rather that multiple classes act in concert. Reviews of the published clinical data show inconsistencies in chemical character- ization and preparation of cranberry products tested for UTI prevention22. The optimal formulation and use of cranberry products for UTI prevention will require further knowledge of the active constituents and mechanisms of action. Until then, cranberry juice or whole berry products might be best used until more is known regarding how and why cranberry may aid in preventing UTIs. Methods G l The collected solids were then dissolved in d.i. H2O and lyophilized. The trituration step was repeated twice prior to further separation by PGC chromatography to provide sample cPOS-t (91.7%, w/w dry). Uronic acid identification of cPOS. The composition of the poly-galacturonic acid backbone of cPOS was determined by acid hydrolysis of cPOS followed by derivatization with 1-phenyl-3-methyl-5-pyrazolone (PMP)47. Briefly, 10 mg cPOS was hydrolyzed in 1 mL 2 M TFA at 90 °C for 5 h. After cooling to room temperature, the reaction mixture was centrifuged at 3000 rpm for 5 min. The supernatant was evaporated in vacuo to remove residual TFA. The hydrolysate mixture was dissolved in 1 mL distilled water and treated with 30 µL NaOH (0.3 M) and 20 µL PMP solution (0.5 M in methanol) for derivatization. The mixture was incubated at 70 °C for 60 min, cooled to room temperature and neutralized with 30 µL of HCl (0.3 M). The mixture was then extracted with 1 mL chloroform. The aqueous layer was filtered by passing through a 0.45 µm syringe filter. Glucuronic acid (2.0 mM), galacturonic acid (2.0 mM) and a distilled water blank were derivatized the same as described above. HPLC anal- yses of PMP-labeled samples were conducted on a Prominence i-series system (Shimadzu) using a C18 column (4.6 mm × 250 mm, 5 𝜇m, J.T. Baker Inc., Phillipsburg, NJ, USA). The injection volume was 20 µL with a flow rate of 1.0 mL/min at 35 °C. Mobile phase A was 10% ACN/water with 0.045% KH2 PO4–0.05% triethylamine buffer (pH 7.5) and mobile phase B was 100% ACN. The column was eluted with 6% B for 4 min, 6–12% for 1 min and then 12% B for 20 min. The UV detection wavelength was 254 nm. Bacterial strains and cultivation. E. coli CFT073 was obtained from a cryogenically frozen stock at the laboratory of P.S.C. and stored at −80 °C in a 1:1 mixture of LB broth and 50% glycerol by volume. LB broth and LB agar were used for routine cultivation. Liquid M9 minimal medium was prepared as described previously16, and M9 minimal medium agar plates were prepared with 1.5% noble agar to avoid impurities present in bacteri- ological agar. In vitro quiescence inhibition assay. The procedure for this assay closely follows a published protocol16. In general, overnight cultures of E. Methods G l coli CFT073 were prepared in 0.4% glucose M9 minimal medium as aforemen- tioned. Bacteria from this culture were diluted to a final concentration of 105 CFU in 4 mL of liquid overlay media (0.2% glucose M9 minimal medium with 0.9% noble agar at 45 °C). Each 4 mL overlay inoculum was poured over a pre-warmed (37 °C) 0.2% glucose M9 minimal media agar plate immediately after inoculation. These plates were allowed to solidify at room temperature with lids slightly ajar. Once solidified, test spots were added to the overlay media and allowed to dry before incubating the plate upside down at 37 °C for 24 h. Test spots were 10 μL of each test solution at 10 mg/mL in M9 minimal media unless indicated otherwise. Non-growing E. coli in the overlay were considered to be quiescent if growth could be induced with the positive control (3 co-spots; 5 μL each of tyrosine, lysine, and methionine solutions at 0.1 mg/mL; co-spots added sequentially to the identical location on the agar). To test the effects in glucose-free conditions, glucose was not added to any of the media components prior to completing the assay. When adding glucose back to the glucose-free plates, a 10 μL spot of 20% glucose in water (2 mg; sufficient to support bacteria growth) was added directly following and to the iden- tical location as the dried test spot. For all quiescence assays, a positive result was defined as observable bacteria growth at the site of a test sample following 24 h incubation. A negative result was defined as no observable bac- teria growth at the site of a test sample. Images of agar plates were made after 24 h using a Molecular imager Gel Doc XR+ (Bio-rad, Hercules, CA, USA) system with Image Lab Software. Persister cell viability assay. The procedure for this assay closely followed a published protocol16. E. coli CFT073 was streaked from cryogenically frozen stocks onto LB agar plates and incubated overnight at 37 °C. A loopful of cells from the plate was added to 10 mL 0.4% glucose M9 minimal media in a 125 mL culture flask and incubated overnight at 37 °C and 200 rpm. Methods G l General experimental procedures. NMR experiments were conducted using an Agilent Inova NMR 500 MHz spectrometer in D2O (99.99%, Sigma-Aldrich, St. Louis, MO, USA) at 25 °C. LC-MS analysis was per- formed on a Prominence UFLC system (Shimadzu, Kyoto, Japan) coupled to a QTRAP 4500 mass spectrom- eter (AB Sciex, Framingham, MA, USA) with electrospray ionization source on negative ion mode with scans from m/z 200 to m/z 2000. High Resolution Electrospray Ionization mass spectra and tandem mass spectra (HR-ESI-MS and HR-ESI-MS/MS) were acquired using a TripleTOF 4600 spectrometer (AB Sciex) operating in negative ion mode. Flash chromatography was completed using an Agilent 971-FP flash purification sys- tem (Agilent Technologies, Santa Clara, CA, USA) with Biotage SNAP KP-C18-HS 120 g cartridges (Biotage, Charlotte, NC, USA). HPLC separations were performed on a Shimadzu Prominence i-series HPLC (Shimadzu). Separation of cranberry pectic oligosaccharides. Cranberry hulls were degraded with pectinase (Klerzyme 150, DSM Food Specialties, Heerlen, Netherlands) and fractionated as previously described with mod- ifications30. Briefly, cranberry pectinase treated hull extract was fractionated using a C18 flash column sequen- tially eluted with 500 mL 100% deionized (d.i.) water, 500 mL 15% methanol/H2O and 500 mL 100% methanol. Fractions from each step-gradient were individually pooled into three major fractions, Cranf1W (38.1%, w/w, dry), Cranf1b (23.8%, w/w, dry) and Cranf1M (28.1%, w/w, dry). Cranf1b was then subjected to separation using a PGC HyperSep Hypercarb SPE cartridge (1 gram, Thermofisher Scientific, Waltham, MA, USA). The PGC car- tridge was firstly conditioned by 50% ACN/water and then equilibrated with 100% d.i. water. Two mL of 20 mg/ mL Cranf1b aqueous solution was then loaded onto the PGC cartridge and eluted sequentially with d.i. water, 10% ACN/H2O/0.1% TFA and 30% ACN/H2O/0.1% TFA. The 10% ACN/H2O/0.1% TFA PGC eluent (cPOS, Scientific Reports \| (2019) 9:19590 \| https://doi.org/10.1038/s41598-019-56005-w www.nature.com/scientificreports/ 15% w/w, dry) was then separated using a TSKgel Amide-80 HR HILIC column (4.6 × 250 mm, 5 μm, TOSOH Bioscience, Tokyo, Japan) at 1 mL/min and 35 °C. The column was eluted with a linear gradient from 80% ACN/ H2O/0.1% formic acid to 40% ACN/H2O/0.1% formic acid over 30 min. uG3m2 and 1 were collected at 10 min and 12.5 min, respectively (Fig. 2). Trituration of Cranf1b to remove iridoid terpenes was accomplished by sonicating 200 mg Cranf1b in 30 mL 95% ethanol/water and then centrifuging the solids at 17,000x g for 8 min. Methods G l Cultures were diluted in fresh 0.2% glucose M9 minimal media supplemented with test samples (1 mg/mL) to an optical density (OD600) of 0.1 (~108 CFU/mL) and grown in the presence of ampicillin sodium at 10x MIC (0.1 mg/mL) to generate persister fractions. Cultures were grown in the absence of ampicillin and/or cranberry constituents as controls. The cultures were incubated shaking (200 rpm) at 37 °C and viable counts were measured at 0, 4, and 24 hours by plating on LB media. The persister cell viability assays were completed in triplicate and analyzed for statistical significance. Statistics. Persister cell viability assays were compared using a two-tailed student’s t test. P-values ≤ 0.05 were considered statistically significant. Received: 6 March 2019; Accepted: 4 December 2019; Published: xx xx xxxx Scientific Reports \| (2019) 9:19590 \| https://doi.org/10.1038/s41598-019-56005-w ( ) 4. Foxman, B. Urinary tract infection syndromes: occurrence, recurrence, bacteriology, risk factors, and disease burden. Infect. Dis Clin. North Am. 28, 1–13 (2014). and treatment options. Nat. Rev. Microbiol. 13, 269–284 (2015). 3. Aydin, A., Ahmed, K., Zaman, I., Khan, M. S. & Dasgupta, P. Recurrent urinary tract infections in women. Int. Urogynecol. J. 26, 795–804 (2015). 1. Stamm, W. E. & Norrby, S. R. Urinary tract infections: disease panorama and challenges. J. Infect. Dis. 183, S1–4 (2001). References 1. Stamm, W. E. & Norrby, S. R. Urinary tract infections: disease panorama and challenges. J. Infect. Dis. 183, S1–4 (2001). 2. Flores-Mireles, A. L., Walker, J. N., Caparon, M. & Hultgren, S. J. Urinary tract infections: epidemiology, mechanisms of infection and treatment options. Nat. Rev. Microbiol. 13, 269–284 (2015). 3. Aydin, A., Ahmed, K., Zaman, I., Khan, M. S. & Dasgupta, P. Recurrent urinary tract infections in women. Int. Urogynecol. J. 26, 795–804 (2015). 4. Foxman, B. Urinary tract infection syndromes: occurrence, recurrence, bacteriology, risk factors, and disease burden. Infect. Dis. Clin. North Am. 28, 1–13 (2014). References Sun, J. et al. Cranberry (Vaccinium macrocarpon) oligosaccharides decrease biofilm formation by uropathogenic Escherichia coli. J Funct. Foods 17, 235–242 (2015).t 1. Coleman, C. M. et al. Arabinoxyloglucan oligosaccharides may contribute to the antiadhesive properties of porcine urine after cranberry consumption. J. Nat. Prod. 82, 589–605 (2019). y p 32. Hotchkiss, A. T. Jr. et al. Cranberry xyloglucan structure and inhibition of Escherichia coli adhesion to epithelial cells. J. Agric. Food Chem. 63, 5622–5633 (2015).it 3. Spiro, M. D. et al. Purification and characterization of biologically active 1, 4-linked α-d-oligogalacturonides after partial digestion of polygalacturonic acid with endopolygalacturonase. Carbohydr. Res. 247, 9–20 (1993). yg yg y 4. Cipriani, T. R. et al. Polygalacturonic acid: Another anti-ulcer polysaccharide from the medicinal plant Maytenus ilicifolia Carbohydr. Polym. 78, 361–363 (2009). 35. Marcon, M. V., Carneiro, P. I. B., Wosiacki, G., Beleski-Carneiro, E. & Petkowicz, C. L. O. Pectins from apple pomace–characterization by 13C and 1H NMR spectroscopy. Ann. Magn. Reson. 4, 56–63 (2005). h l f f d l h d l ( ) y p py g ( ) 36. Cheng, H. N. & Neiss, T. G. Solution NMR spectroscopy of food polysaccharides. Polymer Reviews 52, 81–114 (2012). 37. Jensen, H. D., Krogfelt, K. A., Cornett, C., Hansen, S. H. & Christensen, S. B. Hydrophilic carboxylic acids and iridoid glycosides in the juice of American and European cranberries (Vaccinium macrocarpon and V. oxycoccos), lingonberries (V. vitisidaea), and blueberries (V. myrtillus). J. Agric. Food Chem. 50, 6871–6874 (2002). y g 38. Dinda, B., Debnath, S. & Harigaya, Y. Naturally occurring iridoids. A review, part 1. Chem. Pharm. Bull. 55, 159–222 (2007). Ch dh Afifi Ü S h O kl T 13 f ll d d l d d 38. Dinda, B., Debnath, S. & Harigaya, Y. Naturally occurring irido 39. Chaudhuri, R. K., Afifi-Yazar, F. Ü., Sticher, O. & Winkler, T. 13C their acylated derivatives. Tetrahedron 36, 2317–2326 (1980). 39. Chaudhuri, R. K., Afifi-Yazar, F. Ü., Sticher, O. & Winkler, T. 13C NMR spectroscopy of naturally occurring iridoid glucoside their acylated derivatives. Tetrahedron 36, 2317–2326 (1980).l ii their acylated derivatives. Tetrahedron 36, 2317–2326 (1980) 40. Demi̇rezer, L. Ö., Gürbüz, F., Güvenalp, Z., STRÖCH, K. & Zeeck, A. Iridoids, flavonoids and monoterpene glycosides from Galium verum subsp. verum. Turk. J. Chem. 30, 525–534 (2006). 41. Croxall, G. et al. References mSphere 1, e00055–00015 (2016). g y p 7. Putrinš, M. et al. Phenotypic heterogeneity enables uropathogenic Escherichia coli to evade killing by antibiotics and serum complement. Infect. Immun. 83, 1056–1067 (2015).i f 18. Niu, H. et al. Identification of anti-persister activity against uropathogenic Escherichia coli from a clinical drug library. Antibiotics 4, 179–187 (2015). 19. Orman, M. A. & Brynildsen, M. P. Inhibition of stationary phase respiration impairs persister formation in E. coli. Nat. Commun. 6, 7983 (2015). 20. Kim, J. S. et al. Fumarate-mediated persistence of Escherichia coli against antibiotics. Antimicrob. Agents. Chemother. 60, 2232– (2016).hf 1. Vasileiou, I., Katsargyris, A., Theocharis, S. & Giaginis, C. Current clinical status on the preventive effects of cranberry consumption against urinary tract infections. Nutr. Res. 33, 595–607 (2013). 22. Jepson, R. G., Williams, G. & Craig, J. C. Cranberries for preventing urinary tract infections. Cochrane Database Syst. Rev. 10, Cd001321 (2012). 3. Sobota, A. E. Inhibition of bacterial adherence by cranberry juice: potential use for the treatment of urinary tract infections. J. Urol 131, 1013–1016 (1984).i 131, 1013 1016 (1984). 4. Gupta, K. et al. Cranberry products inhibit adherence of p-fimbriated Escherichia coli to primary cultured bladder and vagina ith li l ll J U l 177 2357 2360 (2007) 4. Gupta, K. et al. Cranberry products inhibit adherence of p-fimbriated Escherichia coli to primary cultured bladder and vagina epithelial cells. J. Urol. 177, 2357–2360 (2007).f p 25. Caljouw, M. A. et al. Effectiveness of cranberry capsules to prevent urinary tract infections in vulnerable older persons: a double- blind randomized placebo-controlled trial in long-term care facilities. J. Am. Geriatr. Soc. 62, 103–110 (2014). 6. Maisuria, V. B., Los Santos, Y. L.-d, Tufenkji, N. & Déziel, E. Cranberry-derived proanthocyanidins impair virulence and inhibi quorum sensing of Pseudomonas aeruginosa. Sci. Rep. 6, 30169 (2016). q g g p 27. Howell, A. B. et al. A-type cranberry proanthocyanidins and uropathogenic bacterial anti-adhesion activity. Phytochemistry 66, 2281–2291 (2005). ( ) 8. Zafriri, D., Ofek, I., Adar, R., Pocino, M. & Sharon, N. Inhibitory activity of cranberry juice on adherence of type 1 and type P fimbriated Escherichia coli to eucaryotic cells. Antimicrob. Agents Chemother. 33, 92–98 (1989).f i 9. Jensen, H. D., Struve, C., Christensen, S. B. & Krogfelt, K. A. Cranberry juice and combinations of its organic acids are effective against experimental urinary tract infection. Front. Microbiol. 8, 542 (2017).i g p y 0. References Scientific Reports \| (2019) 9:19590 \| https://doi.org/10.1038/s41598-019-56005-w www.nature.com/scientificreports/ 5. Foxman, B. & Brown, P. Epidemiology of urinary tract infections: Transmission and risk factors, incidence, and costs. Infect. Dis Clin. North. Am. 17, 227–241 (2003). 6. Hunstad, D. A. & Justice, S. S. Intracellular lifestyles and immune evasion strategies of uropathogenic Escherichia coli. Annu. Rev Microbiol. 64, 203–221 (2010). , ( ) 7. Ejrnaes, K. et al. Pulsed-field gel electrophoresis typing of Escherichia coli strains from samples collected before and after pivmecillinam or placebo treatment of uncomplicated community-acquired urinary tract infection in women. J. Clin. Microbiol. 44, 1776–1781 (2006). 8. Kerrn, M. B., Struve, C., Blom, J., Frimodt-Moller, N. & Krogfelt, K. A. Intracellular persistence of Escherichia coli in urinary bladder from mecillinam-treated mice. J. Antimicrob. Chemother. 55, 383–386 (2005). 9. Rosen, D. A., Hooton, T. M., Stamm, W. E., Humphrey, P. A. & Hultgren, S. J. Detection of intracellular bacterial communities in human urinary tract infection. PLOS Med. 4, e329 (2007). 9. Rosen, D. A., Hooton, T. M., Stamm, W. E., Humphrey, P. A. & Hultgren, S. J. Detection o human urinary tract infection. PLOS Med. 4, e329 (2007). y ( ) 10. Silverman, J. A., Schreiber, H. L., Hooton, T. M. & Hultgren, S. J. From physiology to pharmacy: Developments in the pathoge and treatment of recurrent urinary tract infections Curr Urol Rep 14 448 456 (2013) y 10. Silverman, J. A., Schreiber, H. L., Hooton, T. M. & Hultgren, S. J. From physiology to pharmacy: Developments in the pathogenesis and treatment of recurrent urinary tract infections. Curr. Urol. Rep. 14, 448–456 (2013). y p 11. Shah, D. et al. Persisters: a distinct physiological state of E. coli. BMC Microbiol. 6, 53 (2006). wis, K. Persister cells: molecular mechanisms related to antibiotic to 12. Lewis, K. Persister cells: molecular mechanisms related to antibiotic tolerance. Handb. Exp. Pharmacol., 121–133, (2012). L i K P i ll A R Mi bi l 6 ( ) 13. Lewis, K. Persister cells. Annu. Rev. Microbiol. 64, 357–372 (201 14. Lewis, K. Multidrug tolerance of biofilms and persister cells. Curr. Top. Microbiol. Immunol. 322, 107–131 (2008).hl 15. Joers, A., Kaldalu, N. & Tenson, T. The frequency of persisters in Escherichia coli reflects the kinetics of awakening from dormancy. J. Bacteriol. 192, 3379–3384 (2010). 6. Leatham-Jensen, M. P. et al. Uropathogenic Escherichia coli metabolite-dependent quiescence and persistence may explain antibiotic tolerance during urinary tract infection. Scientific Reports \| (2019) 9:19590 \| https://doi.org/10.1038/s41598-019-56005-w References Molecular epidemiology of extraintestinal pathogenic Escherichia coli isolates from a regional cohort of elderly patients highlights the prevalence of ST131 strains with increased antimicrobial resistance in both community and hospital care settings. J Antimicrob Chemother 66, 2501–2508 (2011). 42. Gibreel, T. M. et al. Population structure, virulence potential and antibiotic susceptibility of uropathogenic Escherichia coli from Northwest England. J Antimicrob Chemother 67, 346–356 (2012). Northwest England. J Antimicrob Chemother 67, 346–356 (201 g 43. Pedrolli, D. B., Monteiro, A. C., Gomes, E. & Carmona, E. C. Pectin and pectinases: production, characterization and indu application of microbial pectinolytic enzymes. Open Biotechnol. J. 3, 9–18 (2009).h 43. Pedrolli, D. B., Monteiro, A. C., Gomes, E. & Carmona, E. C. Pectin and pectinases: production, characterization application of microbial pectinolytic enzymes. Open Biotechnol. J. 3, 9–18 (2009).h pp p y y p 44. Hasegawa, S. & Nagel, C. W. The characterization of an α, β-unsaturated digalacturonic acid. J. Biol. Chem. 237, 619–621 (1962). 44. Hasegawa, S. & Nagel, C. W. The characterization of an α, β-un S. & Nagel, C. W. The characterization of an α, β-unsaturated digala g gh g 45. Linhardt, R., Galliher, P. & Cooney, C. Polysaccharide lyases. Applied Biochem. Biotechnol. 12, 135–176 (1987). y y y pp 46. van Alebeek, G. J., Christensen, T. M., Schols, H. A., Mikkelsen, J. D. & Voragen, A. G. Mode of action of pectin lyase A of Aspergillus niger on differently C(6)-substituted oligogalacturonides. J. Biol. Chem. 277, 25929–25936 (2002).ii f 47. Ruhaak, L. R. et al. Glycan labeling strategies and their use in identification and quantification. Anal. Bioanal. Chem. 397, 3457– (2010). www.nature.com/scientificreports/ Acknowledgementsh g This work was supported, in part, by Ocean Spray Cranberries, Inc. (Lakeville-Middleboro, MA, USA). RD was financially supported by the Omar Magnate Foundation Fellowship. Research reported in this publication was made possible by the use of equipment and services available through the RI-INBRE Centralized Research Core Facility, which is supported by the Institutional Development Award (IDeA) Network for Biomedical Research Excellence from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103430, and by the National Science Foundation EPSCoR Cooperative Agreement #EPS- 1004057. Competing interestsh p g The authors declare no competing interests. Author contributions D.C.R., J.S., R.W.D. and P.S.C. conceived and designed the experiments. J.S. and R.W.D. prepared the manuscript and contributed equally to this work. J.S. and R.W.D. performed chemical purification and characterization of cranberry materials. R.W.D and L.N. performed in vitro quiescence and persister cell assays. Z.P. contributed to Figures 2 and S3. L.N. contributed to Figure 8. C.K., P.S.C., N.P.S and D.C.R edited and approved the final version of the manuscript. Additional information Supplementary information is available for this paper at https://doi.org/10.1038/s41598-019-56005-w. Supplementary information is available for this paper at https://doi.org/10.10 Correspondence and requests for materials should be addressed to D.C.R. 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 Cre- ative 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 per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2019 Scientific Reports \| (2019) 9:19590 \| https://doi.org/10.1038/s41598-019-56005-w
W4385525647.txt	https://rur.oekom.de/index.php/rur/article/download/2203/3394	de		Von der Raumordnung zur Umweltordnung	Raumforschung und Raumordnung	1,994	cc-by	4,160	Ulrich Hodapp: Von der Raumordnung zur Umweltordnung Ulrich Hodapp Von der Raumordnung zur Umweltordnung Planerische Konsequenzen eines ökosystemaren Denkansatzes* Kurzfassung Die Einführung eines ökosystemaren Planungsansatzes und die Bildung von Ökosystemtypen in der Umweltplanung haben gravierende Auswirkungen auf Planungsziele, Planungskonzepte und Planelemente. Als Indikatoren zur Beurteilung der Funktionsfähigkeit von einzelnen Ökosystemen und der Landschaft insgesamt eignen sich stofflich-energetische und biotische Stabilitätskriterien. Ein Einbeziehen anthropogener Ökosysteme und damit letztendlich die Einflußnahme auf das menschliche Wirtschaften ist unverzichtbar. Die Bestimmung eines "ökologischen Nutzbarkeitsrahmens", nur innerhalb dessen der Mensch Ökosysteme und Umweltmedien in Anspruch nehmen kann, und das Konzept einer "naturnahen Kulturlandschaft" sind zwei mögliche Konzeptansätze einer ökosystemar ausgerichteten Umweltplanung. Die Ausgestaltung als Umweltleitplanung ist ein wesentlicher Baustein auf dem Weg von der bestehenden Raumordnung zu einer Umweltordnung. 1 Konzeptionelle Grundlagen 1.1 Die Stellung des Naturund Umweltschutzes Die derzeit vorherrschende Sichtweise in der Umweltpolitik und Umweltplanung betrachtet den Natur- und Umweltschutz als ein eigenständiges Interesse neben vielen anderen (z.B. Industrie, Landwirtschaft). Diese Interessen kämpfen alle gleichermaßen gegeneinander um ihre Durchsetzung. Aus dieser Sichtweise ergeben sich die "unsinnigen" Konfrontationen z.B. um Arbeitsplätze oder Umweltschutz. Notwendig ist aber eine Sichtweise, die den Natur- und Umweltschutz und dam,it die Sicherung der natürlichen Lebensgrundlagen nicht als Konkurrenz, sondern als Basis und elementare Grundlage jeder wirtschaftlichen und gesellschaftlichen Entwicklung betrachtet. "Denn wenn es richtig ist, daß über ökologische Qualitäten unserer Umwelt bereits entschieden wird, indem festgelegt wird, mit welchen Stof- RuR4/5.1994 fen, Energien und Verfahren welche Güter hergestellt werden - und dies ist augenscheinlich der Fall -, dann muß es Aufgabe der Umweltpolitik sein, für die Produktions- und Konsumtionsprozesse der Gesellschaft Rahmen mit dem Ziel zu setzen, daß dabei auch die gewünschten Naturqualitäten erhalten bzw. erzeugt werden" (1). 1.2 Die Einführung von Ökosystemtypen als Planungskategorie Ein ökosystemarer Konzeptansatz muß die getrennte Betrachtung der Umweltmedien Wasser, Boden, Luft/Klima und Flora/Fauna überwinden. Dies ist möglich durch die Abgrenzung einzelner Ökosysteme, innerhalb derer die einzelnen Umweltmedien im jeweiligen Wirkungsgefüge zu berücksichtigen sind. Anthropogene Ökosysteme sind dabei mit einzuschließen. Damit rückt neben dem Wirkungsgefüge der Naturfaktoren untereinander das Wirkungsgefüge zwischen dem Menschen und der Natur in den Mittelpunkt. Das Gesamtsystem unserer Umwelt muß als ein miteinander verflochtenes ökologisch-ökonomisches System betrachtet werden, in dem ein Schutz der Umwelt nicht gegen den Menschen, sondern nur durch umweltverträgliches Wirtschaften des Menschen erreicht wird. Dazu ist sowohl eine flächendeckende wie eine verursacherbezogene Betrachtungsweise notwendig. Die Gliederung der gesamten Landschaft in Ökosystemtypen stellt dafür eine planerisch handhabbare Möglichkeit dar. Die Restbestände der natürlichen/ naturnahen Ökosysteme bilden das ökologische Rückgrat einer jeden Landschaft (Ökosystemkomplex). Sie überlappen sich in mehr oder weniger kleinen Teilbereichen mit den anthropogen geprägten Bioökosystemen (Wald- und Agrarökosysteme) sowie den anthropogenen Techno-Ökosystemen (Urban-industrielle Ökosysteme). Limnische Ökosysteme bleiben außer Betracht. 287 Ulrich Hodapp: Von der Raumordnung zur Umweltordnung Ökosystemkomplex / Landschaft 1.3 Indikatoren einer funktionsfähigen Landschaft: Stofflichenergetische und biotische Stabilität Bei der näheren Betrachtung der in Ökosystemen ablaufenden Prozesse zeigt sich, daß Stoff- und Energieflüsse die zentralen Komponenten innerhalb einzelner Ökosysteme und zwischen verschiedenen Ökosystemen sind. Diesen Komponenten wurde planerisch bisher keinerlei Beachtung geschenkt. Dabei greift der Mensch auf allen Ebenen in die natürlichen, dynamischen Energie- und Stoffflüsse ein und schafft neue (z.B. Abbau von Rohstoffen, Produktion von Gütern, Energieerzeugung). Über die Ausbreitungspfade Luft, Boden und Wasser kommt es, vor allem durch das Einbringen von Stoffen, zu Veränderungen der natürlichen Kreisläufe. Diese Stoffe sind Abbildung 1 Gliederung der Landschaft (Ökosystemkomplex) in Ökosystemtypen nichts anderes als Abfälle der mensch- lieh-energetische und biotische Instalichen Tätigkeiten und Ausdruck der bilitäten in allen Ökosystsemen sind Funktionsunfähigkeit bestehender an- die Folge. thropogener Ökosysteme. Für diese Abfälle fehlen sowohl in den anthropo- Die Lösung der ökologischen Frage genen wie den natürlichen Ökosyste- steht und fällt mit der Schaffung von men die entsprechenden Abbaumög- ökologisch funktionsfähigen anthropolichkeiten in der notwendigen Größen- genen Systemen, d.h. der Ökologisieordnung. Die vielfältigen Mechanis- rung der menschlichen Nutzungs- und men natürlicher Ökosysteme zur Bewirtschaftungsmuster. Aufgabe Selbstregulierung werden überfordert, einer zukünftigen Umweltplanung überlagert oder ausgeschaltet und da- bzw. Umweltordnung muß es daher mit die Funktionsfähigkeit dieser Öko- sein, auf der Grundlage von flächensysteme beeinträchtigt. Die Beseiti- deckend ökosystemar funktionsfähigung und Isolierung natürlicher/natur- gen Ökosystemen für den Menschen naher Ökosysteme durch die Zunahme stabile Wirtschafts- und Produktionsmenschlicher Landnutzungen führt zu bedingungen zu schaffen. Aus der Anaeiner zunehmenden biotischen Insta- lyse natürlicher Funktionsprinzipien in bilität aufgrund der Beseitigung der Ökosystemen ergibt sich, daß zur Beurnatürlichen Diversität. Aus ökosyste- teilung der Funktionsfähigkeit von marer Sicht führt die weitreichende Ökosystemen die stofflich-energetiFunktionsunfähigkeit anthropogener . sche und die biotische Stabilität (im Ökosysteme zwangsläufig auch zu Sinne einer dynamischen Stabilität) funktionsunfähigen natürlichen/natur- herangezogen werden können (vgl. nahen Ökosystemen. Weltweite stoff- Übersicht 1). Übersicht 1 Stofflich-energetische und biotische Stabilität Stofflich-energetische Stabilität • ein Nichtgleichgewichtszustand, bei dem jedoch der systeminteme Zustand (Vorräte an Energie und Stoffen) durch äußere Einflüsse zeitlich konstant gehalten wird; In- und Output sowie Auf- und Abbauprozesse sind ausgewogen; Kreislaufprinzip • ein selbstgeregelter, mehr oder weniger gleichmäßiger Energiedurchsatz und Stoff~msatz mit einem Minimum an Neben- und Wechselwirkungen zwischen den einzelnen Ökosystemen • die stoffliche und energetische Grundlage liegt im Ökosystem selbst Biotische Stabilität • Einhalten der biotischen Mindestanforderungen (Flächen, Populationen, Lebensbedingungen) • kleinteilige, mosaikhafte Vielfalt in den Ökosystemen bzw. im Ökosystemkomplex • Vorhandensein der ganzen biotischen Bandbreite an Biozönosen einschließlich den Lebensräumen und Lebensbedingungen • flächendeckend stabile Ökosystemstrukturen 288 RuR4/5 . l994 Ulrich Hodapp: Von der Raumordnung zur Umweltordnung 2 Konsequenzen für die Planung 2.1 Konsequenzen für die Planungsziele Die zukünftigen Aufgaben und Zielsetzungen einer nach ökosystemaren Gesichtspunkten aufgebauten Umweltleitplanung können folgendermaßen formuliert werden: Umweltleitpläne weisen auf der Grundlage einer Bewertung der bestehenden Umweltsituation, einer Risikoabschätzung, einer als funktion~fähig zu bezeichnenden Vergleichssituation und einer Bewertung der zukünftigen Entwicklungsmöglichkeiten ökologische Leitbilder aus. Diese Leitbilder bilden die Grundlage einer Umweltvorsorgeplanung (im Sinne einer Risikovermeidung und Bewirtschaftung) mit dem Ziel der Sicherung bzw. Wiederherstellung der Funktionsfähigkeit der Ökosysteme und der Ökosystemkomplexe. Dazu sind die notwendigen Schutz-, Entwicklungs- und Sanierungsräume auszuweisen, einschließlich der dazu notwendigen Ziele, Maßnahmen und Prioritäten (fachlich und zeitlich). Im Rahmen dieser Zielsetzungen gilt es vorrangig eine grundlegende Definition der Funktionsfähigkeit der einzelnen Ökosysteme und der Ökosystemkomplexe festzulegen. Im nachfolgenden werden für natürliche/naturnahe und urban-industrielle Ökosysteme beispielhaft Definitionen vorgestellt, die als ein erster Beitrag für eine zukünftige Diskussion zu verstehen sind. herzustellen. Während natürliche Ökosysteme vom Menschen unbeeinflußt bleiben, sind naturnahe Ökosysteme durch direkte menschliche Pflege- und Bewirtschaftungsmaßnahem so zu steuern, daß ihre ökologischen Funktionen optimal und nachhaltig gesichert werden. Mit der Funktionsfähigkeit verbindet sich der Anspruch, daß die in diesen Ökosystemen ablaufenden natürlichen Prozesse durch indirekte Einflüsse anthropogener Ökosysteme nicht beeinträchtigt werden dürfen, so daß eine stofflich-energetische und biotische Stabilität vorliegt. RuR4/5 .1994 (1) die Bestimmung der ökologischen Nutzbarkeitspotentiale von Ökosystemen und einzelnen Umweltmedien (ökologisch verträglicher Nutzbarkeitsrahmen) im Sinne einer Risikovermeidung und Ressourcenbewirtschaftung sowie die Bestimmung der anthropogenen Nutzungsnotwendigkeit für Umweltentlastungspotentiale (Alternativ-, Spar- und Reduktionspotentiale); (2) Urban-industrielle Ökosysteme Urban-industrielle Ökosysteme setzen sich zusammen aus dem kleinräumigen Nebeneinander von natürlichen, naturnahen und umweltverträglich bewirtschafteten bzw. umweltverträglich wirtschaftenden Ökosystemen. Diese Ökosysteme erfüllen im Rahmen ihrer Funktionsfähigkeit festzulegende natürliche ökologische Teilfunktionen im Landschaftshaushalt. Mit der Funktionsfähigkeit verbindet sich der Anspruch, daß im Rahmen umweltverträglichen Wirtschaftens die in diesen Ökosystemen ablaufenden technischen Stoff- und Energieflüsse soweit optimiert bzw. minimiert sind, daß sowohl eine Beeinträchtigung der Funktionsfähigkeit der Bio-Ökosysteme als auch der menschlichen Gesundheit ausgeschlossen werden kann (Einhalten eines ökologisch verträglichen Nutzungsrahmens). 2.2 Konsequenzen für die Planungskonzepte Natürliche/naturnahe Ökosysteme Unter den natürlichen Ökosystemen sind Lebensräume zu verstehen, die den Charakter von Urlandschaften aufweisen und keinerlei menschlicher Nutzung unterliegen. Naturnahe Ökosysteme sind aus der menschlichen Nutzung hervorgegangene Lebensräume, die regelmäßigen menschlichen Eingriffen geringer Intensität unterliegen. Diese Lebensräume sind zur Erfüllung ihrer Funktionen im Landschaftshaushalt in ausreichender Vielfalt, Größe, Zusammensetzung und Verbindung zu erhalten bzw. wieder- grundlegende Anforderungen an zukünftige Planungskonzepte: 2.2.1 Zu berücksichtigende Belange Für die zu berücksichtigenden Belange ergibt sich die Notwendigkeit veränderter und erweiterter Anforderungen. Die Zielsetzung der Schaffung funktionsfähiger Ökosysteme beeinhaltet eine Berücksichtigung sowohl des Wirkungsgefüges der natürlichen Landschaftsfaktoren als auch des Wirkungsgefüges zwischen diesen Landschaftsfaktoren und der menschlichen Gesellschaft (v.a. auch der Stoff- und Energieflüsse). Aus einer solchen Zielsetzung entstehen gleichzeitig zwei die Bestimmung eines ökologisch verträglichen Landnutzungskonzepts. 2.2.2 Bestimmung eines ökologischen Nutzbarkeitsrahmens Die Nutzungserfordernisse des Menschen an die Umweltmedien und die Ökosysteme und die sich daraus ergebenden Beeinträchtigungen sind im wesentlichen zurückzuführen auf die fehlende Selbstregulation der anthropogenen Ökosysteme, den hohen Stoff- und Energieeinsatz in diesen Systemen, die fehlenden Kreisläufe und die völlige Abhängigkeit der urban-industriellen Ökosysteme von den BioÖkosystemen und einzelnen Naturfaktoren als Stoff- und Energieeinfuhrbzw. -austragsräume. Die Schaffung funktionsfähiger anthropogener Ökosysteme macht die Bestimmung eines ökologischen Nutzbarkeitsrahmens, nur innerhalb dessen eine Nutzung ökologischer Potentiale durch den Menschen verträglich ist, zwingend notwendig (vgl. Abb. 2). Unter der übergeordneten Zielsetzung des Erhalts bzw. der Schaffung einer funktionsfähigen Landschaft bildet nicht der menschliche Bedarf, sondern die Empfindlichkeit der einzelnen Ökosysteme den Ausgangspunkt, d.h. die Nutzungsmöglichkeiten orientieren sich an den ökologischen Notwendigkeiten. Ein anthropogenes Potentialnutzungskonzept innerhalb eines solchen ökologischen Nutzbarkeitsrahmens wird an umfassenden Bewirtschaftungskonzepten für die einzelnen Umweltmedien nicht vorbeikommen. 289 Ulrich Hodapp: Von der Raumordnung zur Umweltordnung Abbildung 2 Ökologischer Nutzbarkeitsrahmen IST -Zustand 1 Planerische Aufgabe 1 1 1 Ökologische Zielsetzung Funktionsunfähigkeit der Techno-Ökosysteme Funktionsfähigkeit der Landschaft 1 1 1 Funktionsfähigkeit der Bio -Ökosysteme Funktionsfähigkeit der Techno -Ökosysteme Bestehende Nutzungsansprüche 1 Empfindlichkeit und ökologische Anforderungen von Bio- Ökosystemen Umweltmedien Umweltmedien Luft/ Klima Boden Wasser Flora/ Fauna Luft/ Klima Boden Wasser Flora/ Fauna Diskrepanz SOLL/IST Beeinträchtigungen ökologische Nutzungspotentiale ökologische Nutzungspotentiale Überschreitungen des Nutzungsrahmens - ökologischer Nutzungsrahmen ~ Kollisionen / Konkurrenzen Stoff- und Energieeintrag; Ver- und Gebrauch von Potentialen 1 anthropogen-technische Umweltentlastungspotentiale anthropogene Nutzungsmöglichkeiten Alternativ-, Reduktions- und Sparpotentiale 1 ökologischer Potentialnutzungskonzept Bewirtschaftungskonzepte für Luft/ Klima, Boden, Wasser, Flora/ Fauna Nutzbarkeitsrahmen Maßnahmen zur stufenweisen Umsetzung 1 290 1 RuR4/5.1994 Ulrich Hodapp: Von der Raumordnung zur Umweltordnung 2.2.3 Das Konzept einer "naturnahen Kulturlandschaft" Abbildung3 Naturnahe Kulturlandschaft Funktionsfähiger Ökosystemkomplex naturnahe Kulturlandschaft stofflich-energetische und biotische Stabilität 1 1 funktionsfähige Bio - Ökosysteme 1 1 1 H 1 1 1 1 funktionsfähige urban-industrielle Ökosysteme 1 1 1 Agrarökosysteme Waldökosysteme 1 1 1 1 urban-industrielle Ökosysteme 1 1 umweltverträglich bewirtschaftete Ökosysteme umweltverträglich bewirtschaftete Ökosysteme umweltverträglich bewirtschaftete Ökosysteme 1 1 1 naturnahe Ökosysteme naturnahe Ökosysteme naturnahe Ökosysteme I· 1 1 1 natürliche Ökosysteme 1 natürliche Ökosysteme natürliche Ökosysteme 1 1 1 1 1 1 1 1 ökologischer Potentialnutzungskonzept 1 1 Nutzbarkeitsrahmen Der hier verwendete Begriff der "naturnahen Kulturlandschaft" definiert ein ökologisch verträgliches Landnutzungskonzept. Dessen zentrale Inhalte liegen in einer Veränderung der Flächenverhältnisse zugunsten natürlicher/ naturnaher und zu Lasten anthropogener Ökosysteme (biotische Stabilität), in einer Orientierung von direkten menschlichen Bewirtschaftungsweisen an ökosystemaren Funktionsund Regelungsmechanismen natürlicher Ökosysteme im Sinne einer umweltverträglichen Bewirtschaftung (stofflich-energetische Stabilität) und damit in dem kleinräumigen Nebeneinander von natürlichen, naturnahen und umweltverträglich bewirtschafteten Ökosystemen sowie dem Aus- RuR4/5.1994 schluß von Beeinträchtigungen durch indirekte anthropogene Einflüsse aus den urban-industriellen Ökosystemen (Einhalten des Potentialnutzungsrahmens). 2.3 Konsequenzen für die Planungsinstrumente/Planungselemente 2.3.J Verändertes Raum-, Umweltund Planungsverständnis Der Raum ist in Zukunft als ein zusammenhängendes, komplexes ökosystemares Gefüge zu verstehen. Die sektorale und m_ediale Trennung in einzelne Umweltmedien ist zugunsten einer integrierten Betrachtung dieser Umweltmedien als Bestandteile einzelner Öko- systeme zu überwinden. Dabei müssen die stofflich-energetischen und biotischen Komponenten gleichermaßen und parallel Berücksichtigung finden. Die Berücksichtigung der stofflichenergetischen Komponente wird in einer zukünftigen Raum- und Umweltplanung eine zentrale Bedeutung erlangen. Ohne eine Bilanzierung der Stoffund Energieflüsse der Techno-Ökosysteme und ihre Einbindung in eine Gesamtbilanzierung ist weder eine verursacherbezogene Planung noch eine Wiederherstellung der Funktionsfähigkeit der Landschaften möglich. Das Verständnis der Umwelt als ein dynamisches, sich ständig veränderndes System bedingt die Erkenntnis, daß Planung nur noch als ein sich dauernd ver- 291 Ulrich Hodapp: Von der Raumordnung zur Umweltordnung ändernder Prozeß aufgefaßt werden kann, bei dem einer Berücksichtigung der zeitlichen Komponente eine entscheidende Bedeutung zukommt. 2.3.2 ökologische Leitbilder In der gegenwärtigen Planung ist das Fehlen von konkreten und positiven ökologischen Leitbildern augenscheinlich ein gravierendes Defizit. Die gegenwärtige Planung hat die Funktion eines defensiven Reparaturbetriebs und bestimmt höchstens die ökologischen Rückzugslinien und Mindeststandards. Die Funktion positiver ökologischer Leitbilder kann folgendermaßen beschrieben werden: ökologische Leitbilder haben die Aufgabe, auf den unterschiedlichen Planungsebenen Aussagen zu treffen, wie die jeweiligen Planungsräume ökologisch "optimal" bzw. im Sinne der Funktionsfähigkeit der Öksysternkomplexe "normal" aussehen sollen. 2.3.3 Umweltqualitätsziele Die beschriebenen neuen und erweiterten Aufgaben und Zielsetzungen führen zu völlig veränderten Anforderungen an die qualitativen und quantitativen Aussagen von Planelementen. Neben einer neuen Form räumlicher Ausweisungen kommt vor allem Umweltqualitätszielen als neuer Form qualitativer Planelemente eine entscheidende Bedeutung zu. Für die verschiedenen Planungsschritte und Planungsebenen sind Umweltqualitätsziele unterschiedlichen Konkretheitsgrads zu entwickeln und einzusetzen. An dieser Stelle können inhaltliche Aspekte solcher Planelemente nur grob umrissen werden. Nachfolgend sind beispielhaft einige als notwendig erachtete Bereiche für die Festlegung von Umweltqualitätszielen benannt: Stofjl,ich-energetische Umweltqualitätsziele: Leit- und Zielwerte - zur Bestimmung der Empfindlichkeit einzelner Ökosysteme, - zur Reduktion/Begrenzung von regionalen Stoffumsätzen, - zur Begrenzung von Teil- bzw. Gesamtemissionen, - zur Festlegung des ökologischen Nutzbarkeitsrahmens, - zur Nutzungsintensität von Umweltentlastungspotentialen Biotische Umweltqualitätsziele: Leitund Zielwerte - zur Bestimmung von Arten und Biotopqualitäten, - zur Flächenaufteilung in Ökosystemtypen und zur Festlegung des Flächenbedarfs einzelner Ökosysteme, Vernetzungserfordernisse, - für ökologische Ausstattungsmerkmale der Landschaftsstruktur, - für Extensivierungskonzepte in Agrarökosystemen 2.3.4 Raumbezagene Planelemente Räumliche Ausweisungen auf der Konzept- und Maßnahmenebene sind untrennbar mit auf diese Ausweisungen bezogenen, konkreten qualitativen Festsetzungen bzw. Handlungsanweisungen verbunden. Insoweit haben diese Planelemente die Aussagen von Umweltqualitätszielen räumlich zu verorten. Dabei besteht auch die Notwendigkeit, für die Landschaft als Gan- zes Planelemente festzusetzen. Auf der Ebene eines Maßnahmenplans erscheinen Planelemente, die ein Verschlechterungsverbot bzw. ein ökologisches Verbesserungsgebot der Umweltqualität bzw. einzelner Teilqualitäten festschreiben, zwingend notwendig. Die Grundstruktur zukünftiger räumlicher Ausweisungen eines Konzeptbzw. Maßnahmenplans wird an einem Agrarökosystem beispielhaft dargestellt (vgl. Übersicht 2). 2.3.5 Umweltordnung statt Raumordnung Das Abstecken des ökologisch verträglichen Nutzbarkeitsrahmens der Landschaft bedingt die Erkenntnis in den Vorrang ökologischer Belange vor wirtschaftlichen und gesellschaftlichen Belangen. Eine Gleichrangigkeit der Belange ist nur innerhalb dieses Nutzbarkeitsrahmens möglich. Wird von dieser Erkenntnis ausgegangen, ergibt sich in konsequenter Anwendung, daß festgesetzte Umweltqualitätsziele zur Definition der Funktionsfähigkeit einzelner Ökosysteme sowie der Landschaft insgesamt nicht mehr mit anderen Belangen abwägbar sind. Dies bedingt die Notwendigkeit, daß eine zukünftige Umweltleitplanung - ausgehend vom bestehenden Planungssystem - die Stellung der Raumordnung einnimmt. Dies ist zu verstehen im Sinne einer "Umweltordnung", die verbindliche ökologische Rahmenziele setzt. Damit ist die Frage nach der zukünftigen Stellung und den Aufgaben einer Raumordnung (z.B . Abwägung aller Belange innerhalb des ökologischen Nutzbarkeitsrahmens) zu stellen. Übersicht 2 Konzeptplan Planelement Agrarökosystem mit der ökologischen Hauptfunktion "Landschaftspflege" Ökosystemqualitäten Anteile natürlicher/naturnaher Ökosysteme 35 %; folgende Biotoptypen sollen vorhanden sein: ... ; Anteile an Sukzessionsflächen 7,5 %; Minderung der Bewirtschaftsintensität um 25 % Maßnahmenplan Planelement agrarwirtschaftliches Maßnahmengebiet "Landschaftspflege" Maßnahmen höchste Priorität für die Durchführung eines Extensivierungsprogramms; Flächenstillegung innerhalb von 5 Jahren 20 %; ökologischer Maßnahmenschwerpunkt: Wiederherstellung natürlicher Grundwasserverhältnisse und der entsprechenden natürlichen Lebensräume 292 RuR 4/5.1994 Ulrich Hodapp: Von der Raumordnung zur Umweltordnung Diese Forderung nach einer "Umweltordnung" zieht, neben ihrer tiefgreifenden gesellschaftspolitischen Bedeutung, weitreichende umwelt- und planungsrechtliche Konsequenzen nach sich und ist mit den derzeitigen Rechtsund Verwaltungsstrukturen nicht vereinbar. Die entscheidende Frage bei der Installierung einer Umweltordnung wird sein, welche Institution mit welcher gesellschaftspolitischen und rechtlichen Legitimation in welchem Verfahren die verbindlichen Ziele festlegt. 2.4 Methodischer Konzeptansatz seits getrennt und andererseits im Gesamtzusammenhang des Ökosystemkomplexes zu betrachten. Ausgehend von der Zielsetzung der Funktionsfähigkeit des planerisch abzugrenzenden Ökosystemkomplexes (Landschaft) und der Funktionsfähigkeit der einzelnen, darin enthaltenen Ökosystemtypen, erfolgt im Rahmen der Analyse und Bewertung der Umweltsituation eine planerische Unterscheidung der Ökosystemtypen: - Natürliche/naturnahe Ökosysteme, - Waldökosysteme, - Agrarökosysteme, - Urban-industrielle Ökosysteme, 2.4.1 Gesamtkonzept - Ökosystemkomplex. Im Rahmen eines Gesamtkonzepts stellt sich während der einzelnen Planungsschritte vor allem die Aufgabe, die einzelnen Ökosystemtypen einer- Ausgehend von dieser Analyse und Bewertung wird für den gesamten Ökosystemkomplex durch die Festlegung von "allgemeinen" Umweltqualitätszielen ein Leitbild formuliert, welches durch die Aufstellung von einzelnen Leitbildern für die Wald-, Agrar- und Urbanindustriellen Ökosysteme seine Ausformung erfährt. Ab diesem Planungsschritt werden die natürlichen/naturnahen Ökosysteme nicht mehr als eigenständige Kategorie betrachtet, sondern als integraler Bestandteil der Wald-, Agrar- und urbanindustriellen Ökosysteme, da diese zukünftig aus dem kleinräumigen Nebeneinander von natürlichen, naturnahen und um~eltverträglich wirtschaftenden Ökosystemen gebildet werden. Die räumliche Verortung und die Konkretisierung der Umweltqualitätsziele erfolgt im Rahmen eines Konzeptplanes, der den SOLL-Zustand im Sinne eines funktionsfähigen Ökosystemkomplexes darstellt. Dem Erreichen dieses SOLL-Zustands dient die Erstellung eines Maßnahmensplans. Abbildung4 Methodischer Konzeptansatz einer ökosystembezogenen Umweltleitplanung Analyse, Bewertung Ökosystemkomplex natürliche / naturnahe Ökosysteme Waldökosysteme Agrarökosysteme urban-industrielle Ökosysteme 1 1 Leitbild Ökosystemkomplex Waldkökosystem urban-industrielles Ökosystem Agrarökosystem 1 1 natürliche, naturnahe, umweltverträglich wirtschaftende Ökosysteme 1 1 Konzeptplan Maßnahmen plan RuR4/5 .l994 293 Ulrich Hodapp: Von der Raumordnung zur Umweltordnung Abbildung 5 Methodisches Ablaufschema einer ökosystembezogenen Umweltleitplanung Zielsetzung Funktionsfähigkeit der Landschaft ökosystemare Erkenntnisse 1 Zielsetzung Funktionsfähigkeit der einzelnen Ökosystemtypen 1 1 - 1 1 Ökosystemmodell Waldökosysteme natürliche / naturnahe Ökosysteme Agrarökosysteme urban-industrielle Ökosysteme ÖSIS-Agrar ÖSIS-Urban 1 ÖSIS-Natur ÖSIS-Wald Stoffbilanzen Energiebilanzen Biozönesebilanzen Funktionsbilanzen WAR - Situation IST - Situation KANN - Situation Potential - Situation ö s Ökosystem als Ursache von Beeinträchtigungen - anderer Ökosysteme - des eigenen Ökosystems I - s u - Beeinträchtigungen des Ökosystems durch andere Ökosysteme 1 Wechselwirkungen : 1 M w E L T Analyse stofflich - energetisch biotisch Trendszenario 1 ~ Bewertung Funktionen in der Landschaft H ökosystemarer Bedarf Leitbild Leitbild SOLL-Werte ökologischer Nutzbarkeitsrahmen KONZEPTPLAN KONZEPTPLAN Potentialnutzungskonzept stofflich-energetischer und biotischer SOLL- Zustand MASSNAHMENPLAN MASSNAHMENPLAN Maßnahmenziele, -räume und Vorgaben zeitliche und fachliche Prioritäten 1 - Maßnahmen der Fachbehörden - Maßnahmenplanungen der Fachplanungen ERFOLGS KONTROLLE 294 RuR 4/5.1994 Ulrich Hodapp: Von der Raumordnung zur Umweltordnung 2.4.2 Methodisches Ablaufschema Das in Abbildung 5 dargestellte Ablaufschema basiert im wesentlichen auf dem Ansatz eines Ökosystemmodells. Dessen unverzichtbare Grundlage bildet das Bestehen eines EDV-gestützten "ökosystemaren Informationssystems Umwelt" (ÖSIS-Umwelt) mit den Teilkomponenten ÖSIS-Natur, ÖSISWald, ÖSIS-Agrar und ÖSIS-Urbanindustriell. Gleichermaßen unverzichtbar erscheint die Einbeziehung von entscheidungsunterstützenden Systemen bzw. Expertensystemen auf der Ebene der bewertenden und planenden Teilschritte. Die wesentlichen inhaltlichen Komponenten des Ökosystemmodells sind die Stoff-, Energie-, Biozönose- und Funktionsbilanzen. Stoff- und Energiebilanzen dienen der Erfassung des stofflichenergetischen Zustandes und damit von fehlgesteuerten Stoff- und Energieflüssen, vor allem urban-industrieller Ökosysteme (Verursacherseite) sowie deren Auswirkungen auf die BioÖkosysteme (Wirkungsseite). Im Rahmen der Biozönosebilanzen sind die mit dem Begriff der "biotischen Stabilität" zu beschreibenden Aspekte zu erfassen (z.B . Artenzusammenstellung, Artenvielfalt) und im Rahmen von Funktionsbilanzen die von den Ökosystemen erbrachten ökologischen Funktionen im Ökosystemkomplex. In bezug auf diese inhaltlichen Komponenten bestehen derzeit unbestreitbar vielfältige inhaltliche und methodische Defizite und Probleme, und es steht derzeit kein ausgereifter, planungspraktisch anwendbarer Ansatz zur Verfügung. Zur Erfassung der notwendigen Datengrundlagen ist von der Berücksichtigung einer WAR-, der IST- und der KANN-Situation auszugehen. Die WAR-Situation ist zu verstehen im Sinne einer historischen Vergleichssituation, bei der, insgesamt bzw. hinsichtlich der zu vergleichenden Teilaspekte, von einer Funktionsfähigkeit des Ökosystemkomplexes ausgegangen werden kann. Aus der Diskrepanz zwischen WAR- und IST-Situation lassen sich die ökologischen Verluste und RuR 4/5.1994 Defizite ermitteln (und ggf. durch eine Risikoprognose fortschreiben). Die WAR-Situation liefert gleichzeitig quantitative und qualitative Grundlagendaten für die Bestimmung der SOLL-Werte auf der Leitbildebene. Unter der KANN-Situation sind die quantitativen und qualitativen Entwicklungsmöglichkeiten der Ökosysteme zu verstehen. Durch die Verknüpfung der so erhaltenen Daten einzelner Ökosysteme zu einem Ganzen lassen sich vielfältige Ursache-Wirkungsbeziehungen nicht nur innerhalb einzelner Ökosysteme (Ökosystem als Ursache von Beeinträchtigungen des eigenen Ökosystems), sondern auch zwischen Ökosystemen innerhalb des untersuchten Ökosystemkomplexes und zwischen Ökosystemkomplexen (Beeinträchtigung eines Ökosystems durch andere Ökosysteme) abbilden. Die Zusammenführung der einzelnen Komponenten mündet in der Bewertung der IST/ KANN-Situation einschließlich der IST/KANN-Potentialnutzung. Die Darstellung von Trendszenarien dient einerseits der Risikoabschätzung und stellt andererseits ein wesentliches Element einer möglichen Öffentlichkeitsbeteiligung bzw. Öffentlichkeitsinformation dar. Dies ist notwendig, um den für eine Durchsetzung der ökologischen Zielsetzungen notwendigen breiten gesellschaftlichen Konsens herzustellen. Auf der Leitbildebene werden im wesentlichen die zukünftigen ökologischen Funktionen des Ökosystemkomplexes und der einzelnen Ökosysteme bestimmt und die Funktionsfähigkeit durch Umweltqualitätsziele im Sinne von SOLL-Werten (insbesondere des ökologischen Nutzbarkeitsrahmens) festgelegt. Über die Bewertung von Alternativen erfolgt die Erstellung eines Konzeptplans, der den räumlich verorteten und mit konkreten inhaltlichen Qualitätsangaben ausgefüllten funktionsfähigen SOLL-Zustand darstellt. Die Maßnahmen zum Erreichen dieses SOLL-Zustands sind in einem gesonderten Maßnahmenplan auszuweisen. Durch seine Funktion als "LEIT"-Plan stellt er nicht die vielen Einzelmaßnahmen dar, sondern beschränkt sich darauf, wichtige Maß- nahmengebiete, die darin nötigen wichtigen Maßnahmenziele und die sachlichen und zeitlichen Prioritäten festzulegen. Damit gibt der Maßnahmenplan vor allem Vorgaben an die einzelnen (Fach-)Behörden, denen die Um- und Durchsetzung der Maßnahmen obliegt. 2.5 Ansätze für planerische Umsetzungsmöglichkeiten 2.5.1 Die Bestimmung des ökologischen Nutzbarkeitsrahmens von Grundwasserpotentialen Auf der Grundlage des methodischen Ansatzes zur Bestimmung des ökologischen Nutzbarkeitsrahmens von Potentialen stellt Abbildung 6 eine planerische Umsetzungsmöglichkeit für die Bestimmung von Grundwasserpotentialen dar. Die zentralen Elemente sind dabei die dynamische landschaftsökologische Risikoanalyse und die dynamische Bewertung der qualitativen Eignung. Die dynamische landschaftsökologische Risikoanalyse bezieht nicht nur die Empfindlichkeit der bestehenden Ökosysteme gegen Grundwasserentnahmen ein, sondern berücksichtigt auch die zukünftigen Anforderungen von Ökosystemen im Hinblick auf den angestrebten funktionsfähigen SOLLZustand. So kann die Zielsetzung der Wiederherstellung natürlicher Grundwasserverhältnisse derzeitige Nutzungspotentiale zukünftig von einer Grundwasserentnahme ausschließen. Veränderungen der Grundwassereignungen aufgrund der zeitlichen Dynamik werden planerisch bisher kaum berücksichtigt. Derzeit bestehende qualitative Beeinträchtigungen führen zu einem Ausscheiden dieser Gebiete bzw. Potentiale aus den planerischen Überlegungen. Dies betrifft sowohl Beeinträchtigungen aufgrund atmogenen Stoffeintrags (Versauerung) als auch Beeinträchtigungen aus der landwirtschaftlichen (z.B. Nitrate) und industriellen Nutzung (z.B. Schwermetalle). Eine Beseitigung solcher Beeinträchtigungen ist derzeit noch kein planerisches Ziel. 295 Ulrich Hodapp: Von der Raumordnung zur Umweltordnung Abbildung 6 Vorgehensweise zur Bestimmung von Grundwasserpotentialen Funktionsfiihigkeit der Landschaft 1 IST-Situation natürliche Eignung SOLL-Situation Leitbild / Konzept 1 1 des Grundwassers (Quantität/Qualität) zur Trink- und Brauchwassemutzung 1 landschaftsökologlsche Risikoanalyse Empfindlichkeit der Ökosysteme gegenüber bestehenden und zukünftigen Wasserentnahmen SOLL-Situation IST-Situation 1 Negativräume 1 Positivräume 1 ~ 1 Negativräume 1 1 1 ökologisch geeignete Gebiete zur Entnahme 1 1 bestehende qualitative Beeinträchtigung derzeit qualitativ bestehende Eignung 1 1 atmogener Stoffeintrag unmittelbare Raumnutzung Sanierungsmodelle Risikoanalyse 1 1 1 1 Negativräume 1 Negativräume 1 1 1 Positivräume 1 zukünftig ökologisch und qualitativ geeignete Gebiete und Mengen für eine Grundwasserentnahme 1 ökologischer Potentialnutzungskonzept bestehende Nachfrage Spar- und Reduktionspotentiale J Nutzbarkeitsrahmen 1 Maßnahmen 1 1 • Verbrauchreduzierung Änderung Entnehmeorte und -mengen • Sanierung durch Nutzungsänderung • 296 RuR4/5.1994 Ulrich Hodapp: Von der Raumordnung zur Umweltordnung Der in dieser Arbeit entwickelte ökosystemare Planungsansatz ist verursacherbezogen und sieht damit eine wesentliche Zielsetzung in der Beseitigung von Beeinträchtigungen. Dadurch werden derzeit ungeeignete Potentiale langfristig wieder geeignet. So kann die Schaffung natürlicher/naturnaher Ökosysteme zu einer Extensivierung bzw. Einstellung der landwirtschaftlichen Produktion führen , wodurch langfristig eine "Sanierung" des Grundwassers möglich ist. Diese zeitliche Dynamik wird durch die Einbeziehung von Sanierungsmodellen in die Eignungsbewertung berücksichtigt. 2.5.2 Methodisches Vorgehen zur Einbeziehung der Waldschadensproblematik Für die in dieser Arbeit konzipierte Umweltleitplanung muß der Anspruch formuliert werden, den Problembereich der Waldschäden planerisch anzugehen. Dazu ist die Erstellung eines Waldökosystemmodells notwendig, in welchem die wesentlichen relevanten Ökosystemkomponenten, Biozönosebilanzen, Stoffhaushalts- und Energiehaushaltsbilanzen sowie Funktionsbilanzen im Landschaftshaushalt zusammengeführt werden. Die Grundlage bildet dabei das von ULRJCH (2) im Rahmen eines "forstwirtschaftlichen Informationssystems" entwickelte Konzept von Stoffhaushaltsbilanzen (vgl. Abb. 7). Die im Rahmen eines solchen Ökosystemmodells zur Verfügung stehenden Daten scheinen geeignet, detailliertere Erkenntnisse über den Ursachenkomplex von Waldschäden zu gewinnen und gleichzeitig verursacherbezogene planerische Handlungsmög- Abbildung7 Differenzierung eines Waldökosystemmodells lichkeiten aufzuzeigen. Abbildung 8 stellt in groben Zügen einen Ansatz dar, wie eine planerische Umsetzbarkeit aussehen könnte. Auf der Analyseebene stellt sich die Notwendigkeit der Bildung von Raumkategorien zur Beschreibung des Grads der Funktions(un)fähigkeit sowie die Benennung der wesentlichen Ursachefaktoren. Auf der Ebene des Leitbilds und des Konzeptplans müssen die, auf der Grundlage eines Modellszenarios, ermittelten und die Funktionsfähigkeit beschreibenden Immissionsleitwerte dargestellt werden. Diese stellen gleichzeitig die Voraussetzung dar, um über Modellrechnungen den ökologischen Nutzbarkeitsrahmen im Sinne einer Luftbewirtschaftung festzulegen . Ök\)systemmodell faunistische Typen Biozönose~pen L c._________.1 IST WAR floristische Typen Waldschadenstypen Beeinträchtigungstypen faunistische Modelle IST Biozönosebilanzen >- KANN SOLL floristische Modelle Waldschadensmodelle Beeinträchtigungsmodelle Wasserhaushaltstypen IST WAR Stoffhaushaltstypen Energiehaushalt Stoffeintragstypen Bodenzustandstypen Stoffaustragstypen Wachstumsmodelle Nährstofmodelle IST Stoff- und Energiebilanzen KANN SOLL Niederschlags- und Depositionsmodelle Wasserhaushaltsmodelle Stoffaustragsmodelle ökophysiolog. Parameter Funktionsbilanzen im Landschaftshaushalt '-- RuR4/5 .1994 Funktionstypen IST WAR Landschaftshaushalt KANN SOLL Landschaftshaushalt Funktionstypen 297 Ulrich Hodapp: Von der Raumordnung zur Umweltordnung Abbildung8 Methodisches Vorgehen zur Einbeziehung der Waldschadensproblematik ÖSIS-Wald wesentliche Ökosystemkomponenten 1 / • Wasserhaushaltstypen • Stoffeintragstypen • Bodenzustandstypen • Waldschadenstypen • Bestandswachstumsmodelle • Depositionsmodelle • Wasserhaushaltsmodelle Modell • faunistisches • floristisches Modell • ökophysiologische Parameter 1 Bewertung Ist-Situation - Trendszenario Risikoanalyse >-- ökologischer Bedarf 1 Modellszenario Funktionsfähigkeit 1 Planelemente funktionsfähige Räume Risikoräume akute Gefährdungsräume Verlusträume Planelemente funktionsfähige Räume Risikoräume akute Gefährdungsräume Verlusträume Leitbild Immissionsleitwerte Modellrechnungen Ursache-Wirkung 1 ökologischer Nutzbarkeitsrahmen Luftbewirtschaftung 1 Konzeptplan Maßnahmenkonzept 1 Planelemente räumlich konkretisierte Immissionsleitwerte Planelemente Maßnahmengebiet "stoffliche Sanierung"; mit den Sanierungszielen, zeitlichen Zielen, Prioritäten Flächen zur Heraus• nahme aus der Bewirtschaftung zur • Maßnahmen Luftbewirtschaftung 298 RuR4/5 .1994 Ulrich Hodapp: Von der Raumordnung zur Umweltordnung Anmerkungen *) Dieser Beitrag ist die vom Autor überarbeitete Version eines durch den Förderkreis für Raum- und Umweltforschung - Vereinigung von Freunden der Akademie für Raumforschung und Landesplanung e.V. (FRU) ausgezeichneten Beitrags, einer kurzen Darstellung wichtiger Teilergebnisse einer Diplomarbeit an der Universität Kaiserslautern mit dem Titel "Beitrag zur Diskussion um eine Umweltleitplanung - Ansätze für eine Binnendifferenzierung nach ökosytemaren Gesichtspunkten", vorgelegt im Juni 1992 am Fachgebiet Siedlungswasserwirtschaft bei Herrn Prof. Dr.-Ing. JACOBITZ unter Betreuung von Herrn Dr.-Ing. BURDE. (1) HOFMEISTER, S.; HOBLER. H.-H.: Stoff- und Energiebilanzen als Instrument der räumlichen Planung. Hrsg.: Akademie für Raumforschung und Landesplanung. - Hannover 1990. = Beiträge 118, S. 14 (2) ULRICH, B.: Grundzüge eines Forstökologischen Informationssytems FIS-Ö. - Göttingen 1991. = Schreiben an die Forstbehörden Dipl.-Ing. Ulrich Hodapp Mozartstraße 37 67 655 Kaiserslautern RuR4/5.1994 299
https://openalex.org/W1967795498	https://europepmc.org/articles/pmc4254922?pdf=render	English	null	The Myocardial Ischemia Evaluated by Real-Time Contrast Echocardiography May Predict the Response to Cardiac Resynchronization Therapy: A Large Animal Study	PloS one	2,014	cc-by	6,162	RESEARCH ARTICLE Yongle Chen, Leilei Cheng, Haohua Yao, Haiyan Chen, Yongshi Wang, Weipeng Zhao, Cuizhen Pan, Xianhong Shu Department of Echocardiography, Zhongshan Hospital, Fudan University, Shanghai Institute of Cardiovascular Diseases, Shanghai Institute of Medical Imaging, Shanghai, PR China shu.xianhong@zs-hospital.sh.cn (XS); cheng.leilei@zs-hospital.sh.cn (LC) shu.xianhong@zs-hospital.sh.cn (XS); cheng.leilei@zs-hospital.sh.cn (LC) The Myocardial Ischemia Evaluated by Real-Time Contrast Echocardiography May Predict the Response to Cardiac Resynchronization Therapy: A Large Animal Study Yongle Chen, Leilei Cheng, Haohua Yao, Haiyan Chen, Yongshi Wang, Weipeng Zhao, Cuizhen Pan, Xianhong Shu Echocardiography and CRT compared with the control group after 4 weeks of pacing (49.8¡15.5 dB/s vs. compared with the control group after 4 weeks of pacing (49.8¡15.5 dB/s vs. 28.5¡4.6 dB/s, p,0.05). Therefore, we considered that myocardial perfusion may be superior to standard metrics of LV synchrony in selecting appropriate candidates for CRT. In addition, CRT can improve myocardial perfusion in addition to cardiac synchrony, especially in the setting of ischemic cardiomyopathy. 28.5¡4.6 dB/s, p,0.05). Therefore, we considered that myocardial perfusion may be superior to standard metrics of LV synchrony in selecting appropriate candidates for CRT. In addition, CRT can improve myocardial perfusion in addition to cardiac synchrony, especially in the setting of ischemic cardiomyopathy. Introduction Heart failure, a complex clinical syndrome that encompasses a host of abnormalities in cardiac structure and function, is a major public health epidemic that accounts for a high degree of mortality and morbidity worldwide [1]. Beyond routine oral medication, cardiac resynchronization therapy (CRT) is a viable treatment strategy for patients with refractory heart failure [2]. CRT by means of bi-ventricular pacing can support ventricular contraction, increase cardiac output, relieve heart failure symptoms, improve quality of life of patients, and reduce their overall mortality. Despite these highly encouraging effects, the overall efficacy of CRT is hampered by the fact that approximately 30% of patients who are administered the therapy do not benefit from bi-ventricular pacing and in some cases may exhibit worsening of their heart failure symptoms [3–5]. Although ischemic cardiomyopathy is clearly one of the most common heart failure etiologies, evidence-based criteria for applying CRT in patients with ischemic cardiomyopathy are still scarce. Two-dimensional speckle tracking imaging (2D-STI) is a novel technique for evaluating cardiac motion after CRT [6]. Real-time myocardial contrast echocardiography (RT-MCE) has been used in vivo for decades to quantitatively assess myocardial perfusion by calculating myocardial blood flow (MBF). Accordingly, the aim of the present animal study was twofold: (1) to validate that changes in myocardial perfusion may be key factors in predicting the outcome of CRT in the setting of ischemic cardiomyopathy and (2) to explore that CRT can improve myocardial perfusion in this common disease etiology. OPEN ACCESS Evidence-based criteria for applying cardiac resynchronization therapy (CRT) in patients with ischemic cardiomyopathy are still scarce. The aim of the present study was to evaluate the predictive value of real-time myocardial contrast echocardiography (RT-MCE) in a preclinical canine model of ischemic cardiomyopathy who received CRT. Ischemic cardiomyopathy was produced by ligating the first diagonal branch in 20 beagles. Dogs were subsequently divided into two groups that were either treated with bi-ventricular pacing (CRT group) or left untreated (control group). RT-MCE was performed at baseline, before CRT, and 4 weeks after CRT. Two-dimensional speckle tracking imaging was used to evaluate the standard deviation of circumferential (Cir12SD), radial (R12SD), and longitudinal (L12SD) strains of left ventricular segments at basal as well as middle levels. Four weeks later, the Cir12SD, R12SD, and myocardial blood flow (MBF) of the treated group were significantly improved compared to their non-CRT counterparts. Furthermore, MBF values measured before CRT were significantly higher in responders than in non-responders to bi-ventricular pacing. Meanwhile, no significant differences were observed between the responder and non- responder groups in terms of Cir12SD, R12SD, and L12SD. A high degree of correlation was found between MBF values before CRTand LVEF after CRT. When MBF value.24.9 dB/s was defined as a cut-off point before CRT, the sensitivity and specificity of RT-MCE in predicting the response to CRT were 83.3% and 100%, respectively. Besides, MBF values increased significantly in the CRT group Citation: Chen Y, Cheng L, Yao H, Chen H, Wang Y, et al. (2014) The Myocardial Ischemia Evaluated by Real-Time Contrast Echocardiography May Predict the Response to Cardiac Resynchronization Therapy: A Large Animal Study. PLoS ONE 9(12): e113992. doi:10.1371/ journal.pone.0113992 Copyright: 2014 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 repro- duction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper. Funding: The work was supported by Chinese National Scientific Fund (No. 81371576 and No. 81201095) http://www.nsfc.gov.cn/. The funder 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. 1 / 12 PLOS ONE \| DOI:10.1371/journal.pone.0113992 December 3, 2014 Echocardiography and CRT branch, resulting in myocardial infarction (MI). An epicardial lead (CAPSURE EPI 4965, Medtronic, USA) was sutured to the surface of the marginal area of the MI. All beagles were followed for 2 weeks to document the presence and severity of heart failure after the MI procedure. Right atrial (MEMBRANE EX 1474K, Medtronic, USA) and ventricular leads (CAPSURE SENSE 4074, Medtronic, USA) were subsequently implanted along with the pacemaker unit (INSYNC III 8042, Medtronic, USA); meanwhile, the epicardial lead was connected to the pacemaker. Pacing was initiated in the CRT group only. After 4 weeks of pacing, animals receiving CRT were further divided into two subgroups on the basis of their response to CRT. The left ventricular ejection fraction (LVEF) of dogs in the control group was spontaneously improved after MI and peaked at 45% by 4 weeks after the intervention. Thus, LVEF >45% was considered a positive response to CRT after 4 weeks of resynchronization therapy in the CRT group. Animals in CRT group were then classified as ‘‘responders’’ or ‘‘non-responders’’ to the bi-ventricular treatment depending on their hemodynamic function after 4 weeks. branch, resulting in myocardial infarction (MI). An epicardial lead (CAPSURE EPI 4965, Medtronic, USA) was sutured to the surface of the marginal area of the MI. All beagles were followed for 2 weeks to document the presence and severity of heart failure after the MI procedure. Right atrial (MEMBRANE EX 1474K, Medtronic, USA) and ventricular leads (CAPSURE SENSE 4074, Medtronic, USA) were subsequently implanted along with the pacemaker unit (INSYNC III 8042, Medtronic, USA); meanwhile, the epicardial lead was connected to the pacemaker. Pacing was initiated in the CRT group only. After 4 weeks of pacing, animals receiving CRT were further divided into two subgroups on the basis of their response to CRT. The left ventricular ejection fraction (LVEF) of dogs in the control group was spontaneously improved after MI and peaked at 45% by 4 weeks after the intervention. Thus, LVEF >45% was considered a positive response to CRT after 4 weeks of resynchronization therapy in the CRT group. Animals in CRT group were then classified as ‘‘responders’’ or ‘‘non-responders’’ to the bi-ventricular treatment depending on their hemodynamic function after 4 weeks. Canine model of ischemic cardiomyopathy Canine model of ischemic cardiomyopathy All protocols were approved by the Animal Care and Use Committee of Zhongshan Hospital of Fudan University. All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. Totally 20 adult female beagles were divided into two groups that either underwent bi-ventricular pacing (CRT group, n510) or did not (control group, n510). All the animals were anesthetized (3% sodium pentobarbital, 30 mg/kg) and intubated. Their chests were opened allowing ligation of their first diagonal All protocols were approved by the Animal Care and Use Committee of Zhongshan Hospital of Fudan University. All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. Totally 20 adult female beagles were divided into two groups that either underwent bi-ventricular pacing (CRT group, n510) or did not (control group, n510). All the animals were anesthetized (3% sodium pentobarbital, 30 mg/kg) and intubated. Their chests were opened allowing ligation of their first diagonal 2 / 12 PLOS ONE \| DOI:10.1371/journal.pone.0113992 December 3, 2014 Statistical analysis All results were summarized as mean ¡ SD. The independent Student’s t test was used for comparing results of the CRT and control groups. The paired Student’s t test was used for comparing changes within each group. A Pearson correlation coefficient was used in testing the relationship between MBF and LVEF after CRT. Results were considered statistically significant for two-sided p,0.05. All data were analyzed using SPSS 16.0 software (SPSS Inc, Chicago, IL, USA). Echocardiography and CRT sequence of basal and middle views at short-axis of left ventricle were captured in disc for subsequent off-line analysis. The MBF value, a reliable measure of myocardial perfusion, was calculated from the product of the plateau video intensity (A) and the rate constant rise of the plateau video intensity (b), as previously reported [7]. Baseline characters of the canine model Two dogs of CRT group and one dog of control group died for ventricular fibrillation following the surgical procedure. All other canine successfully completed the study (CRT group, 8 dogs; control group, 9 dogs). Since the LVEF of dogs in the control group was spontaneously improved after MI and peaked at 45% by 4 weeks after the intervention, we defined LVEF >45% was a positive response to CRT for the beagles in the bi-ventricular pacing group. Then, animals in CRT group were classified as ‘‘responders’’ or ‘‘non-responders’’ depending on their hemodynamic function after 4 weeks of resynchronization treatment. Accordingly, 2 of 8 dogs in the CRT group showed no significant response to CRT and were therefore classified as non-responders, whereas the other 6 dogs exhibited significant benefits. As compared, there was no significant difference in LVEF, LVEDV, LVESV, MBF, Cir12SD, R12SD, or L12SD between the CRT and control groups at baseline and following MI (i.e., before CRT treatment; Table 1). Comparison of LVEF, LVEDV, and LVESV between the two groups after CRT Echocardiography protocol The commercially available echocardiographic systems (Philips IE 33, Philips Medical Systems Corporation, MA, USA) was used, equipped with a 1–5 MHz transducer (S5-1) and a 1–3 MHz transducer (X3-1), by two experienced cardiologists. At baseline, 2 weeks after MI (before CRT), and 4 weeks after CRT was initiated, all beagles underwent two-dimensional, three-dimensional echo- cardiography as well as RT-MCE measurements. The data were stored in DICOM format. Furthermore, all the images were analyzed using Philips Q-Lab 8.1 work station (Philips Medical Systems Corporation, MA, USA). The LVEF, left ventricular end diastolic volume (LVEDV), and left ventricular end systolic volume (LVESV) were obtained from three-dimensional images of apical four-chamber views. Left ventricular synchrony was quantified with 2D- STI, which were traced and analyzed automatically from apical 4-, 3-, and 2- chamber views (longitudinal strain), as well as the parasternal short axis views (radial and circumferential strain). The standard deviations of the time-to-peak of the transmural regional circumferential strain (Cir12SD), radial strain (R12SD), and longitudinal strain (L12SD) for 12 segments of basal as well as middle level of left ventricle were determined. For RT-MCE, the contrast agent applied was the commercially available SonoVue (Brocco, Milan, Italy). The contrast agent 59 mg SonoVue was diluted in 5 ml saline and injected via anterior auricular veins slowly (2.5 mL per 1 mL/ min). When imaging performed, the focus was initially set at two-thirds of the depth of the image and then moved at the level of myocardial segment to be examined. Mechanical index was set at 1.7 for flash images and 0.1 for real-time images. The definitive setting of the echocardiographic images was optimized after initial infusion, kept constant throughout the study, and matched at follow-up MCE study. At least 15 consecutive heart beats of every destruction-replenishment 3 / 12 PLOS ONE \| DOI:10.1371/journal.pone.0113992 December 3, 2014 Echocardiography and CRT Comparison of left ventricular synchrony index between the two groups after CRT No significant differences in L12SD were observed between the groups before or after the intervention. In contrast, significant differences in Cir12SD and R12SD were found in the CRT group before and after bi-ventricular pacing (40.8 vs. 31.2 ms and 78.1 vs. 43.9 ms, respectively, all p,0.05). Furthermore, both Cir12SD and R12SD were more significantly improved in the CRT group compared to the control group (31.2 vs. 41.7 ms and 43.9 vs. 65.7 ms, respectively, all p,0.05; Table 1, Figure 1). Comparison of MBF between the two groups The MBF value in normal beagles was 73.5¡10.5 dB/s. No difference was observed between the two groups at baseline (70.2¡11.3 dB/s vs. 76.4¡9.3 dB/s, p.0.05) or at 2 weeks after MI and before CRT (25.9¡5.9 dB/s vs. 23.6¡5.7 dB/ s). However, MBF values increased significantly in the CRT group compared with the control group after 4 weeks of pacing (49.8¡15.5 dB/s vs. 28.5¡4.6 dB/s, p,0.05; Table 1). These results are consistent with CRT-mediated effect of myocardial perfusion in ischemic heart failure (Figure 2). Comparisons between responders and non-responders in the CRT group Since the LVEF of dogs in the control group was spontaneously improved after MI and peaked at 45% by 4 weeks after the intervention, we defined LVEF >45% as the cut-off point for distinguishing the responders or non-responders to CRT. Six dogs (75%) in the CRT group were found to be effectively treated by the therapy, the other 2 canine were sorted to as unsuccessful models. Obviously, LVESV and Table 1. The echocardiographic parameters of the two groups at baseline, before, and after CRT. control group (n59) CRT group (n58) Baseline Before CRT 4 w after CRT Baseline Before CRT 4 w after CRT LVEF (%) 71.9¡6.1 30.0¡3.0 39.0¡5.9 73.0¡5.9 31.9¡4.4 53.3¡12.6{ LVEDV (ml) 26.8¡3.5 34.6¡2.7 34.0¡2.1 27.1¡3.8 35.3¡2.1 30.0¡4.1{ LVESV (ml) 9.6¡2.6 22.0¡4.0 22.8¡4.3 9.5¡3.7 18.2¡3.8 14.3¡2.5{ Cir12SD (ms) 19.7¡3.5 44.2¡9.7 41.7¡10.8 18.8¡3.3 40.8¡5.4 31.2¡6.9{ R12SD (ms) 25.9¡5.0 70.3¡12.6 65.7¡9.7 29.3¡4.9 78.1¡15.1 43.9¡17.1{ L12SD (ms) 31.3¡9.9 55.3¡18.0 55.9¡15.0 35.6¡7.6 59.4¡16.1 55.4¡9.7 MBF (dB/s) 76.4¡9.3 23.6¡5.7 28.5¡4.6 70.2¡11.3 25.9¡5.9 49.8¡15.5{ p,0.05 compared to that before CRT; {p,0.05 compared to control group. doi:10.1371/journal.pone.0113992.t001 Table 1. The echocardiographic parameters of the two groups at baseline, before, and after CRT. Comparison of LVEF, LVEDV, and LVESV between the two groups after CRT LVEF showed a greater significant increase in the CRT group (from 31.9% to 53.3%) than in the control group by 4 weeks after the initiation of bi-ventricular pacing in the CRT group (p,0.05). Similarly, animals in the CRT group exhibited a significantly greater reduction in LVEDV than those in the control group (p,0.05). As shown in Table 1, changes in LVESV were also consistent with greater improvement in the CRT group (p,0.05), thereby explaining the selective improvement in LVEF. 4 / 12 PLOS ONE \| DOI:10.1371/journal.pone.0113992 December 3, 2014 Comparison of MBF between the two groups The MBF value in normal beagles was 73.5¡10.5 dB/s. No difference was observed between the two groups at baseline (70.2¡11.3 dB/s vs. 76.4¡9.3 dB/s, p.0.05) or at 2 weeks after MI and before CRT (25.9¡5.9 dB/s vs. 23.6¡5.7 dB/ s). However, MBF values increased significantly in the CRT group compared with the control group after 4 weeks of pacing (49.8¡15.5 dB/s vs. 28.5¡4.6 dB/s, p,0.05; Table 1). These results are consistent with CRT-mediated effect of myocardial perfusion in ischemic heart failure (Figure 2). Comparison of left ventricular synchrony index between the two groups after CRT No significant differences in L12SD were observed between the groups before or after the intervention. In contrast, significant differences in Cir12SD and R12SD were found in the CRT group before and after bi-ventricular pacing (40.8 vs. 31.2 ms and 78.1 vs. 43.9 ms, respectively, all p,0.05). Furthermore, both Cir12SD and R12SD were more significantly improved in the CRT group compared to the control group (31.2 vs. 41.7 ms and 43.9 vs. 65.7 ms, respectively, all p,0.05; Table 1, Figure 1). doi:10.1371/journal.pone.0113992.t001 Comparisons between responders and non-responders in the CRT group Since the LVEF of dogs in the control group was spontaneously improved after MI and peaked at 45% by 4 weeks after the intervention, we defined LVEF >45% as the cut-off point for distinguishing the responders or non-responders to CRT. Six dogs (75%) in the CRT group were found to be effectively treated by the therapy, the other 2 canine were sorted to as unsuccessful models. Obviously, LVESV and MBF differed significantly between the responders and non-responders before CRT (LVESV: 19.5¡3.4 ml vs. 14.4¡1.3 ml; MBF: 28.4¡4.9 dB/s vs. 5 / 12 PLOS ONE \| DOI:10.1371/journal.pone.0113992 December 3, 2014 Echocardiography and CRT Figure 1. An illustration of LVEF, LVEDV, LVESV, Cir12SD, R12SD, L12SD, and MBF between control group and CRT group at 4 weeks after CRT. The LVEF, LVESV, Cir12SD, R12SD, and MBF were improved in CRT group. LVEF5left ventricular ejection fraction; LVEDV5left ventricular end diastolic volume; LVESV5left ventricular end systolic volume; Cir12SD5standard deviation of the time-to-peak of the transmural regional circumferential strain; R12SD5standard deviation of the time-to-peak of the transmural regional radial strain; L12SD5standard deviation of the time-to-peak of the transmural regional longitudinal strain; MBF5myocardial blood flow. Figure 1. An illustration of LVEF, LVEDV, LVESV, Cir12SD, R12SD, L12SD, and MBF between control group and CRT group at 4 weeks after CRT. The LVEF, LVESV, Cir12SD, R12SD, and MBF were improved in CRT group. LVEF5left ventricular ejection fraction; LVEDV5left ventricular end diastolic volume; LVESV5left ventricular end systolic volume; Cir12SD5standard deviation of the time-to-peak of the transmural regional circumferential strain; R12SD5standard deviation of the time-to-peak of the transmural regional radial strain; L12SD5standard deviation of the time-to-peak of the transmural regional longitudinal strain; MBF5myocardial blood flow. doi:10.1371/journal.pone.0113992.g001 Figure 2. An illustration of RT-MCE between the CRT and control groups. The arrows indicate the ischemic areas. The images in A and B show the improvement of myocardial perfusion before and after bi- ventricular pacing in the CRT group (MBF: 23 dB/S vs. 58 dB/s). The images in C and D show no significant improvement of myocardial perfusion in the ischemic area of the control group (MBF: 19 dB/S vs. 23 dB/s). doi:10.1371/journal.pone.0113992.g001 doi:10.1371/journal.pone.0113992.g001 Figure 2. An illustration of RT-MCE between the CRT and control groups. The arrows indicate the ischemic areas. The images in A and B show the improvement of myocardial perfusion before and after bi- ventricular pacing in the CRT group (MBF: 23 dB/S vs. 58 dB/s). Comparisons between responders and non-responders in the CRT group The images in C and D show no significant improvement of myocardial perfusion in the ischemic area of the control group (MBF: 19 dB/S vs. 23 dB/s). doi:10.1371/journal.pone.0113992.g002 6 / 12 PLOS ONE \| DOI:10.1371/journal.pone.0113992 December 3, 2014 19.4¡4.3 dB/s, all p,0.05), whereas no significant differences in LVEDV, Cir12SD, R12SD, or L12SD were observed (Table 2, Figure 3). On the other hand, while the myocardial perfusion was analyzed, the correlation coefficient between MBF before CRT and LVEF in responders after 4 weeks of CRT was 0.917 (p,0.05). When MBF.24.9 dB/s was defined as a cut-off value before CRT, the sensitivity and the specificity of predicting the response to CRT were found to be 83.3% and 100%, respectively. Echocardiography and CRT 19.4¡4.3 dB/s, all p,0.05), whereas no significant differences in LVEDV, Cir12SD, R12SD, or L12SD were observed (Table 2, Figure 3). On the other hand, while the myocardial perfusion was analyzed, the correlation coefficient between MBF before CRT and LVEF in responders after 4 weeks of CRT was 0.917 (p,0.05). When MBF.24.9 dB/s was defined as a cut-off value before CRT, the sensitivity and the specificity of predicting the response to CRT were found to be 83.3% and 100%, respectively. Table 2. The echocardiographic parameters between responders and non-responders. Response group (n56) Non-response group (n52) Baseline Before CRT 4 w after CRT Baseline Before CRT 4 w after CRT LVEF (%) 71.7¡4.2 32.2¡4.8 58.8¡8.2{ 72.0¡2.8 31.0¡4.2 36.5¡4.9 LVEDV (ml) 26.8¡4.4 34.9¡2.1 28.2¡2.9{ 27.1¡0.5 36.7¡1.8 35.4¡0.9 LVESV (ml) 9.1¡5.3 19.5¡3.4 { 13.9¡2.6 9.4¡2.5 14.4¡1.3 15.6¡2.4 Cir12SD (ms) 18.4¡3.8 40.8¡5.8 29.1¡6.6* 20.1¡0.6 40.6¡6.2 37.3¡3.1 R12SD (ms) 28.5¡5.3 79.9¡17.5 36.1¡10.7{ 32.0¡3.2 72.7¡1.7 67.4¡0.1 L12SD (ms) 36.2¡7.6 56.8¡17.9 54.3¡10.8 33.8¡10.4 67.3¡7.4 58.7¡7.4 MBF (dB/s) 67.6¡12.1 28.4¡4.9 { 57.2¡8.6{ 77.8¡4.0 19.4¡4.3 27.6¡0.4 p,0.05 compared to that before CRT; {p,0.05 compared to non-response group. doi:10.1371/journal.pone.0113992.t002 Table 2. The echocardiographic parameters between responders and non-responders. Response group (n56) Non-response group (n52) Baseline Before CRT 4 w after CRT Baseline Before CRT 4 w after CRT LVEF (%) 71.7¡4.2 32.2¡4.8 58.8¡8.2{ 72.0¡2.8 31.0¡4.2 36.5¡4.9 LVEDV (ml) 26.8¡4.4 34.9¡2.1 28.2¡2.9{ 27.1¡0.5 36.7¡1.8 35.4¡0.9 LVESV (ml) 9.1¡5.3 19.5¡3.4 { 13.9¡2.6 9.4¡2.5 14.4¡1.3 15.6¡2.4 Cir12SD (ms) 18.4¡3.8 40.8¡5.8 29.1¡6.6* 20.1¡0.6 40.6¡6.2 37.3¡3.1 R12SD (ms) 28.5¡5.3 79.9¡17.5 36.1¡10.7{ 32.0¡3.2 72.7¡1.7 67.4¡0.1 L12SD (ms) 36.2¡7.6 56.8¡17.9 54.3¡10.8 33.8¡10.4 67.3¡7.4 58.7¡7.4 MBF (dB/s) 67.6¡12.1 28.4¡4.9 { 57.2¡8.6{ 77.8¡4.0 19.4¡4.3 27.6¡0.4 *p,0.05 compared to that before CRT; {p,0.05 compared to non-response group. doi:10.1371/journal.pone.0113992.t002 Table 2. The echocardiographic parameters between responders and non-responders 19.4¡4.3 dB/s, all p,0.05), whereas no significant differences in LVEDV, Cir12SD, R12SD, or L12SD were observed (Table 2, Figure 3). On the other hand, while the myocardial perfusion was analyzed, the correlation coefficient between MBF before CRT and LVEF in responders after 4 weeks of CRT was 0.917 (p,0.05). When MBF.24.9 dB/s was defined as a cut-off value before CRT, the sensitivity and the specificity of predicting the response to CRT were found to be 83.3% and 100%, respectively. 19.4¡4.3 dB/s, all p,0.05), whereas no significant differences in LVEDV, Cir12SD, R12SD, or L12SD were observed (Table 2, Figure 3). PLOS ONE \| DOI:10.1371/journal.pone.0113992 December 3, 2014 doi:10.1371/journal.pone.0113992.t002 Discussion Bi-ventricular pacing has been shown to improve cardiac function, and CRT is an efficient therapeutic choice for patients with heart failure [8]. Most previous studies have focused on the positioning of the pacing electrode, the optimization of the A–V delay, the QRS duration, and the evaluation of LV synchrony [9–12]. Meanwhile, effective myocardial perfusion may play an important role in this procedure. To our knowledge, this is the first large animal study which has determined the relationship between myocardial blood circulation evaluated by RT-MCE and the outcome of CRT, in addition, considered the impact of bi- ventricular pacing on myocardial perfusion. In the first place, our present finding of improved LVEDV in CRT responders after 4 weeks of bi-ventricular pacing, which is consistent with some [13] but not all previous observations [14]. Specifically, Chung et al. recently reported that chronic bi-ventricular pacing in the infarct region fails to improve LVEDV in patients after 1 year follow-up. Interestingly, the LV leads in that particular study were placed in the peri-infarct or infarct area. Indeed, pacing in this region, which is known to exhibit reduced excitability, impaired contractile function, excessive scar formation, and gap junction remodeling, may be suboptimal, potentially explaining the lack of improvement that they observed [15]. Another possible explanation for the divergent outcomes between our present study and those of Chung et al may be related to important differences in the measurements of 7 / 12 Echocardiography and CRT Figure 3. An illustration of LVEF, LVEDV, LVESV, Cir12SD, R12SD, L12SD, and MBF between response group and non-response group. The MBF was significantly improved in response group. LVEF5left ventricular ejection fraction; LVEDV5left ventricular end diastolic volume; LVESV5left ventricular end systolic volume; Cir12SD5standard deviation of the time-to-peak of the transmural regional circumferential strain; R12SD5standard deviation of the time-to-peak of the transmural regional radial strain; L12SD5standard deviation of the time-to-peak of the transmural regional longitudinal strain; MBF5myocardial blood flow. doi:10.1371/journal.pone.0113992.g003 Figure 3. An illustration of LVEF, LVEDV, LVESV, Cir12SD, R12SD, L12SD, and MBF between response group and non-response group. The MBF was significantly improved in response group. LVEF5left ventricular ejection fraction; LVEDV5left ventricular end diastolic volume; LVESV5left ventricular end systolic volume; Cir12SD5standard deviation of the time-to-peak of the transmural regional circumferential strain; R12SD5standard deviation of the time-to-peak of the transmural regional radial strain; L12SD5standard deviation of the time-to-peak of the transmural regional longitudinal strain; MBF5myocardial blood flow. doi:10.1371/journal.pone.0113992.g003 doi:10.1371/journal.pone.0113992.g003 doi:10.1371/journal.pone.0113992.g003 LVEDV. Echocardiography and CRT [21], only the first diagonal branch was affected in our present study. For all we know, there are few studies addressing the relationship between coronary artery distribution and longitudinal, circumferential, or radial strain. We do believe that further studies are needed to clarify these relationships. Thirdly, it has been widely accepted that RT-MCE is a non-invasive technique that can be performed at the bedside to assess LV function and coronary flow reserve (CFR) in patients with heart failure [22]. MCE is considered to be a powerful predictor of functional recovery after acute MI [23–24]. Recently, Anantharam et al. reported that CFR, calculated as the ratio of MBF during stress to that at rest, is a powerful predictor of mortality in patients with heart failure [22]. Moreover, previous studies indicated that CRT not only improves microcirculatory function [25–26] but also increases the left anterior descending coronary artery flow, which is associated with an improvement in regional myocardial contraction [27]. In our study, our findings are clear that MBF at baseline were highly correlated with the efficacy of bi-ventricular treatment and were consistent with CRT induced amendment on myocardial microcirculation. Therefore, as an easy and repeatable means, the evaluation of myocardial perfusion by RT-MCE may be vital to the implementation and optimization of CRT. In brief, we established an in vivo canine model of ischemic cardiomyopathy, in which we demonstrated significant improvement of MBF by CRT. We uncovered important differences in basal (pre-CRT) MBF values between responders and non-responders. The significance of MBF as a predictor of CRT outcome is further highlighted by the fact that no significant differences were observed for the other measured indices of cardiac function, including LVEF, LVEDV, Cir12SD, R12SD, and L12SD. The high degree of correlation between MBF measured before CRT and LVEF measured in the responder subgroup after 4 weeks of CRT is agreed with the notion that MBF may be mechanistically related to CRT success in ischemic cardiomyopathy. In other words, our findings indicate that assessment of myocardial perfusion may be more important than LV dyssynchrony (Cir12SD, R12SD, and L12SD) in selecting appropriate individuals for CRT. Besides, our findings may also explain the high sensitivity of MBF in detecting hibernating myocardium which has been reported by Fernandes et al. already [28], as well as Hickman et al. The latter concluded that resting MBF could distinguish hibernating myocardium from non-viable myocardium [29]. Discussion Chung and colleagues used Biplane, A4C, and A2C to determine LVEDV, whereas we relied on a robust 3D imaging approach acquired from a full volume set of apical four-chamber views which would be more accuracy. y Secondly, 2D-STI is a relatively new technique for the evaluation of prognosis for bi-ventricular pacing. The impact of CRT on longitudinal, radial, and circumferential strains has been documented in multiple studies [16–17]. However, the predictive value of these metrics remains controversial. In fact, up to the present, studies addressing the predictive value of LV synchrony in ischemic cardiomyopathy are scarce. Previously, Parsai et al found that CRT response is dictated by normalization of multiple independent mechanisms, of which LV dyssynchrony is only one. They found that long axis dyssynchrony alone failed to detect 40% of responders [18]. And, Gorcsan et al endeavored themselves to combine longitudinal and radial dyssynchrony detection to evaluate the effect of resynchronization therapy [19]. A more recent study showed that CRT resulted in a significant improvement in both LV dyssynchrony and contractile function as measured by global longitudinal strain [20]. In the present study, a significant improvement of radial dyssynchrony after CRT was observed. One potential reason for the divergent outcomes across various studies may be related to key anatomical differences in the occluded coronary arteries, which may have led to differences in the location and size of the MI. For example, whereas the left main and three vessel coronary artery disease were involved in the study by Choi et al 8 / 12 PLOS ONE \| DOI:10.1371/journal.pone.0113992 December 3, 2014 Conclusions The MBF value is positively correlated with the responsiveness to CRT and, therefore, should be considered an important predictor of CRT outcome in the setting of ischemic cardiomyopathy. Meanwhile, we’re pretty sure that CRT can improve both cardiac synchrony and myocardial perfusion in this clinically relevant large animal model of ischemic cardiomyopathy. Acknowledgments We are grateful for the support of the entire staff of the echocardiography and cardiology of Zhongshan Hospital, Fudan University, Shanghai Institute of Cardiovascular Diseases and Shanghai Institute of Medical Imaging. Limitations The present study has some important limitations that require mention. First of all, we relied on a clinically relevant large animal model. Therefore, the number of subjects per group (especially the non-responder subgroup in CRT group) was by necessity limited when compared to rodent studies. In the next place, our study focused on the global dyssynchrony of the left ventricle, was not designed to uncover potentially important differences in intraventricular properties between various segments within the LV. Thirdly, despite its clinical relevance, our model of MI involved ligation of the first diagonal branch, and therefore, future studies in which the severity of MI can be modulated by ligating additional coronary arteries are warranted. Fourthly, as we mentioned, the pathophysiological process of CRT remains unclear. Varies studies are required to determine the cellular and molecular mechanisms that underlie the observations of the present study. Finally, as with any other animal study, our preclinical findings will require validation in humans before they can be appropriately translated to clinical applications. Echocardiography and CRT that neurohumoral activation, including free fatty acid and glucose metabolism as well as insulin resistance, may be involved in the beneficial effects of CRT [32]. Indeed, all of these factors may be mechanistically related to the improvement in MBF by CRT. But, further studies are required to determine the individual contributions of these mechanisms to CRT-mediated changes in MBF. In our study, MBF after CRT was significantly improved in the responder subgroup, implying that CRT may play a key role in reversing the pathophysiological processes associated with ischemic HF. For example as evidence, Aiba et al. investigated that CRT partially restores ion channel remodeling, abnormal Ca2+ homeostasis, and regional heterogeneity of action potential duration induced by the dyssynchronous mechanical activation of the failing LV in a canine model of left bundle branch block coupled with tachypacing induced heart failure [30]. Additionally, Chakir et al. revealed that CRT reverses regional and global molecular remodeling, generating more homogeneous activation of stress kinases and reducing apoptosis in the same model [31]. Moreover, Ashrafian illustrated PLOS ONE \| DOI:10.1371/journal.pone.0113992 December 3, 2014 9 / 12 References 1. Shah AM, Mann DL (2011) In search of new therapeutic targets and strategies for heart failure: recent advances in basic science. Lancet 378: 704–712. 2. Holzmeister J, Leclercq C (2011) Implantable cardioverter defibrillators and cardiac resynchronisation therapy. Lancet 378: 722–730. 3. Cleland JG, Daubert JC, Erdmann E, Freemantle N, Kappenberger L, et al. (2005) The effect of cardiac resynchronization on morbidity and mortality in heart failure. N Engl J Med 352: 1539–1549. 4. Kydd AC, Khan FZ, Watson WD, Pugh PJ, Virdee MS, et al. (2014) Prognostic Benefit of Optimum Left Ventricular Lead Position in Cardiac Resynchronization Therapy: Follow-Up of the TARGET Study Cohort (Targeted Left Ventricular Lead Placement to guide Cardiac Resynchronization Therapy). JACC Heart Fail 2: 205–212. 5. Chen Y, Duan C, Liu F, Shen S, Chen P, et al. (2014) Impact of etiology on the outcomes in heart failure patients treated with cardiac resynchronization therapy: a meta-analysis. PLoS One 9: e94614. 6. Biswas M, Sudhakar S, Nanda NC, Buckberg G, Pradhan M, et al. (2013) Two- and Three- dimensional speckle tracking echocardiography: clinical and future directions. Echocardiography 30: 88– 105. 7. Wei K, Jayaweera AR, Firoozan S, Linka A, Skyba DM, et al. (1998) Quantification of myocardial blood flow with ultrasound-induced destruction of microbubbles administered as a constant venous infusion. Circulation 97: 473–483. 8. Shanks M, Bertini M, Delgado V, Ng AC, Nucifora G, et al. (2010) Effect of biventricular pacing on diastolic dyssynchrony. J Am Coll Cardiol 56: 1567–1575. 9. Kass DA (2003) Predicting cardiac resynchronization response by QRS duration: The long and short of it. J Am Coll Cardiol 42: 2125–2127. 10. Bleeker GB, Holman ER, Steendijk P, Boersma E, van der Wall EE, et al. (2006) Cardiac resynchronization therapy in patients with a narrow QRS complex. J Am Coll Cardiol 48: 2243–2250. 11. Beshai JF, Grimm RA, Nagueh SF, Baker JH 2nd, Beau SL, et al. (2007) Cardiac-resynchronization therapy in heart failure with narrow QRS complexes. N Engl J Med 357: 2461–2471. 12. Bax JJ, Bleeker GB, Marwick TH, Molhoek SG, Boersma E, et al. (2004) Left ventricular dyssynchrony predicts response and prognosis after cardiac resynchronization therapy. J Am Coll Cardiol 44: 1834–1840. 13. Chung ES, Menon SG, Weiss R, Schloss EJ, Chow T, et al. (2007) Feasibility of biventricular pacing in patients with recent myocardial infarction: impact on ventricular remodeling. Congest Heart Fail 13: 9– 15. 14. Author Contributions Conceived and designed the experiments: XHS LLC. Performed the experiments: YLC HYC. Analyzed the data: HHY YSW. Contributed reagents/materials/analysis tools: WPZ CZP. Wrote the paper: YLC LLC. 10 / 12 PLOS ONE \| DOI:10.1371/journal.pone.0113992 December 3, 2014 Echocardiography and CRT Echocardiography and CRT 20. Pouleur AC, Knappe D, Shah AM, Uno H, Bourgoun M, et al. (2011) Relationship between improvement in left ventricular dyssynchrony and contractile function and clinical outcome with cardiac resynchronization therapy: the MADIT-CRT trial. Eur Heart J 32: 1720–1729. 21. Choi JO, Cho SW, Song YB, Cho SJ, Song BG, et al. (2009) Longitudinal 2D strain at rest predicts the presence of left main and three vessel coronary artery disease in patients without regional wall motion abnormality. Eur J Echocardiogr 10: 695–701. 22. Anantharam B, Janardhanan R, Hayat S, Hickman M, Chahal N, et al. (2011) Coronary flow reserve assessed by myocardial contrast echocardiography predicts mortality in patients with heart failure. Eur J Echocardiogr 12: 69–75. 23. Hillis GS, Mulvagh SL, Gunda M, Hagen ME, Reeder GS, et al. (2003) Contrast echocardiography using intravenous octafluoropropane and real-time perfusion imaging predicts functional recovery after acute myocardial infarction. J Am Soc Echocardiogr 16: 638–645. 24. Olszowska M, Kostkiewicz M, Podolec P, Rubis P, Tracz W. (2010) Myocardial viability detected by myocardial contrast echocardiography-prognostic value in patients after myocardial infarction. Echocardiography 27: 430–434. 25. Erol-Yilmaz A, Atasever B, Mathura K, Lindeboom J, Wilde A, et al. (2007) Cardiac resynchronization improves microcirculation. J Card Fail 13: 95–99. 26. Flevari P, Theodorakis G, Paraskevaidis I, Kolokathis F, Kostopoulou A, et al. (2006) Coronary and peripheral blood flow changes following biventricular pacing and their relation to heart failure improvement. Europace 8: 44–50. 27. Valzania C, Gadler F, Winter R, Braunschweig F, Brodin LA, et al. (2008) Effects of cardiac resynchronization therapy on coronary blood flow: Evaluation by transthoracic Doppler echocardiography. Eur J Heart Fail 10: 514–520. 28. Fernandes DR, Tsutsui JM, Bocchi EA, Cesar LA, Sbano JC, et al. (2011) Qualitative and quantitative real time myocardial contrast echocardiography for detecting hibernating myocardium. Echocardiography 28: 342–349. 29. Hickman M, Chelliah R, Burden L, Senior R (2010) Resting myocardial blood flow, coronary flow reserve, and contractile reserve in hibernating myocardium: implications for using resting myocardial contrast echocardiography vs. dobutamine echocardiography for the detection of hibernating myocardium. Eur J Echocardiogr 11: 756–762. 30. Aiba T, Hesketh GG, Barth AS, Liu T, Daya S, et al. (2009) Electrophysiological Consequences of Dyssynchronous Heart Failure and Its Restoration by Resynchronization Therapy. Circulation 119: 1220–1230. 31. Chakir K, Daya SK, Tunin RS, Helm RH, Byrne MJ, et al. PLOS ONE \| DOI:10.1371/journal.pone.0113992 December 3, 2014 References Chung ES, Dan D, Solomn SD, Bank AJ, Pastore J, et al. (2007) Effect of peri-infarct pacing early after myocardial infarction: results of the prevention of myocardial enlargement and dilatation post myocardial infarction study. Circ Heart Fail 3: 650–658. 15. Pfeffer MA, Braunwald E. (1990) Ventricular remodeling after myocardial infarction. Experimental observations and clinical implications. Circulation 81: 1161–1172. 16. Helm RH, Leclercq C, Faris OP, Ozturk C, McVeigh E, et al. (2005) Cardiac dyssynchrony analysis using circumferential versus longitudinal strain: Implications for assessing cardiac resynchronization. Circulation 111: 2760–2767. 17. Dohi K, Suffoletto MS, Schwartzman D, Ganz L, Pinsky MR, et al. (2005) Utility of echocardiographic radial strain imaging to quantify left ventricular dyssynchrony and predict acute response to cardiac resynchronization therapy. Am J Cardiol 96: 112–116. 18. Parsai C, Bijnens B, Sutherland GR, Baltabaeva A, Claus P, et al. (2009) Toward understanding response to cardiac resynchronization therapy: left ventricular dyssynchrony is only one of multiple mechanisms. Eur Heart J 30: 940–949. 19. Gorcsan J 3rd, Tanabe M, Bleeker GB, Suffoletto MS, Thomas NC, et al. (2007) Combined Longitudinal and Radial dyssynchrony predicts ventricular response after resynchronization therapy. J Am Coll Cardiol 50: 1476–1483. 11 / 12 PLOS ONE \| DOI:10.1371/journal.pone.0113992 December 3, 2014 (2008) Reversal of global apoptosis and regional stress kinase activation by cardiac resynchronization. Circulation 117: 1369–1377. 32. Ashrafian H, Frenneaux MP, Opie LH (2007) Metabolic mechanisms in heart failure. Circulation 116: 434–448. 12 / 12
https://openalex.org/W2891704156	https://research.chalmers.se/publication/505234/file/505234_Fulltext.pdf	English	null	Leaching and Recovery of Rare-Earth Elements from Neodymium Magnet Waste Using Organic Acids	Metals	2,018	cc-by	14,574	Leaching and recovery of rare-earth elements from neodymium magnet waste using organic acids Downloaded from: https://research.chalmers.se, 2024-10-24 05:54 UTC Citation for the original published paper (version of record): Gergoric, M., Ravaux, C., Steenari, B. et al (2018). Leaching and recovery of rare-earth elements from neodymium magnet waste using organic acids. Metals, 8(9). http://dx.doi.org/10.3390/met8090721 N.B. When citing this work, cite the original published paper. research.chalmers.se offers the possibility of retrieving research publications produced at Chalmers University of Technology. It covers all kind of research output: articles, dissertations, conference papers, reports etc. since 2004. research.chalmers.se is administrated and maintained by Chalmers Library research.chalmers.se offers the possibility of retrieving research publications produced at Chalmers University of Technology. It covers all kind of research output: articles, dissertations, conference papers, reports etc. since 2004. research.chalmers.se is administrated and maintained by Chalmers Library Leaching and recovery of rare-earth elements from neodymium magnet waste using organic acids Received: 20 August 2018; Accepted: 11 September 2018; Published: 13 September 2018 Abstract: Over the last decade, rare-earth elements (REEs) have become critical in the European Union (EU) in terms of supply risk, and they remain critical to this day. End-of-life electronic scrap (e-scrap) recycling can provide a partial solution to the supply of REEs in the EU. One such product is end-of-life neodymium (NdFeB) magnets, which can be a feasible source of Nd, Dy, and Pr. REEs are normally leached out of NdFeB magnet waste using strong mineral acids, which can have an adverse impact on the environment in case of accidental release. Organic acids can be a solution to this problem due to easier handling, degradability, and less poisonous gas evolution during leaching. However, the literature on leaching NdFeB magnets waste with organic acids is very scarce and poorly investigated. This paper investigates the recovery of Nd, Pr, and Dy from NdFeB magnets waste powder using leaching and solvent extraction. The goal was to determine potential selectivity between the recovery of REEs and other impurities in the material. Citric acid and acetic acid were used as leaching agents, while di-(2-ethylhexyl) phosphoric acid (D2EHPA) was used for preliminary solvent extraction tests. The highest leaching efﬁciencies were achieved with 1 mol/L citric acid (where almost 100% of the REEs were leached after 24 h) and 1 mol/L acetic acid (where >95% of the REEs were leached). Fe and Co—two major impurities—were co-leached into the solution, and no leaching selectivity was achieved between the impurities and the REEs. The solvent extraction experiments with D2EHPA in Solvent 70 on 1 mol/L leachates of both acetic acid and citric acid showed much higher afﬁnity for Nd than Fe, with better extraction properties observed in acetic acid leachate. The results showed that acetic acid and citric acid are feasible for the recovery of REEs out of NdFeB waste under certain conditions. Keywords: rare earths; neodymium magnets; solvent extraction; leaching; citric acid; acetic acid Leaching and Recovery of Rare-Earth Elements fro Neodymium Magnet Waste Using Organic Acids Marino Gergoric 1,* , Christophe Ravaux 2, Britt-Marie Steenari 1, Fredrik Espegren 1 and Teodora Retegan 1 1 Nuclear Chemistry and Industrial Materials Recycling, Chemistry and Chemical Engineering, Chalmers University of Technology, 412 96 Göteborg, Sweden; bms@chalmers.se (B.-M.S.); espegren@chalmers.se (F.E.); tretegan@chalmers.se (T.R.) 1 Nuclear Chemistry and Industrial Materials Recycling, Chemistry and Chemical Engineering, Chalmers University of Technology, 412 96 Göteborg, Sweden; bms@chalmers.se (B.-M.S.); espegren@chalmers.se (F.E.); tretegan@chalmers.se (T.R.) p g ( ); g ( ) 2 Decontamination and Environmental Management, National School of Chemistry Montpellier, 34090 Montpellier, France; christophe.ravaux@enscm.fr * Correspondence: marino@chalmers.se; Tel.: +46-(0)31-772-28-42 34090 Montpellier, France; christophe.ravaux@enscm.fr * Correspondence: marino@chalmers.se; Tel.: +46-(0)31-772-28-42 * Correspondence: marino@chalmers.se; Tel.: +46-(0)31-772-28-42 Received: 20 August 2018; Accepted: 11 September 2018; Published: 13 September 2018 (article starts on next page) metals 1. Introduction Furthermore, according to predicted growth [8], REE magnets and dysprosium are expected to be the most in-demand materials (in tons) due to the development of green technologies, with an average annual growth of 5.3% between 2010 and 2035 [7]. Electronic devices, such as speakers, mobile devices, and hard disk drives, are available as waste today and can be readily recycled unlike large magnets found in wind turbines and electric cars that have a long life span [3]. Hydrometallurgical methods, such as leaching, solvent extraction, and precipitation, are attractive and efﬁcient methods for recovery of REEs out of NdFeB magnets [3]. The elements contained in the magnet are normally dissolved/leached into an aqueous solution, followed by solvent extraction (occasionally ion-exchange) from the leachate and back-extraction into a new aqueous solution for further reprocessing. Various leaching processes for recovery of REEs out of NdFeB magnets have been developed. In 2016, Önal et al. [9] developed a selective leaching process for REEs by leaching Fe in the solid residue. The NdFeB magnet powder sample was turned into a sulfate mixture by mixing with concentrated H2SO4 (12–16 mol/L) in crucibles. After drying and high-temperature treatment, the powder was leached in demineralized water for 15 min to 24 h, which led to >95% recovery of REEs while Fe remained in the solid residue in the form of a sulfate. In 2014, Yoon et al. [10] studied the leaching of NdFeB magnet scrap using H2SO4 at different temperatures and concentrations. The optimal leaching conditions were determined to be 70 ◦C and 3 mol/L H2SO4 with a leaching time of 4 h. In 2013, Lee et al. [11] carried out an investigation on leaching of NdFeB magnets using H2SO4, HCl, HNO3, and NaOH. Out of the four leaching agents, HCl and H2SO4 showed the best leaching performance. Optimum conditions for this process were S:L (solid-to-liquid ratio) ratio of 20 g/L, leaching time of 15 min, and concentrations of 3 mol/L HCl and 1.5 mol/L H2SO4. The vast majority of research so far has been conducted using strong mineral acids for leaching [12–15]. 1. Introduction Rare-earth elements (REEs) possess excellent physical and chemical properties, which is why they are used on a large scale in permanent magnets, lamp phosphors, NiMH batteries, catalysts, various mechanical and electric devices as well as electric motors and generators, which are important for transition to a greener economy. Their demand in industry is thus growing rapidly today [1]. Mined mostly in China (over 95% of global supply), REEs are still considered the most critical raw materials in the European Union (EU) in terms of supply risk [2–4]. Due to the geopolitical situation, even the most optimistic predictions cannot rule out a REE market crisis on a global level, similar to the one that occurred in 2011 [5]. However, an imminent crisis in REEs supply in the EU is not expected. Metals 2018, 8, 721; doi:10.3390/met8090721 www.mdpi.com/journal/metals 2 of 17 Metals 2018, 8, 721 Metals 2018, 8, 721 The processes for the production of pure REEs out of ores use highly concentrated mineral acids, which take a toll on the environment, including acids leakage and release of toxic gasses generated during leaching [6]. Furthermore, large amounts of solvents are used for the recovery of REEs out of the obtained leachates. Despite the environmental issues, there are very few ongoing large-scale processes for recycling REEs from end-of-life products, mainly due to price stabilization since the crisis in 2011 and due to varying environmental legislation across the globe [7]. Development of a sustainable and economically feasible recycling process for the recovery of the REEs out of secondary sources is needed in order to decrease the dependence of the industry on mining. As of today, the recycling of REEs in the EU is still in its early stages, with only 7% of the light REEs and 6% of the heavy REEs recovered from secondary sources to meet the EU’s demand for REEs [2]. NdFeB scrap magnets are a viable source of some REEs. They consist mainly of Nd (around 30%) and Fe (around 64%), with trace amounts of B (around 0.5%) [6]. Small admixtures of Pr and Dy are also often present [3], with Dy being especially important because it is added to increase the temperature stability against demagnetization. 1. Introduction It is largely produced by methanol carbonylation [27], and the price is comparable with that of mineral acids, making it attractive for usage as a leaching agent. In the work of Zanonato et al. [28], it was shown that Nd3+ forms at least successive mononuclear complexes according to the following equilibrium: ssociation constants being pKa1 = 3.13, pKa2 = 4.76, and pKa3 = 6.40 at 25 C [23]. It is naturally und in citrus fruits but is usually produced through a cheaper process by fermentation of sugars sing Aspergilus niger bacteria [24]. As citric acid is in high demand both in chemical and food dustry, the price is comparable to that of mineral acids. Two possible mechanisms of dissolution ith citric acid include metal ion displacement with hydronium ions and formation of soluble metal– gand complexes by metal chelation [25]. In a study by Brown et al. [26], it was shown that Nd3+ omplex with citrate counterion is formed according to the following equilibrium: Nd3+ + jCH3COO ⇆ Nd (OOCCH3)j(3 j) +, j = 1, 2, 3 (2) that acetic acid could be used for the recovery of Nd and possibly other REEs fro Nd3+ + jCH3COO− nNd3+ + jH+ + kCit3− ⇆ NdkHjCitk(3n+j−3k) (1) Nd (OOCCH3)j(3−j)+, j = 1, 2, 3 (2) ( ) Es fro (2) electronic waste. Carboxylic acids have been previously used for actinide and lanthanide separations [29,30] a The existence of NdCit, NdHCit, NdHCit2, and NdCit2 was confirmed by the solution in the pH nge 2–5, which proved that citric acid could be used for the leaching of Nd and other REEs out of A d d b l h I l l This indicates that acetic acid could be used for the recovery of Nd and possibly other REEs from electronic waste. electronic waste. Carboxylic acids have been previously used for actinide and lanthanide separations [29,30] a The existence of NdCit, NdHCit, NdHCit2, and NdCit2 was confirmed by the solution in the pH nge 2–5, which proved that citric acid could be used for the leaching of Nd and other REEs out of a A eti a id i a o a i a id o tai i o e a bo yli ou ith Ka 4 76 It i la ely This indicates that acetic acid could be used for the recovery of Nd and possibly other REEs from electronic waste. 1. Introduction could be suitable for the recovery of REEs out of NdFeB magnet waste as they can form solu complexes with REEs. However, the leaching of neodymium magnets with organic acids is ve scarcely investigated. In 2016, Behera et al. [6] investigated in detail the kinetics of extraction of N out of NdFeB waste using acetic acid. The study showed that acetic acid in concentrations higher th 0.4 mol/L at 800 rpm, 1% (w/v) and 308 K was effective at leaching Nd and Fe out of the was However, in this study—as well as in other works—little attention was paid to the recovery of oth REEs, meaning no detailed kinetic or temperature gradient study was performed for vital REEs l Dy and Pr in the waste; the behavior of other impurities such as Fe, B and Co during the leachi process was not completely addressed either. The feasibility of further extraction of REEs out leachates of organic acids was also not addressed. In this study, leaching of a high‐temperature‐treated neodymium magnet powder w i ti t d i dil t d it i id d ti id Th l hi ti i d i d scrap. Acetic acid is an organic acid containing one carboxylic group with pKa = 4.76. It is largely roduced by methanol carbonylation [27], and the price is comparable with that of mineral acids, aking it attractive for usage as a leaching agent. In the work of Zanonato et al. [28], it was shown at Nd3+ forms at least successive mononuclear complexes according to the following equilibrium: Nd3+ + jCH3COO− ⇆ Nd (OOCCH3)j(3−j) +, j = 1, 2, 3 (2) This indicates that acetic acid could be used for the recovery of Nd and possibly other REEs from ectronic waste. Carboxylic acids have been previously used for actinide and lanthanide separations [29,30] and ould be suitable for the recovery of REEs out of NdFeB magnet waste as they can form soluble omplexes with REEs. However, the leaching of neodymium magnets with organic acids is very arcely investigated In 2016 Behera et al [6] investigated in detail the kinetics of extraction of Nd Carboxylic acids have been previously used for actinide and lanthanide separations [29,30] and could be suitable for the recovery of REEs out of NdFeB magnet waste as they can form soluble complexes with REEs. 1. Introduction However, the leaching of neodymium magnets with organic acids is very scarcely investigated. In 2016, Behera et al. [6] investigated in detail the kinetics of extraction of Nd out of NdFeB waste using acetic acid. The study showed that acetic acid in concentrations higher than 0.4 mol/L at 800 rpm, 1% (w/v) and 308 K was effective at leaching Nd and Fe out of the waste. However, in this study—as well as in other works—little attention was paid to the recovery of other REEs, meaning no detailed kinetic or temperature gradient study was performed for vital REEs like Dy and Pr in the waste; the behavior of other impurities such as Fe, B and Co during the leaching process was not completely addressed either. The feasibility of further extraction of REEs out of leachates of organic acids was also not addressed. investigated using diluted citric acid and acetic acid. The leaching process was optimized in order achieve the maximum REEs recovery by varying leaching time, acids concentration, and S:L rat Before the leaching experiments, the powder before and after roasting treatment was analyzed usi scanning electron microscopy coupled with energy dispersive X‐ray spectroscopy (SEM/ED analysis, x‐ray diffraction (XRD) , and total dissolution using aqua regia. Solvent extraction w D2EHPA was performed on the obtained leachates to test the feasibility of REEs recovery from su leachates using conventional methods. 2 Materials and Methods arcely investigated. In 2016, Behera et al. [6] investigated in detail the kinetics of extraction of Nd ut of NdFeB waste using acetic acid. The study showed that acetic acid in concentrations higher than 4 mol/L at 800 rpm, 1% (w/v) and 308 K was effective at leaching Nd and Fe out of the waste. owever, in this study—as well as in other works—little attention was paid to the recovery of other EEs, meaning no detailed kinetic or temperature gradient study was performed for vital REEs like y and Pr in the waste; the behavior of other impurities such as Fe, B and Co during the leaching rocess was not completely addressed either. The feasibility of further extraction of REEs out of achates of organic acids was also not addressed. In this study, leaching of a high‐temperature‐treated neodymium magnet powder was In this study, leaching of a high-temperature-treated neodymium magnet powder was investigated using diluted citric acid and acetic acid. 1. Introduction The extraction of the metals of interest after leaching is most commonly done using solvent extraction agents like di-(2-ethylhexyl) phosphoric acid (D2EHPA), tributyl phosphate (TBP) and 2-ethylhexyl phosphonic acid (HEHEHP) [16], although a lot of research in recent years has been focused on the investigation of solvent extraction using ionic liquids as promising new, environmentally friendly extractants [3,17]. Even though efﬁcient, hydrometallurgical methods have drawbacks. As already mentioned, large amount of waste is generated during the processes and toxic chemicals are often used. This has to be mitigated using more environmentally friendly chemicals and decreasing the quantities of both the aqueous and the organic waste produced. Despite possessing excellent properties for leaching REEs out of NdFeB magnets, strong mineral acids such as HCl, HNO3 and H2SO4 pose the risk of adverse impact on the environment [6,18,19] because of issues with regeneration of the used acids, while handling can also pose a great challenge in some situations. In cases where highly concentrated acids are released to the environment, they could acidify the soil and would require further soil treatment to neutralize the acidiﬁed soil [20]. Moreover, when using strong mineral acids, evolution of poisonous gasses is often a problem. Using organic acids or signiﬁcantly more diluted mineral acids [21] could have advantages in this regard because of Metals 2018, 8, 721 3 of 17 easier handling, less poisonous gas evolution due to lower acidities, and much easier degradability. Some researchers even claim that a more sustainable REE-leaching process could be achieved using some bacteria like Shewanella putrefaciens [22]. Metals 2018, 8, x FOR PEER REVIEW 3 o achieved using some bacteria like Shewanella putrefaciens [22] Citric acid is an organic acid that contains three carboxylic groups, with the three hydrogen dissociation constants being pKa1 = 3.13, pKa2 = 4.76, and pKa3 = 6.40 at 25 ◦C [23]. It is naturally found in citrus fruits but is usually produced through a cheaper process by fermentation of sugars using Aspergilus niger bacteria [24]. As citric acid is in high demand both in chemical and food industry, the price is comparable to that of mineral acids. Two possible mechanisms of dissolution with citric acid include metal ion displacement with hydronium ions and formation of soluble metal–ligand complexes by metal chelation [25]. In a study by Brown et al. 1. Introduction [26], it was shown that Nd3+ complex with citrate counterion is formed according to the following equilibrium: achieved using some bacteria like Shewanella putrefaciens [22]. Citric acid is an organic acid that contains three carboxylic groups, with the three hydrog dissociation constants being pKa1 = 3.13, pKa2 = 4.76, and pKa3 = 6.40 at 25 °C [23]. It is natura found in citrus fruits but is usually produced through a cheaper process by fermentation of sug using Aspergilus niger bacteria [24]. As citric acid is in high demand both in chemical and fo industry, the price is comparable to that of mineral acids. Two possible mechanisms of dissoluti with citric acid include metal ion displacement with hydronium ions and formation of soluble met ligand complexes by metal chelation [25]. In a study by Brown et al. [26], it was shown that N complex with citrate counterion is formed according to the following equilibrium: etals 2018, 8, x FOR PEER REVIEW 3 of 17 nNd3+ + jH+ + kCit3− nNd3+ + jH+ + kCit3− ⇆ NdkHjCitk(3n+j−3k) (1) NdkHjCitk (3n+j−3k) (1) like Shewanella putrefaciens [22]. acid that contains three carboxylic groups, with the three hydrogen K 1 3 13 K 2 4 76 d K 3 6 40 t 25 °C [23] It i t ll nNd3+ + jH+ + kCit3− nNd3+ + jH+ + kCit3− ⇆ NdkHjCitk(3n+j−3k) NdkHjCitk (3n+j−3k) ike Shewanella putrefaciens [22]. acid that contains three carboxylic groups, w (1) (1) n The existence of NdCit, NdHCit, NdHCit2, and NdCit2 was confirmed by the solution in the p range 2–5, which proved that citric acid could be used for the leaching of Nd and other REEs out e‐scrap. Acetic acid is an organic acid containing one carboxylic group with pKa = 4.76. It is larg produced by methanol carbonylation [27], and the price is comparable with that of mineral aci making it attractive for usage as a leaching agent. In the work of Zanonato et al. [28], it was show that Nd3+ forms at least successive mononuclear complexes according to the following equilibriu The existence of NdCit, NdHCit, NdHCit2, and NdCit2 was conﬁrmed by the solution in the pH range 2–5, which proved that citric acid could be used for the leaching of Nd and other REEs out of e-scrap. Acetic acid is an organic acid containing one carboxylic group with pKa = 4.76. 1. Introduction The leaching process was optimized in order to achieve the maximum REEs recovery by varying leaching time, acids concentration, and S:L ratio. Before the leaching experiments, the powder before and after roasting treatment was analyzed using scanning electron microscopy coupled with energy dispersive X-ray spectroscopy (SEM/EDX) analysis, X-ray diffraction (XRD), and total dissolution using aqua regia. Solvent extraction with D2EHPA was performed on the obtained leachates to test the feasibility of REEs recovery from such leachates using conventional methods. The hydrogen decre efore the leaching experiments i l t i 2.1. Material Characterization y g p ( ) p g p Pretreating was done by roasting at 400 °C for 1.5 h in an Entech muffle furnace (LF2) in a cera crucible. After roasting, the powder was sieved to a particle size of <355 µm using a Retsch AS 2 (Retsch, Haan, Germany) vibratory sieve shaker. Throughout this research, these two form d d ill b f d t HD Ndf B d d t d NdF B d T d t i t anning electron microscopy coupled with energy dispersive X‐ray spectroscopy (SEM/EDX) nalysis, x‐ray diffraction (XRD) , and total dissolution using aqua regia. Solvent extraction with 2EHPA was performed on the obtained leachates to test the feasibility of REEs recovery from such achates using conventional methods. The hydrogen decrepitated (HD) NdFeB powder was used in both original and pretreated form. Pretreating was done by roasting at 400 ◦C for 1.5 h in an Entech mufﬂe furnace (LF2) in a ceramic crucible. After roasting, the powder was sieved to a particle size of <355 µm using a Retsch AS 4 of 17 Metals 2018, 8, 721 200 (Retsch, Haan, Germany) vibratory sieve shaker. Throughout this research, these two forms of powder used will be referred to as HD NdfeB powder and roasted NdFeB powder. To determine the elemental composition, 0.5 g of the powder was dissolved in 25 mL aqua regia (V (conc. HCl): V (conc. HNO3) = 3:1) at 23 ± 1 ◦C for 6 hours and ﬁltered with Whatman microﬁber ﬁlters (GF/A 125 and 65 mm diameter). After ﬁltration, the solution was diluted using 0.5 mol/L HNO3 (suprapur® 65%, Merck, Kenilworth, NJ, USA). No residues were observed on the ﬁlter paper after ﬁltration. The composition of the prepared solution was measured using ICP-OES (inductively coupled plasma optical emission spectroscopy, Thermo Scientiﬁc iCAP 600 Series ICP Spectrometer, Thermo Fisher, Cambridge, UK) and recalculated into weight percentages of the elements in the roasted powder. The same procedure was performed on the nonroasted HD NdFeB powder. The same measuring method was used throughout this paper. All experiments were done in triplicate to ensure statistical reliability of the results. The hydrogen decre efore the leaching experiments i l t i 2.1. Material Characterization SEM/EDX analysis was carried out on the HD NdFeB before and after roasting using a Phenom ProX (Thermo Fisher Scientiﬁc, Phenom-World B.V., Eindhoven, The Netherlands) scanning electron microscope with Phenom ProSuite (Thermo Fisher Scientiﬁc, Phenom-World B.V., Eindhoven, The Netherlands) software and Element Identiﬁcation module in order to determine whether any surface modiﬁcations might have occurred during roasting or if there was any elemental redistribution in the particles. The chemical composition of both the HD and roasted NdFeB powders were further ascertained using XRD Bruker D8 Advance (Bruker Corporation, Billerica, MA, USA) with Cu Kα used to examine the phase content. The 2θ range was 20–80◦and step size 0.04◦/s. 2.2. Leaching Kinetics Acetic acid (Sigma-Aldrich, ≥99.5%) and citric acid (Sigma-Aldrich, ≥99.5%) were used as leaching agents, prepared by diluting the concentrated acids with MiliQ water (Merck Millipore Q-POD) to a desired concentration. In order to choose the optimal leaching conditions, the ﬁrst variables investigated were time and concentration. Each sample was reacting in a closed 100 mL PP plastic cup. Solid to liquid ratio was kept at 0.5 g of magnet powder per 25 mL of acid, the temperature at 23 ± 1 ◦C, and stirring speed at 400 rpm. The samples were leached for 100, 200, 300 min, and 24 h. The starting concentrations of the organic acids used were 0.1, 0.2, 0.4, 0.8, and 1 mol/L. Each experiment was done in triplicate. The heating and stirring plate used was an IKA® RT15. Powder was weighed on a Fisher MH-214 Analytical scale before the reaction and was used in leaching efﬁciency calculation. The sampled leachates were diluted in 1 mol/L HNO3 (suprapur® 65%, Merck, Kenilworth, NJ, USA), and the metal concentrations were measured using ICP-OES. The pH values of the acids before leaching and leachates after 24 h leaching time was measured using MeterLab™PHM 240 pH/ion Meter pH electrode (Radiometer, Copenhagen, Denmark). 2.3. Solid to Liquid Ratio Study After the determination of the optimal leaching time and acid concentration, the solid to liquid ratio for leaching was investigated. For this study 1/30, 1/50, and 1/80 g/mL S:L ratio was chosen. The volume of the leaching agent was kept at 25 mL in all cases, and the mass of the NdFeB powder was varied. The samples were reacted with 1 mol/L citric and 1 mol/L acetic acid for 300 min in PP plastic cups. The rotation speed was kept constant at 400 rpm, and the temperature was kept at 23 ± 1 ◦C. After the reaction, the leachates were sampled, diluted in 1 mol/L nitric acid, and the metal concentrations were measured using ICP-OES. p p y leachates of organic acids wa 3.1. Material Characterization In this study, leaching of a high‐temperature‐treated neodymium magnet powder was investigated using diluted citric acid and acetic acid. The leaching process was optimized in order to achieve the maximum REEs recovery by varying leaching time, acids concentration, and S:L ratio. Before the leaching experiments, the powder before and after roasting treatment was analyzed using scanning electron microscopy coupled with energy dispersive X‐ray spectroscopy (SEM/EDX) analysis, x‐ray diffraction (XRD) , and total dissolution using aqua regia. Solvent extraction with D2EHPA was performed on the obtained leachates to test the feasibility of REEs recovery from such leachates using conventional methods. 2. Materials and Methods 2.1. Material Characterization The composition of the HD NdFeB powder used in this research was determined in a previous work performed by our group [13], where HNO3 was used to leach the HD NdFeB and TODGA (N, N, N′, N′-tetraoctyl diglycolamide) was used to extract the REEs from the acquired leachate. The results were in accordance with the composition of neodymium magnets found on the market [3,7,32]. Preliminary leaching tests were performed on the HD NdFeB powder using 0.4–1.6 mol/L citric acid and acetic acid at S:L ratio 1/50 g/mL and 25 ± 1 ◦C. REEs exhibited very slow leaching kinetics (less than 20% REEs leached after 8 hours, even at concentrations above 1.2 mol/L, while it took roughly ﬁve days to leach 80% of Nd and 60% of Pr and a mere 20% of Dy). Thus, the HD NdFeB powder was roasted and demagnetized to potentially increase its leaching properties. Roasting was used to change the powder chemically and demagnetization to minimize the amount of powder clinging to the stirring magnet. After roasting, the composition of the roasted powder was determined by dissolving it in aqua regia (results shown in Table 1). y g p ( ) p g p etreating was done by roasting at 400 °C for 1.5 h in an Entech muffle furnace (LF2) in a cer ucible. After roasting, the powder was sieved to a particle size of <355 µm using a Retsch AS etsch, Haan, Germany) vibratory sieve shaker. Throughout this research, these two form wder used will be referred to as HD NdfeB powder and roasted NdFeB powder To determine Table 1. 2.4. Solvent Extraction Carboxylic acids have been previously used for actinide and lanthanide separations [29,30] and could be suitable for the recovery of REEs out of NdFeB magnet waste as they can form soluble complexes with REEs However, the leaching of neodymium magnets with organic acids is very where [A]org and [A]aq are the equilibrium concentration of the metal of interest in all its existing species in the organic and aqueous phase, respectively. Separation factor represents the degree of separation between species A and B in the extraction system and is calculated according to the following equation. electronic waste. Carboxylic acids have been previously used for actinide and lanthanide separations [29,30] and could be suitable for the recovery of REEs out of NdFeB magnet waste as they can form soluble complexes with REEs However the leaching of neodymium magnets with organic acids is very where [A]org and [A]aq are the equilibrium concentration of the metal of interest in all its existing species in the organic and aqueous phase, respectively. Separation factor represents the degree of separation between species A and B in the extraction system and is calculated according to the following equation. g y g g y [6] investigated in detail the kinetics of extraction of Nd tudy showed that acetic acid in concentrations higher than SF = DA DB (5) f Nd than (5) 0.4 mol/L at 800 rpm, 1% (w/v) and 308 K was effective at leaching Nd and Fe out of the waste. However, in this study—as well as in other works—little attention was paid to the recovery of other REEs meaning no detailed kinetic or temperature gradient study was performed for vital REEs like The equilibrium pH values in the aqueous phase were measured using MeterLab™PHM 240 pH/ion Meter pH electrode. 2.4. Solvent Extraction To extract metals from the aqueous leachate, D2EHPA (Sigma-Aldrich 97%) in Solvent 70 was used. D2EHPA is a widely used industrial extracting agent, which was used in the case of 1 mol/L citric acid and acetic acid leachates to determine the possible use for the recovery of REEs and other 5 of 17 nd food olution Metals 2018, 8, 721 using Aspergilu elements out of organic acids leachates using solvent extraction. The usual extraction reaction of lanthanides using acidic extractants like D2EHPA [31] is of ion-exchange character: with citric acid include metal ion displacement with hydronium ions and formation of soluble metal– ligand complexes by metal chelation [25]. In a study by Brown et al. [26], it was shown that Nd3+ complex with citrate counterion is formed according to the following equilibrium: Ln3+ + 3(HX)2 Nd3+ + jH+ + kCit3− ⇆ NdkHjCitk(3n+j−3k) (1) LnX3(HX)3 + 3H+ (3) Ln3+ + 3(HX)2 Nd3+ + jH+ + kCit3− ⇆ NdkHjCitk(3n+j−3k) LnX3(HX)3 + 3H+ ) (3) The existence of NdCit, NdHCit, NdHCit2, and NdCit2 was confirmed by the solution in the pH range 2–5, which proved that citric acid could be used for the leaching of Nd and other REEs out of e‐scrap. Acetic acid is an organic acid containing one carboxylic group with pKa = 4.76. It is largely produced by methanol carbonylation [27], and the price is comparable with that of mineral acids, making it attractive for usage as a leaching agent. In the work of Zanonato et al. [28], it was shown that Nd3+ forms at least successive mononuclear complexes according to the following equilibrium: where Ln represents the lanthanide ion and HX represents a molecule of D2EHPA. The experiments were performed on the Ika Vibrax Vxr basic shaking machine in 3.5 mL glass shaking vials, for 1 h and the Vorg: Vaq = 1. After the extraction, the aqueous phase was sampled, diluted in 1 mol/L HNO3, and measured using the ICP-OES. The distribution ratios (D) were calculated as the mass balance in the aqueous phase before and after the extraction. Distribution ratios were calculated according to the following equation: O− ⇆ Nd (OOCCH3)j(3−j) +, j = 1, 2, 3 (2) e used for the recovery of Nd and possibly other REEs from DA = [A]org [A]aq (4) ) f (4) electronic waste. p p y leachates of organic acids wa 3.1. Material Characterization The protective coating is not, however, of interest here from a recycling standpoint as it does not contain REEs. Lastly, the difference from 100% can be attributed to the experimental errors while performing the experiments. Metals 2018, 8, x FOR PEER REVIEW 6 of 17 that are similar to the ones in the HD NdFeB powder. Some impurities present in the NdFeB magnet structure, such as Co and B, were also present, but in percentages lower than 2%. Fe and Nd were represented in slightly lower percentages than expected, which can be attributed to the reaction of oxygen and other atmospheric gases with the original powder during roasting as the oven did not have a controlled atmosphere. Due to the inability to measure the content of oxygen and other air components using the ICP‐OES, the composition of these could not be reported by aqua regia dissolution. Some percentages of Fe and Ni (normally present in the magnet coating) might have been lost during the sieving of the roasted powder as larger chunk‐like chips from the protective coating of the original magnet were removed. The protective coating is not, however, of interest here from a recycling standpoint as it does not contain REEs. Lastly, the difference from 100% can be To further characterize the waste, SEM/EDX analysis was performed. This was done in order to determine the surface changes that occurred in the HD NdFeB powder during roasting and to examine possible redistributions of elements during roasting and sieving. Figure 1 shows the SEM and EDX of the HD NdFeB powder (a and b) and the roasted NdFeB powder (c and d). attributed to the experimental errors while performing the experiments. To further characterize the waste, SEM/EDX analysis was performed. This was done in order to determine the surface changes that occurred in the HD NdFeB powder during roasting and to examine possible redistributions of elements during roasting and sieving. Figure 1 shows the SEM and EDX of the HD NdFeB powder (a and b) and the roasted NdFeB powder (c and d) . (a) (b) (c) (d) Figure 1. Four different SEM magnifications (4100×, 10 kV‐Map) HD NdFeB powder in two different locations (a,b) and roasted neodymium magnet powder sample in two locations (c,d) . The crosses and lines on the SEM pictures show the locations where the EDX analysis was performed. p p y leachates of organic acids wa 3.1. Material Characterization Elemental composition, in wt %, of the (a) hydrogen decrepitated (HD) NdFeB totally dissolved in aqua regia for 1 h at 80 ± 1 ◦C and S:L = 1/10 g/mL and (b) roasted NdFeB powder dissolved in aqua regia for approximately six hours at temperature 23 ± 1 ◦C and S:L = 0.1/5 g/mL. No residues were left after ﬁltration. ntal composition, 0.5 g of the powder was dissolved in 25 mL aqua regia (V (conc. HNO3) = 3:1) at 23 ± 1 °C for 6 hours and filtered with Whatman microfiber filters (G 5 mm diameter) . After filtration, the solution was diluted using 0.5 mol/L HNO3 (su Element (a) Mass Fraction in HD NdFeB Magnet Powder/wt % [13] (b) Mass Fraction in Roasted NdFeB Magnet Powder/wt % Fe 61.1 ± 1.0 56.4 ± 0.3 Nd 25.3 ± 0.7 22.7 ± 0.0 Pr 2.62 ± 0.17 2.73 ± 0.05 Dy 1.08 ± 0.27 0.77 ± 0.01 Co 1.42 ± 0.07 1.25 ± 0.03 B 1.00 ± 0.02 0.74 ± 0.01 Metals 2018, 8, 721 6 of 17 Over one quarter of the roasted NdFeB powder weight fraction consists of REEs, which makes it a feasible source for REEs recovery. As expected, the two major components of the roasted neodymium magnet powder were Fe and Nd, making up 56.4 ± 0.3% and 22.7 ± 0.0% of the powder, respectively. Pr made up 2.73 ± 0.05% and Dy 0.77 ± 0.01% of the overall composition—percentages that are similar to the ones in the HD NdFeB powder. Some impurities present in the NdFeB magnet structure, such as Co and B, were also present, but in percentages lower than 2%. Fe and Nd were represented in slightly lower percentages than expected, which can be attributed to the reaction of oxygen and other atmospheric gases with the original powder during roasting as the oven did not have a controlled atmosphere. Due to the inability to measure the content of oxygen and other air components using the ICP-OES, the composition of these could not be reported by aqua regia dissolution. Some percentages of Fe and Ni (normally present in the magnet coating) might have been lost during the sieving of the roasted powder as larger chunk-like chips from the protective coating of the original magnet were removed. p p y leachates of organic acids wa 3.1. Material Characterization In Figure 1a,b, it can be observed that the morphology of the powder looked even except for the Figure 1. Four different SEM magniﬁcations (4100×, 10 kV-Map) HD NdFeB powder in two different locations (a,b) and roasted neodymium magnet powder sample in two locations (c,d). The crosses and lines on the SEM pictures show the locations where the EDX analysis was performed. (b) (a) (b) (a) (a) (a) (c) (b) (d) (d) (c) Figure 1. Four different SEM magnifications (4100×, 10 kV‐Map) HD NdFeB powder in two different locations (a,b) and roasted neodymium magnet powder sample in two locations (c,d) . The crosses and lines on the SEM pictures show the locations where the EDX analysis was performed. Figure 1. Four different SEM magniﬁcations (4100×, 10 kV-Map) HD NdFeB powder in two different locations (a,b) and roasted neodymium magnet powder sample in two locations (c,d). The crosses and lines on the SEM pictures show the locations where the EDX analysis was performed. 7 of 17 Metals 2018, 8, 721 In Figure 1a,b, it can be observed that the morphology of the powder looked even except for the particle size, which varied and ranged from 2–20 µm. EDX analysis was also performed on the same powder shown in Figure 1. The EDX results suggested that the HD NdFeB powder elemental composition correlated to the composition of NdFeB magnets found in the market [7]. No clusters with higher concentration of certain elements than in the bulk material were found. Some locations on the powder—like the one represented by Line 1 in Figure 1a and point 2 in Figure 1b—showed higher amounts of oxygen, which can be attributed to the fact that the REE-rich grain boundary phase oxidizes fast when exposed to air during the HD process [33]. The results of the SEM/EDX analysis of the roasted NdFeB powder showed different results. In Figure 1c,d, it can be observed that the morphology of the powder was significantly more diverse now than it was before the roasting treatment. Three types of particles could be observed: the flat surface particles sized around 10 µm in diameter, the bulkier particles with a distinct rugged morphology with the diameter size of around 10–20 µm, and the small white specs less than 5 µm in diameter. EDX was performed in order to determine the elemental composition of the various particle types. p p y leachates of organic acids wa 3.1. Material Characterization The ﬂat surface particles were largely composed of iron and oxygen (spots 1 and 2 in Figure 1d). EDX analysis of the white specs showed that these consisted mainly of Nd, Pr, and O, pointing to the creation of clusters of REE oxides during the roasting treatment. The large particles with the rugged surface consisted mostly of Fe and O and smaller amounts of REEs. The weight percentage ﬂuctuations were probably due to variations in the penetration depth of the electrons during the EDX analysis, which can vary depending on the surface topography and composition. Because the electrons penetrate at an approximately constant mass, spatial resolution is a function of density. From the SEM images, it can be observed that the morphology of the hydrogen decrepitated and roasted powder differed signiﬁcantly, which was expected. In the case of the HD NdFeB powder, the surfaces of the particles were relatively similar, with most of the particles looking homogeneous and having sharp edges. On the other hand, the roasted powder was heterogeneous. It was conﬁrmed using the EDX analysis that the heterogeneity was also present from a particle composition point of view as it was shown that the composition varied signiﬁcantly across the particle size and shape in the roasted NdFeB powder. To determine the chemical species that can be found in the powder before and after treatment, as well as the changes that occurred in the HD NdFeB power during the roasting process, both powders were analyzed using XRD. The XRD pattern of the HD NdFeB powder (Figure 2a) showed the presence of Nd2Fe14BH3, a hydride formed during the hydrogen decrepitation process. Apart from the main Nd2Fe14BH3, the presence of Fe2B and NdFe1.14Co0.76 was observed. The XRD pattern also showed the presence of traces of Ni. The XRD pattern of the roasted NdFeB powder (Figure 2b) showed the presence of Fe2O3 and Nd2O3, which were formed during the roasting process in the presence of air atmosphere. Ni was also present in higher amounts in elemental form. Apart from these main components there were also present traces of Pr2O3 and Fe in elemental form. The results of the XRD correlated with the results of the SEM-EDX analysis. 8 of 17 Metals 2018, 8, 721 (a) (b) Figure 2. XRD pattern of the (a) hydrogen decrepitated NdFeB powder and (b) roasted NdFeB powder. Figure 2. p p y leachates of organic acids wa 3.1. Material Characterization XRD pattern of the (a) hydrogen decrepitated NdFeB powder and (b) roasted NdFeB powder. 2 L hi Ki ti (a) (b) Figure 2. XRD pattern of the (a) hydrogen decrepitated NdFeB powder and (b) roasted NdFeB powder. (a) ( ) (a) (b) (a) (b) (b) (b) Figure 2. XRD pattern of the (a) hydrogen decrepitated NdFeB powder and (b) roasted NdFeB powder Figure 2. XRD pattern of the (a) hydrogen decrepitated NdFeB powder and (b) roasted NdFeB powder. Figure 2. XRD pattern of the (a) hydrogen decrepitated NdFeB powder and (b) roasted NdFeB d Figure 2. XRD pattern of the (a) hydrogen decrepitated NdFeB powder and (b) roasted NdFeB powder. 3 2 L hi Ki i Figure 2. XRD pattern of the (a) hydrogen decrepitated NdFeB powder and (b) roasted NdFeB powder. 2. Leaching Kinetics Figure 2. XRD pattern of the (a) hydrogen decrepitated NdFeB powder and (b) roasted NdFeB powder. p 3 2 Leaching Kinetic 3.2. Leaching Kinetics powder. 3 2 L hi Ki ti p 3 2 Leaching Kinetic 3.2. Leaching Kinetics po e 3 2 L hi Ki ti 3.2. Leaching Kinetics Apart from the REEs, Fe and Co leaching efficiencies were also monitored as they make up a sufficient percentage of the material to compromise the leaching process. Citric acid and acetic acid showed good leaching properties for REEs out of the roasted magnet powder waste. The results from leaching of roasted neodymium magnet powder with acetic acid and citric acid are shown in Figures 3 and 4, respectively. The leaching kinetics of the REEs, Fe, and Co are represented as an average of a triplicate experiment. Apart from the REEs, Fe and Co leaching efﬁciencies were also monitored as they make up a sufﬁcient percentage of the material to compromise the leaching process. Citric acid and acetic acid showed good leaching properties for REEs out of the roasted magnet powder waste. The results from leaching of roasted neodymium magnet powder with acetic acid and citric acid are shown in Figures 3 and 4, respectively. The leaching kinetics of the REEs, Fe, and Co are represented as an average of a triplicate experiment. 3.2. Leaching Kinetics Apart from the REEs, Fe and Co leaching efficiencies were also monitored as they make up a sufficient percentage of the material to compromise the leaching process. Citric acid and acetic acid showed good leaching properties for REEs out of the roasted magnet powder waste. The results from leaching of roasted neodymium magnet powder with acetic acid and citric acid are shown in Figures 3 and 4, respectively. The leaching kinetics of the REEs, Fe, and Co are represented as an average of a triplicate experiment. (a) (a) Figure 3. Cont. 9 of 17 Metals 2018, 8, 721 , , (b) (c) (d) (e) Figure 3. Effect of the concentration of acetic acid (0.1, 0.2, 0.4, 0.8 and 1 mol/L) and leaching time (100 min, 200 min, 300 min and 24 h) on the leaching efﬁciency of (a) Nd, (b) Pr, (c) Dy, (d) Fe, and (e) Co. The temperature was kept at 25 ± 1 ◦C, magnetic stirring at 400 rpm, and S:L ratio at 1/50 g/mL. (b) (e) Figure 3. Effect of the concentration of acetic acid (0.1, 0.2, 0.4, 0.8 and 1 mol/L) and leaching time (100 min, 200 min, 300 min and 24 h) on the leaching efﬁciency of (a) Nd, (b) Pr, (c) Dy, (d) Fe, and (e) Co. Metals 2018, 8, 721 Figure 3. Metals 2018, 8, 721 Figure 3. 10 of 17 Leaching of Nd, Pr, Dy, Fe, Co, and B at varying concentrations of acetic acid were studied. Leaching time varied from 0–24 h, while the temperature and S:L ratio were kept constant. The leaching efﬁciency was found to be signiﬁcantly dependent on the acetic acid concentration as well as the leaching time. It can be observed that changing the acetic acid concentration from 0.05 to 1 mol/L and using a 24 h leaching time increased the leaching efﬁciency from <20% to >95% for all REEs. Thus, acetic acid in concentrations higher than 1 mol/L is recommended for the efﬁcient recovery of REEs from roasted neodymium magnet powder. After 24 h of leaching with 1 mol/L acetic acid, almost all of Dy, Pr, Fe, Co, and B were leached into the solution, while over 95% of Nd was recovered. Diluting acetic acid from 1 mol/L to 0.1 mol/L decreased the pH value from 2.2 to 2.8. The pH values of the leachates after 24 h leaching were 4.1, 4.2, 4.6, 5.2, 5.6, and 5.9 for 1 mol/L, 0.8 mol/L, 0.6 mol/L, 0.4 mol/L, 0.2 mol/L, and 0.1 mol/L leachates, respectively, pointing at proton starvation during leaching. Co. The temperature was kept at 25 ± 1 °C, magnetic stirring at 400 rpm, and S:L ratio at 1/50 g/mL. Leaching of Nd, Pr, Dy, Fe, Co, and B at varying concentrations of acetic acid were studied. Leaching time varied from 0–24 h, while the temperature and S:L ratio were kept constant. The leaching efficiency was found to be significantly dependent on the acetic acid concentration as well as the leaching time. It can be observed that changing the acetic acid concentration from 0.05 to 1 mol/L and using a 24 h leaching time increased the leaching efficiency from <20% to >95% for all REEs. Thus, acetic acid in concentrations higher than 1 mol/L is recommended for the efficient recovery of REEs from roasted neodymium magnet powder. After 24 h of leaching with 1 mol/L acetic acid, almost all of Dy, Pr, Fe, Co, and B were leached into the solution, while over 95% of Nd was recovered. Diluting acetic acid from 1 mol/L to 0.1 mol/L decreased the pH value from 2.2 to 2.8. p 3 2 Leaching Kinetic 3.2. Leaching Kinetics po e 3 2 L hi Ki ti The temperature was kept at 25 ± 1 ◦C, magnetic stirring at 400 rpm, and S:L ratio at 1/50 g/mL. Metals 2018, 8, 721 Figure 3. The pH values of the leachates after 24 h leaching were 4.1, 4.2, 4.6, 5.2, 5.6, and 5.9 for 1 mol/L, 0.8 mol/L, 0.6 mol/L, 0.4 mol/L, 0.2 mol/L, and 0.1 mol/L leachates, respectively, pointing at proton starvation during leaching. Fe, B, and Co showed similar trends and no selectivity for REEs was observed, meaning that the increase in leaching of the REEs was also followed by the co-leaching of Fe, B, and Co into the solution. As 1 mol/L acetic acid was needed for quantitative leaching of REEs, this concentration was used for further experiments. g g Fe, B, and Co showed similar trends and no selectivity for REEs was observed, meaning that the increase in leaching of the REEs was also followed by the co‐leaching of Fe, B, and Co into the solution. As 1 mol/L acetic acid was needed for quantitative leaching of REEs, this concentration was used for further experiments. (a) (b) Figure 4. Cont. Figure 4. Cont. 11 of 17 17 11 of 17 17 Metals 2018, 8, 721 M t l 2018 8 F (c) (d) (e) Figure 4. Effect of the concentration of citric acid (0.1, 0.2, 0.4, 0.8 and 1 mol/L) and leaching time (100 min, 200 min, 300 min and 24 h) on the leaching efficiency of (a) Nd, (b) Pr, (c) Dy, (d) Fe, and (e) Co. The temperature was kept at 25 ± 1 °C by, magnetic stirring at 400 rpm, and S:L ratio at 1/50 g/mL. As in the case of acetic acid, the effect of variation in the concentration of citric acid was observed to influence the leaching efficiency of the components of the roasted neodymium powder (Figure 4) . Increasing the concentration of citric acid from 0.1 mol/L to 1 mol/L increased the leaching efficiency for the REEs from <20% to >95%, reaching the highest leaching efficiency after 24 h. The increase in Figure 4. Effect of the concentration of citric acid (0.1, 0.2, 0.4, 0.8 and 1 mol/L) and leaching time (100 min, 200 min, 300 min and 24 h) on the leaching efﬁciency of (a) Nd, (b) Pr, (c) Dy, (d) Fe, and (e) Co. The temperature was kept at 25 ± 1 ◦C by, magnetic stirring at 400 rpm, and S:L ratio at 1/50 g/mL. Metals 2018, 8, 721 Figure 3. As in the case of acetic acid, the effect of variation in the concentration of citric acid was observ o influence the leaching efficiency of the components of the roasted neodymium powder (Figure ncreasing the concentration of citric acid from 0.1 mol/L to 1 mol/L increased the leaching efficien or the REEs from <20% to >95%, reaching the highest leaching efficiency after 24 h. The increase he leaching efficiency of REEs was accompanied by the co-leaching of Fe, B, and Co into the solutio (e) Figure 4. Effect of the concentration of citric acid (0.1, 0.2, 0.4, 0.8 and 1 mol/L) and leaching time (100 min, 200 min, 300 min and 24 h) on the leaching efficiency of (a) Nd, (b) Pr, (c) Dy, (d) Fe, and (e) Co. The temperature was kept at 25 ± 1 °C by, magnetic stirring at 400 rpm, and S:L ratio at 1/50 g/mL. Figure 4. Effect of the concentration of citric acid (0.1, 0.2, 0.4, 0.8 and 1 mol/L) and leaching time (100 min, 200 min, 300 min and 24 h) on the leaching efﬁciency of (a) Nd, (b) Pr, (c) Dy, (d) Fe, and (e) Co. The temperature was kept at 25 ± 1 ◦C by, magnetic stirring at 400 rpm, and S:L ratio at 1/50 g/mL. As in the case of acetic acid, the effect of variation in the concentration of citric acid was observed to influence the leaching efficiency of the components of the roasted neodymium powder (Figure 4) . Increasing the concentration of citric acid from 0.1 mol/L to 1 mol/L increased the leaching efficiency for the REEs from <20% to >95%, reaching the highest leaching efficiency after 24 h. The increase in As in the case of acetic acid, the effect of variation in the concentration of citric acid was observed to influence the leaching efficiency of the components of the roasted neodymium powder (Figure 4). Increasing the concentration of citric acid from 0.1 mol/L to 1 mol/L increased the leaching efficiency for the REEs from <20% to >95%, reaching the highest leaching efficiency after 24 h. The increase in the leaching efficiency of REEs was accompanied by the co-leaching of Fe, B, and Co into the solution. 3.3. Solid to Liquid Ratio Study The leaching efficiencies The leaching efficiencies of Nr, Pr, Dy, Fe, and Co in 1 mol/L citric acid and acetic acid as a function of solid to liquid ratio are shown in Figure 5a,b. The S:L ratios used were 1/30, 1/50, and 1/80 g/mL. In the case of 1 mol/L citric acid, changing the S:L ratio from 1/30 to 1/80 g/mL had negligible effects on the leaching efficiencies of all the elements present. Dy showed slightly higher leaching efficiency at 1/80 S:L ratio, but all within the statistical error. On the other hand, in the case of 1 mol/L acetic acid, the change of S:L ratio from 1/30 to 1/80 g/mL had a significant effect on the leaching of Fe, which was leached at 61.69 ± 6.58% at 1/30 g/mL and 79.30 ± 2.28% at 1/80 g/mL. This can be attributed to the strength of the acids. Acetic acid is a monoprotic acid with pKa = 4.76, while citric acid is a triprotic acid with pKa1 = 3.13, pKa2 = 4.76 and pKa3 = 6.39, which can explain why the leaching efficiency of Fe was lower due to proton starvation in the leachate. Exact amounts of H+ were not recalculated from the pKa values as activity coefficients would need to be determined in that case. The leaching efficiency of Co was also slightly affected by increasing the S:L ratio; it increased from 86.11 ± 0.38% at 1/30 g/mL to 93.92 ± 1.70% at 1/80 g/mL. For industrial purposes, it can be extracted from the leachate using Cyanex 272 (di-(2,4,4-trimethylpentyl) phosphinic acid) at equilibrium pH 4–5 [34]. Changing the S:L ratio from 1/30 to 1/80 g/mL did not, however, affect the leaching efficiency of Nd, Pr, and Dy, which stayed the same across the S:L ratio. B was found to be leached quantitatively at all S/L ratios investigated. For complete extraction of B from the leachate, aliphatic 1,3-diols can be used [35]. function of solid to liquid ratio are shown in Figure 5a,b. The S:L ratios used were 1/30, 1/50, and 1/80 g/mL. In the case of 1 mol/L citric acid, changing the S:L ratio from 1/30 to 1/80 g/mL had negligible effects on the leaching efficiencies of all the elements present. Dy showed slightly higher leaching efficiency at 1/80 S:L ratio, but all within the statistical error. Metals 2018, 8, 721 Figure 3. The pH value of the leaching acid measured increased from 1.23 to 2.5 from 1 mol/L to 0.1 mol/L, while the values after 24 h leaching were 2.3, 2.6, 3.4, 4.5, and 5.3 for 1 mol/L, 0.8 mol/L, 0.6 mol/L, As in the case of acetic acid, the effect of variation in the concentration of citric acid was observed to influence the leaching efficiency of the components of the roasted neodymium powder (Figure 4) . Increasing the concentration of citric acid from 0.1 mol/L to 1 mol/L increased the leaching efficiency for the REEs from <20% to >95%, reaching the highest leaching efficiency after 24 h. The increase in As in the case of acetic acid, the effect of variation in the concentration of citric acid was observed to influence the leaching efficiency of the components of the roasted neodymium powder (Figure 4). Increasing the concentration of citric acid from 0.1 mol/L to 1 mol/L increased the leaching efficiency for the REEs from <20% to >95%, reaching the highest leaching efficiency after 24 h. The increase in the leaching efficiency of REEs was accompanied by the co-leaching of Fe, B, and Co into the solution. The pH value of the leaching acid measured increased from 1.23 to 2.5 from 1 mol/L to 0.1 mol/L, while the values after 24 h leaching were 2.3, 2.6, 3.4, 4.5, and 5.3 for 1 mol/L, 0.8 mol/L, 0.6 mol/L, 12 of 17 / , ol/L, 0.4 i th Metals 2018, 8, 721 p while the value 0.4 mol/L, 0.2 mol/L, and 0.1 mol/L of citric acid, respectively. An interesting observation here is the decrease in leaching efficiency of the Nd, Pr, and Dy that occurred after 24 h leaching with 0.1 mol/L citric acid. Fe and Co did not exhibit the same behavior—the leaching efficiency reached equilibrium after 300 min for Fe, while the leaching efficiency for Co increased another 10% after 24 h. This can be explained by the pH increase up to 5.3 in the 0.1 mol/L, which led to the precipitation of the REEs, most likely as citrates as citrates of REEs are known to be soluble in aqueous solutions around pH 2–5 [26]. decrease in leaching efficiency of the Nd, Pr, and Dy that occurred after 24 h leaching with 0.1 mol/L citric acid. Metals 2018, 8, 721 Figure 3. Fe and Co did not exhibit the same behavior—the leaching efficiency reached equilibrium after 300 min for Fe, while the leaching efficiency for Co increased another 10% after 24 h. This can be explained by the pH increase up to 5.3 in the 0.1 mol/L, which led to the precipitation of the REEs, most likely as citrates as citrates of REEs are known to be soluble in aqueous solutions around pH 2– 5 [26]. Both acetic acid and citric acid showed much better leaching properties when the roasted and sieved NdFeB powder was used than was the case for the HD NdFeB magnet It was shown that p Both acetic acid and citric acid showed much better leaching properties when the roasted and sieved NdFeB powder was used than was the case for the HD NdFeB magnet. It was shown that roasting and the elemental redistributions that occurred during the process (molar volume changes during the oxidation and pore formation that make the inner surfaces of the powder more available for leaching) beneﬁted the leaching properties. Sieving the powder into smaller particles also improved the leaching process, which was also shown in a previous work by Behera et al. [6]. The ideal conditions for the leaching of Nd were determined to be 1 mol/L of either citric or acetic acid, and further experiments were conducted with these concentrations. sieved NdFeB powder was used than was the case for the HD NdFeB magnet. It was shown that roasting and the elemental redistributions that occurred during the process (molar volume changes during the oxidation and pore formation that make the inner surfaces of the powder more available for leaching) benefited the leaching properties. Sieving the powder into smaller particles also improved the leaching process, which was also shown in a previous work by Behera et al. [6]. The ideal conditions for the leaching of Nd were determined to be 1 mol/L of either citric or acetic acid, and further experiments were conducted with these concentrations. 3.3. Solid to Liquid Ratio Study 3 4 Solvent Extraction 3.4. Solvent Extraction 3.4. Solvent Extraction A screening investigation regarding the possible separation of the leached metal ions by solvent extraction was made on the leachates obtained after 24 h leaching of the roasted magnet powder using 1 mol/L citric acid and acetic acid. D2EHPA dissolved in Solvent 70 in concentrations of 0.2, 0.4, 0.6, 0.8, and 1 mol/L was used as the organic phase for the extraction of the REEs out of the leachates. Figure 6 represents the D values for Fe and Nd as they are the major components of the leachate. The distribution ratios of other REEs were shown to behave very similarly to the distribution ratios of Nd, and these metals were completely extracted at higher D2EHPA concentrations. The distribution ratios of REEs increased with increasing D2EHPA concentration in the organic phase, as expected. The distribution ratios of Fe were much lower compared to Nd, so high separation factors were expected to be found, the highest being the ones at 0.2 acetic acid extraction with 1 mol/L D2EHPA, which reached the value of 161.9 ± 21.0. The distribution ratios were one order of magnitude higher in the acetic acid media than in the citric acid solutions. This can be attributed to the equilibrium pH values, which were around 3.5 after the extraction from 1 mol/L acetic acid leachate and slowly increased with the decreasing D2EHPA concentration but stayed around 1.5 after the extraction from 1 mol/L citric acid leachate. As shown in a previous study [36], higher equilibrium pH values gave higher distribution ratios for REEs due to REE–D2EHPA complex formation, which is favored in higher equilibrium pH media as D2EHPA is an acidic extractant that exhibits the ion‐ exchange mechanism, with release of H+ ions after the complex has been formed (Equation (3) ) . The behavior of B and Co was also monitored in the extraction process. It was found that at investigated conditions (0.2–1 M D2EHPA in Solvent 70) , less than 1% of both the B and Co were extracted from the leachates, which makes this system even more attractive in terms of selective REEs recovery. 3 4 Solvent Extraction 3.4. Solvent Extraction According to Table 2, separation factors between Nd and Fe were generally higher in the case of 1 M acetic acid leachate but had reached a value of 129 ± 55 in extraction from 1 M citric acid leachate, ki th t i i i ti l bl l t lik Nd f l t f F A screening investigation regarding the possible separation of the leached metal ions by solvent extraction was made on the leachates obtained after 24 h leaching of the roasted magnet powder using 1 mol/L citric acid and acetic acid. D2EHPA dissolved in Solvent 70 in concentrations of 0.2, 0.4, 0.6, 0.8, and 1 mol/L was used as the organic phase for the extraction of the REEs out of the leachates. Figure 6 represents the D values for Fe and Nd as they are the major components of the leachate. The distribution ratios of other REEs were shown to behave very similarly to the distribution ratios of Nd, and these metals were completely extracted at higher D2EHPA concentrations. The distribution ratios of REEs increased with increasing D2EHPA concentration in the organic phase, as expected. The distribution ratios of Fe were much lower compared to Nd, so high separation factors were expected to be found, the highest being the ones at 0.2 acetic acid extraction with 1 mol/L D2EHPA, which reached the value of 161.9 ± 21.0. The distribution ratios were one order of magnitude higher in the acetic acid media than in the citric acid solutions. This can be attributed to the equilibrium pH values, which were around 3.5 after the extraction from 1 mol/L acetic acid leachate and slowly increased with the decreasing D2EHPA concentration but stayed around 1.5 after the extraction from 1 mol/L citric acid leachate. As shown in a previous study [36], higher equilibrium pH values gave higher distribution ratios for REEs due to REE–D2EHPA complex formation, which is favored in higher equilibrium pH media as D2EHPA is an acidic extractant that exhibits the ion-exchange mechanism, with release of H+ ions after the complex has been formed (Equation (3)). The behavior of B and Co was also monitored in the extraction process. 3.3. Solid to Liquid Ratio Study The leaching efficiencies On the other hand, in the case of 1 mol/L acetic acid, the change of S:L ratio from 1/30 to 1/80 g/mL had a significant effect on the leaching of Fe, which was leached at 61.69 ± 6.58% at 1/30 g/mL and 79.30 ± 2.28% at 1/80 g/mL. This can be attributed to the strength of the acids. Acetic acid is a monoprotic acid with pKa = 4.76, while citric acid is a triprotic acid with pKa1 = 3.13, pKa2 = 4.76 and pKa3 = 6.39, which can explain why the leaching efficiency of Fe was lower due to proton starvation in the leachate. Exact amounts of H+ were not recalculated from the pKa values as activity coefficients would need to be determined in that case. The leaching efficiency of Co was also slightly affected by increasing the S:L ratio; it increased from 86.11 ± 0.38% at 1/30 g/mL to 93.92 ± 1.70% at 1/80 g/mL. For industrial purposes, it can be extracted from the leachate using Cyanex 272 (di‐(2,4,4‐trimethylpentyl) phosphinic acid) at equilibrium pH 4– 5 [34]. Changing the S:L ratio from 1/30 to 1/80 g/mL did not, however, affect the leaching efficiency of Nd, Pr, and Dy, which stayed the same across the S:L ratio. B was found to be leached quantitatively at all S/L ratios investigated. For complete extraction of B from the leachate, aliphatic 1,3‐diols can be used [35]. (a) Figure 5. Cont. Figure 5. Cont. 13 of 17 13 of 17 Metals 2018, 8, 721 Metals 2018 8 x FO (b) Figure 5. The dependence of the amount of Nd leached on the S:L ratio at 25 ± 1 °C using (a) 1 mol/L citric acid and (b) 1 mol/L acetic acid with 400 rpm stirring for 24 h in both cases. Figure 5. The dependence of the amount of Nd leached on the S:L ratio at 25 ± 1 ◦C using (a) 1 mol/L citric acid and (b) 1 mol/L acetic acid with 400 rpm stirring for 24 h in both cases. (b) Figure 5. The dependence of the amount of Nd leached on the S:L ratio at 25 ± 1 °C using (a) 1 mol/L citric acid and (b) 1 mol/L acetic acid with 400 rpm stirring for 24 h in both cases. Figure 5. 3.3. Solid to Liquid Ratio Study The leaching efficiencies The dependence of the amount of Nd leached on the S:L ratio at 25 ± 1 ◦C using (a) 1 mol/L citric acid and (b) 1 mol/L acetic acid with 400 rpm stirring for 24 h in both cases. 4. Conclusions 4. Conclusions In the present work, the leaching of REEs, Fe, and Co from roasted NdFeB magnet powder was studied using citric acid and acetic acid. The treated NdFeB powder was characterized prior to the leaching experiments by total dissolution with aqua regia and SEM/EDX analysis. Kinetics of the leaching process at different concentrations of acids and S:L ratios were studied. After the leaching experiments, solvent extraction was performed using D2EHPA in Solvent 70 as the organic phase and the leachates of citric acid and acetic acid to determine the feasibility of the extraction of REEs from such leachates as well as possible further developments of the process. In the present work, the leaching of REEs, Fe, and Co from roasted NdFeB magnet powder was studied using citric acid and acetic acid. The treated NdFeB powder was characterized prior to the leaching experiments by total dissolution with aqua regia and SEM/EDX analysis. Kinetics of the leaching process at different concentrations of acids and S:L ratios were studied. After the leaching experiments, solvent extraction was performed using D2EHPA in Solvent 70 as the organic phase and the leachates of citric acid and acetic acid to determine the feasibility of the extraction of REEs from such leachates as well as possible further developments of the process. from such leachates as well as possible further developments of the process. By total dissolution of the roasted NdFeB magnet powder in aqua regia, the composition of the magnet was determined to be consistent with that found in the market. Lower percentages of some elements, such as Fe, pointed towards the presence of oxygen due to air contact during roasting. SEM analysis of the neodymium magnet powder before and after roasting showed significant changes in the morphology of the powder. EDX showed the composition of the powder was composed of NdFeB matrix and further confirmed the redistribution of the elemental composition that ensued after roasting. In the HD NdFeB magnet powder, the elemental composition was even throughout the particles observed before roasting. By contrast, in the roasted NdFeB magnet powder, elemental composition was significantly more uneven, with some parts containing significantly higher amounts of REEs, while others contained higher amounts of Fe. 3 4 Solvent Extraction 3.4. Solvent Extraction Separation factors between Nd and Fe after extraction with 0.2, 0.4, 0.6, 0.8, and 1 mol/L D2EHPA in Solvent 70. The aqueous phases used were 1 mol/L citric acid and acetic acid leachates previously obtained. Phase ratios were kept at Vorg/Vaq = 1 during 30 min shaking at 1500 rpm on the custom made shaking machine Temperature was thermostatically kept at 25 ± 1 °C Table 2. Separation factors between Nd and Fe after extraction with 0.2, 0.4, 0.6, 0.8, and 1 mol/L D2EHPA in Solvent 70. The aqueous phases used were 1 mol/L citric acid and acetic acid leachates previously obtained. Phase ratios were kept at Vorg/Vaq = 1 during 30 min shaking at 1500 rpm on the custom-made shaking machine. Temperature was thermostatically kept at 25 ± 1 ◦C. usto ade s aki g ac i e. e pe atu e was t e ostatica y kept at 25 C. c (D2EHPA)/mol/L SFNd/Fe (1 mol/L Acetic Acid) SFNd/Fe (1 mol/L Citric Acid) 0.2 161.9 ± 21.0 0.39 ± 0.10 0.4 94.9 ± 9.4 2.07 ± 0.86 0.6 64.5 ± 13.4 17.0 ± 2.4 0.8 64.1 ± 16.0 76.7 ± 5.9 1 82.2 ± 21.0 129 ± 55 c (D2EHPA)/mol/L SFNd/Fe (1 mol/L Acetic Acid) SFNd/Fe (1 mol/L Citric Acid) 0.2 161.9 ± 21.0 0.39 ± 0.10 0.4 94.9 ± 9.4 2.07 ± 0.86 0.6 64.5 ± 13.4 17.0 ± 2.4 0.8 64.1 ± 16.0 76.7 ± 5.9 1 82.2 ± 21.0 129 ± 55 g p y p c (D2EHPA)/mol/L SFNd/Fe (1 mol/L Acetic Acid) SFNd/Fe (1 mol/L Citric Acid) c (D2EHPA)/mol/L SFNd/Fe (1 mol/L Acetic Acid) SFNd/Fe (1 mol/L Citric Acid) 3 4 Solvent Extraction 3.4. Solvent Extraction Distribution ratio of Nd and Fe as a function of the concentration of D2EHPA in a small-scale solvent extraction set-up (Vorg/Vaq = 1; 1 h shaking at 1500 rpm on an adaptable vortex mixer). The aqueous phase was a leachate (citric acid and acetic acid 1 M; S:L 1g /50 mL; 25 ◦C; 24 h; 400 rpm stirring) using D2EHPA in Solvent 70 solutions as extracting agents, while temperature was thermostatically kept at 25 ± 1 ◦C. Figure 6. Distribution ratio of Nd and Fe as a function of the concentration of D2EHPA in a small‐ Figure 6. Distribution ratio of Nd and Fe as a function of the concentration of D2EHPA in a small-scale Figure 6. Distribution ratio of Nd and Fe as a function of the concentration of D2EHPA in a small‐ scale solvent extraction set‐up (Vorg/Vaq = 1; 1 h shaking at 1500 rpm on an adaptable vortex mixer) . The aqueous phase was a leachate (citric acid and acetic acid 1 M; S:L 1g /50 mL; 25 °C; 24 h; 400 rpm stirring) using D2EHPA in Solvent 70 solutions as extracting agents, while temperature was thermostatically kept at 25 ± 1 °C. Figure 6. Distribution ratio of Nd and Fe as a function of the concentration of D2EHPA in a small-scale solvent extraction set-up (Vorg/Vaq = 1; 1 h shaking at 1500 rpm on an adaptable vortex mixer). The aqueous phase was a leachate (citric acid and acetic acid 1 M; S:L 1g /50 mL; 25 ◦C; 24 h; 400 rpm stirring) using D2EHPA in Solvent 70 solutions as extracting agents, while temperature was thermostatically kept at 25 ± 1 ◦C. g scale solvent extraction set‐up (Vorg/Vaq = 1; 1 h shaking at 1500 rpm on an adaptable vortex mixer) . The aqueous phase was a leachate (citric acid and acetic acid 1 M; S:L 1g /50 mL; 25 °C; 24 h; 400 rpm stirring) using D2EHPA in Solvent 70 solutions as extracting agents, while temperature was thermostatically kept at 25 ± 1 °C. solvent extraction set-up (Vorg/Vaq = 1; 1 h shaking at 1500 rpm on an adaptable vortex mixer). The aqueous phase was a leachate (citric acid and acetic acid 1 M; S:L 1g /50 mL; 25 ◦C; 24 h; 400 rpm stirring) using D2EHPA in Solvent 70 solutions as extracting agents, while temperature was thermostatically kept at 25 ± 1 ◦C. Table 2. 3 4 Solvent Extraction 3.4. Solvent Extraction It was found that at investigated conditions (0.2–1 M D2EHPA in Solvent 70), less than 1% of both the B and Co were extracted from the leachates, which makes this system even more attractive in terms of selective REEs recovery. According to Table 2, separation factors between Nd and Fe were generally higher in the case of 1 M acetic acid leachate but had reached a value of 129 ± 55 in extraction from 1 M citric acid leachate, making these systems promising in separating valuable elements like Nd from large amounts of Fe usually contained in NdFeB magnets. tals 2018, 8, 721 14 o tals 2018, 8, x FOR PEER REVIEW 14 o Figure 6. Distribution ratio of Nd and Fe as a function of the concentration of D2EHPA in a small‐ scale solvent extraction set‐up (Vorg/Vaq = 1; 1 h shaking at 1500 rpm on an adaptable vortex mixer) . The aqueous phase was a leachate (citric acid and acetic acid 1 M; S:L 1g /50 mL; 25 °C; 24 h; 400 rpm stirring) using D2EHPA in Solvent 70 solutions as extracting agents, while temperature was thermostatically kept at 25 ± 1 °C. Figure 6. Distribution ratio of Nd and Fe as a function of the concentration of D2EHPA in a small-scale solvent extraction set-up (Vorg/Vaq = 1; 1 h shaking at 1500 rpm on an adaptable vortex mixer). The aqueous phase was a leachate (citric acid and acetic acid 1 M; S:L 1g /50 mL; 25 ◦C; 24 h; 400 rpm stirring) using D2EHPA in Solvent 70 solutions as extracting agents, while temperature was thermostatically kept at 25 ± 1 ◦C. 14 of 17 14 of 17 Metals 2018, 8, 721 Metals 2018 8 x FO etals 2018, 8, 721 14 of etals 2018, 8, x FOR PEER REVIEW 14 o Figure 6. Distribution ratio of Nd and Fe as a function of the concentration of D2EHPA in a small‐ scale solvent extraction set‐up (Vorg/Vaq = 1; 1 h shaking at 1500 rpm on an adaptable vortex mixer) . The aqueous phase was a leachate (citric acid and acetic acid 1 M; S:L 1g /50 mL; 25 °C; 24 h; 400 rpm stirring) using D2EHPA in Solvent 70 solutions as extracting agents, while temperature was thermostatically kept at 25 ± 1 °C. Figure 6. 4. Conclusions 4. Conclusions Faster leaching kinetics observed in the case of acetic acid and citric acid can be attributed to the elemental redistribution during roasting and By total dissolution of the roasted NdFeB magnet powder in aqua regia, the composition of the magnet was determined to be consistent with that found in the market. Lower percentages of some elements, such as Fe, pointed towards the presence of oxygen due to air contact during roasting. SEM analysis of the neodymium magnet powder before and after roasting showed signiﬁcant changes in the morphology of the powder. EDX showed the composition of the powder was composed of NdFeB matrix and further conﬁrmed the redistribution of the elemental composition that ensued after roasting. In the HD NdFeB magnet powder, the elemental composition was even throughout the particles observed before roasting. By contrast, in the roasted NdFeB magnet powder, elemental composition was signiﬁcantly more uneven, with some parts containing signiﬁcantly higher amounts of REEs, while others contained higher amounts of Fe. Faster leaching kinetics observed in the case of acetic acid and citric acid can be attributed to the elemental redistribution during roasting and sieving. of acetic acid and citric acid can be attributed to the elemental redistribution during roasting and sieving. Acetic acid and citric acid showed good leaching properties for REEs, Fe, and Co. Of all the Acetic acid and citric acid showed good leaching properties for REEs, Fe, and Co. Of all the concentrations studied, 1 mol/L citric acid and 1 mol/L acetic acid exhibited the best leaching Metals 2018, 8, 721 15 of 17 properties. Over 95% of REEs were leached after 24 h with 1 mol/L acetic acid, while REEs were leached almost quantitatively after 24 h with 1 mol/L citric acid. Co-leaching of Fe and Co into the solution could not be avoided due to similar dissolution properties as the REEs, and both were leached >95% in both 1 mol/L citric acid and 1 mol/L acetic acid after 24 h. Changing the S:L ratio from 1/30 to 1/80 g/mL in 1 mol/L citric acid after 24 h showed no difference in leaching efficiency, while 1 mol/L acetic acid after 24 h showed significantly less Fe leached with a S:L ratio 1/30 g/mL. 4. Conclusions 4. Conclusions When making preliminary solvent extraction tests with 1 mol/L D2EHPA in Solvent 70 on the leachates in 1 mol/L citric acid and acetic acid, the REEs distribution ratios increased with increasing D2EHPA concentration from 0.2 mol/L to 1 mol/L. Higher distribution ratios were achieved in acetic acid with higher SF between Nd and Fe, but good separation between Nd and Fe was achieved in extraction from 1 M citric acid leachate. The study showed significant results with respect to organic acids leaching and further extraction of REEs from the leachate using D2EHPA. A further development in this direction would be beneficial, especially using mixer-settler bench scale systems, to further determine the process parameters for efficient separation of REEs from Fe. Author Contributions: Conceptualization: M.G., C.R.; Methodology: M.G., C.R.; Software: F.E., B.-M.S.; Validation: M.G., T.R.; Formal analysis: M.G., C.R., F.E.; Investigation: M.G., C.R.; Resources: T.R., B.-M.S.; Data curation: M.G., C.R., F.E.; Writing—original draft preparation: M.G.; Writing—review and editing: M.G., B.-M.S., T.R.; Visualization: M.G., C.R.; Supervision: T.R.; Project administration: B.-M.S.; Funding acquisition: T.R., B.-M.S. Funding: The research leading to these results has received funding from the European Community’s Seventh Framework Programme ([FP7/2007–2013]) under grant number No. 607411 (MC-ITN EREAN: European Rare Earth Magnet Recycling Network). This publication reﬂects only the authors’ views, exempting the Community from any liability. Project website: http://www.erean.eu. The authors would like to extend special thanks to the ENVIREE project (Environmentally friendly and efﬁcient methods for extraction of rare-earth elements (REE) from secondary sources-ERA-MIN project 2015–2017) contribution with additional funding. Acknowledgments: The authors would like to extend special thanks to Burçak Ebin for the help w XRD measurements. Conﬂicts of Interest: The authors declare no conﬂict of interest. Author Contributions: Conceptualization: M.G., C.R.; Methodology: M.G., C.R.; Software: F.E., B.-M.S.; Validation: M.G., T.R.; Formal analysis: M.G., C.R., F.E.; Investigation: M.G., C.R.; Resources: T.R., B.-M.S.; Data curation: M.G., C.R., F.E.; Writing—original draft preparation: M.G.; Writing—review and editing: M.G., B.-M.S., T.R.; Visualization: M.G., C.R.; Supervision: T.R.; Project administration: B.-M.S.; Funding acquisition: T.R., B.-M.S. References 1. Firdaus, M.; Rhamdhani, M.A.; Durandet, Y.; Rankin, W.J.; McGregor, K. Review of High-Temperature Recovery of Rare Earth (Nd/Dy) from Magnet Waste. J. Sustain. Metall. 2016, 2, 276–295. [CrossRef] 1. Firdaus, M.; Rhamdhani, M.A.; Durandet, Y.; Rankin, W.J.; McGregor, K. Review of High-Temperature Recovery of Rare Earth (Nd/Dy) from Magnet Waste. J. Sustain. Metall. 2016, 2, 276–295. [CrossRef] Firdaus, M.; Rhamdhani, M.A.; Durandet, Y.; Rankin, W.J.; McGregor, K. Review of High-Temperature Firdaus, M.; Rhamdhani, M.A.; Durandet, Y.; Rankin, W.J.; McGregor, K. Review of High-Temperature Recovery of Rare Earth (Nd/Dy) from Magnet Waste. J. Sustain. Metall. 2016, 2, 276–295. [CrossRef] g g p Recovery of Rare Earth (Nd/Dy) from Magnet Waste. J. Sustain. Metall. 2016, 2, 276–295. [CrossRef 2. European Commission. Critical Raw Materials for the EU; Report of the Ad-hoc Working Group on Deﬁning Critical Raw Materials: Brussels, Belgium, 2018. 2. European Commission. Critical Raw Materials for the EU; Report of the Ad-hoc Working Group on Deﬁning Critical Raw Materials: Brussels, Belgium, 2018. g 3. Binnemans, K.; Jones, P.T.; Blanpain, B.; Van Gerven, T.; Yang, Y.; Walton, A.; Buchert, M. Recycling of rare earths: A critical review. J. Clean. Prod. 2013, 51, 1–22. [CrossRef] 3. Binnemans, K.; Jones, P.T.; Blanpain, B.; Van Gerven, T.; Yang, Y.; Walton, A.; Buchert, M. Recycling of rare earths: A critical review. J. Clean. Prod. 2013, 51, 1–22. [CrossRef] 4. Guyonnet, D.; Planchon, M.; Rollat, A.; Escalon, V.; Tuduri, J.; Charles, N.; Vaxelaire, S.; Dubois, D.; Fargier, H. Material ﬂow analysis applied to rare earth elements in Europe. J. Clean. Prod. 2015, 107, 215–228. [CrossRef] 4. Guyonnet, D.; Planchon, M.; Rollat, A.; Escalon, V.; Tuduri, J.; Charles, N.; Vaxelaire, S.; Dubois, D.; Fargier, H. Material ﬂow analysis applied to rare earth elements in Europe. J. Clean. Prod. 2015, 107, 215–228. [CrossRef] 5. Rollat, A.; Guyonnet, D.; Planchon, M.; Tuduri, J. Prospective analysis of the ﬂows of certain rare earths in 4. Guyonnet, D.; Planchon, M.; Rollat, A.; Escalon, V.; Tuduri, J.; Charles, N.; Vaxelaire, S.; Dubois, D.; Fargier, H. Material ﬂow analysis applied to rare earth elements in Europe. J. Clean. Prod. 2015, 107, 215–228. [CrossRef] 5. Rollat, A.; Guyonnet, D.; Planchon, M.; Tuduri, J. Prospective analysis of the ﬂows of certain rare earths in Europe at the 2020 horizon. Waste Manag. 2016, 49, 427–436. [CrossRef] [PubMed] 5. Rollat, A.; Guyonnet, D.; Planchon, M.; Tuduri, J. References Önal, M.A.R.; Chenna, E.A.; Borra, R.; Blanpain, B.; Gerven, T.V.; Guo, M. Recycling of NdFeB magnets using nitration, calcination and water leaching for REE recovery. Hydrometallurgy 2017, 167, 115–123. [CrossRef] nitration, calcination and water leaching for REE recovery. Hydrometallurgy 2017, 167, 115–123. [CrossRef] 16. Xie, F.; Zhang, T.A.; Dreisinger, D.; Doyle, F. A critical review on solvent extraction of rare earths from aqueous solutions. Miner. Eng. 2014, 56, 10–28. [CrossRef] 16. Xie, F.; Zhang, T.A.; Dreisinger, D.; Doyle, F. A critical review on solvent extraction of rare earths from aqueous solutions. Miner. Eng. 2014, 56, 10–28. [CrossRef] 17. Riano, S.; Binnemans, K. Extration and separation of neodymium and dysprosium from used NdFeB magnets: an application of ionic liquids in solvent extraction towards the recycling of magnets. Green Chem. 2015, 17, 2931–2942. [CrossRef] 18. Shabani, M.A.; Irannajad, M.; Azadmehr, A.R. Investigation on leaching of malachite by citric acid. Int. J. Miner. Metall. Mater. 2012, 19, 782–786. [CrossRef] 19. Astuti, W.; Hirajima, T.H.; Sasaki, K.; Okibe, N. Comparison of effectiveness of citric acid and other acids in leaching of low-grade Indonesian saprolitic ores. Miner. Eng. 2016, 85, 1–16. [CrossRef] 20. Park, H.; Jung, K.; Alorro, R.D.; Yoo, K. Leaching Behavior of Copper, Zinc and Lead from Contaminated Soil with Citric Acid. Mater. Trans. 2013, 54, 1220–1223. [CrossRef] 21. Josso, P.; Roberts, S.; Teagle, D.A.H.; Pourret, O.; Herrington, R.; Albarran, P.L. Extraction and separation of rare earth elements from hydrothermal metalliferous sediments. Miner. Eng. 2018, 118, 106–121. [CrossRef] 22. Fujimoto, J.; Tanaka, K.; Watanabe, N.; Takahashi, Y. Simultaneous recovery and separation of rare earth elements in ferromanganese nodules by using Shewanella putrefaciens. Hydrometallurgy 2016, 166, 80–86. [CrossRef] 23. Astuti, W.; Hriajima, T.; Sasaki, K.; Okibe, N. Comparison of atmospheric citric acid leaching kinetics of nickel from different Indonesian saprolitic ores. Hydrometallurgy 2016, 161, 138–151. [CrossRef] 24. Vandenberghe, L.P.S.; Soccol, S.R.; Pandey, A.; Lebeault, J.M. Microbial production of citric acid. Braz. Arch. Biol. Technol. 1999, 42, 263–276. [CrossRef] 25. McDonald, R.G.; Whittington, B.I. Atmospheric acid leaching of nickel laterites review: Part I. Sulphuric acid technologies. Hydrometallurgy 2008, 91, 35–55. [CrossRef] 26. Brown, M.A.; Kropf, A.J.; Paulenova, A.; Gelis, A.V. Aqueous complexation of citrate with neodymium(iii) and americium(iii): A study by potentiometry, absorption spectrophotometry, microcalorimetry, and XAFS. Dalton Trans. 2014, 43, 6446–6454. [CrossRef] [PubMed] 27. Nam, J.S.; Kim, A.R.; Kim, D.M.; Chang, T.S.; Kim, B.S.; Bae, J.W. References Prospective analysis of the ﬂows of certain rare earths in Europe at the 2020 horizon. Waste Manag. 2016, 49, 427–436. [CrossRef] [PubMed] 6. Behera, S.S.; Parhi, P.K. Leaching kinetics study of neodymium from the scrap magnet using acetic acid. Sep. Purif. Technol. 2016, 160, 59–66. [CrossRef] 6. Behera, S.S.; Parhi, P.K. Leaching kinetics study of neodymium from the scrap magnet using acetic acid. Sep. Purif. Technol. 2016, 160, 59–66. [CrossRef] 7. Yang, Y.; Walton, A.; Sheridan, R.; Güth, K.; Gauß, R.; Gutﬂeisch, O.; Buchert, M.; Steenari, B.M.; Gerven, T.V.; Jones, P.T.; et al. REE Recovery from End-of-Life NdFeB Permanent Magnet Scrap: A Critical Review. J. Sustain. Metall. 2017, 3, 122–149. [CrossRef] 8. Alonso, E.; Sherman, A.M.; Wallington, T.J.; Everson, M.P.; Field, F.R.; Roth, R.; Kirchain, R.E. Evaluating Rare Earth Element Availability: A Case with Revolutionary Demand from Clean Technologies. Environ. Sci. Technol. 2012, 46, 3406–3414. [CrossRef] [PubMed] 9. Önal, M.A.R.; Borra, C.R.; Guo, M.; Blanpain, B.; Van Gerven, T. Recycling of NdFeB Magnets Using Sulfation, Selective Roasting, and Water Leaching. J. Sustain. Metall. 2015, 1, 199–215. [CrossRef] 16 of 17 16 of 17 Metals 2018, 8, 721 10. Yoon, H.-S.; Kim, C.; Chung, K.W.; Lee, S.-J.; Joe, A.-R.; Shin, Y.-H.; Lee, S.; Yoo, S.-J.; Kim, J.-G. Leaching kinetics of neodymium in sulfuric acid from E-scrap of NdFeB permanent magnet. Korean J. Chem. Eng. 2014, 31, 706–711. [CrossRef] 1. Lee, C.; Chen, Y.; Liao, C.; Popuri, S.R.; Tsai, S.; Hung, C. Selective Leaching Process for Neodym Recovery from Scrap Nd-Fe-B Magnet. Metall. Mater. Trans. 2013, 44, 5825–5833. [CrossRef] 12. Lyman, J.W.; Palmer, G.R. Scrap Treatment Method for Rare Earth Transition Metal Alloys. U.S. Patent 5,129,945, 14 July 1992. 13. Gergoric, M.; Ekberg, C.; Foreman, M.R.J.; Steenari, B.M.; Retegan, T. Characterization and Leaching of Neodymium Magnet Waste and Solvent Extraction of the Rare-Earth Elements Using TODGA. J. Sustain. Metall. 2017, 3, 638–645. [CrossRef] 14. Parhi, P.K.; Sethy, T.R.; Rout, P.C.; Saranji, K. Separation and recovery of neodymium and praseodymium from permanent magnet scrap through the hydrometallurgical route. Sep. Sci. Technol. 2016, 51, 2232–2241. [CrossRef] 15. Önal, M.A.R.; Chenna, E.A.; Borra, R.; Blanpain, B.; Gerven, T.V.; Guo, M. Recycling of NdFeB magnets using nitration, calcination and water leaching for REE recovery. Hydrometallurgy 2017, 167, 115–123. [CrossRef] 16. Xie, F.; Zhang, T.A.; Dreisinger, D.; Doyle, F. A critical review on solvent extraction of rare earths from 15. References Novel heterogeneous Rh-incorporated graphitic-carbon nitride for liquid-phase carbonylation of methanol to acetic acid. Catal. Commun. 2017, 99, 141–145. [CrossRef] 28. Zanonato, P.L.; Bernardo, P.; Bismondo, A.; Rao, L.; Choppin, G.R. Thermodynamic Studies of the Complexation between Neodymium and Acetate at Elevated Temperatures. J. Solut. Chem. 2001, 30, 1–18. [CrossRef] 29. Cotton, S. Lanthanide and Actinide Chemistry; John Wiley & Sons: Chichester, UK, 2006; ISBN 9780470010082. 30. Nash, K.L.; Jensen, M.P. Analytical-scale separations of the lanthanides: A review of techniques and fundamentals. Separ. Sci. Technol. 2001, 36, 1257–1282. [CrossRef] 31. Geist, A.; Nitsch, W.; Kim, J. On the kinetics of rare-earth extraction into D2EHPA. Chem. Eng. Sci. 1999, 54, 1903–1907. [CrossRef] Metals 2018, 8, 721 17 of 17 32. Tunsu, C.; Petranikova, M.; Gergori´c, M.; Ekberg, C.; Retegan, T. Reclaiming rare earth elements from end-of-life products: A review of the perspectives for urban mining using hydrometallurgical unit operations. Hydrometallurgy 2015, 156, 239–258. [CrossRef] 33. Awais, M.; Coelho, F.; Degri, M.; Herraiz, E.; Walton, A.; Rowson, N. Hydrocyclone Separation of Hy Decrepitated NdFeB. Recycling 2017, 2, 22. [CrossRef] 34. Swain, B.; Cho, S.S.; Lee, G.H.; Lee, C.G.; Uhm, S. Extraction/Separation of Cobalt by Solvent Extraction: A Review. Korean Soc. Ind. Eng. Chem. 2015, 26, 631–639. 35. Karakaplan, M.; Tural, S.; Tural, B.; Turgut, Y.; Hosgoren, H. The Solvent Extraction of Boron with Synthesized Aliphatic 1,3-Diols: Stripping and Extraction Behavior of Boron by 2,2,5-Trimethyl-1,3-hexanediol. Solvent Extr. Ion Exch. 2004, 22, 897–911. [CrossRef] 36. Gergoric, M.; Ekberg, C.; Steenari, B.M.; Retegan, T. Separation of Heavy Rare-Earth Elements from Light Rare-Earth Elements Via Solvent Extraction from a Neodymium Magnet Leachate and the Effects of Diluents. J. Sustain. Metall. 2017, 3, 601–610. [CrossRef] © 2018 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/W2091086070	https://zenodo.org/records/2490933/files/article.pdf	English	null	Unique Equipment for the Unwateringg of New York City's Great Aqueduct, The Problem of Mexico, and more	Scientific American	1,916	public-domain	6,966	By Robert G. Skerrett M DCH has been written about the vari ous phases of the great aqueduct which is to .carry water from the Catskill mountains to New York city, a hundred and more miles away. General interest has beeD aroused in this engineering project which, in some particulars, is a more difficult undertaking than the Pan ama Canal; but the public has been largely absorbed in the structural details, pure and simple. The technical task, however, is not that alone of providing a conduit for the water and then expecting that beneficent flood to flow on through stone-hewn arteries for generations to come without further consideration. Within this boat is installed the electric pumping plant with a capacity of some thing like 5,500,000 gallons daily. It con tains two 4-stage vertical, centrifugal pumps, each provided with a direct-con nected electric motor. These pumps dis charge into a 10-inch pipe which rises vertically from the center of the boat. The discharge pipe is connected, by means of a special gooseneck pipe, to a small chamber from which the water flows away into a nearby stream or sewer. The manner of operating the pumps and the boat. arranging the exhaust conduit is interest ing. When everything is ready, and the operator or operators in charge of the boat are at their stations, the pumps are started, and at once they begin the task of removing the water beneath the boat and thus promoting its gradual descent into the shaft. Power plant of the Hudson River siphon Note the reel of cable supplying energy for the pumps and I1ghts carried by the boat. Power plant of the Hudson River siphon ble supplying energy for the pumps and I1ghts c As a matter of fact, the aqueduct must be available for inspection throughout every part of it, and this is the engineer's defense against deterioration, breakdown, and the menace to health if not the denial of sufficient water to the ceaselessly increasing millions of the na tion's Metropolis.. So, too, from time to time various sections of the aqueduct must be subjected' to one test or another, and the.;;e should properly simulate the cir cumstances or stresses of maximum service. By Robert G. Skerrett Because of these requirements it· will be necessary at intervals to unwater or drain great stretches of the aqueduct and to withdraw from its tunnels and shafts and pipe lines millions upon millions of gallons of water, and to do this with all reasonable dispatch. The pumping boat at the bottom of the drainage shaft of the Hudson River siphon. It carries four two-stage vertical centrifugal pumps In this fashion, the water is lowered about 20 feet. Then, the discharge pipe Is disconnected from the gooseneck pipe which has followed it down, and another 20-foot length of discharge pipe is interposed in the line, the gooseneck joined to tbe upper end of the new section, and pumping resumed. Again, when the boat has gone down a score more feet, then the joint with the gooseneck is broken and an additional link in the discharge pipe inserted. The making and breaking of the joints is effected quickly, and the pumping operations are continued day and night until the water is removed in this way from the shafts and tunnels. The engineers of the Board of Water Supply of the city of New York have made preparation for just such contingencies, and the drainage equipment for the Rondout pressure tunnel and the Hudson River siphon are distinctly unique. 'l'hey are this because of the conditions imposed and the facilities devised to meet successfully these unusual circumstances. The Rondout pressure tunnel contains 30,000,000 gallons of water, and to drain that portion of the aqueduct the greater part of that water must be lifted 500 feet! Again, in dealing with the Hudson River siphon, the pumping out in that case finally imposes the unparalleled task of raising the water from a depth of 1,114 feet below tide level. It called for some study upon the part of Mr. J. Waldo Smith, the Chief Engineer, and his able asso ciates before the plan ultimately adopted was decided upon. f One of our illustrations shows the pumping equip ment at the bottom of the Hudson River shaft, nearly 1,150 feet below the top of the chamber, and supporting the pipe column which is absolutely watertight. In working against this great head, the pumps have been operated in series after reaching a depth of 650 feet, i. Unique Equipment for the Unwatering of New York City's Great Aqueduct a cylindrical boat or float. This carries the pumping outfit. It is 12 feet 9 inches in diameter. with it total depth of 30 feet, and when bearing its final load will have a freeboard of a little short of 2% feet above the surface of the water. Power plant of the Hudson River siphon Note the reel of cable supplying energy for the pumps and I1ghts carried by the boat. By Robert G. Skerrett By Robert G. Skerrett e., one pump exhausts into the other, and by this step·up process the water is finally raised to the ground level. Some idea of the service required can be gath ered from the fact that the discharge end of one of the pumps reaches a pressure of about 525 pounds to the square inch. To prevent excessive leakage due to this high pressure the engineers of the Water Supply Board were obliged to devise a special form of metal packing for the stuffing-boxes on the shafts of the pumps. Noth ing suitable for the work was available in the market. Th f th l t i d th t f The pumping boat at the bottom of the drainage shaft of the Hudson River siphon. It carries four two-stage vertical centrifugal pumps The gooseneck secured to the drainage pipe leading up from the boat in the shaft. Note the crane and counterweight of the gooseneck There were many different schemes suggested, and these eventually narrowed down to the following: (1) Use of compressed air. (2) Automatic bailing device, such as commonly used in mines. (3) Artesian well pumps in separate shafts. (3) Artesian well pumps in separate shafts. (4) Mine sinking pumps (4) Mine sinking pumps. (5) Pumping equipment to be located on a float and follow the water down. Of these the last method 'was chosen because it promised to be the most suitable for the problem, both on account of the minimum amount of machinery needed and the cost of masonry work involved. The drainage shaft in the case of the Hudson River siphon is the exploratory shaft on the east side of the river which was originally driven in order to determine how deep it would be necessary to go in order to get thoroughly sound rock for the tunnel running from shore to shore beneath the river. This shaft really forms an integral part in the water supply system. At a depth of 192 feet below tide level there is a connecting tunnel reaching back into the mountain for 800 feet and there linked up with the aqueduct extending southward to the city. Of these the last method 'was chosen because it promised to be the most suitable for the problem, both on account of the minimum amount of machinery needed and the cost of masonry work involved. The Problem of Mexico Our national history has proved ' that the militia, as at present organized, is a force of ,doubtful utility; and to spend an additional $75,000,000 a year upon an experiment which has proved to be a failure is the supremest folly. Furthermore, as we noted in our issue of April 15, throughout all this at tempted army legislation there has been maintained a powerful lobby of militiamen in Washington. Hence the House and the Senate now stand committed to a paid militia with all the political abuses which will inevitably arise, should this element in the bills be jammed'through the House and Senate Conference Committee. The military propaganda in Washington, emboldened by its success, would now seek to ,consolidate itself preparatory to a fresh advance. This has ever been the history of pernicious political movements of this character. We repeat that, unless the country wakes up to the sinister meaning of what has hap pened, representative government itself will' be threat ened, and the only militarism which we have to fear, namely, a military force using political influence to gain for its members special consideration under the law, will not only be found in our midst, but will soon set itself to the task of becoming the dominant political power. On the other hand there are certain purposes for which it is essential that the record of time shall fur nish some indication of the local hour·angle of the sun. Suppose, for example, it is a question of drawing a curve to show the daily march of temperature. The time coordinates of such a curve would be meaningless for both practical and scientific purposes if labeled in Greenwich hours; unless, of course, the place to which the data referred happened to lie on the meridian of Greenwich. It should be noted that the standard time now' in vogue is also, though in a less degree, inappro priate for such uses, except in reference to events that occur on one of the standard meridians. Indeed, for refined observations we need not only local time, but "apparent" (or sundial) time. Our record in the Philippines stands as a proof of our good faith, which we commend to the con sideration of the Mexicans and the smaller American republics. A Federalization that W outd Be Fatal Charles Allen Munn. President. Frederick C. Beach, Secretary, Orson D. Munn. Treasurer. all at 233 Broadway T , r T HE Hay bill and the Chamberlain bill have been , passed respectively by the House and the Senate. In the Senate, where the present diplomatic con· troversy with Germany undoubtedly had its effect, the Chamberlain bill was carried with scarcely a dozen Senators opposed to it, and it went through without a roll call. This bill authorizes a regular army of 250,000 men as against the 140,000 provided in the Hay bill; 261,000 volunteers, and 275,000 state militia sub ject to federal call. The bill also provides a system of military training for students in schools and colleges, application for the training to be purely voluntary, with the understanding that all students over eighteen years of age, by volunteering, obligate themselves to answer any fe,deral call to the colors which may be issueq. by the President during their training period. Entered at the Post Ot'l!ce ot New York, N. Y .. as Second Class Ma,tter Entered as Second Class Matter at the Post Ot'l!ce Department, Cauada Trade Mark Registered in the United States Patent Ot'l!ce Copyright 1916 by [lunn & Co., Inc. Great Britain rights reserved a a g Illustrated articles must not be reproduced without permission y The answer is easy. We have inherited from our remote ancestors their primitive conceptions of the time 0' day, just as we have inherited many other primitive conceptions which, but for the mental inertia of our species, would have passed into oblivion long ago. Early man found himself provided with a ready-made timepiece in the shape of the sun. Sunrise, high noon, and sunset were the events that marked off the progress of the day. The daylight period, or "natural day," which circumscribed most human activities, was the all-important time-interval. Even when the ancients divided their day into hours, it was the natural and not the civil day that was so divided. In Greece and Rome there were 12 hours between sunrise and sunset -hours that were long in summer and short in winter. The Problem of Mexico T T HE drift of events during the past few years, and particularly during the past few weeks, has forced the conviction that some day, sooner or later, and rather sooner than later, the United St!!tes will be driven to the necessity of bringing order out ,of the universal chaos which prevails in Mexico; anq In this connection we direct special attention to tɀe article in the present issue on that country, written by ,one wJ?o has made a special study of the complicated problems of that sorely distracted land. The fact that the author of the article is a military man lends particular significance to his conviction that the restoration of Mexico is as much, if not more, a question of civil reform than of military force. As a matter of fact it calls for the exe .. ·cise of both, military force being employed only in such sufficiency as would provide for the introduction and permanent operation of civil reforms. Under the suggested arrangement the amount appro priated annually for the militia would have to be increased from the present sum of $6,000,000 to a total for the proposed militia of 275,000 of $75,000,000. Now $75,000,000 would build, equip and maintain three battle-cruisers a year, and we ask those members of the Senate who have voted for this section of the bill, whether a steady addition of three battle-cruisers a year to our fieet would not be a far more powerful deterrent to any possible enemy than the existence of a body of what any European army would call com paratively raw ,and untrained troops; for such the militia always has been and in the very nature of things must ever be. Let us, for example, take the case of Germany, whose troops undergo two years. qf continuous intensive training. To her military mind, our militia, who ' receive only forty-eight drills at night in armories and not to ,exceed ten days a year in camp, are not trained soldiers and under existing conditions never can be regarded as first-line effectives. This is not the fault of the militia, but of the system upon which it has been planned and under which it exists. We have the greatest regard for the militiaman as such, and if he is not. The Problem of Mexico Our success in the pacification of the Philippines constitutes a high tribute to the character and ability of our officials. The dishonest white found himself in Bilibed prison as quickly as did the dishonest native; the self·seeking American office holder was soon given his papers; the inefficients were sidetracked. The insular government, acting as would a great sieve con tinually shaken with violence, retained only the men of large caliber and broad outlook; a few years of that strenuous administration left only the big men in office. The Philippine occupation forms one of the brightest chapters in the history of our civil and military admin istration. There was evidence of unselfish devotion to a high ideal-as witness the case of one Governor-General who turned his salary back into public works. It is a fact that, in all our years of occupation, not a single American official down there has accumulated even a small competence. One might hazard the prediction that the day will come when only one kind of standard time will be used throughout the world-viz., that of the meridian of Greenwich-but this does not mean that we can do without local time. Probably the present system of time zones, with their very irregular boundaries (de termined in America by the exigencies of the railways and in Europe by political frontiers), could be aban doned altogether. The clocks in railway stations and telegraph offices might keep identical time in England, India, China and America. The curious fictIon of the In ternational Date Line could be given up. We might even apply the nomenclature of the Greenwich clock to our office hours and our meal hours. It would, at first, seem strange to the New Yorker to begin work at 4 A. M. instead of 9 A.M., and dine at 2 P.M. instead of 7 P.M., but as these changes would be merely nom inal and involve no dislocation of his habits with re spect to daylight and darkness, he would soon become accustomed to them. , In the present defenseless condition of the country every dollar that is spent for defense should be spent to the best possible advantage. The Problem of Mexico as he cannot in the nature of things be, a first-class soldier, since he lacks the soldier's training and experience, it is not the militia- man's fault. , It has been predicted that the complete conquest of Mexico would need the employment of five hun dred thousand troops. Probably it would take that number ' to Belgiumize Mexico, but the United States has neither the heart, nor the wish, nor the peculiar form of genius for a task of that kind. When we take hold of the Mexican problem with a firm hand and a definite purpose, we must act in the same spirit in which we entered Cuba and the Philippines. Our policy and its results in those once unhappy islands are of record. The motives were humanitarian, and the splen did results, as given in the article above referred to, stand as a proof of our altruistic attitude to the Philip- pine peopie. , But only ·the astronomer and the mariner have com pletely broken away from the parochial time-keeping system of antiquity, and then only for certain purposes. Greenwich time prevails in the observatory, but must frequently be translated into local time. Ships' chron ometers keep Greenwich time, but ships' bells a make shift local time. The idea of making Greenwich mean time universal is not a new one. When standard time was adopted in America there were many advocates of the plan of using one kind of time instead of five. The International Time Conference which met in Paris in 1912 declared that" the universal time shall be that of Greenwich," and the wireless time signals of the world are now based upon this decision. All humanity is actuated 1;ly the same basic impulses, and of all the emotions the love of offspring is the strongest and most nearly universal. Hence the se curity of the home is the only assured foundation of government. Our success in the Philippines was directly due to the recognition of these fundamental conditions by those who had charge of our affairs out there. It took but a few years for the United States government to bring peace after a long period of alien domination. Compare thi/! with the situation in Formosa, where the natives are still resisting Japan after all her lopg years of occupation. A Federalization that W outd Be Fatal The advantages of using hours of uniform length were so little appreciated, that the more elaborate forms of water-clock, or clepsydra, were, with great ingenuity, constructed so as to vary their pace with the season; an expedient comparable in its naivetll with the at tempts made in comparatively modern times to con struct mechanical clocks that should take account of the equation of time and keep pace with the sun·dial. One can imagine the perplexity in which an ancient Greek or Roman horologist would have found himself involved if he had traveled to circumpolar ,latitudes, where the "natural" day, at its maximum, lasts for weeks or months, and each of its "hours" (according to the classical conception of this time-unit) would be several modern days in length. l'he object of this jO!wnal is to record accurately and lucidly the latest scientific, mechanical and industrial news of the day. As a weekly journal, it is in a post tion to announce interesting developments before they are published elsewhere. The Editor is glad to have submitted to him timely articles suitable for these columns, especially when such articles are accompanied by photographs. We shuld feel more inclined to congratulate the Sena'te on the passage of this bill, were it not that it contains the same serious defect that mars the Hay bill, as passed by the House. In both cases, the so called federalization of the militia, which includes among other provisions the payment of that body, is distinctly unconstitutional. The Judge Advocate Gen eral of the army has given it as his opinion that dis obedience on the part of a member of such a federalized ilitia would not constitute an act for which the man ,could be tried by federal court-martial, and this because of the inevitable clash between the proposed federal and the existing state authority. By Robert G. Skerrett The drainage shaft in the case of the Hudson River siphon is the exploratory shaft on the east side of the river which was originally driven in order to determine how deep it would be necessary to go in order to get thoroughly sound rock for the tunnel running from shore to shore beneath the river. This shaft really forms an integral part in the water supply system. At a depth of 192 feet below tide level there is a connecting tunnel reaching back into the mountain for 800 feet and there linked up with the aqueduct extending southward to the city. The boat is steadied vertically by means of concrete guides formed upon the surface of the shaft. This prevents any rotary motion. The boat with its equip ment can be brought to the' surface again when the siphon is refilled or it can be hoisted out. When this is done the equipment is dismantled at the bottom of the shaft and raised piece by piece. The final step con sists in bringing up the float itself, which weighs about 14 tons. The pumping machinery can be used else where, and is designed to be taken from place to place and reinstalled in other floats for the same service. The gooseneck secured to the drainage pipe leading up from the boat in the shaft. Note the crane and counterweight of the gooseneck Ordinarily this shaft on the eastern shore of the Hudson is entirely filled with water, and the top of it is under a " head" of a little Qver 400 feet, because of the height of the water in the aqueduct passing through the mountains east and west of the river. Therefore, it is necessary to cap and seal this shaft by a huge dome of steel cast in one piece. This is removed when the shaft is to be drained. But before this is done the water in the system above this point is allowed to escape through blow-outs until the level is brought down to that of the tide. Then, something like 40,000,000 gal lons remain to be pumped out in order to drain the siphon and the tributary parts of the aqueduct lying below the tide level. The drainage shaft has a diameter of 14 feet. In a chamber just above tide level is stored © 1916 SCIENTIFIC AMERICAN, INC SCIENTIFIC AMERICAN curious is the result of telegraphing in the opposite direction. A message is wired from Greenwich at noon, and it reaches New York at seven o'clock in the morn ing of the same day-five hours earlier than it was sent! elsewhere in this issue, there is not a branch of the government )vhich these men, rich in their Philippine experience, could not direct with a minimum of friction and administer with supreme honesty and with a single eye to the ultimate benefit of Mexico. These statements involve contradictions in terms with which we have become so familiar that they have lost their strangeness. Nevertheless the contradictions re main. When it is natO in London it is neither more nor less than now in Calcutta and New York. Call it what you like, the time remains identical. But why this bewildering diversity of "times, ': when there is really only one time? The Problem of Mexico Should the course of events (or Destiny if you will) necessitate our pacification of Mexico, we have not only the power to take over and administer the country but we have the men, I,tll well known, most of them available, and few of whom would not be willing to undertake any personal sacrifice if called upon by the President. There are men who have lived for years among Spanish speaking and Spanish thinking people; men who have made the trial, partially failed, profited by their failures and tried again. Should we under take such a limited military occupation as is suggested SCIENTIFIC AMERICAN April 29, 1916 444 Aeronautics Noel Pemberton-Billing, lately elected on the air preparedness issue, stated that a series of casualties with a total of 150 dead, 150 wounded and 105 missing was due to the sending of British aviators to the front in aeroplanes which were outclassed hope lessly by German machines. Harold J. Tennant, Parlia mentary Secretary ,of the War Office, made conciliatory answers to these charges and assured Mr. Pemberton Billing that he was wholly misinformed, although ad mitting that at present a majority of German machines were probably faster than the bulk of the British ma chines. He added, however, that the disadvantage was only a temporary one, and the near future would wit ness the British aviators on superior mounts. A Divided Exhaust Manifold.-What appears to be an excellent idea is the divided exhaust manifold that l':\s been adopted by one manufacturer. It has been more than suspected that the ordinary type of exhaust manifold is not conducive to a free escape of the gases, as the violent discharge of cylinder nearest the outlet of the manifold is likely to back up the previously dis charged gases from cylinders nearer the dead end of the manifold. In the design in question, which is for a four-cylinder engine, the manifold is composed of two separate passages contained in a single casting, which combine in a single outlet. Cylinders 1 and 4 connect with one of these passages, and cylinders 3 and 2 with the other, which would appear to give the gases an ample opportunity to establish a· flow in the right direc tion before they are broken up by an opposing discharge. Heart Action at Great Altitudes.- Dr. G. Ferry, a Frenchman enjoying the rank of Aide-Major de 2e classe au Pare d'Aviation, brings forth some interesting facts regarding blood pressure at- various heights in a recent issue of La Presse Medical, which are based on careful records made during a number of flights. His conclusions are: The pulse becomes more and more rapid from the ground up to a height of 750 meters. From this height to 1,250 meters it still augments, but less rapidly. Above this height it again accelerates more rapidly. The period of slower acceleration seems to be explained by the fact that between 750 meters and 1,250 meters the air is usually calmer than at lower altitudes, and the wind more regular. Aeronautics "Fat Sparks."-A great deal is written about the efficacy of a "big, hot spark" in producing increased motor efficiency and speed; but reduced to actual facts the increasingly hot spark from a magneto, resulting from higher motor speed, is only of advantage in as far as it tends to clear the soot from the plugs. This hotter spark is the rȾsult of higher motor speed, and not the cause. The Gilbert and Ellice Islands, in the Pacillc Ocean, which were proclaimed a British protectorate in 1892, have now been annexed to the British Empire as a crown colony. The population of these islands was reported in 1911 to be 26,417 natives and 446 for eigners. An Aerial Ambulance, it is reported, is being built by a California manufacturer, and army aviators at San Diego, Cal., have been permitted to see it. Under the body of the aeroplane is slung a small cot, which is so fastened and constructed that it will be impossiJle for the occupant to fall out or even be shaken when the aeroplane volplanes to earth. It is understood that while the craft is en route to the hospital a trained at tendant will be enabled to give first aid to the patient. The Ecological SOCiety of America, which was or ganized at the time of the Columbus meeting of the American Association for the Advancement of Science, last December, has already enrolled more than 200 members. Dr. Victor E. Shelford, of the University of Illinois, is president; Prof. W. M. Wheeler, of Harvard University, vice-president; and Dr. Forrest Shreve, of the Carnegie Desert Laboratory, Tucson, Ariz., secre tary-treasurer. The SOCiety will hold an annual meet ing for the reading of papers, and will also organize field meetings from time to time in various parts of the country. During the present year field meetings will be held in the neighborhood of Chicago in June and at San Diego in August. Simplified Lubrication.-In view of comments re cently made in this column in regard to the innumerable little spots around controlling levers, brake rigging, etc. Aeronautics Above this height the cold becomes a great factor in acceleration. Each time a gust strikes the aeroplane the pulse accelerates. During a flight at a particular altitude the pulse re mains constant. When descent begins there is again for a very short period a quickening of the pulse, due, it is thought, to the thrill of excitement experienced when the engine is shut off. After this the frequency falls in a regular manner during a slow descent. Each " event" in the descent causes an acceleration, short, but definite. The pulse at the end of the flight is always more rapid than at the beginning. The Growing Love of Wild Birds among the people or this country is commented upon in the last annual report of the U. S. Biological Survey. Everywhere efforts are being made to increase the number of birds and attract them to the vicinity of homes. This move· ment is said to be partly !esthetic, but also partly due to a growing appreciation of the usefulness of birds as insect destroyers. The report declares that "the increase of interest in wild birds throughout the United States during the past decade has been phe nomenal; and organizations having for their chief ob ject the care and protection of birds are numbered by hundreds, if not thousands." Efforts to attract birds to city parks and suburbs have been made by many civic leagues and women's clubs. In order to aid this interesting movement the Department of Agriculture issued last year two publications, entitled "Bird Houses and How to Build Them " and "How to Attract Birds in the .Northeastern United States." The latter is the first of a series planned to cover all parts pf the country. ,These publications devote speCial attention to the kinds of fruit-bearing shrubs and trees that are important as furnishing food for birds. Ignorant Drivers.-The number of furiously steam ing radiators to be noticed, even on the coldest days, calls attention to the fact that the average driver of a motor vehicle has very vague ideas about operating his engine-and this is only one point in which he is lacking. The prevailing error seems to be in running with full throttle and retarded spark, a sure method of overheating the engine, and one whicli would soon result in disaster if the cooling apparatus was ·not ex tremely efficient. Aeronautics , which ordinarily depend for their lubrication on a varied assortment of oil and grease cups, carefully tlicked away in unsuspected corners where many of them are never found after the machine leaves the shop, H is interesting to note that an oil-less bearing has been d!:.veloped which, it is claimed, takes care of these small, but important points, and relieves the owner of much trouble and worry. A German Anti-Aircraft Gun.-The Germans are using at the present time a 104 mm. anti-aircraft Krupp gun, 45 calibers long, which sends a projectile weighing 15% kilogrammes, with a muzzle velocity of 800 meters, to a height of 4,000 meters. It can be fired at the rate of 15 rounds per minute. The shrapnel shell which it fires is said to burst into 625 fragments. Guns of this type, as well as those of 120 mm.. are the ordnance which defends Ostend. German Aviatik of New Type.-A new type of A viatik biplane was recently brought down behind the French lines. Its wings, which no longer sweep back as in the earlier types, measure 41 feet in span, with a chord of 6.4 feet. The structure is of oval steel tubing throughout. The engine is a 170 horse-poweI' Mercedes driving a Garuda tractor. The weight, empty, works out at 1,600 pounds, and the useful load, includ ing armament, amounts to approximately 1,300 pound3. The machine is exceptionally fast and has a Climbing speed of 4,000 to 4,500 feet in fifteen minutes. A Suggestion in Preparedness.-In the discussions .aL;out preparedness it has been prominently suggested tbat a belt line road be built around the United States, Hear the sea coast. Such a road would be of undoubted value; but enthusiasts in this direction apparently lose sight of the fundamental fact that, either for war or peace, we need good roads everywhere. Another lesson that can be drawn from the experience of Europe of late is to so build the roads that they will stand up in time of need. If our special war roads were built by the average county official they would probably not l;Iurvive actual war conditions over a week. War Changes in Geographic Names.-It is an nounced in La Geographie that the French admiralty has replaced the numerous German geographic names in the French subantarctic island of Kerguelen by names of French origin. Aeronautics 40-Cent Gasoline.- It is freely predicted, and with every possibility of coming true, that the price of gasoline will go to 40 cents within a short time, and this can hardly be regarded as less than a catastrophe, especially at this time when business men are just beginning to appreCiate the value of motor trucks for commercial purposes. This situation raises a point on which makers of motor vehicles have been noticeably silent, and that is the use of heavier fuels, which seel)1S to be vastly more important than some of the improve ments that have been occupying attention of late. An engine operating on distillate, kerosene or some other cheap fuel would be the salvation of the commercial vehicle business, and would appear to be absolutely vital where the much talked of farm tractors are con cerned. Zoological Station in British Guiana.- A new un dertaking of the New York ZoOlogical Society is the establishment of a tropical station in British Guiana, for the study of the evolution and life histories of the local fauna, especially birds. Funds for the first year's work of the station were furnished by Messrs. Cleve land H. Dodge, Mortimer L. Schiff, C. Ledyard Blair, James J. Hill, and George J. Gould; while the govern ment of British Guiana has offered the use of its botanical gardens and wild government land. The officers of the new station, who sailed from New York January 22d, comprise C. William Beebe, curator of birds in the New York ZoOlogical Park, in charge; C. Inness Hartley, P. G. Howes, and Donald Carter. It is proposed to build a bungalow at the edge of the jungle and equip it as a laboratory. One of the first birds to be studied will be the hoazin (Opisthocomus cris tatus), of which there are no specimens in captivity, and concerning which there has been much contro versy. One function of the station will be to collect and forward regular supplies of living animals for the New York" Zoo." The Causes of British Casualties among aviators was a topic of keen discussion in the House of Com mons recently. Aeronautics We join heartily in the wish expressed by our British contemporary Nature that similar practices may not be carried too far. Any change of this sort is a potential cause of confusion, not to mention the expense entailed upon the publishers of geographical and other reference books. We have already noted in these columns several previous changes in names due to the war. In this connection it is in teresting to record that German writers and publishers have almost unanimously ignored the change in the name of the Russian capital to "Petrograd." Fifteen-Ton Flying Boats for British Admiralty. The British Admiralty has ordered 20 triplanes similar to the 15-ton Curtis flying yacht which is now being completed at a plant near Buffalo, according to a recent statement of Henry Woodhouse, governor of the Aero Club of America. The spread of the planes of these machines will be 133 feet, and the propulsion will be supplied by four 12-cylinder, 250 horse-power motors. An auxiliary motor will be provided to drive a screw propeller, so that the craft can be navigated at slow speeds on the surface of the water. Motor Vehicles in War.- It has been quite generally recognized that France was saved, in the early days of the war, by the aid of motor vehicles, which enabled rapid mobilization of troops and supplies to be effected; and since that time, in this mechanical war, the uses, and the necessity for motor vehicles have grown to such an extent that it is fully recognized that the army that is best supplied with transportation facilities, both for men and supplies, easily dominates one of equal strength that is not so equipped. It is humiliating that our government is so oblivious to the world's progress, but some of our motor associat10ns and our National Guards, in some states, are agitating the matter of organizing military motor transportation squadrons for the purpose of study and experiment. The Dangers of Wood Alcohol are just now attract ing much attention, not only on the part of the public but also, fortunately, of legislative bodies. A note in Public Health Reports records the progress of the cam paign against this substance during the year 1915. Universal'.Time H H AVING annihilated space by means of the electric telegraph, man found himself face to face with a daily and hourly paradox. A cablegram leaves England at noon, and in the prov erbial "twinkling of an eye" arrives at its destination in India. The Indian operator looks at the clock. Does it mark the noon hour? Not at all. Notwithstanding practically instantaneous transmission, the deepatch does not reach India until 5 :30 P. M. Even more Two corollaries suggest themselves. First, universal time has no use for," A.M." and " P.M." The hours should be numbered from 1 to 24 (as they are already in many countries). Second, the phraseology employed in naming the time of day should indicate whether universal or local time is meant by some briefer method than is now available. © 1916 SCIENTIFIC AMERICAN, INC April 29, 1916 April 29, 1916 SCIENTIFIC AMERICAN 445 Aeronautics The National Association of Retail Druggists adopted a resolution opposing "the use of wood alcohol in medic inal preparations to be used by human beings," and in favor of such labeling as would protect the public against its harmful use. Two states, New Hampshire and South Dakota, each enacted a law restricting the sale of wood alcohol and prescribing a form of label to be used. The South Dakota law debars its use in any food, drink, medicine or toilet preparation intended for human use, internally or externally. The cities of New York, Chicago and Montclair, N. J., have adopted regulations or ordinances restricting its use. Transatlantic Flight by Aeroplane.- It is reported that Rodman Wanamaker is preparing again for a flight across the Atlantic Ocean in an aeroplane. A ma chine for the purpose is now building; it is said to be a giant triplane of larger proportions than anything hitherto attempted, mounting motors capable of develop ing 1,800 horse-power. The present understanding is that the flight is contemplated for the early part of thQ coming summer, and that the piȿot for the trip has not yet been decided upon. The course, too, is still unde. cided, although it will probably be laid from St. Johns, N. F. The fact that details concerning both the aircraft being built and the contemplated journey are being withheld from the public, renders impossible any com ment on the undertaking, for the present at least. Transatlantic Flight by Aeroplane.- It is reported that Rodman Wanamaker is preparing again for a flight across the Atlantic Ocean in an aeroplane. A ma chine for the purpose is now building; it is said to be a giant triplane of larger proportions than anything hitherto attempted, mounting motors capable of develop ing 1,800 horse-power. The present understanding is that the flight is contemplated for the early part of thQ coming summer, and that the piȿot for the trip has not yet been decided upon. The course, too, is still unde. cided, although it will probably be laid from St. Johns, N. F. The fact that details concerning both the aircraft being built and the contemplated journey are being withheld from the public, renders impossible any com ment on the undertaking, for the present at least. Aeronautics Even as it is such practice does the engine no good. When speed is to be reduced the first operation is to check the throttle until there are signs of knocking, when the spark may be gradually re tarded; and when increasing speed the operation is reversed, the spark being first advanced, followed by opening the throttle. Many a lowly motorcyclist, with his air cooled motor, could give points to the superior automobilist on efficient and economical driving. © 1916 SCIENTIFIC AMERICAN, INC
https://openalex.org/W2750387695	https://jps.library.utoronto.ca/index.php/bsc/article/download/17182/14122	English	null	<i>Index to Douglas Library Notes</i>: Volume 1(1951)-Volume 21 (1971), compiled by A.R. Hazelgrove	Papers of the Bibliographical Society of Canada	1,977	cc-by	760	This massive volume is printed on good paper, and strongly bound in buckram; and for what one gets, it is indeed moderately priced. This massive volume is printed on good paper, and strongly bound in buckram; and for what one gets, it is indeed moderately priced. have provided a more useful solution. It should be noted that the entries are filed letter-by-letter (an indexer's introduction would have been most welcome) and that 'Mc' and Mac' are separately filed. l h h Reviw Edtor p y Although further editing of the DLN index would have been welcome, Mr. Hazel- grove's work will prove useful to researchers, librarians, and the DLN's former readers. WVhile the style and format of the publication is compatible with other works issued in the Douglas Library Occasional Papers series, it is unfortunate that the index could not have been produced to match the size and e cslsofe ta rinl o h rsn odtoso faca stringency'? Index to Douglas Library Notes: Volume 1 (1951) - Vrolume 21 (1971), compiled by A.R. Hazelgrove. Kingston, Ont.: Special Collections, Douglas Library, Queen's Uni- versity, 1976. 26 p. (Douglas Library Occa- sional Papers: no. 4), paper, free. Th emise of the Douglassibry nNoo e not only "one of the first signs of the present régime of financial stringency" for the Douglas Library and many other Canadian libraries, but also a sad occasion for the journal's loyal readers. Throughout its twenty year history DLN was a consistant model, one of the few in Canada then or now, of what a scholarly library house organ should be. DLN's four successive editors provided its readers with reviews and short erudite studies on literary, historical and bibliogra- phical matters connected with the library, its acquisitions, and its collection. Peter E. Greig (Mr. Greig- is Secretary of the National Library Adi'sory Board's Committee on Bibliographical Services for Canada, Indexer to the Bibli'ograh~ical Society of Canada, and Chairman of the Society's Standzing Committee on "Bibli'ography of Canadian Bibli'ograpihies. ") (Mr. Greig- is Secretary of the National Library Adi'sory Board's Committee on Bibliographical Services for Canada, Indexer to the Bibli'ograh~ical Society of Canada, and Chairman of the Society's Standzing Committee on "Bibli'ography of Canadian Bibli'ograpihies. ") q , Mr. A.R. Hazelgrove has published other indexes on Kingston materials through the Kingston Historical Society (Agnes Maule Machar: The Story of Old Kingston: Index, 1971; and Hi'stori'c Kzingston: Vrolumes 1-20, 1952-19 72: Index, 1973, for example). This massive volume is printed on good paper, and strongly bound in buckram; and for what one gets, it is indeed moderately priced. While the DLN index adequately reflects the diversity of subject matter in the parent journal, it does not do full justice to the compiler's experience and skill. The addition of birth and death dates in the personal name entries is occasionally inconsistent and incomplete. The practice of using a surname as the access point for a string of forenames is to be deprecated, and may even serve to confuse future researchers. Cross references for personal names are also a possible source of puzzlement for future users; the preferred entry is given in natural order and not in the inverted order in which it will actually be found. Capitalization is unreliable (MacPhail) and inconsistent (Dalhousie Library vs. Dalhousie library). Ti·tle entries, surprisingly, are the least successful, perhaps because the indexer has attempted to use them in a key-word fashion. The recall value of finding Li~fe in America under 'America' and LzIfe in Reu ters, A under 'Life' is dubious. Proper subject entries in either case would A Lithuanuan Bibliography. Adam and Filomena Kantautas. Edmonton: University of Alberta Press, 1975. xxxix, 725 p., cloth, $10.00. ISBN 0-88864-010-2 The magnitude and scope of this checklist of books and articles held by the major libraries of Canada and the United States is very impressive. If one wants to play the numbers game there are game, there are 10168 entries, arranged in double columns 536 pages of subject entries 149 pages of Author and Title index 38 pages of periodicals consulted 39 pages of an analytic table of contents with notes plus a list of 43 locations in Canada; 457 in the US and 11 libraries in other countries other countries 3587 different authors This work, which was prepared to foster interest in the heritage, history, and tradition of Lithuania, will be of interest and of value to serious researchers as well as to casual brow- sers. A glance at the broad subject headings will quickly reveal the close relationships and interaction with neighbouring countries - 69
https://openalex.org/W2591766135	https://www.frontiersin.org/articles/10.3389/fphys.2017.00134/pdf	English	null	Alteration in Taste Perception in Cancer: Causes and Strategies of Treatment	Frontiers in physiology	2,017	cc-by	9,514	Edited by: Ghanshyam Upadhyay, City College of New York-City University of New York, USA Keywords: taste modalities, cancer, inﬂammation, metabolism, treatment Reviewed by: Giamila Fantuzzi, University of Illinois at Chicago, USA Muheeb Beg, Weill Cornell Medical College, USA Reviewed by: Giamila Fantuzzi, University of Illinois at Chicago, USA Muheeb Beg, Weill Cornell Medical College, USA Correspondence: Naim A. Khan naim.khan@u-bourgogne.fr Alteration in Taste Perception in Cancer: Causes and Strategies of Treatment Babar Murtaza 1, Aziz Hichami 2, Amira S. Khan 3, François Ghiringhelli 2 and Naim A. Khan 1 1 Physiologie de la Nutrition and Toxicologie, UMR U866 Institut National de la Santé et de la Recherche Médicale/Université de Bourgogne-Franche Compté/Agro-Sup, Dijon, France, 2 UMR U866 Institut National de la Santé et de la Recherche Médicale/Université de Bourgogne-Franche Compté, Chimiothérapie et Réponse Anti-tumorale, Dijon, France, 3 Département de Biochimie, Biologie Cellulaire & Moléculaire, Université de Constantine 1, Constantine, Alegria The sense of taste is responsible for the detection and ingestion of food to cover energetic requirements in health and disease. The change in taste perception might lead to malnutrition that is usually one of the frequent causes of morbidity and mortality in patients with cancer. In this review, we summarize the mechanisms of taste perception and how they are altered in cancer. We also address the question of the implication of inﬂammation, responsible for the alterations in taste modalities. We highlight the role of radio- and chemotherapy in the modulation of taste physiology. Other several factors like damage to taste progenitor cells and disruption of gut microbiota are also dealt with relation to taste perception in cancer. We further shed light on how to restore taste acuity, by using different preventive methods, dietary modiﬁcations and pharmacotherapy in subjects with advanced cancer state. REVIEW published: 08 March 2017 doi: 10.3389/fphys.2017.00134 REVIEW published: 08 March 2017 doi: 10.3389/fphys.2017.00134 Correspondence: Naim A. Khan Specialty section: This article was submitted to Gastrointestinal Sciences, a section of the journal Frontiers in Physiology Received: 24 October 2016 Accepted: 22 February 2017 Published: 08 March 2017 Changes in taste perception are especially important in diseases like cancer, which is one of the main causes of morbidity and mortality throughout the world. Altered taste perception in cancer subjects is usually ignored by clinicians as this aspect does not represent the life-threatening events. There are some indications that taste alteration might be an alarming early sign of tumor cell invasion in cancer patients (Sherry, 2001). Indeed, the most distressing symptom in patients with advanced cancer is gastrointestinal abnormalities, whereas the change in taste is the fourth most common symptom after dry mouth, weight loss, and early satiety (Komurcu et al., 2002). Some Keywords: taste modalities, cancer, inﬂammation, metabolism, treatment INTRODUCTION Weill Cornell Medical College, USA The dietary habits are governed, in part, by oro-sensory detection of taste. In fact, the taste of nutrients leads human beings to decide quickly to accept or reject a food. Basically, there are ﬁve taste modalities, i.e., sweet, sour, bitter, salty, umami and perhaps, a sixth fat taste (Heinze et al. (2015). Beside the fact that taste is essential for life because it regulates food intake, taste also provides hedonic pleasure from eating. The taste perception also activates neuronal pathways, leading to the preparation for digestion, absorption, and storage of nutrients (Brondel et al., 2013). A dysfunction of taste perception (dysgeusia) may impair the quality of life by aﬀecting appetite, body weight, and psychological well-being (Deems et al., 1991). There are several factors that may aﬀect taste perception, including medication, nutrition, lesions in the oral mucosa, prolonged exposure to radiations and chemotherapy, smoking, chronic hepatitis, renal dysfunction, aging, and perturbation in hormonal secretions (Maﬀeis and Silva-Netto, 1989). Correspondence: Naim A. Khan naim.khan@u-bourgogne.fr MECHANISM OF TASTE PERCEPTION The TRCs or type II cells are sensitive to sweet, bitter and umami substances via the receptor activation of T1R and T2R family. These cells secrete ATP in the interstitial medium through pannexin channel (Romanov et al., 2008). The ATP binds to the P2Y receptors on the presynaptic or type III cells. This mechanism results in the release of serotonin, thus activating the postsynaptic receptors which are involved in the transmission of the taste information to brain. The type III cells are sensitive to acidic substances (Yang et al., 2000; Brondel et al., 2013; Roland and and Rémi, 2013). The fourth type of taste cells are termed as type IV cells which are basal, non-polarized, presumably undiﬀerentiated cells and serve as progenitor cells for other three types of taste cells (Miura et al., 2006). The comprehensive reviews on diﬀerent taste modalities and the activation of diﬀerent brain areas can be consulted elsewhere (Bermúdez-Rattoni, 2004; Chaudhari and Roper, 2010; Roper, 2013; Besnard et al., 2016). However, in brief, we would like to outline the implication of TRCs in diﬀerent taste perception. Salt activates sodium channels, while the acidic compounds induce the depolarization via by blocking potassium channels (Kinnamon et al., 1988). Sweet, bitter, fat, and umami Citation: studies suggests that 15 to 100 percent of cancer patients may suﬀer from a taste change (Lockhart and Clark, 1989; Ripamonti et al., 1998). Citation: Murtaza B, Hichami A, Khan AS, Ghiringhelli F and Khan NA (2017) Alteration in Taste Perception in Cancer: Causes and Strategies of Treatment. Front. Physiol. 8:134. doi: 10.3389/fphys.2017.00134 March 2017 \| Volume 8 \| Article 134 Frontiers in Physiology \| www.frontiersin.org 1 Taste and Cancer Murtaza et al. taste involve metabotropic receptors (Medler, 2011). CD36 and GPR120 play non-overlapping roles during orosensory detection of dietary fats (Ozdener et al., 2014). Two large families of receptors have been identiﬁed: T1R and T2R. The T2R receptors are involved in the perception of bitter taste (Chandrashekar et al., 2000). The heterodimeric T1R2/T1R3 detects the sweet taste (Montmayeur et al., 2001), whereas the heterodimeric T1R1/T1R3 is sensitive to umami taste (Chaudhari et al., 2000). The binding of sapid molecules to these taste receptors activates a G-protein, called Gustducin (Wong et al., 1996). The alpha- subunit of gustducin activates the PLCβ2and generates inositol- trisphosphate (IP3) and diacylglycerol. All these intracellular mechanisms lead to an increase in intracellular calcium and neurotransmitter release. A diagrammatical representation of various types of taste cells and receptors involved in taste perception are presented in Figure 2. The neurotransmitters released by the TRC activate the aﬀerent nerve ﬁbers that carry taste information to the CNS. These aﬀerent nerves are: (i) the cord of the eardrum (gustatory branch of the facial nerve) which connects the fungiform papillae of the anterior two thirds of the tongue, (ii) the glossopharyngeal nerve (IX) that connects the goblet buds in the posterior third, and (iii) the superior laryngeal vagus nerve (nerve X) that transmits oropharyngeal sensitivity. Other nerves, such as the trigeminal nerve, are also incidentally involved in taste perception. Neurons conduct a ﬁrst relay in the solitary nucleus, located in the dorsolateral part of the bulb, and then make a second relay in the ventromedial nucleus of the thalamus before they project into the cortical or other area, involved in identifying taste (type and intensity). The nucleus accumbens and the ventral tegmental area are also activated following food ingestion. They are involved in hedonic responses (pleasure) and the reward circuit (Kettaneh et al., 2002; Brondel et al., 2013; Roland and and Rémi, 2013). studies suggests that 15 to 100 percent of cancer patients may suﬀer from a taste change (Lockhart and Clark, 1989; Ripamonti et al., 1998). MECHANISM OF TASTE PERCEPTION Before discussing the relationship between cancer and taste changes, we must have an idea of oro-sensory perception of taste. Taste is a complex entity that interacts with other senses: Hearing, touch, smell, and vision. All the information from the sensory organs is ﬁnally analyzed by the central nervous system (CNS). During mastication, foods is mixed with saliva which is secreted by mandibular, sublingual, and parotid salivary glands. Saliva dilutes and disseminates palatable molecules to the taste receptors on the tongue, palate, larynx, pharynx, and the upper third of the esophagus (Matsuo, 2000). Taste receptors or taste receptor cells (TRCs) have been identiﬁed on tongue epithelium and throughout the digestive tract (Rozengurt and Sternini, 2007), but we will not discuss the latter part in this review article. Human beings have around 5,000 taste buds. Of these, 30% are in the fungiform papillae, 30% in foliate papillae and 40% in circumvallate papilla (Suzuki, 2007). The ﬁliform papillae contain no taste buds. Goblet or circumvallate buds are located in the posterior position of the tongue forming an inverted "V" which are nine in humans. The foliate papillae are located in posterior lateral position of the tongue. The fungiform and ﬁliform papillae are present on the apical surface of the tongue. Each taste bud contains 50–100 taste cells, surrounded by supporting cells which are renewed after every 10 days (Wakisaka, 2005). The localization of taste buds and TRCs on human tongue are shown in Figure 1. Three cell types have been identiﬁed, based on morphological criteria (Takeda and Hoshino, 1975). Glial or Type I cells that assure homeostasis in the taste bud, are sensitive to salty substances and possess ATPase activity (Bartel et al., 2006), which is crucial to degrade high concentrations of extracellular ATP. The TRCs or type II cells are sensitive to sweet, bitter and umami substances via the receptor activation of T1R and T2R family. These cells secrete ATP in the interstitial medium through pannexin channel (Romanov et al., 2008). The ATP binds to the P2Y receptors on the presynaptic or type III cells. This mechanism results in the release of serotonin, thus activating the postsynaptic receptors which are involved in the transmission of the taste information to brain. The type III cells are sensitive to acidic substances (Yang et al., 2000; Brondel et al., 2013; Roland and and Rémi, 2013). Frontiers in Physiology \| www.frontiersin.org MECHANISM OF TASTE PERCEPTION The fourth type of taste cells are termed as type IV cells which are basal, non-polarized, presumably undiﬀerentiated cells and serve as progenitor cells for other three types of taste cells (Miura et al., 2006). The comprehensive reviews on diﬀerent taste modalities and the activation of diﬀerent brain areas can be consulted elsewhere (Bermúdez-Rattoni, 2004; Chaudhari and Roper, 2010; Roper, 2013; Besnard et al., 2016). However, in brief, we would like to outline the implication of TRCs in diﬀerent taste perception. Salt activates sodium channels, while the acidic compounds induce the depolarization via by blocking potassium Before discussing the relationship between cancer and taste changes, we must have an idea of oro-sensory perception of taste. Taste is a complex entity that interacts with other senses: Hearing, touch, smell, and vision. All the information from the sensory organs is ﬁnally analyzed by the central nervous system (CNS). During mastication, foods is mixed with saliva which is secreted by mandibular, sublingual, and parotid salivary glands. Saliva dilutes and disseminates palatable molecules to the taste receptors on the tongue, palate, larynx, pharynx, and the upper third of the esophagus (Matsuo, 2000). Taste receptors or taste receptor cells (TRCs) have been identiﬁed on tongue epithelium and throughout the digestive tract (Rozengurt and Sternini, 2007), but we will not discuss the latter part in this review article. Human beings have around 5,000 taste buds. Of these, 30% are in the fungiform papillae, 30% in foliate papillae and 40% in circumvallate papilla (Suzuki, 2007). The ﬁliform papillae contain no taste buds. Goblet or circumvallate buds are located in the posterior position of the tongue forming an inverted "V" which are nine in humans. The foliate papillae are located in posterior lateral position of the tongue. The fungiform and ﬁliform papillae are present on the apical surface of the tongue. Each taste bud contains 50–100 taste cells, surrounded by supporting cells which are renewed after every 10 days (Wakisaka, 2005). The localization of taste buds and TRCs on human tongue are shown in Figure 1. Three cell types have been identiﬁed, based on morphological criteria (Takeda and Hoshino, 1975). Glial or Type I cells that assure homeostasis in the taste bud, are sensitive to salty substances and possess ATPase activity (Bartel et al., 2006), which is crucial to degrade high concentrations of extracellular ATP. CANCER: INFLAMMATION AND TASTE ALTERATIONS As regards the association between inﬂammation and taste bud dysfunction that might result into altered taste perception, we can cite the study of Wang et al. (2009). These investigators have reported the expression of Toll-like receptors (TLRs), type I and II interferon (IFN) receptors, and their downstream signaling components in taste tissue. Some TLRs appear to be selectively or more abundantly expressed in taste buds than in non-gustatory lingual epithelium. Immunohistochemical observations have conﬁrmed the presence of these receptor proteins in taste bud cells, of which TLRs 2, 3, and 4 are expressed in type II cells. Administration of TLR ligands and lipopolysaccharides activated IFN-g signaling pathways, up- regulated the expression of IFN-g-inducible genes, and down- regulated the expression of c-fos in taste buds. Interestingly, systemic administration of IFNs triggered apoptosis of taste bud cells in mice and, consequently, contributed to the development of taste disorders. It has been shown that MRL/lpr mice have a high concentration of IFNγ and TNF-a, and INFγ would There also seems a relationship between inﬂammation and hyperglycemia in cancer since carbohydrate metabolism is altered in cancer patients (Duan et al., 2014), as is the case of type 2 diabetes. The high glucose metabolism in cancer cells might be due to high expression of glucose transporters (Walenta et al., 2000; Hauptmann et al., 2005). Gondivkar et al. (2009) have reported that the subjects with type 2 diabetes may also suﬀer from alterations in taste thresholds for diﬀerent taste modalities. Hence, it is possible that the alteration in carbohydrate metabolism and taste perception may be a common mechanism between type 2 diabetes and cancer. However, this statement requires further studies in future. CANCER: INFLAMMATION AND TASTE ALTERATIONS One of the striking features of advanced cancer is the inﬂammatory state that is generally associated with an infection (Coussens and Werb, 2002). Rapidly proliferating cancer cells release a number of cytokines/chemokines which favor the recruitment of macrophages and neutrophils, which, in turn, produce a series of cytokines and cytotoxic mediators including prostaglandins (Kuper et al., 2000). It is mention worthy that 15% of cancer are associated with an infection that might trigger a sustained inﬂammatory state (Kuper et al., 2000). In neck cancer, the level of pro-inﬂammatory cytokines, i.e., IL-1α, RANTES, MIG, G-CSF, GM-CSF, INF-γ, TNF-a, IL-17, IL-4, IL-6, and IL-10, in the body is increased by several fold (Johnson et al., 2014). The cytokines like IL-12 and IFNγ have an anti-tumor role, while the cytokines like IL-6, IL-17, and IL-23 are pro- tumor (Lin and Karin, 2007). The cytokines IL-6 and IL-10 are associated with poor prognosis in all types of cancer (Lippitz, 2013). Inﬂammation plays a crucial role in cachexia (Epstein and Barasch, 2010). Conversely, cancer cachexia is associated with an increase in blood levels of C-reactive protein, cytokines March 2017 \| Volume 8 \| Article 134 Frontiers in Physiology \| www.frontiersin.org 2 Murtaza et al. Taste and Cancer FIGURE 1 \| Human taste system: (A) Localization of different types of gustatory papillae onto the human tongue. (B) Enlarged section of circumvallate papillae showing taste bud cells. FIGURE 1 \| Human taste system: (A) Localization of different types of gustatory papillae onto the human tongue. (B) Enlarged section of circumvallate papillae showing taste bud cells. (interleukin 1b, interleukin-6, TNF-α, and leukemia inhibitor factor, LIF) and other tumor derived factors like lipid mobilizing factor (LMF) and protein mobilizing factor (PMF). The LMF and PMF can directly mobilize fatty acids and amino acids, respectively, from adipose tissue and skeletal muscle. Ming-Hua et al. (2016) have recently conﬁrmed that high concentrations of IL-1b, IL-6, TNF-α, and LMF are directly involved in cancer cachexia. Indeed, LMF initiates ubiquitin-dependent catabolic pathway and contributes to weight loss during cancer cachexia (Dimitriu et al., 2005) which is, somehow, associated with loss of taste perception as demonstrated by Maschke et al. (2017). The inﬂammatory markers via blood circulation may also exert their action in the brain and modulate the areas involved in the control of feeding behavior including smell and taste perception (Argilés et al., 2014). CANCER: INFLAMMATION AND TASTE ALTERATIONS These observations suggest that alteration in taste in cancer patients might be controlled both at taste bud and brain levels. As regards the association between inﬂammation and taste bud dysfunction that might result into altered taste perception, we can cite the study of Wang et al. (2009). These investigators have reported the expression of Toll-like receptors (TLRs), type I and II interferon (IFN) receptors, and their downstream signaling components in taste tissue. Some TLRs appear to be selectively or more abundantly expressed in taste buds than in non-gustatory lingual epithelium. Immunohistochemical observations have conﬁrmed the presence of these receptor proteins in taste bud cells, of which TLRs 2, 3, and 4 are expressed in type II cells. Administration of TLR ligands and lipopolysaccharides activated IFN-g signaling pathways, up- regulated the expression of IFN-g-inducible genes, and down- regulated the expression of c-fos in taste buds. Interestingly, systemic administration of IFNs triggered apoptosis of taste bud cells in mice and, consequently, contributed to the development of taste disorders. It has been shown that MRL/lpr mice have a high concentration of IFNγ and TNF-a, and INFγ would induce the apoptosis of TRCs. The perception of bitter, sweet and umami taste is also reduced in these mice. This alteration of taste perception is associated with a decrease in the number of gustducin positive cells and renewal of TRCs (Kim et al., 2012). (interleukin 1b, interleukin-6, TNF-α, and leukemia inhibitor factor, LIF) and other tumor derived factors like lipid mobilizing factor (LMF) and protein mobilizing factor (PMF). The LMF and PMF can directly mobilize fatty acids and amino acids, respectively, from adipose tissue and skeletal muscle. Ming-Hua et al. (2016) have recently conﬁrmed that high concentrations of IL-1b, IL-6, TNF-α, and LMF are directly involved in cancer cachexia. Indeed, LMF initiates ubiquitin-dependent catabolic pathway and contributes to weight loss during cancer cachexia (Dimitriu et al., 2005) which is, somehow, associated with loss of taste perception as demonstrated by Maschke et al. (2017). The inﬂammatory markers via blood circulation may also exert their action in the brain and modulate the areas involved in the control of feeding behavior including smell and taste perception (Argilés et al., 2014). These observations suggest that alteration in taste in cancer patients might be controlled both at taste bud and brain levels. Frontiers in Physiology \| www.frontiersin.org MODIFICATION OF MICROBIOTA AND TASTE ALTERATIONS IN CANCER Roughly, there are ∼1014 microbes residing in human intestine with genetic content almost 100 times higher than that of the human genome (Ley et al., 2006). These microbiota are present throughout the gastrointestinal tract, starting from mouth till the terminal part of large intestine (Rozengurt and Sternini, 2007). Schmidt et al. (2014) have shown that the abundance of oral microbiota was varied in individuals during oral cancer. In another study, it was observed that some bacterial species in the buccal cavity of patients with oral squamous cell carcinoma diﬀered from that of normal volunteers (Mager et al., 2005). Interestingly, the chnages in gut microbiota are closely related with alterations in taste preference in mice (Duca et al., 2012; Swartz et al., 2012). March 2017 \| Volume 8 \| Article 134 Frontiers in Physiology \| www.frontiersin.org 3 Murtaza et al. Taste and Cancer FIGURE 2 \| Intercellular communications involved in taste perception. Receptors for sweet, umami, fat, and bitter taste are expressed on type-II cells while type III cells express receptors for sour taste and type-I cells are believed to be involved in salt taste perception. Binding of tastants (1) on type-II and type-III cells ultimately leads to an increase in intracellular calcium levels (2). ATP is excreted by type-II(3) cells which binds with P2Y receptors on type-III cells (4) and causes the release of 5-HT (5). 5-HT causes afferent nerve endings to carry taste message to speciﬁc brain areas on one side (5), and other the other side, causes feedback inhibition of ATP release (6). Excess ATP is also degrades by ATPase present on type-I cells (not shown). Type IV cells are progenitor cells that differentiate into other three types of cells. (ATP, adenosine triphosphate; 5-HT, 5-hydroxytryptamine or serotonin). FIGURE 2 \| Intercellular communications involved in taste perception. Receptors for sweet, umami, fat, and bitter taste are expressed on type-II cells while type III cells express receptors for sour taste and type-I cells are believed to be involved in salt taste perception. Binding of tastants (1) on type-II and type-III cells ultimately leads to an increase in intracellular calcium levels (2). ATP is excreted by type-II(3) cells which binds with P2Y receptors on type-III cells (4) and causes the release of 5-HT (5). MODIFICATION OF MICROBIOTA AND TASTE ALTERATIONS IN CANCER 5-HT causes afferent nerve endings to carry taste message to speciﬁc brain areas on one side (5), and other the other side, causes feedback inhibition of ATP release (6). Excess ATP is also degrades by ATPase present on type-I cells (not shown). Type IV cells are progenitor cells that differentiate into other three types of cells. (ATP, adenosine triphosphate; 5-HT, 5-hydroxytryptamine or serotonin). CANCER THERAPY AND CHANGES IN TASTE One possible mechanism might be that the modiﬁcations of gut microbiota might inﬂuence the expression of gut G-protein coupled receptors (Rousseaux et al., 2007), beside manipulating host feeding behavior through hormonal and neural mechanisms (Alcock et al., 2014). Oral mucositis, which is closely related with alterations in taste, is frequently reported in cancer patients undergoing chemotherapy (Knox et al., 2000). Wang et al. (2015) have proposed that disruption of oral microbiota could result in chemotherapy-induced inﬂammation through toll- like receptors (TLRs) and nucleotide oligomerization domain (NOD)-like receptors (NLRs). Ligands for these receptors like peptidoglycan, lipopolysaccharide, bacterial DNA, and protein ﬂagellin are frequently provided by disrupted microbiota, which results in induction of inﬂammatory process. As cancer therapy is frequently associated with disrupted microbiota, while alterations of microbiota leads to inﬂammation and changes is taste perception, it would be natural to assume that disruption of gut microbiota might result in taste changes in cancer patients. However, this argument needs to be studied in future. Frontiers in Physiology \| www.frontiersin.org Chemotherapy Chemotherapy may aﬀect the taste perception (Berteretche et al., 2004; Rehwaldt et al., 2009). In a study conducted on cancer patients undergoing chemotherapy, the prevalence of taste alterations was reported to be as high as 69.9%, and a signiﬁcant association was found between taste alterations and a change in patient’s quality of life such as appetite and fatigue (Zabernigg et al., 2010). In a study, performed on pediatric patients, undergoing chemotherapy, the altered taste perception caused problems in feeding behavior (Skolin et al., 2006). It was suggested that changes had occurred both in the primary gustatory sense as well as in food perception in these patients. Taste-test showed that these patients had increased thresholds for bitter taste (Skolin et al., 2006). Such changes in taste may be resolved within several months after the completion of chemotherapy (Bernhardson et al., 2007). Zinc is an important March 2017 \| Volume 8 \| Article 134 4 Murtaza et al. Taste and Cancer 1978) by damaging salivary glands. A brief summary of the factors that result in taste change has been shown in Figure 3. micronutrient which also plays a role in the perception of taste. Studies suggest that Zinc depletion is closely related to a change of taste in cancer patients (Heyneman, 1996; Yamagata et al., 2003). A possible mechanism by which the drugs treating cancer could cause Zinc deﬁciency may involve the binding and chelation of Zinc and other heavy metals by sulfhydryl group in their structures, leading to Zinc depletion and loss of taste (Comeau et al., 2001). Plausible Effect of Radio- and Chemotherapy on Taste Progenitor Cells Radiation or chemotherapy might target both taste progenitor as well as already existing taste cells. How these treatments aﬀect diﬀerentiation or reduce the cell number by targeting apoptotic or particular pathways are poorly understood. There are pathways known for diﬀerentiation of taste bud cells such as sonic hedgehog (SHH) and notch pathways (Kapsimali and Barlow, 2013) which are modulated during cancer (Merchant and Matsui, 2010). In particular, SHH is exclusively expressed in type IV taste cells, which are undiﬀerentiated basal cells and the precursors of the other three types of taste cells (Miura et al., 2006). Use of SHH pathway inhibitor, Vismodegib, in cancer patients is often associated with profound alterations in taste sensation (Von Hoﬀet al., 2009; Sekulic et al., 2012; Tang et al., 2012). Yang et al. (2015) have shown that Vismodegib treatment in mice resulted in an altered taste perception. Signiﬁcant reductions in taste bud size, numbers of taste cells per taste bud, and numbers of SHH-expressing cells in taste bud were also observed in these mice. Furthermore, the numbers of phospholipase Cβ2- and α-gustducin- T1R3, glucagon-like peptide-1, and glucagon-expressing cells were also reduced in the taste bud of these mice. Similar ﬁndings were observed by Kumari et al. (2015), who reported that the blockade of hedgehog pathway by cancer drug LDE225 resulted in the disruption of taste cells and taste perception in mice. All these ﬁndings suggest that modulation of SHH by cancer therapy might also be implicated in taste alterations in cancer patients. Radiation Therapy Taste disorders are quite frequent in patients undergoing radiation therapy for head and neck cancers (Zheng et al., 2002). In a study, it was reported that bitter taste was the most aﬀected taste, while sweet taste was aﬀected to a lesser extent (Zheng et al., 2002). Another study showed that bitter and salty taste were the earliest and the most aﬀected taste modalities, while sweet taste was the least aﬀected as a result of radiation therapy (Mossman and Henkin, 1978). Taste impairment may start a few weeks after the beginning of radiation treatment, while it may recover to its previous levels 6 months to 1 year after the treatment has been stopped but some patients may suﬀer from permanent loss of taste (Ruo Redda and Allis, 2006). Impairment of ummami taste as a result of radiation therapy in cancer patients has also been reported (Shi et al., 2004). Changes in taste are mainly due to the damage caused by radiation ﬁeld to taste cells and the pattern of the taste disorder is heavily inﬂuenced by the distribution of the taste buds damaged during the radiation therapy (Yamashita et al., 2006). Also, radiation and chemotherapy induce apoptosis of TRCs and inhibit taste progenitor/stem cell proliferation. Role of dry mouth (xerostomia) has also been implicated as a factor contributing to taste change, as radiation therapy frequently aﬀects saliva quantity and composition (Mossman and Henkin, Miura and Barlow (2010) have proposed that in head and neck cancer patients, taste bud progenitors are more vulnerable to FIGURE 3 \| Factors contributing to alterations in taste perception in cancer. (TRCs, taste receptor cells; TBCs, taste bud cells). GURE 3 \| Factors contributing to alterations in taste perception in cancer. (TRCs, taste receptor cells; TBCs, taste bud cells Frontiers in Physiology \| www.frontiersin.org March 2017 \| Volume 8 \| Article 134 Taste and Cancer Murtaza et al. paclitaxel chemotherapy, included a number of strategies like changing food habits by adding new recipes (Speck et al., 2013). Wickham et al. (1999) proposed to increase food palatability by adding artiﬁcial falvours. Rehwaldt et al. (2009) have reported that each patient should have its own particular strategy to adapt taste alterations. PREVENTION AND TREATMENT OF TASTE ALTERATIONS Patient counseling can be started in advance to prepare patient mentally before time. Rhodes et al. (1994) have demonstarted if the patients are prepared psychologically for taste alterations, they can tolerate taste changes easily. Ravasco et al. (2003) have stressed on counseling the cancer patients to face the altered changes in taste perception. Before chemotherapy, the patients can be encouraged to try new food products or supplements (Capra et al., 2001). Lemon juice and chewing gum could be used prior to meals to make the meals more pleasant. Small but frequent meals should be encouraged as they are better tolerated by the patients (Ravasco, 2005). Patients can also be asked to maintain good oral hygiene as it may also contribute to changes in taste. To assess whether changes in taste perception in cancer patients are causing taste aversion and, consequently, to malnutrition, the clinicians may use diﬀerent approaches, like Malnutrition Screening Tool (Ferguson et al., 1999), Interdisciplinary Nutrition Care Plan (Capra et al., 2001), or the Patient-Generated Subjective Global Assessment (Ferguson, 2003). A close contact and relationship between health care professionals and patients is highly desirable in order to assess the nutritional status and improve the quality of life of the patients. Zinc supplementation can be valuable for patients undergoing cancer chemotherapy. The results of a pilot study suggested that after 2 weeks of chemotherapy, the intake of Zinc protected the cancer patients against taste disorders (Yamagata et al., 2003). In another study, a Zinc containing formulation known as Polaprezinc was able to improve the taste alterations in 70 percent of the patients, though an early administration has been suggested (Mizukami et al., 2016). Furthermore, the results of a randomized and controlled clinical trial on the eﬀects of Zinc sulfate have clearly demonstrated that exogenous Zinc improved taste acuity (Ripamonti et al., 1998). Similar ﬁndings were observed in a randomized placebo-controlled trial where Zinc sulfate prevented the radiation-induced taste changes in head/neck cancer patients (Najaﬁzade et al., 2013). Halyard et al. (2007) conducted a clinical study on Zinc sulfate vs. placebo and concluded that intake of Zinc oﬀered protection against Radiation Therapy Hence, more than 50% of the patients reported that they tried selected one of the following strategies: More fats and sauces, eating smaller and more frequent meals, using more condiments, eating blander foods, adding something sweet to meats, sucking on hard candy, eating more boiled foods, and avoiding beef. These strategies were helpful for the majority (74–87%) of patients who tried them. Nonetheless, self- care strategies are diﬃcult to be practized due to psychological constraints. the eﬀect of fractionated radiotherapy as compared to terminally diﬀerentiated taste cells. They have compared this model with irradiated skin epithelium, where proliferating skin cells are more prone to radiation induced DNA damage than the post-mitotic cells. Following irradiation, the progenitor cells try to repair damaged DNA, and if they fail, they start apoptosis. Repeated irradiation causes increased loss of progenitor cells (Dörr et al., 2000; Potten et al., 2002). As chemotherapy also targets rapidly dividing cells, consequently, taste alterations are observed in patients undergoing chemotherapy for non-head/neck cancers as well. MEASUREMENT OF TASTE IMPAIRMENT AND DETECTION THRESHOLD IN CANCER Diﬀerent methods have been used for the measurement of taste impairment and detection thresholds in cancer patients. A detailed account of the methods will be out of scope of this article; hence, only a brief summary has been given. An electro- gustometer can be used for the detection of taste thresholds (Williams and Cohen, 1978). One electrode is the tongue electrode, while the other reference electrode is placed on dorsal side or the wrist. Electrical current is applied in diﬀerent steps and the lowest current intensity, perceived by the subject, is taken as the detection threshold (Berteretche et al., 2004). Chemical detection involves using tastant solution with suprathreshold concentrations for each of the basic taste modalities (Trant et al., 1982; Bossola et al., 2007). The subject’s mouth is rinsed with a sip of distilled water prior to testing each sample (Sánchez- Lara et al., 2010). The lowest concentration of solute at which the individual consistently recognizes the taste is considered as taste threshold (Gallagher and Tweedle, 1983; Steinbach et al., 2009). A 3-armed forced choice (3-AFC) method can be applied to monitor the subject’s response for detection and reorganization of taste (Mossman and Henkin, 1978). Subjects can be asked to place a cross-hatch on a 10-cm line labeled at each end (0 = dislike extremely, no sweetness, sourness, saltiness, or bitterness, and 10 = like extremely, extremely sweet, sour, salty, or bitter) to indicate degree of liking and intensity of each sample (Trant et al., 1982). Alternatively, a scale ranging from 0 (total taste loss) to 3 (no taste loss) can be employed to classify the subjects, depending upon if they recognize no or all concentrations of a particular taste (Maes et al., 2002). Questionnaires have also been used by diﬀerent researchers to determine the patient’s self- reported taste changes and their eﬀects on their quality of life (Chencharick and Mossman, 1983; Huldij et al., 1986; Bjordal et al., 1994; Bernhardson et al., 2008). The choice of a particular test depends on the practitioner. REFERENCES Besnard, P., Passilly-Degrace, P., and Khan, N. A. (2016). Taste of fat: a sixth taste modality? Physiol. Rev. 96, 151–176. doi: 10.1152/physrev.00002.2015 Alcock, J., Maley, C. C., and Aktipis, C. (2014). Is eating behavior manipulated by the gastrointestinal microbiota? Evolutionary pressures and potential mechanisms. Bioessays 36, 940–949. doi: 10.1002/bies.201400071 Bjordal, K., Ahlner-Elmqvist, M., Tollesson, E., Jensen, A. B., Razavi, D., Maher, E. J., et al. (1994). Development of a European Organization for Research and Treatment of Cancer (EORTC) questionnaire module to be used in quality of life assessments in head and neck cancer patients. Acta Oncol. 33, 879–885. doi: 10.3109/02841869409098450 Argilés, J. M., Busquets, S., Stemmler, B., and López-Soriano, F. J. (2014). Cancer cachexia: understanding the molecular basis. Nat. Rev. Cancer 14, 754–762. doi: 10.1038/nrc3829 Bohuslavizki, K. H., Klutmann, S., Brenner, W., Mester, J., Henze, E., and Clausen, M. (1998). Salivary gland protection by amifostine in high-dose radioiodine treatment: results of a double-blind placebo-controlled study. J. Clin. Oncol. 16, 3542–3549. doi: 10.1200/JCO.1998.16.11.3542 Bartel, D. L., Sullivan, S. L., Lavoie, É. G., Sévigny, J., and Finger, T. E. (2006). Nucleoside triphosphate diphosphohydrolase-2 is the ecto-ATPase of type I cells in taste buds. J. Comp. Neurol. 497, 1–12. doi: 10.1002/cne. 20954 Bossola, M., Cadoni, G., Bellantone, R., Carriero, C., Carriero, E., Ottaviani, F., et al. (2007). Taste intensity and hedonic responses to simple beverages in gastrointestinal cancer patients. J. Pain Symptom Manage. 34, 505–512. doi: 10.1016/j.jpainsymman.2006.12.013 Bermúdez-Rattoni, F. (2004). Molecular mechanisms of taste-recognition memory. Nat. Rev. Neurosci. 5, 209–217. doi: 10.1038/nrn1344 Bernhardson, B.-M., Tishelman, C., and Rutqvist, L. E. (2007). Chemosensory changes experienced by patients undergoing cancer chemotherapy: a qualitative interview study. J. Pain Symptom Manage. 34, 403–412. doi: 10.1016/j.jpainsymman.2006.12.010 Brondel, L., Jacquin, A., Meillon, S., and Pénicaud, L. (2013). Le goût: physiologie, rôles et dysfonctionnements. Nutr. Clin. Métabol. 27, 123–133. doi: 10.1016/j.nupar.2013.06.002 Bernhardson, B.-M., Tishelman, C., and Rutqvist, L. E. (2008). Self-reported taste and smell changes during cancer chemotherapy. Support. Care Cancer 16, 275–283. doi: 10.1007/s00520-007-0319-7 Capra, S., Ferguson, M., and Ried, K. (2001). Cancer: impact of nutrition intervention outcome—nutrition issues for patients. Nutrition 17, 769–772. doi: 10.1016/S0899-9007(01)00632-3 Chandrashekar, J., Mueller, K. L., Hoon, M. A., Adler, E., Feng, L., Guo, W., et al. (2000). T2Rs function as bitter taste receptors. Cell 100, 703–711. doi: 10.1016/S0092-8674(00)80706-0 Berteretche, M., Dalix, A., d’ornano, A. C., Bellisle, F., Khayat, D., and Faurion, A. (2004). Decreased taste sensitivity in cancer patients under chemotherapy. Support. AUTHOR CONTRIBUTIONS NK and FG proposed the topic, supervised and ﬁnally reviewed. BM, AH, and AK participated equally in searching the data and writing of the review. FUTURE PROSPECTS The pathophysiology of taste alterations diﬀers with relation to the phase of the disease and speciﬁc treatment. Unfortunately, this aspect has been ignored in most of the publications. Researchers should take into account the therapy and stages of disease while collecting the data so that more speciﬁc patient care protocols can be devised in order to minimize the eﬀect of a change in taste on patient’s quality of life. Manufacturers must take into account the taste alterations that frequently accompany cancer and should design and provide food supplements accordingly to make them more palatable. An interesting idea can be to develop “taste enhancers” which, once added to food, could enhance its palatability by acting as agonist at speciﬁc taste receptor. Such agonists can be developed for each taste modality. After identiﬁcation of particular taste impairment, the enhancers for that taste can be added to make food more pleasurable for patients. The eﬃcacy of two drugs, i.e., pilocarpine and bethanechol, that increase production of saliva was tested upon saliva secretion in cancer patients with hypo-salivation following radiation therapy (Gorsky et al., 2004). Though the patients reported improvement in saliva secretion by both the agents, only bethanechol improved taste perception. A pilot investigation was conducted to assess the role of serotonergic blockade by using ondansetron, an antagonist of serotonin type-3 receptor (Edelman et al., 1999). This study included metastatic cancer patients who were not undergoing chemotherapy or radiotherapy. Utilizing an “extensive battery of taste tests,” it was concluded that ondansetron brought about signiﬁcant improvements in patients to enjoy food. A major drawback in this study was that it was not a blinded trial, and, hence, placebo eﬀects could not be fully excluded (Edelman et al., 1999). Moreover, it was not clear whether the enhanced enjoyment from eating was due to improvements in taste. In rodent model, it seems that 5-HT acts as a paracrine inhibitory feedback signal manifested by the inhibition of ATP secretion from TRCs. Taste buds express 5-HT1A receptor and the use of its speciﬁc agonist, 8-OH-DPAT, inhibited taste- evoked Ca2+ release in TRCs and also curtailed ATP release. Servier medical art was used as a part of some of the ﬁgures. Servier medical art was used as a part of some of the ﬁgures. Servier medical art was used as a part of some of the ﬁgures. SELF-CARE STRATEGIES BY PATIENTS TO MINIMIZE ALTERATIONS IN TASTE The patients themselves can assess the changes in thresholds for diﬀerent taste modalities. Consequently, they may change either their feeding habits or adopt appropriate palatable strategies. For example, breast cancer patients, undergoing docetaxel or March 2017 \| Volume 8 \| Article 134 Frontiers in Physiology \| www.frontiersin.org 6 Taste and Cancer Murtaza et al. alterations in taste modalities. Hence, the routine use of Zinc sulfate may be suggested for patients undergoing cancer therapy. Conversely, blocking the action of endogenous 5-HT in taste bud cells by using WAY100635, a selective 5-HT1A receptor antagonist, increased taste-evoked ATP release (Huang et al., 2009). However, in-depth studies aiming to reproduce the afore- mentioned observations in humans are awaited. Amifostine, an organic thiophosphate, has been shown to protect normal tissues from damage caused by radiation and chemotherapy (Kouvaris et al., 2007). Amifostine may also play a role in the protection of salivary glands and, thus leading to improvement of xerostomia. In a phase II randomized trial, amifostine has been used to assess protection against the toxic eﬀects of carboplatin (Bohuslavizki et al., 1998). Münter et al. (2007) also came to the similar conclusion that amifostine may prevent reduced salivary gland function in patients, subjected to radiotherapy. REFERENCES Clin. Invest. 117, 1175–1183. doi: 10.1172/JCI31537 Gallagher, P., and Tweedle, D. E. (1983). Taste threshold and acceptability of commercial diets in cancer patients. J. Parenter. Enteral Nutr. 7, 361–363. doi: 10.1177/0148607183007004361 Lippitz, B. E. (2013). Cytokine patterns in patients with cancer: a systematic review. Lancet Oncol. 14, e218–e228. doi: 10.1016/S1470-2045(12)70582-X Lockhart, P. B., and Clark, J. R. (1989). Oral complications following neoadjuvant chemotherapy in patients with head and neck cancer. NCI monogr. 9, 99–101. Gondivkar, S. M., Indurkar, A., Degwekar, S., and Bhowate, R. (2009). Evaluation of gustatory function in patients with diabetes mellitus type 2. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endodontol. 108, 876–880. doi: 10.1016/j.tripleo.2009.08.015 Maes, A., Huygh, I., Weltens, C., Vandevelde, G., Delaere, P., Evers, G., et al. (2002). De Gustibus: time scale of loss and recovery of tastes caused by radiotherapy. Radiother. Oncol. 63, 195–201. doi: 10.1016/S0167-8140(02)00025-7 Gorsky, M., Epstein, J. B., Parry, J., Epstein, M. S., Le, N. D., and Silverman, S. (2004). The eﬃcacy of pilocarpine and bethanechol upon saliva production in cancer patients with hyposalivation following radiation therapy. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endodontol. 97, 190–195. doi: 10.1016/j.tripleo.2003.08.031 Maﬀeis, E. R., and Silva-Netto, C. R. (1989). Factors that alter taste perception. Rev. Faculdade Odontol. Lins 3, 28–32. Mager, D. L., Haﬀajee, A. D., Devlin, P. M., Norris, C. M., Posner, M. R., and Goodson, J. M. (2005). The salivary microbiota as a diagnostic indicator of oral cancer: a descriptive, non-randomized study of cancer-free and oral squamous cell carcinoma subjects. J. Transl. Med. 3:27. doi: 10.1186/1479-5876-3-27 Halyard, M. Y., Jatoi, A., Sloan, J. A., Bearden, J. D. III, Vora, S. A., Atherton, P. J., et al. (2007). Does zinc sulfate prevent therapy-induced taste alterations in head and neck cancer patients? Results of phase III double-blind, placebo-controlled trial from the North Central Cancer Treatment Group (N01C4). Int. J. Radiat. Oncol 67, 1318–1322. doi: 10.1016/j.ijrobp.2006.10.046 Maschke, J., Kruk, U., Kastrati, K., Kleeberg, J., Buchholz, D., Erickson, N., et al. (2017). Nutritional care of cancer patients: a survey on patients’ needs and medical care in reality. Int. J. Clin. Oncol. 22, 200–206. doi: 10.1007/s10147-016-1025-6 Matsuo, R. (2000). Role of saliva in the maintenance of taste sensitivity. Crit. Rev. Oral Biol. Med. 11, 216–229. doi: 10.1177/10454411000110020501 Hauptmann, S., Grünewald, V., Molls, D., Schmitt, W. D., Köbel, M., Kriese, K., et al. (2005). REFERENCES Care ancer 12, 571–576. doi: 10.1007/s00520-004-0589-2 March 2017 \| Volume 8 \| Article 134 Frontiers in Physiology \| www.frontiersin.org 7 Murtaza et al. Taste and Cancer Chaudhari, N., and Roper, S. D. (2010). The cell biology of taste. J. Cell Biol. 190, 285–296. doi: 10.1083/jcb.201003144 Huang, Y. A., Dando, R., and Roper, S. D. (2009). Autocrine and paracrine roles for ATP and Serotonin in mouse taste buds. J. Neurosci. 29:13909. doi: 10.1523/JNEUROSCI.2351-09.2009 Chaudhari, N., Landin, A. M., and Roper, S. D. (2000). A metabotropic glutamate receptor variant functions as a taste receptor. Nat. Neurosci. 3, 113–119. doi: 10.1038/72053 Huldij, A., Giesbers, A., Poelhuis, E. H., Hart, A. A., Hulshof, K. F., and Bruning, P. F. (1986). Alterations in taste appreciation in cancer patients during treatment. Cancer Nurs. 9, 38–42. doi: 10.1097/00002820-198602000-00006 Chencharick, J. D., and Mossman, K. L. (1983). Nutritional consequences of the radiotherapy of head and neck cancer. Cancer 51, 811–815. doi: 10.1002/1097- 0142(19830301)51:5<811::AID-CNCR2820510511>3.0.CO;2-M Johnson, S. D., De Costa, A.-M., and Young, M. R. (2014). Eﬀect of the premalignant and tumor microenvironment on immune cell cytokine production in head and neck cancer. Cancers 6, 756–770. doi: 10.3390/cancers6020756 Comeau, T. B., Epstein, J. B., and Migas, C. (2001). Taste and smell dysfunction in patients receiving chemotherapy: a review of current knowledge. Support. Care Cancer 9, 575–580. doi: 10.1007/s005200100279 Kapsimali, M., and Barlow, L. A. (2013). Developing a sense of taste. Semin. Cell Dev. Biol. 24, 200–209. doi: 10.1016/j.semcdb.2012.11.002 Coussens, L. M., and Werb, Z. (2002). Inﬂammation and cancer. Nature 420, 860–867. doi: 10.1038/nature01322 Kettaneh, A., Fain, O., Stirnemann, J., and Thomas, M. (2002). Les troubles du goût. La Revue Méd. Interne 23, 622–631. doi: 10.1016/S0248-8663(02) 00623-9 Deems, D. A., Doty, R. L., Settle, R. G., Moore-Gillon, V., Shaman, P., Mester, A. F., et al. (1991). Smell and taste disorders, a study of 750 patients from the University of Pennsylvania Smell and Taste Center. Arch. Otol. Head Neck Surg. 117, 519–528. doi: 10.1001/archotol.1991.01870170065015 Kim, A., Feng, P., Ohkuri, T., Sauers, D., Cohn, Z. J., Chai, J., et al. (2012). Defects in the peripheral taste structure and function in the MRL/lpr mouse model of autoimmune disease. PLoS ONE 7:e35588. doi: 10.1371/journal.pone.0035588 117, 519–528. doi: 10.1001/archotol.1991.01870170065015 Dimitriu, C., Martignoni, M. E., Bachmann, J., Fröhlich, B., Tintarescu, G., Buliga, T., et al. (2005). Clinical impact of cachexia on survival and outcome of cancer patients. Rom. J. Int. Med. 43, 173–185. Kinnamon, S. REFERENCES C., Dionne, V. E., and Beam, K. G. (1988). Apical localization of K+ channels in taste cells provides the basis for sour taste transduction. Proc. Nat. Acad. Sci.U.S.A. 85, 7023–7027. doi: 10.1073/pnas.85.18.7023 Dörr, K. B., Brankovic, K., and Hartmann, W (2000). Repopulation in mouse oral mucosa: changes in the eﬀect of dose fractionation. Int. J. Radiat. Biol. 76, 383–390. doi: 10.1080/095530000138727 Knox, J. J., Puodziunas, A. L., and Feld, R. (2000). Chemotherapy-induced oral mucositis. Drugs Aging 17, 257–267. doi: 10.2165/00002512-200017040-00002 Duan, W., Shen, X., Lei, J., Xu, Q., Yu, Y., Li, R., et al. (2014). Hyperglycemia, a neglected factor during cancer progression. Biomed Res. Int. 2014:461917. doi: 10.1155/2014/461917 Komurcu, S., Nelson, K. A., Walsh, D., Ford, R. B., and Rybicki, L. A. (2002). Gastrointestinal symptoms among inpatients with advanced cancer. Am. J. Hospice Palliat. Med. 19, 351–355. doi: 10.1177/104990910201900513 Duca, F. A., Swartz, T. D., Sakar, Y., and Covasa, M. (2012). Increased oral detection, but decreased intestinal signaling for fats in mice lacking gut microbiota. PLoS ONE 7:e39748. doi: 10.1371/journal.pone.00 39748 Kouvaris, J. R., Kouloulias, V. E., and Vlahos, L. J. (2007). Amifostine: the ﬁrst selective-target and broad-spectrum radioprotector. Oncologist 12, 738–747. doi: 10.1634/theoncologist.12-6-738 Kumari, A., Ermilov, A. N., Allen, B. L., Bradley, R. M., Dlugosz, A. A., and Mistretta, C. M. (2015). Hedgehog pathway blockade with the cancer drug LDE225 disrupts taste organs and taste sensation. J. Neurophysiol. 113, 1034–1040. doi: 10.1152/jn.00822.2014 Edelman, M. J., Gandara, D. R., Meyers, F. J., Ishii, R., O’mahony, M., Uhrich, M., et al. (1999). Serotonergic blockade in the treatment of the cancer anorexia-cachexia syndrome. Cancer 86, 684–688.doi: 10.1002/(SICI)1097- 0142(19990815)86:4<684::AID-CNCR18>3.0.CO;2-6 Kuper, H., Adami, H. O., and Trichopoulos, D. (2000). Infections as a major preventable cause of human cancer. J. Intern. Med. 248, 171–183. doi: 10.1046/j.1365-2796.2000.00742.x Epstein, J. B., and Barasch, A. (2010). Taste disorders in cancer patients: pathogenesis, and approach to assessment and management. Oral Oncol. 46, 77–81. doi: 10.1016/j.oraloncology.2009.11.008 Ley, R. E., Peterson, D. A., and Gordon, J. I. (2006). Ecological and evolutionary forces shaping microbial diversity in the human intestine. Cell 124, 837–848. doi: 10.1016/j.cell.2006.02.017 Ferguson, M. (2003). Patient-generated subjective global assessment. Oncology 17, 13–14; discussion 14–16. Ferguson, M., Capra, S., Bauer, J., and Banks, M. (1999). Development of a valid and reliable malnutrition screening tool for adult acute hospital patients. Nutrition 15, 458–464. doi: 10.1016/S0899-9007(99)00084-2 Lin, W.-W., and Karin, M. (2007). A cytokine-mediated link between innate immunity, inﬂammation, and cancer. J. REFERENCES 05.002 Mossman, K. L., and Henkin, R. I. (1978). Radiation-induced changes in taste acuity in cancer patients. Int. J. Radiat. Oncol. 4, 663–670. doi: 10.1016/0360-3016(78)90190-6 Skolin, I., Wahlin, Y. B., Broman, D. A., Koivisto Hursti, U.-K., Larsson, M. V., and Hernell, O. (2006). Altered food intake and taste perception in children with cancer after start of chemotherapy: perspectives of children, parents and nurses. Support. Care Cancer 14, 369–378. doi: 10.1007/s00520-005-0904-6 Münter, M. W., Hoﬀner, S., Hof, H., Herfarth, K. K., Haberkorn, U., Rudat, V., et al. (2007). Changes in salivary gland function after radiotherapy of head and neck tumors measured by quantitative pertechnetate scintigraphy: comparison of intensity-modulated radiotherapy and conventional radiation therapy with and without Amifostine. Int. J. Radiat. Oncol. 67, 651–659. doi: 10.1016/j.ijrobp.2006.09.035 Speck, R. M., Demichele, A., Farrar, J. T., Hennessy, S., Mao, J. J., Stineman, M. G., et al. (2013). Taste alteration in breast cancer patients treated with taxane chemotherapy: experience, eﬀect, and coping strategies. Support. Care Cancer 21, 549–555. doi: 10.1007/s00520-012-1551-3 Najaﬁzade, N., Hemati, S., Gookizade, A., Berjis, N., Hashemi, M., Vejdani, S., et al. (2013). Preventive eﬀects of zinc sulfate on taste alterations in patients under irradiation for head and neck cancers: a randomized placebo-controlled trial. J. Res. Med. Sci. 18, 123–126. Steinbach, S., Hummel, T., Böhner, C., Berktold, S., Hundt, W., Kriner, M., et al. (2009). Qualitative and quantitative assessment of taste and smell changes in patients undergoing chemotherapy for breast cancer or gynecologic malignancies. J. Clin. Oncol. 27, 1899–1905. doi: 10.1200/JCO.2008.19.2690 Ozdener, M. H., Subramaniam, S., Sundaresan, S., Sery, O., Hashimoto, T., Asakawa, Y., et al. (2014). CD36- and GPR120-mediated Ca(2)(+) signaling in human taste bud cells mediates diﬀerential responses to fatty acids and is altered in obese mice. Gastroenterology 146, 995–1005. doi: 10.1053/j.gastro.2014.01.006 Suzuki, T. (2007). Cellular mechanisms in taste buds. Bull. Tokyo Dent. Coll. 48, 151–161. doi: 10.2209/tdcpublication.48.151 Swartz, T. D., Duca, F. A., de Wouters, T., Sakar, Y., and Covasa, M. (2012). Up- regulation of intestinal type 1 taste receptor 3 and sodium glucose luminal transporter-1 expression and increased sucrose intake in mice lacking gut microbiota. Br. J. Nutr. 107, 621–630. doi: 10.1017/S0007114511003412 Potten, C. S., Booth, D., Cragg, N. J., Tudor, G. L., O’shea, J. A., Booth, C., et al. (2002). Cell kinetic studies in the murine ventral tongue epithelium: mucositis induced by radiation and its protection by pretreatment with keratinocyte growth factor (KGF). Cell Prolif. 35, 32–47. REFERENCES Glucose transporter GLUT1 in colorectal adenocarcinoma cell lines is inversely correlated with tumour cell proliferation. Anticancer Res. 25, 3431–3436. Medler, K. F. (2011). Multiple roles for TRPs in the taste system: not your typical TRPs. Adv. Exp. Med. Biol. 704, 831–846. doi: 10.1007/978-94-007-0265-3_43 Heinze, J. M., Preissl, H., Fritsche, A., and Frank, S. (2015). Controversies in fat perception. Physiol. Behav. 152, 479–493. doi: 10.1016/j.physbeh.2015. 08.033 Merchant, A. A., and Matsui, W. (2010). Targeting Hedgehog— a cancer stem cell pathway. Clin. Cancer Res. 16, 3130–3140. doi: 10.1158/1078-0432.CCR-09-2846 Ming-Hua, C., Bao-Hua, Z., and Lei, Y. (2016). Mechanisms of anorexia cancer cachexia syndrome and potential beneﬁts of traditional Heyneman, C. A. (1996). Zinc deﬁciency and taste disorders. Ann. Pharmacother. 30, 186–187. doi: 10.1177/106002809603000215 March 2017 \| Volume 8 \| Article 134 Frontiers in Physiology \| www.frontiersin.org 8 Murtaza et al. Taste and Cancer dietary behaviors in cancer patients under chemotherapy. Nutr. J. 9:15. doi: 10.1186/1475-2891-9-15 medicine and natural herbs. Curr. Pharm. Biotechnol. 17, 1147–1152. doi: 10.2174/1389201017666161018123311 medicine and natural herbs. Curr. Pharm. Biotechnol. 17, 1147–1152. doi: 10.2174/1389201017666161018123311 Schmidt, B. L., Kuczynski, J., Bhattacharya, A., Huey, B., Corby, P. M., Queiroz, E. L., et al. (2014). Changes in abundance of oral microbiota associated with oral cancer. PLoS ONE 9:e98741. doi: 10.1371/journal.pone.0098741 Miura, H., and Barlow, L. A. (2010). Taste bud regeneration and the search for taste progenitor cells. Arch. Ital. Biol. 148, 107. Miura, H., Kusakabe, Y., and Harada, S. (2006). Cell lineage and diﬀerentiation in taste buds. Arch. Histol. Cytol. 69, 209–225. doi: 10.1679/aohc.69.209 Sekulic, A., Migden, M. R., Oro, A. E., Dirix, L., Lewis, K. D., Hainsworth, J. D., et al. (2012). Eﬃcacy and safety of vismodegib in advanced basal- cell carcinoma. N. Engl. J. Med. 366, 2171–2179. doi: 10.1056/NEJMoa11 13713 Mizukami, Y., Sato, J., Nihei, S., Kashiwaba, M., Kudo, K., Okuyama, H., et al. (2016). The eﬀectiveness of polaprezinc preparation for taste alteration in cancer chemotherapy. Gan To Kagaku Ryoho 43, 979–983. Sherry, V. (2001). Taste alterations among patients with cancer. Clin. J. Oncol. Nurs. 6, 73–77. doi: 10.1188/02.CJON.73-77 Montmayeur, J.-P., Liberles, S. D., Matsunami, H., and Buck, L. B. (2001). A candidate taste receptor gene near a sweet taste locus. Nat. Neurosci. 4, 492–498. doi: 10.1038/87440 Shi, H.-B., Masuda, M., Umezaki, T., Kuratomi, Y., Kumamoto, Y., Yamamoto, T., et al. (2004). Irradiation impairment of umami taste in patients with head and neck cancer. Auris Nasus Larynx 31, 401–406. doi: 10.1016/j.anl.2004. REFERENCES doi: 10.1046/j.1365-2184.35. s1.4.x Takeda, M., and Hoshino, T. (1975). Fine structure of taste buds in the rat. Arch. Histol. Japonicum 37, 395–413. doi: 10.1679/aohc1950.37.395 Ravasco, P. (2005). Aspects of taste and compliance in patients with cancer. Eur. J. Oncol. Nurs. 9, S84–S91. doi: 10.1016/j.ejon.2005.09.003 Tang, J. Y., Mackay-Wiggan, J. M., Aszterbaum, M., Yauch, R. L., Lindgren, J., Chang, K., et al. (2012). Inhibiting the hedgehog pathway in patients with the basal-cell nevus syndrome. N. Engl. J. Med. 366, 2180–2188. doi: 10.1056/NEJMoa1113538 Ravasco, P., Monteiro-Grillo, I., and Camilo, M. E. (2003). Does nutrition inﬂuence quality of life in cancer patients undergoing radiotherapy? Radiother. Oncol. 67, 213–220. doi: 10.1016/S0167-8140(03)00040-9 Trant, A. S., Serin, J., and Douglass, H. O. (1982). Is taste related to anorexia in cancer patients? Am. J. Clin. Nutr. 36, 45–58. Rehwaldt, M., Wickham, R., Purl, S., Tariman, J., Blendowski, C., Shott, S., et al. (2009). Self-care strategies to cope with taste changes after chemotherapy. Oncol. Nurs. Forum 36, E47–E56. doi: 10.1188/09.ONF.E47-E56 Von Hoﬀ, D. D., Lorusso, P. M., Rudin, C. M., Reddy, J. C., Yauch, R. L., Tibes, R., et al. (2009). Inhibition of the hedgehog pathway in advanced basal-cell carcinoma. N. Engl. J. Med. 361, 1164–1172. doi: 10.1056/NEJMoa0905360 Von Hoﬀ, D. D., Lorusso, P. M., Rudin, C. M., Reddy, J. C., Yauch, R. L., Tibes, R., et al. (2009). Inhibition of the hedgehog pathway in advanced basal-cell g g p y carcinoma. N. Engl. J. Med. 361, 1164–1172. doi: 10.1056/NEJMoa0905360 Rhodes, V. A., Mcdaniel, R. W., Hanson, B., Markway, E., and Johnson, M. (1994). Sensory perception of patients on selected antineoplastic chemotherapy protocols. Cancer Nurs. 17, 45–51. Wakisaka, S. (2005). Lectin histochemistry of taste buds in the circumvallate papilla of the rat. Chem. Senses 30, i46–i47. doi: 10.1093/chemse/bjh106 Ripamonti, C., Zecca, E., Brunelli, C., Fulfaro, F., Villa, S., Balzarini, A., et al. (1998). A randomized, controlled clinical trial to evaluate the eﬀects of zinc sulfate on cancer patients with taste alterations caused by head and neck irradiation. Cancer 82, 1938–1945. doi: 10.1002/(SICI)1097- 0142(19980515)82:10<1938::AID-CNCR18>3.0.CO;2-U Walenta, S., Wetterling, M., Lehrke, M., Schwickert, G., Sundfør, K., Rofstad, E. K., et al. (2000). High lactate levels predict likelihood of metastases, tumor recurrence, and restricted patient survival in human cervical cancers. Cancer Res. 60, 916–921. Wang, H., Zhou, M., Brand, J., and Huang, L. (2009). Inﬂammation and taste disorders: mechanisms in taste buds. Ann. N. Y. Acad. Sci. 1170, 596–603. REFERENCES doi: 10.1111/j.1749-6632.2009.04480.x Roland, S., and Rémi, G. (2013). Odorat et Goût: De la Neurobiologie des Sens Chimiques Aux Applications. Versailles: Editions Quae. Wang, Y., Zhou, X., and Xu, X. (2015). Oral microbiota: an overlooked etiology for chemotherapy-induced oral mucositis? J. Formosan Med. Assoc. 114, 297–299. doi: 10.1016/j.jfma.2013.10.014 Romanov, R. A., Rogachevskaja, O. A., Khokhlov, A. A., and Kolesnikov, S. S. (2008). Voltage dependence of ATP secretion in mammalian taste cells. J. Gen. Physiol. 132, 731–744. doi: 10.1085/jgp.200810108 Roper, S. D. (2013). Taste buds as peripheral chemosensory processors. Semin. Cell Dev. Biol. 24, 71–79. doi: 10.1016/j.semcdb.2012.12.002 Wickham, R. S., Rehwaldt, M., Kefer, C., Shott, S., Abbas, K., Glynn-Tucker, E., et al. (1999). Taste changes experienced by patients receiving chemotherapy. Oncol. Nurs. Forum 26, 697–706. Rousseaux, C., Thuru, X., Gelot, A., Barnich, N., Neut, C., Dubuquoy, L., et al. (2007). Lactobacillus acidophilus modulates intestinal pain and induces opioid and cannabinoid receptors. Nat. Med. 13, 35–37. doi: 10.1038/nm1521 Williams, L. R., and Cohen, M. H. (1978). Altered taste thresholds in lung cancer. Am. J. Clin. Nutr. 31, 122–125. Rozengurt, E., and Sternini, C. (2007). Taste receptor signaling in the mammalian gut. Curr. Opin. Pharmacol. 7, 557–562. doi: 10.1016/j.coph.2007. 10.002 Wong, G. T., Gannon, K. S., and Margolskee, R. F. (1996). Transduction of bitter and sweet taste by gustducin. Nature 381, 796–800. doi: 10.1038/381796a0 Yamagata, T., Nakamura, Y., Yamagata, Y., Nakanishi, M., Matsunaga, K., Nakanishi, H., et al. (2003). The pilot trial of the prevention of the increase in electrical taste thresholds by zinc containing ﬂuid infusion during chemotherapy to treat primary lung cancer. J. Exp. Clin. Cancer Res. 22, 557–563. Ruo Redda, M. G., and Allis, S. (2006). Radiotherapy-induced taste impairment. Cancer Treat. Rev. 32, 541–547. doi: 10.1016/j.ctrv.2006.06.003 Sánchez-Lara, K., Sosa-Sánchez, R., Green-Renner, D., Rodríguez, C., Laviano, Sánchez-Lara, K., Sosa-Sánchez, R., Green-Renner, D., Rodríguez, C., Laviano, A., Motola-Kuba, D., et al. (2010). Inﬂuence of taste disorders on Sánchez Lara, K., Sosa Sánchez, R., Green Renner, D., Rodríguez, C., Laviano, A., Motola-Kuba, D., et al. (2010). Inﬂuence of taste disorders on March 2017 \| Volume 8 \| Article 134 Frontiers in Physiology \| www.frontiersin.org 9 Murtaza et al. Taste and Cancer Zheng, W. K., Inokuchi, A., Yamamoto, T., and Komiyama, S. (2002). Taste dysfunction in irradiated patients with head and neck cancer. Hukuoka Acta Med. 93, 64–76. Yamashita, H., Nakagawa, K., Tago, M., Nakamura, N., Shiraishi, K., Eda, M., et al. (2006). Frontiers in Physiology \| www.frontiersin.org REFERENCES Taste dysfunction in patients receiving radiotherapy. Head Neck 28, 508–516. doi: 10.1002/hed.20347 Yang, H., Cong, W. N., Yoon, J. S., and Egan, J. M. (2015). Vismodegib, an antagonist of hedgehog signaling, directly alters taste molecular signaling in taste buds. Cancer Med. 4, 245–252. doi: 10.1002/ cam4.350 Conﬂict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Yang, R., Crowley, H. H., Rock, M. E., and Kinnamon, J. C. (2000). Taste cells with synapses in rat circumvallate papillae display SNAP-25-like immunoreactivity. J. Comp. Neurol. 424, 205–215. doi: 10.1002/1096-9861(20000821)424:2<205::AID-CNE2>3.0.CO;2-F Copyright © 2017 Murtaza, Hichami, Khan, Ghiringhelli and Khan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Zabernigg, A., Gamper, E.-M., Giesinger, J. M., Rumpold, G., Kemmler, G., Gattringer, K., et al. (2010). Taste alterations in cancer patients receiving chemotherapy: a neglected side eﬀect? Oncologist 15, 913–920. doi: 10.1634/theoncologist.2009-0333 March 2017 \| Volume 8 \| Article 134 Frontiers in Physiology \| www.frontiersin.org 10
https://openalex.org/W2148520888	https://escholarship.org/content/qt8xb8462c/qt8xb8462c.pdf?t=ppugo0	English	null	Laminar specific attachment and neurite outgrowth of thalamic neurons on cultured slices of developing cerebral neocortex	Development	1,994	cc-by	13,715	UC Irvine UC Irvine Previously Published Works Title Laminar specific attachment and neurite outgrowth of thalamic neurons on cultured slices of developing cerebral neocortex. Permalink Permalink https://escholarship.org/uc/item/8xb8462c Journal Development (Cambridge, England), 120(10) Journal Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed INTRODUCTION cortical layers 2-6, which form in an ‘inside-out’ fashion (reviewed by DeCarlos and O’Leary, 1992; Erzurumlu and Jhaveri, 1992; Kageyama and Robertson, 1993). After entering the cortical plate, thalamic growth cones migrate toward the pial surface, passing through the infragranular laminae (5/6), to the granular layer (4) or the lower portion of layer 3 where most axons terminate and arborize (Miller et al., 1993; Kageyama and Robertson, 1993; Agmon et al., 1993). During brain development, the growth cones of advancing axons often travel relatively long distances to reach their targets. Evidently, this process involves speciﬁc decisions made by growth cones as they navigate (reviewed by Dodd and Jessell, 1988; Hynes and Lander, 1992). The formation of con- nections between the thalamus and the cerebral cortex is an attractive model for studying axonal development within the mammalian brain because the anatomy and timing of the growth of this pathway is relatively well characterized (reviewed by O’Leary and Koester, 1993). g y g There is both in vivo and in vitro evidence suggesting that the pathway decisions made by thalamocortical afferents are controlled by cues residing in speciﬁc cortical layers. For example, Ghosh et al. (1990) have shown that ablating subplate neurons in developing animals prevents the innervation of the cortical plate by thalamic afferents. Studies of long-term, thal- amocortical co-cultures (Yamamoto et al., 1989, 1992; Molnár and Blakemore, 1991; Bolz et al., 1992; Götz et al., 1992) have demonstrated that axons extending from thalamic explants onto postnatal cortical slices generally stop growth in or near layer 4, the major target of these axons in vivo. In rodents, thalamic growth cones leave the thalamus and travel through the ganglionic eminence (anlage of the basal forebrain), to enter the developing cerebral cortex well before most of their target cells are born. The growth cones then travel mostly within the intermediate zone and subplate in a pathway that runs tangential to the cortical surface (the pia) and opposite in direction to the simultaneous growth of cortical efferents. Concurrently, the overlying cortical plate thickens as postmi- totic neurons, including the target cells of thalamic axons, arrive from their birthplace in the ventricular zone. SUMMARY In nervous system development, the growth cones of advancing axons are thought to navigate to their targets by recognizing cell-surface and extracellular matrix molecules that act as speciﬁc guidance cues. To identify and map cues that guide the growth of a particular axonal system, the thalamocortical afferents, an assay was devised to examine short-term interactions of dissociated embryonic thalamic cells with living, ~150 µm slices of developing mouse forebrain. Thalamic cells rapidly (<3 hours) and efficiently attached to and extended neurites on pre- and postnatal slices, but a broad zone throughout the neocortex was generally non-permissive for both thalamic cell attachment and the ingrowth of neurites. This zone coincided with the cortical plate at early stages (embryonic day 15), but later became restricted, in rostral-to-caudal fashion, to cortical laminae 2/3. Thus, at each stage, thalamic cells in vitro avoided just that area that thalamic axons confront, but generally do not enter, in vivo. In addition, neurites that extended on some layers were found to be signiﬁcantly oriented in directions that coincide with the pathways that thalamic axons follow in vivo. These results imply that local adhesive cues and signals that affect process outgrowth are distributed among developing cortical laminae in a manner that could underlie much of the temporal and spatial pat- terning of thalamocortical innervation. Key words: cortical development, thalamic innervation, adhesion, slice culture, neurite outgrowth, axonal development, axon guidance Laminar speciﬁc attachment and neurite outgrowth of thalamic neurons on cultured slices of developing cerebral neocortex Daniel E. Emerling1 and Arthur D. Lander1,2,* 1Department of Brain and Cognitive Sciences and 2Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA Daniel E. Emerling1 and Arthur D. Lander1,2,* 1Department of Brain and Cognitive Sciences and 2Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA Author for correspondence at address1 Author for correspondence at address1 *Author for correspondence at address1 Powered by the California Digital Library University of California eScholarship.org eScholarship.org 2811 Development 120, 2811-2822 (1994) Development 120, 2811-2822 (1994) Printed in Great Britain © The Company of Biologists Limited 1994 p ( ) Printed in Great Britain © The Company of Biologists Limited 1994 Key words: cortical development, thalamic innervation, adhesion, slice culture, neurite outgrowth, axonal development, axon guidance INTRODUCTION Thalamic growth cones project into the cell dense cortical plate only after the latter has begun to differentiate into distinguishable laminae (Lund and Mustari, 1977), eventually becoming We wished to exploit the simplicity of the in vitro approach to look for some of the cues that guide thalamocortical afferents, especially those that might only be present tran- siently and would therefore be difficult to identify in long-term explant co-cultures. Consequently, embryonic thalamic cells were dissociated, ﬂuorescently labeled and plated directly onto 2812 D. E. Emerling and A. D. Lander living, vibratome slices of mouse forebrain taken from different developmental stages. Cells were examined both for attachment and neurite outgrowth during one day in culture. centration was increased to 0.19 mg/ml and the suspension was incubated for another 5 minutes at 37°C. Tissue was triturated gently ﬁve times with a ﬂame-polished pasteur pipette that was coated with a solution of 4% BSA in CMF-HBSS (brought to pH of 7.3 with 1 N NaOH). The suspension was brought to a ﬁnal volume of approx. 10 ml with HHBSS, undissociated tissue was allowed to settle (approx. 5 minutes) and the supernatant was transferred to a new tube. The cell suspension was centrifuged at 100 g for 10 minutes and the pellet gently resuspended in Labeling Medium which consisted of 30 µM Cell Tracker™CMTMR (Molecular Probes, Eugene, OR) in CM; prepared, centrifuged to remove undissolved dye and ﬁlter sterilized just before use. Cells were incubated at 37°C for 30-50 minutes. Two volumes of 37°C CM were added to the suspension along with DNAse (0.12 mg/ml) and incubation continued for 5-15 minutes. Several volumes of HHBSS were added and the suspension was underlayed with a 2 ml cushion of 4% BSA (crystalline BSA in CMF-HBSS; brought to pH 7.3 with 1 N NaOH). Cells were then centrifuged at 150 g for 10 minutes. The pellet was resuspended in CM and cells were checked for viability using Trypan Blue. Routinely, >95% of cells excluded Trypan Blue. The cell suspension was diluted with CM to a concentration of ≈2.5×106 cells/ml. Within 1 hour of coculture, cells were observed to attach well to all embryonic (E15) cortical layers except for the cortical plate, which supported poor attachment. Neurite outgrowth was observed within 3 hours and, on some laminae, exhibited a signiﬁcant tangential orientation. However, no neurites were observed to enter the cortical plate. INTRODUCTION As slices from older animals were used, the non-permissiveness of the cortical plate gradually changed, in a manner that correlated both spatially and temporally with ongoing events in thalamo- cortical development. These results indicate that lamina- speciﬁc signals that affect thalamic neuron behavior can be detected in as little as 3 hours in vitro. Moreover, they suggest that as simple a mechanism as local differences in cell adhesion could play a major role in dictating the timing and location of thalamocortical innervation. Dissection of tissue Random-bred Swiss mice (CD-1, Charles River Laboratories) were naturally mated. Day of identiﬁcation of vaginal plug was considered E0 and day of birth P0. For embryonic tissue, pregnant animals were killed by cervical dislocation and embryos removed. For postnatal tissue, animals were anesthetized by cooling on ice and brains were immediately removed. Brains were dissected in ice-cold phosphate- buffered saline (PBS: 137 mM NaCl, 2.68 mM KCl, 7.83 mM Na2HPO4, 1.47 mM KH2PO4) and prepared for sectioning (see below) or used to obtain tissue for dissociated cell preparations. In the latter case, further dissection of brains into regions (e.g. thalamus) was performed in ice-cold PBS supplemented with 4% calf serum (HyClone Laboratories), 5.6 mg/ml glucose, 25 i.u./ml penicillin, 25 µg/ml streptomycin and 0.001% phenol red, and the Atlas of the Prenatal Mouse Brain (Schambra et al., 1992) was used as a guide. The boundaries used in dissecting the thalamus were, in the rostro- caudal direction, the habenular recess and the dorsal diencephalic sulcus of the third ventricle while, in the dorsoventral direction, the boundaries were the epithalamus and a point just dorsal to the ventral diencephalic (hypothalamic) sulcus. Careful attention was paid to the removal of tissue from the mammillary, preoptic and tegmental nuclei as well of from the epi- and hypothalamus. Dissected tissue was immediately rinsed in multiple volumes of ice-cold HHBSS: calcium- magnesium free, Hank’s Balanced Salt Solution (CMF-HBSS) sup- plemented with 10 mM Hepes, pH 7.2. Preparation and ﬁxation of living slice cultures p g Brains were placed in 37°C, molten, low melting point agarose solution (Gibco BRL; 2% in PBS supplemented with 3 g/L glucose) which was immediately placed on ice to harden. Agarose blocks con- taining brains were cut to size (approx. 1.5 cm3), glued to a vibratome tray (Technical Products International, Inc., St. Louis), immersed in ice-cold PBS (supplemented with 0.45% glucose and 1 mM sodium pyruvate) and the tray was subsequently surrounded by an ice bath. Under the PBS bath, brains were vibratome-sectioned (150-200 µm thick) and slices were placed on nitrocellulose (0.45 µm; Sartorius, Bohemia, NY) ﬁlter disks (12 mm diameter) that had previously been sterilized, incubated in a sterile solution of concanavalin A (1.3 mg/ml in glutamine free DMEM) for 4-18 hours at 4°C and rinsed several times in HHBSS before use. Disks with mounted slices were removed from the bath, placed on sterilized paraﬁlm in plastic Petri dishes and 100 µl of ice-cold CM was added, forming a bubble over the slices. Slices were kept on ice until all had been cut, medium was removed, 45 µl of fresh CM added and then slices were moved to a 37°C humid- iﬁed incubator with a 8% CO2 atmosphere. Cells were plated onto slices by carefully pipetting 45 µl of cells into the medium already on slices (≈105 cells/ﬁlter disk; ≈1000-2000 cells/mm2). Cells were allowed to settle at 37°C and incubated for various periods. Cultures were transferred to a large volume of 37°C HHBSS and gently rinsed to remove unattached cells (for some experiments, cells were visual- ized by ﬂuorescence microscopy before the transfer and rinse procedure). Cultures were then added to warm (37°C) ﬁxative: 4% paraformaldehyde (J. T. Baker, Phillipsburg, NJ) in 0.2 M sodium phosphate buffer (pH 7.5). After 15-30 minutes, the ﬁxed tissue was rinsed in PBS and counterstained (using 10 µg/ml bisbenzimide [Hoechst 33258] in PBS). Cultures were mounted on glass slides in a saturated sucrose solution containing 0.1% sodium azide (Fluka AG) and coverslipped. All salts, sucrose and glucose were purchased from Mallinckrodt. Tissue culture media, sodium pyruvate, penicillin and streptomycin were purchased from Mediatech. All other reagents were purchased from Sigma unless otherwise indicated. 281 Thalamic cell behavior on cortical slices 2813 Thalamic cell behavior on cortical slices jugated, goat anti-mouse IgM, µ-chain speciﬁc (TAGO, Burlingame, CA), diluted 1:80 in wash solution. Cultures were washed several times, rinsed in PBS, counterstained with bisbenzamide, rinsed in 0.1 M NaHCO3 (pH 9.2) and mounted in a saturated sucrose solution con- taining 0.1 M NaHCO3 (pH 8.5) and 0.1% sodium azide. jugated, goat anti-mouse IgM, µ-chain speciﬁc (TAGO, Burlingame, CA), diluted 1:80 in wash solution. Cultures were washed several times, rinsed in PBS, counterstained with bisbenzamide, rinsed in 0.1 M NaHCO3 (pH 9.2) and mounted in a saturated sucrose solution con- taining 0.1 M NaHCO3 (pH 8.5) and 0.1% sodium azide. was superimposed over the ﬂuorescence image of the culture within the microscope objectives, was drawn using the hardware and software of the Neuron Tracing System (Eutectics Electronics Inc., Raleigh, NC). Cortical layers and their borders were identiﬁed and traced under UV ﬂuorescence while labeled and attached thalamic cells were individu- ally traced under rhodamine ﬂuorescence. Using an automated, cali- brated microscope stage and both ﬂuorescence channels, a composite image of slice anatomy, attached cells and neurite morphology was drawn to scale across several microscope ﬁelds. For staining of dissociated cells, the following methods were used. Cells were plated onto acid-washed glass coverslips (12 mm diameter) that had been treated with polylysine (100 µg/ml in PBS) overnight at 4°C, washed several times with PBS, incubated with concanavalin A (400 µg/ml in HHBSS) for 4 hours at 37°C and rinsed several times in HHBSS. Plated cells were left for 3.5 hours at 37°C in a humidi- ﬁed incubator (8% CO2 atmosphere), ﬁxed for 22 minutes with 4% paraformaldehyde in 5% sucrose/PBS and rinsed with PBS. For staining with antibodies to intracellular epitopes, cells were treated at this point with 0.5% Triton X-100 in PBS for 5 minutes at room tem- perature and, for all experiments, cells were blocked with a solution of 5% goat serum, 5% calf serum and 0.1% sodium azide in PBS. This solution was used for all subsequent washes and antibody dilutions. Anti-neuroﬁlament (monoclonal antibody RT97, 1:666 dilution of ascites ﬂuid [Wood and Anderton 1981]); anti-neuron speciﬁc β-tubulin (monoclonal antibody TUJ1, 1:200 dilution of con- centration culture supernatant [Easter et al., 1993]) and anti-mouse NCAM (monoclonal antibody H28, 1:1 dilution of culture supernatant [Gennarini et al., 1984]) were applied at 4°C overnight. 281 Thalamic cell behavior on cortical slices FITC- (Anti- bodies Inc., Davis, CA) and rhodamine-conjugated (Kirkegaard & Perry Labs Inc., Gaithersburg, MD) secondary antibodies were used for visualization. Cell nuclei were counterstained with bisbenzamide and coverslips mounted in 80% glycerol in PBS or 0.1 M NaHCO3. Neurite data were collected from experiments using sagittal slices. Neurites were traced only if their growth cones and soma of origin could be clearly demarcated and if they were at least one soma diameter in length (approx. 10 µm). Since neurites often projected downward, into the slice tissue, they were traced in three dimensions (automated and calibrated control of the focus knob allowed for images to be traced through focal planes). Some cells had two neurites longer than a cell diameter; in these cases, both processes were traced. No neurites had branches which themselves met the length criterion. Neurite data analysis Neurite lengths were calculated by the system software as the 3-D contour distance between a neurite’s origin (point of connection to the soma) and its endpoint (center of the growth cone). Orientations for marginal zone, subplate/intermediate zone and ventricular zone neurites were calculated with respect to the marginal zone/cortical plate, cortical plate/subplate and intermediate zone/ventricular zone borders respectively. Values consisted of the angle between the reference border (or, if curved, a tangent to the border) closest to the neurite’s origin and a line deﬁned by the neurite’s origin and its end point. Only coordinates along the rostral-caudal and pial-to-ventricu- lar axes were used for this calculation. A neurite projecting rostrally, parallel with its reference border, deﬁned 0°. Projections directed toward the pia, perpendicular to the reference border, were assigned 90° and those directed toward the ventricle were assigned −90°. Preparation and ﬁxation of cryostat section cultures Dissected brains were snap frozen in isopentane kept in a dry- ice/ethanol bath. Frozen brains were equilibrated to −20°C and mounted in OCT compound (Miles, Inc., Elkhart, IN). 20 µm slices were cut on a Reichert-Jung 2800 Frigocut-E cryostat. Slices were warmed onto ProbeOn Plus™glass slides (FisherBiotech) and air dried. OCT was peeled off and a well was created (approx. 12 mm diameter) around the tissue slice with rubber cement (Dennison Sta- tionary, Framingham, MA). Slices were then rehydrated with PBS, rinsed with CM and 45 µl of fresh medium was added to the well. Slices were kept at 37°C for 30 minutes before 45 µl of cell suspen- sion was added to the medium on each slice (approx. 105 cells/well) and cultures were incubated at 37°C for 3 hours. Unattached cells were gently rinsed off with 37°C HHBSS. The rubber cement was removed and warm (37°C) ﬁxative was added for 10-20 minutes. After rinsing with PBS and counterstaining (10 µg/ml bisbenzimide in PBS), cultures were visualized by ﬂuorescence microscopy. Attachment data analysis The surface area of and the number of cells on each layer were computed based on stored digital images and attachment densities were calculated from these values. Distances from the cortical plate/subplate border of cells attached to the subplate and intermedi- ate zone were calculated by the system software as the distance, within a plane parallel to the plane of the slice, between the center of each attached cell and the closest point on the cortical plate/subplate border to that cell. Attachment data for different cortical layers were compared by a single factor analysis of variance (ANOV) to obtain reported P values. A Newman-Keuls test was used to verify the outlier values (Zar, 1974). Data collection The selection of ﬁxed slice cultures for quantiﬁcation was based solely on the integrity of slice anatomy, as visualized by bisbenza- mide staining of cell nuclei. Cultures were chosen only if the laminar borders could be clearly discerned and the identity of individual laminae unambiguously determined. Borders were delineated by dif- ferences in the density of cell nuclei and laminae were identiﬁed in some cases by speciﬁc landmarks, such as barrels within the granular layer of the P7 somatosensory cortex. Slices that had been folded or torn while being placed onto the nitrocellulose substrata, or had otherwise become distorted due to stretching or ﬂattening, were not quantiﬁed. Importantly, slice selection was carried out under UV (bis- benzamide) ﬂuorescence only. Since Cell Tracker™CMTMR-ﬂuo- rescence is not observed in this channel, it was possible to be conﬁdent that the selection of slices was carried out blind to patterns of thalamic cell attachment. g For polar plots, neurites were separated according to their angle into twelve 30° interval bins. Neurites that fell on the border between two bins were split with a value of 0.5 assigned to each bin. Each plot contains twelve points which consist of the angle of the center of the bin (θ=−30°, 0°, 30, 60°, ...) and the number of neurites in that bin (r). A chi-square analysis was used to test whether neurite data ﬁt a uniform (random) circular distribution by comparing binned, neurite samples to a hypothetical sample consisting of the same number of neurites evenly distributed among all 12 bins (Zar, 1974). For polar plots, neurites were separated according to their angle into twelve 30° interval bins. Neurites that fell on the border between two bins were split with a value of 0.5 assigned to each bin. Each plot contains twelve points which consist of the angle of the center of the bin (θ=−30°, 0°, 30, 60°, ...) and the number of neurites in that bin (r). A chi-square analysis was used to test whether neurite data ﬁt a uniform (random) circular distribution by comparing binned, neurite samples to a hypothetical sample consisting of the same number of neurites evenly distributed among all 12 bins (Zar, 1974). Immunohistochemistry Thalami were incubated in HHBSS with trypsin (0.18 mg/ml) and DNAse (0.08 mg/ml) in a 15 ml polypropylene, conical tube for approx. 10 minutes at 37°C with occasional, gentle mixing. The tissue suspension was then diluted with 1/3 volume of 37°C Complete Medium (CM): Dulbecco’s modiﬁed Eagle’s medium (DMEM: glutamine free, 4.5 g/L glucose) supplemented with 10 µg/ml trans- ferrin, 5 mg/ml crystalline grade bovine serum albumin (BSA; ICN Biochemicals), 20 nM progesterone, 30 nM sodium selenite, 100 µM putrescine, 10 µg/ml bovine insulin, 1 mM sodium pyruvate, 50 i.u./ml penicillin, 50 µg/ml streptomycin and 25 mM Hepes (pH 7.2). Soybean trypsin inhibitor (0.28 mg/ml) was added, the DNAse con- Slice cultures were prepared, rinsed and ﬁxed as above except that ﬁxation was limited to 18 minutes. Cultures were then washed several times in PBS and treated with 1% Triton X-100 in PBS for 7 minutes at room temperature. The following incubations were done at 4°C. Tissue was blocked (40% goat serum, 40% calf serum, 0.1% Triton X-100, 0.1% sodium azide in PBS; ﬁltered) for 19 hours, washed (5% goat serum, 5% calf serum, 0.1% sodium azide in PBS; ﬁltered) for 3 hours and ﬁrst antibody was added: CS-56 monoclonal mouse anti- chondroitin sulfate (Sigma), ascites ﬂuid diluted in wash solution, 1:500. After 48 hours, cultures were washed several times over 20 hours and then incubated with second antibody for 4 hours: FITC-con- 2814 D. E. Emerling and A. D. Lander (E14-15) mouse thalamic cells were dissociated, labeled with a vital ﬂuorescent dye, washed into serum-free culture medium and allowed to settle onto freshly cut vibratome slices (150-200 µm) of developing mouse forebrain. After 3 hours at 37°C, the slices were gently rinsed to remove unattached cells and then ﬁxed, counterstained with bisbenzimide (Hoechst 33258) and visualized by ﬂuorescence microscopy (see Methods). To avoid the possibility of dye-transfer from thalamic cells to intrinsic cells of the slice, thalamic cells were labeled with a thiol-reactive ﬂuorescent dye, CellTracker™CMTMR, which diffuses into cells, becomes covalently coupled to intracellular components (e.g. glutathione) and is ﬁxable by cross-linking agents. Immunocytochemical evaluation of dissociated thalamic cells prior to plating onto slices indicated that 91% (±3.3%, n=904) expressed neuron-speciﬁc class III β-tubulin and 70% (±3.5%, n=828) contained neuroﬁlament protein. Thus, the vast majority of the cells were neurons. In addition, 83% (±4.0%, n=616) were immunoreactive for NCAM. found on each of the other cortical layers – the marginal, inter- mediate and ventricular zones. On most subcortical regions (e.g. caudoputamen, globus pallidus, preoptic and supraoptic areas, hypothalamus, thalamus), there was also a high density of thalamic cells, although on some (the anterior commissure, the claustrum, the amygdaloid area and olfactory nuclei of the ventral forebrain and parts of the septum) few cells were seen (data not shown). ( ) In some experiments, slices were examined just prior to rinsing and ﬁxation. In these cases, thalamic cells appeared to be uniformly distributed over each slice. Thus, the differences in ﬂuorescent cell density that were seen with rinsed, ﬁxed slices apparently reﬂected region-speciﬁc cell attachment, rather than non-uniform plating of cells or migration of cells from some regions to others during the 3 hour culture period. In other experiments, thalamic cells were plated and cultured on forebrain slices for different periods of time before rinsing and ﬁxation. Patterns of cell attachment similar to that in Fig. 1 were obtained whether rinsing and ﬁxation were carried out as early as 1 hour, or as late as 24 hours, after plating (data not shown). We also found that the preincubation of slices for up to 5 hours at 37°C prior to the plating of cells produced the same pattern of cell attachment as seen in the previous exper- iments (data not shown). Region-speciﬁc attachment of thalamic cells to living slices of E15 forebrain Measurements of cell attachment, neurite length and neurite orienta- tion were obtained from digitized images of ﬁxed cultures. Images were collected using a Zeiss ﬂuorescence microscope ﬁtted with camera lucida optics that were set to view a video monitor. A video trace, which To examine how thalamic neurons behave when confronted in vitro with different brain environments, embryonic day 14 or 15 2814 D. E. Emerling and A. D. Lander When thalamic cells were prepared and plated in this manner onto E15 forebrain slices, large differences were evident in the numbers of ﬂuorescent cells that were found on different parts of the slice. The most striking differences were in the cerebral cortex, the anatomical layers of which could be identiﬁed by nuclear density (Fig. 1). Speciﬁcally, very few thalamic cells were found on the cortical plate, whereas numerous cells were To quantify thalamic cell attachment to the different layers Fig. 1. Laminar speciﬁc attachment of dissociated thalamic cells to slices of embryonic cortex. (A) Laminae of an E15 cortex can be seen under UV ﬂuorescence as differences in nuclear density after a sagittal slice is cultured with thalamic cells (3 hours), ﬁxed and stained with bisbenzamide (see methods). Rostral is to the right, caudal to the left. (B) The same slice viewed under rhodamine optics shows the distribution of attached thalamic cells. Some attached cells are in clusters, which appear as larger, bright dots. The pial and ventricular edges of the cortex are demarcated by dotted white lines at the top and bottom of each ﬁgure. Lines are also positioned in the two photos to reference the same points in the two views of the slice. Very few thalamic cells attach to the cortical plate (CP) while more attach to the intermediate zone (IZ), marginal zone (MZ) and ventricular zone (VZ) as well as to the substratum off the slice (seen at the edges of the photos). Density of attached cells is greatest on the intermediate zone just subjacent to the cortical plate. Scale bar, 500 µm. Fig. 1. Laminar speciﬁc attachment of dissociated thalamic cells to slices of embryonic cortex. (A) Laminae of an E15 cortex can be seen under UV ﬂuorescence as differences in nuclear density after a sagittal slice is cultured with thalamic cells (3 hours), ﬁxed and stained with bisbenzamide (see methods). Rostral is to the right, caudal to the left. (B) The same slice viewed under rhodamine optics shows the distribution of attached thalamic cells. Some attached cells are in clusters, which appear as larger, bright dots. The pial and ventricular edges of the cortex are demarcated by dotted white lines at the top and bottom of each ﬁgure. Lines are also positioned in the two photos to reference the same points in the two views of the slice. Neurite outgrowth by thalamic neurons on living slices of E15 neocortex In photographs such as Fig. 1, the contrast in thalamic cell density between the cortical plate and intermediate zone is par- ticularly noticeable because thalamic cells on the intermediate zone are especially concentrated near the border with the cortical plate (also see Fig. 3). This can also be illustrated by plotting cell density in the intermediate zone as a function of distance from the cortical plate (Fig. 2, inset). These data suggest that thalamic cell attachment is especially high in the subplate, the region of the intermediate zone subjacent to the cortical plate (Kostovic and Molliver, 1974; Luskin and Shatz, Even though thalamic cells were typically plated onto forebrain slices for only 3 hours, substantial neurite outgrowth occurred on all forebrain regions where thalamic cells attached, including the marginal, intermediate and ven- tricular zones of the developing cortex (too few cells attached to the cortical plate to permit conclusions to be drawn about neurite outgrowth on this layer). There were no signiﬁcant differences in the lengths of neurites on cortical layers (marginal zone: 16.1 µm [s.d.=5.7; n=85]; intermediate zone: 16.1 µm [s.d.=6.5; n=114]; ventricular zone 15.8 µm [s.d.=6.8; n=52]). Interestingly, the orientation of neurites on the intermediate zone appeared non-random. On this layer, most neurites projected parallel to the cortical laminae (i.e. along the rostrocaudal axis), while few neurites projected radially (Fig. 3). Fig. 2. Densities of thalamic cells attached to embryonic cortical layers. The densities of cells attached to each of the major embryonic cortical layers – the marginal zone (MZ), cortical plate (CP), intermediate zone (IZ) and ventricular zone (VZ) – were determined for three coronal (light bars) and ﬁve sagittal (darker bars) slices. Black bars represent the means (± standard error) from the 8 samples. Data were collected from the portion of sagittal slices indicated by dotted lines in Fig. 1 or from the dorsolateral part of coronal slices because laminae could be unambiguously distinguished within these regions (total number of cells counted from the 8 slices: nmz=1818, ncp=316, niz=2001, nvz=1647, noff slice=2583). ‘Off Slice’ data were collected from the culture substratum, adjacent to the pial surface. Mean values are signiﬁcantly different among the four layers (Analysis of Variance test: P<0.0005). A Newman-Keuls analysis veriﬁed the value of the mean density on the cortical plate as a signiﬁcant outlier (µiz = µmz ≠ µvz ≠µcp). 2814 D. E. Emerling and A. D. Lander Very few thalamic cells attach to the cortical plate (CP) while more attach to the intermediate zone (IZ), marginal zone (MZ) and ventricular zone (VZ) as well as to the substratum off the slice (seen at the edges of the photos). Density of attached cells is greatest on the intermediate zone just subjacent to the cortical plate. Scale bar, 500 µm. 2815 Thalamic cell behavior on cortical slices 2815 Thalamic cell behavior on cortical slices of the E15 neocortex, measurements of the density of labeled cells on each layer were made for 8 slices and are shown in Fig. 2. Mean thalamic cell attachment to the marginal, inter- mediate and ventricular zones was in the range of 1300-2300 cells/mm2 and was close to the density of cells that was initially plated onto the slices (~1000-2000 cells/mm2; see methods). In contrast, thalamic cell attachment to the cortical plate was substantially lower (243 ± 60 cells/mm2). This result was obtained regardless of whether coronal or sagittal forebrain slices were used (Fig. 2) and was qualitatively similar through- out the rostrocaudal and dorsoventral extent of the neocortex. 1985). Because the rodent subplate is only a few cell layers thick (Valverde et al., 1989; Bayer and Altman, 1990), its boundaries cannot be accurately determined in our thick slices by nuclear staining alone. In some experiments, the subplate was visualized by chondroitin sulfate immunohistochemistry (Sheppard et al., 1991) and was found to correspond to the part of the intermediate zone extending up to ~25 µm from the cortical plate border (data not shown), i.e., containing that region which supports the highest density of attached cells (Fig. 2, inset). Neurite outgrowth by thalamic neurons on living slices of E15 neocortex INSET shows that cells attached to the intermediate zone are not uniformly distributed on this layer. Cells that attached to the intermediate zone were binned according to their distance from the cortical plate border and the data, averaged from the 8 slices, were plotted as the proportion of total cells attached to this layer. On average, over half of the cells that attached to the intermediate zone are located within 25 µm of the cortical plate border. Fig. 2. Densities of thalamic cells attached to embryonic cortical layers. The densities of cells attached to each of the major embryonic cortical layers – the marginal zone (MZ), cortical plate (CP), Fig. 4 shows the ranges of neurite angles (as measured with respect to nearby cortical layer borders; see Methods) that were observed for thalamic cells attached to marginal, intermediate and ventricular zones. The data conﬁrm that a disproportion- ate number of intermediate zone neurites are rostrocaudally oriented (P<0.001), with no signiﬁcant bias toward the rostral or caudal direction (52% and 48% of sample, respectively; n=114). In contrast, neurites extended by thalamic cells on the marginal and ventricular zones followed a much more uniform distribution, not signiﬁcantly different from random (see Fig. 4, legend). g ) Since the subplate was included as part of the intermediate zone in the above analysis, an attempt was made to analyze subplate and non-subplate neurites separately. Neurites seen on the intermediate zone were therefore divided into two popula- tions – those originating within one average neurite length (16.1 µm) of the cortical plate (probable subplate neurites) and those originating in the rest of the intermediate zone. As shown in Fig. 5, both sets of neurites exhibited orientations that were signiﬁcantly biased toward the horizontal, i.e. along the ros- trocaudal axis. Interestingly, among the putative subplate neurites (Fig. 5A), extremely few were oriented superﬁcially (i.e., toward the cortical plate). Consistent with this observa- tion, in no case were neurites observed that crossed from the subplate onto the cortical plate. Indeed, subplate neurites were occasionally seen that extended a short distance superﬁcially, then turned sharply at the cortical plate border (not shown). In contrast, neurites were often seen that crossed the border between the intermediate and ventricular zones, without evidence of turning behaviors. Although these data raise the possibility that thalamic neurites actively avoid the cortical plate, time-lapse cinematographic observations will be required to establish such a conclusion. Behavior of thalamic cells on frozen sections of E15 forebrain it has become too thin to distinguish clearly). Moving toward the rostral end of the slice, however, one sees increasing numbers of labeled cells attached to the cortical plate, espe- cially the deeper regions of the cortical plate. Since the matu- ration of the neocortex is known to follow a rostral-to-caudal gradient (Smart, 1983; Bayer and Altman, 1991), these results suggest that the cortical plate may become permissive for thalamic cell attachment as the cortex matures. Others have found that neurons can exhibit region-selective attachment and/or neurite outgrowth on frozen (cryostat) sections of brain, nerve or muscle (Carbonetto et al., 1987; Sandrock and Matthew, 1987; Covault et al., 1987; Watanabe and Murakami, 1989; Savio and Schwab, 1989; Crutcher, 1989; Geisert, 1991; Tuttle and Matthew, 1991). To determine whether this method would reveal thalamic cell behaviors similar to those observed using living slices, E15 thalamic cells were prepared and labeled as before, but plated onto air-dried 20 µm cryostat sections of fresh-frozen E15 forebrain. After 3 hours at 37°C, non-attached cells were rinsed off and cultures were ﬁxed and counterstained. Compared to previous experi- ments with living sections, the absolute density of attached cells was much lower (Fig. 6). Moreover, attachment appeared to be fairly uniform across all cortical layers. In addition, little neurite outgrowth from cells attached to the cryostat sections was seen. The fact that frozen sections could not substitute for living slices in revealing cortical layer-speciﬁc behaviors of thalamic neurons raises the possibility that those behaviors require living cortical cells. However, when vibratome slices were lightly ﬁxed with formaldehyde before thalamic neurons were plated onto them, thalamic cells displayed layer-speciﬁc attachment much like that which was seen when they were plated onto living slices (data not shown). Thus, it appears that cortical cell viability is not required to obtain cortical layer-speciﬁc thalamic cell attach- ment. It seems more likely that critical molecules or structures that are preserved in vibratome sections are lost or disrupted in frozen sections. In support of this idea, when E14-15 thalamic cells were plated onto P7 sagittal slices, cell attachment to the cortical plate was observed throughout the rostrocaudal extent of the slices (Fig. 8). As was seen on E15 and P1 slices, thalamic cells also attached well to cortical layer I and the developing white matter. Neurite outgrowth by thalamic neurons on living slices of E15 neocortex Fig. 2. Densities of thalamic cells attached to embryonic cortical layers. The densities of cells attached to each of the major embryonic cortical layers – the marginal zone (MZ), cortical plate (CP), intermediate zone (IZ) and ventricular zone (VZ) – were determined for three coronal (light bars) and ﬁve sagittal (darker bars) slices. Black bars represent the means (± standard error) from the 8 samples. Data were collected from the portion of sagittal slices indicated by dotted lines in Fig. 1 or from the dorsolateral part of coronal slices because laminae could be unambiguously distinguished within these regions (total number of cells counted from the 8 slices: nmz=1818, ncp=316, niz=2001, nvz=1647, noff slice=2583). ‘Off Slice’ data were collected from the culture substratum, adjacent to the pial surface. Mean values are signiﬁcantly different among the four layers (Analysis of Variance test: P<0.0005). A Newman-Keuls analysis veriﬁed the value of the mean density on the cortical plate as a signiﬁcant outlier (µiz = µmz ≠ µvz ≠µcp). INSET shows that cells attached to the intermediate zone are not uniformly distributed on this layer. Cells that attached to the intermediate zone were binned according to their distance from the cortical plate border and the data, averaged from the 8 slices, were plotted as the proportion of total cells attached to this layer. On average, over half of the cells that attached to the intermediate zone are located within 25 µm of the cortical plate border. Fig. 2. Densities of thalamic cells attached to embryonic cortical layers. The densities of cells attached to each of the major embryonic cortical layers – the marginal zone (MZ), cortical plate (CP), 2816 D. E. Emerling and A. D. Lander Behavior of thalamic cells on frozen sections of E15 forebrain Unlike what was observed using E15 slices, labeled cells on the developing white matter (former intermediate zone) were not concentrated near the cortical plate border (site of the embryonic subplate). The data on thalamic cell attachment to P7 slices are presented quantitatively in Fig. 9. When these data are compared with Fig. 2, the most striking difference is the increase in the level of thalamic cell attachment to the P7 cortical plate relative to other cortical layers. Closer inspection of the sections revealed, however, that it is not the entire P7 cortical plate that supports thalamic cell attachment, but only its deeper layers. Using nuclear staining alone, it was possible to divide the P7 cortical plate into supragranular (laminae 2/3), Fig. 3. Neurite extension on E15 cortex. (A) High-magniﬁcation, UV ﬂuorescence micrograph of the bisbenzamide-stained cortex, from an experiment similar to that in Fig. 1, shows the cortical plate (CP), the cell poor intermediate zone (IZ) and the ventricular zone (VZ) as indicated at left. (B) The same ﬁeld seen under rhodamine optics reveals attached thalamic cells and their neurites. Fluorescent cells (ﬁlled arrows) attached to the intermediate zone extend three neurites in directions parallel to the CP/IZ border. On the ventricular zone, neurites (open arrows) project both parallel to the cortical layers and along the radial axis (perpendicular to the lamina). Growth cones of those neurites projecting radially cannot be seen because the neurites extend into the slice and out of the plane of focus. Filled arrows and arrowheads in A point to nuclei whose corresponding cells are marked by the same labels in B. These nuclei are out of focus because attached cells lie in a focal plane slightly above that of the cells of the slice. Scale bar, 10 µm. 2817 Thalamic cell behavior on cortical slices Fig. 5. Neurites on the E15 intermediate zone and subplate do not cross onto the cortical plate. The intermediate zone neurite sample shown in Fig. 4B was subdivided into two samples and replotted using the same graphing procedure. (A) The upper sample consists of those intermediate zone neurites (n=37) that originate within 16 µm (one mean neurite length) of the cortical plate. This sample approximates the neurites that are generated on the subplate region of the embryonic cortex. (B) The lower sample represents the rest of the intermediate zone neurites (n=77). Both samples are rostrocaudally oriented (parallel to the cortical lamina) and are signiﬁcantly different from uniform circular distributions (Pupper<0.001, Plower<0.01). Although both samples contain some radially directed projections (along vertical axis), there are almost no neurites on the upper intermediate zone that project superﬁcially, toward the cortical plate. Neurites projecting on the lower intermediate zone are not biased toward either direction along the radial (vertical) axis. Axis tick marks represent a count of 5 neurites. Fig. 4. Distribution of thalamic neurite angles on the different embryonic cortical layers. Polar plots show the angular distribution of neurites projected by thalamic cells on the marginal (A), intermediate (B) and ventricular (C) zones of E15 cortical slices. Neurite angles were calculated with respect to laminar borders and then segregated into twelve, 30° interval bins (see methods). Plotted points represent the center angle of each bin (θ) and the number of neurites (r) in the sample with angles that are within that bin interval. Axis tick marks represent a count of 5 neurites in A, 5 in B and 2 in C. The shapes of the plots reﬂect the predominant directions in which neurites project on a given layer. Plot frames indicate the angle of each bin’s center except for the bins centered on the axes, which are marked with cardinal directions based on cortical anatomy (90° and −90° represent projections toward the overlying and subjacent layers or tissue edges). Marginal (A) and ventricular (C) zone neurites project both rostrocaudally and radially (toward the pia and ventricle) and are not signiﬁcantly different from a uniform circular distribution (chi-square to uniform sample: Pmz>0.1, Pvz>0.5). Intermediate zone neurites mostly project rostrocaudally and are different from a uniform distribution (Piz<0.001). Data were taken from 4 sagittal slices (total number of neurites counted: nmz=85, niz=114, nvz=52) Fig. 5. 2817 Thalamic cell behavior on cortical slices Neurites on the E15 intermediate zone and subplate do not cross onto the cortical plate. The intermediate zone neurite sample shown in Fig. 4B was subdivided into two samples and replotted using the same graphing procedure. (A) The upper sample consists of those intermediate zone neurites (n=37) that originate within 16 µm (one mean neurite length) of the cortical plate. This sample approximates the neurites that are generated on the subplate region of the embryonic cortex. (B) The lower sample represents the rest of the intermediate zone neurites (n=77). Both samples are rostrocaudally oriented (parallel to the cortical lamina) and are signiﬁcantly different from uniform circular distributions (Pupper<0.001, Plower<0.01). Although both samples contain some radially directed projections (along vertical axis), there are almost no neurites on the upper intermediate zone that project superﬁcially, toward the cortical plate. Neurites projecting on the lower intermediate zone are not biased toward either direction along the radial (vertical) axis. Axis tick marks represent a count of 5 neurites. Fig. 4. Distribution of thalamic neurite angles on the different embryonic cortical layers. Polar plots show the angular distribution of neurites projected by thalamic cells on the marginal (A), intermediate (B) and ventricular (C) zones of E15 cortical slices. Neurite angles were calculated with respect to laminar borders and then segregated into twelve, 30° interval bins (see methods). Plotted points represent the center angle of each bin (θ) and the number of neurites (r) in the sample with angles that are within that bin interval. Axis tick marks represent a count of 5 neurites in A, 5 in B and 2 in C. The shapes of the plots reﬂect the predominant directions in which neurites project on a given layer. Plot frames indicate the angle of each bin’s center except for the bins centered on the axes, which are marked with cardinal directions based on cortical anatomy (90° and −90° represent projections toward the overlying and subjacent layers or tissue edges). Marginal (A) and ventricular (C) zone neurites project both rostrocaudally and radially (toward the pia and ventricle) and are not signiﬁcantly different from a uniform circular distribution (chi-square to uniform sample: Pmz>0.1, Pvz>0.5). Intermediate zone neurites mostly project rostrocaudally and are different from a uniform distribution (Piz<0.001). Data were taken from 4 sagittal slices (total number of neurites counted: nmz=85, niz=114, nvz=52). Fig. 4. Distribution of thalamic neurite angles on the different embryonic cortical layers. Developmental changes in the attachment and neurite outgrowth activities of neocortex At E15, the cerebral cortex is at a relatively early stage in its development. To determine whether the laminar-speciﬁc differences in the behavior of embryonic thalamic neurons change as the cortex matures, embryonic (E14-15) thalamic neurons, prepared as before, were plated onto living slices cut from older brains. Postnatal days 1 and 7 (P1 and P7) were chosen to represent intermedi- ate and late stages in cortical histogenesis (reviewed by Bayer and Altman, 1991) and thalamocortical innervation (reviewed by Erzurumlu and Jhaveri, 1992; Kageyama and Robertson, 1993). Fig. 3. Neurite extension on E15 cortex. (A) High-magniﬁcation, UV ﬂuorescence micrograph of the bisbenzamide-stained cortex, from an experiment similar to that in Fig. 1, shows the cortical plate (CP), the cell poor intermediate zone (IZ) and the ventricular zone (VZ) as indicated at left. (B) The same ﬁeld seen under rhodamine optics reveals attached thalamic cells and their neurites. Fluorescent cells (ﬁlled arrows) attached to the intermediate zone extend three neurites in directions parallel to the CP/IZ border. On the ventricular zone, neurites (open arrows) project both parallel to the cortical layers and along the radial axis (perpendicular to the lamina). Growth cones of those neurites projecting radially cannot be seen because the neurites extend into the slice and out of the plane of focus. Filled arrows and arrowheads in A point to nuclei whose corresponding cells are marked by the same labels in B. These nuclei are out of focus because attached cells lie in a focal plane slightly above that of the cells of the slice. Scale bar, 10 µm. Fig. 7 shows the results of a typical exper- iment involving a sagittal slice from a P1 animal. At the caudal end of the slice, the pattern of thalamic cell attachment is similar to that observed with E15 slices: cells attached to the marginal zone (i.e., cortical layer I) and the intermediate zone (i.e., the developing white matter), but not to the cortical plate, which at this age consists of developing layers 2-6 (the ventricular zone is not indicated in Fig. 7 because at this age 2817 Thalamic cell behavior on cortical slices 2817 Thalamic cell behavior on cortical slices Are thalamic cells detecting axon guidance cues? It is striking that regions of cortex that thalamic axons confront, but into which they do not normally grow, are readily detected in culture as regions to which dissociated thalamic cells do not attach. This observation raises the possibility that the adhesive behavior of neuronal cell bodies can, in some cases at least, reveal the locations of axonal guidance cues. Such a view has also been suggested by investigators studying the developing chick retinotectal system (Barbera et al., 1973; Gottlieb et al., 1976). 2817 Thalamic cell behavior on cortical slices Markings on the two sides of the photo are of different scales due to the thinning of the telencephalon that occurs along the ventrodorsal axis of coronal sections. Scale bar, 100 µm. Fig. 6. Cryostat sections of E15 forebrain support only a low density of thalamic cell attachment which is uniform across embryonic cortical layers. Both the laminar structure of the E15 cortex and attached thalamic cells can be visualized in this photo taken under both phase-contrast transmittance and rhodamine ﬂuorescence microscopy. The larger, ﬂuorescent dots are clusters of 2-4 cells. Cells can be seen to attach to all embryonic cortical layers, i.e. the marginal zone (MZ), cortical plate (CP), intermediate zone (IZ) and ventricular zone (VZ). At the top of the photo, adjacent to the marginal zone, ﬂuorescently labeled cells can be seen that are attached to the substratum of the slide. The photo is taken from the lateral cortex of a coronal section. Ventral is to the left, dorsal to the right. Markings on the two sides of the photo are of different scales due to the thinning of the telencephalon that occurs along the ventrodorsal axis of coronal sections. Scale bar, 100 µm. Finally, by P7 in vivo, thalamocortical axons have invaded the cortical plate throughout the rostrocaudal extent of the cortex, with most axons terminating in layer 4 and the deepest aspect of layer 3 (Naegele et al., 1988; Miller et al., 1993; Kageyama and Robertson, 1993; Agmon et al., 1993). In vitro, thalamic cells also attached to and extended neurites on the cortical plate throughout the rostrocaudal extent of the cortex, but attachment and neurite outgrowth within the cortical plate was limited to layers 4-6 (Figs 8, 9), i.e. the layers through which thalamic axons grow in vivo. DISCUSSION At each of these stages, thalamic cells in vitro also attached to and extended neurites on the marginal zone (layer I) and non-cortical areas (Figs 1, 2, 8, 9 and data not shown). Thus, territories not occupied by thalamic axons in vivo can be per- missive for thalamic cell attachment and neurite outgrowth in vitro. In contrast, territories that mark the boundaries of thala- mocortical axon growth in vivo (i.e. the entire cortical plate at early stages; layers 2 and 3 at later stages) were consistently non-permissive for thalamic cell attachment and neurite outgrowth in vitro. These experiments demonstrate that living, but not cryostat, sections of cerebral cortex support selective attachment of and neurite outgrowth from embryonic thalamic neurons in a manner that depends upon the developmental state of the cortex. Embryonic cortical plate and postnatal supragranular layers act as in vitro substrata which, compared to all other cortical laminae, support little neuronal attachment. Neurites of cells attached to the embryonic subplate and intermediate zone project mostly parallel to the cortical layers and neurites on the subplate do not cross onto the cortical plate. These results can be obtained in serum-free medium and are apparent after very short periods in culture (3 hours). 2817 Thalamic cell behavior on cortical slices Cells can be seen to attach to all embryonic cortical layers, i.e. the marginal zone (MZ), cortical plate (CP), intermediate zone (IZ) and ventricular zone (VZ). At the top of the photo, adjacent to the marginal zone, ﬂuorescently labeled cells can be seen that are attached to the substratum of the slide. The photo is taken from the lateral cortex of a coronal section. Ventral is to the left, dorsal to the right. Markings on the two sides of the photo are of different scales due to the thinning of the telencephalon that occurs along the ventrodorsal axis of coronal sections. Scale bar, 100 µm. For example, at the earliest stage examined in this study (mouse E15, which is roughly equivalent to rat E17), tangen- tially oriented thalamic axons normally occur abundantly in the cortical subplate, but are absent from the adjacent cortical plate. Thalamic axons are also present in the deeper interme- diate zone and are occasionally seen in the ventricular zone, (Crandall and Caviness, 1984; Reinoso and O’Leary, 1988; Catalano et al., 1991; Erzurumlu and Jhaveri, 1992; Kageyama and Robertson, 1993). In vitro, embryonic thalamic cells seeded onto E15 cortical slices attached especially well to the superﬁcial intermediate zone (the presumptive subplate), as well as throughout the intermediate zone and, to a lesser extent, the ventricular zone, but they attached very poorly to the cortical plate (Figs 1, 2). Neurites of thalamic cells that attached to the intermediate zone (including the subplate) tended to be oriented tangentially and, like thalamocortical axons in vivo, failed to enter the cortical plate (Figs 3, 4B, 5). For example, at the earliest stage examined in this study (mouse E15, which is roughly equivalent to rat E17), tangen- tially oriented thalamic axons normally occur abundantly in the cortical subplate, but are absent from the adjacent cortical plate. Thalamic axons are also present in the deeper interme- diate zone and are occasionally seen in the ventricular zone, (Crandall and Caviness, 1984; Reinoso and O’Leary, 1988; Catalano et al., 1991; Erzurumlu and Jhaveri, 1992; Kageyama and Robertson, 1993). In vitro, embryonic thalamic cells seeded onto E15 cortical slices attached especially well to the superﬁcial intermediate zone (the presumptive subplate), as well as throughout the intermediate zone and, to a lesser extent, the ventricular zone, but they attached very poorly to the cortical plate (Figs 1, 2). 2817 Thalamic cell behavior on cortical slices Neurites of thalamic cells that attached to the intermediate zone (including the subplate) tended to be oriented tangentially and, like thalamocortical axons in vivo, failed to enter the cortical plate (Figs 3, 4B, 5). At P1 in vivo, thalamocortical axons from the subplate have begun to invade the cortical plate in at least parts of the cortex (Agmon et al., 1993). In vitro, thalamic cells attached to much the same areas of the P1 cortex as observed with E15-E16 slices, except that, at this stage, the cortical plate in the rostral, but not caudal, cortex also supported thalamic cell attachment (Fig. 7). This rostral-to-caudal gradient of attachment to the P1 cortical plate in vitro parallels the general rostral-to-caudal gradient by which cortical histogenesis (Smart, 1983; Bayer and Altman, 1991) and innervation of the cortical plate (cf. Reinoso and O’Leary, 1988) occur in vivo. At P1 in vivo, thalamocortical axons from the subplate have begun to invade the cortical plate in at least parts of the cortex (Agmon et al., 1993). In vitro, thalamic cells attached to much the same areas of the P1 cortex as observed with E15-E16 slices, except that, at this stage, the cortical plate in the rostral, but not caudal, cortex also supported thalamic cell attachment (Fig. 7). This rostral-to-caudal gradient of attachment to the P1 cortical plate in vitro parallels the general rostral-to-caudal gradient by which cortical histogenesis (Smart, 1983; Bayer and Altman, 1991) and innervation of the cortical plate (cf. Reinoso and O’Leary, 1988) occur in vivo. Fig. 6. Cryostat sections of E15 forebrain support only a low density of thalamic cell attachment which is uniform across embryonic cortical layers. Both the laminar structure of the E15 cortex and attached thalamic cells can be visualized in this photo taken under both phase-contrast transmittance and rhodamine ﬂuorescence microscopy. The larger, ﬂuorescent dots are clusters of 2-4 cells. Cells can be seen to attach to all embryonic cortical layers, i.e. the marginal zone (MZ), cortical plate (CP), intermediate zone (IZ) and ventricular zone (VZ). At the top of the photo, adjacent to the marginal zone, ﬂuorescently labeled cells can be seen that are attached to the substratum of the slide. The photo is taken from the lateral cortex of a coronal section. Ventral is to the left, dorsal to the right. 2817 Thalamic cell behavior on cortical slices Polar plots show the angular distribution of neurites projected by thalamic cells on the marginal (A), intermediate (B) and ventricular (C) zones of E15 cortical slices. Neurite angles were calculated with respect to laminar borders and then segregated into twelve, 30° interval bins (see methods). Plotted points represent the center angle of each bin (θ) and the number of neurites (r) in the sample with angles that are within that bin interval. Axis tick marks represent a count of 5 neurites in A, 5 in B and 2 in C. The shapes of the plots reﬂect the predominant directions in which neurites project on a given layer. Plot frames indicate the angle of each bin’s center except for the bins centered on the axes, which are marked with cardinal directions based on cortical anatomy (90° and −90° represent projections toward the overlying and subjacent layers or tissue edges). Marginal (A) and ventricular (C) zone neurites project both rostrocaudally and radially (toward the pia and ventricle) and are not signiﬁcantly different from a uniform circular distribution (chi-square to uniform sample: Pmz>0.1, Pvz>0.5). Intermediate zone neurites mostly project rostrocaudally and are different from a uniform distribution (Piz<0.001). Data were taken from 4 sagittal slices (total number of neurites counted: nmz=85, niz=114, nvz=52). granular (lamina 4) and infragranular (laminae 5/6) layers and to measure cell attachment separately for each layer. As shown in Fig. 9 (inset), thalamic cell attachment to the P7 cortical plate was largely restricted to the granular and infragranular layers (laminae 4-6). As with E15 slices, thalamic cells plated onto P7 slices were observed to extend neurites on all cortical layers to which they attached (including, in this case the cortical plate). Although measurements of neurite angles were not made, no obvious ori- entation of neurites on any layer was observed. Signiﬁcantly, neurites were found that extended between the developing white matter and layer 6 of the cortical plate, without evidence of turning at this border (data not shown). 2818 D. E. Emerling and A. D. Lander Fig. 6. Cryostat sections of E15 forebrain support only a low density of thalamic cell attachment which is uniform across embryonic cortical layers. Both the laminar structure of the E15 cortex and attached thalamic cells can be visualized in this photo taken under both phase-contrast transmittance and rhodamine ﬂuorescence microscopy. The larger, ﬂuorescent dots are clusters of 2-4 cells. The correlations between in vitro behavior of thalamic neurons and in vivo development Dotted white lines indicate the edges of the cortex (pia at the top) and reference the same points of the slice in both photos. Scale bar, 500 µm. Fig. 7. Thalamic cell attachment to P1 cortex. (A) Laminae of a P1 cortex can be seen under UV ﬂuorescence after a sagittal slice is cultured with thalamic cells (3 hours), ﬁxed and stained with bisbenzamide. By this age, the cortical plate (CP) has matured and some differentiated laminae can be distinguished within it. (B) The same slice viewed under rhodamine optics shows the distribution of ﬂuorescently labeled and attached thalamic cells. Many cells attached to the developing white matter (WM, formerly the intermediate zone) and layer 1 (‘1’, formerly the marginal zone). Cells also attach to the cortical plate (not seen on E15 sections), but in a non-uniform manner. The rostral portion (left) supports the greatest level of attachment while the caudal portion (right) is almost devoid of ﬂuorescent cells. Dotted white lines indicate the edges of the cortex (pia at the top) and reference the same points of the slice in both photos. Scale bar, 500 µm. siveness of the cortical plate (and subsequently layers 2/3) is a property that only thalamic neurons can recognize, or whether other classes of neurons might be able to detect the same cues. Although extensive studies have not been carried out, preliminary experiments support the latter view. To date, dissociated cells from E15 cerebral cortex, E14 ganglionic eminence and P4 cerebellum have been labeled and plated onto E15 forebrain slices, and all showed markedly poorer attach- ment to the cortical plate than to other layers (data not shown; for cerebellar cells, the difference between attachment to the cortical plate and to other layers was apparently not as great as for thalamic cells, but was still noticeable). These results suggest that the non-adhesive properties of the cortical plate are quite general and even extend to cell types that never contact the cortex. Such results are perhaps not surprising, given that many axon guidance cues are known to act on cells that normally never see those cues. The correlations between in vitro behavior of thalamic neurons and in vivo development Both the patterns and timing of thalamic cell attachment and neurite outgrowth in this in vitro assay correlate well with in vivo patterns and timing of thalamocortical development: At each developmental stage examined, thalamic cells failed to attach well to, and failed to extend neurites onto, essentially those territories that thalamic axons encounter, but avoid, in vivo. It is interesting in this regard to compare the conclusions of the present study with observations by other investigators on 2819 Thalamic cell behavior on cortical slices 2819 Thalamic cell behavior on cortical slices Fig. 7. Thalamic cell attachment to P1 cortex. (A) Laminae of a P1 cortex can be seen under UV ﬂuorescence after a sagittal slice is cultured with thalamic cells (3 hours), ﬁxed and stained with bisbenzamide. By this age, the cortical plate (CP) has matured and some differentiated laminae can be distinguished within it. (B) The same slice viewed under rhodamine optics shows the distribution of ﬂuorescently labeled and attached thalamic cells. Many cells attached to the developing white matter (WM, formerly the intermediate zone) and layer 1 (‘1’, formerly the marginal zone). Cells also attach to the cortical plate (not seen on E15 sections), but in a non-uniform manner. The rostral portion (left) supports the greatest level of attachment while the caudal portion (right) is almost devoid of ﬂuorescent cells. Dotted white lines indicate the edges of the cortex (pia at the top) and reference the same points of the slice in both photos. Scale bar, 500 µm. Fig. 7. Thalamic cell attachment to P1 cortex. (A) Laminae of a P1 cortex can be seen under UV ﬂuorescence after a sagittal slice is cultured with thalamic cells (3 hours), ﬁxed and stained with bisbenzamide. By this age, the cortical plate (CP) has matured and some differentiated laminae can be distinguished within it. (B) The same slice viewed under rhodamine optics shows the distribution of ﬂuorescently labeled and attached thalamic cells. Many cells attached to the developing white matter (WM, formerly the intermediate zone) and layer 1 (‘1’, formerly the marginal zone). Cells also attach to the cortical plate (not seen on E15 sections), but in a non-uniform manner. The rostral portion (left) supports the greatest level of attachment while the caudal portion (right) is almost devoid of ﬂuorescent cells. D. E. Emerling and A. D. Lander Fig. 8. Thalamic cell attachment to P7 cortex. (A) Laminae of a P7 cortex can be seen under UV ﬂuorescence after a sagittal slice is cultured with thalamic cells (3 hours), ﬁxed and stained with bisbenzamide. The cell-dense, barrel walls of the somatosensory cortex can be seen and used to identify the granular layer 4 (Woolsey and Van Der Loos, 1970; Rice and Van Der Loos, 1977). Rostral is to the left, caudal to the right. (B) The same slice viewed under rhodamine optics shows the distribution of ﬂuorescently labeled and attached thalamic cells. As in the E15 and P1 experiments, cells attached well to layer 1 (‘1’, formerly the marginal zone) and the white matter (WM, formerly the intermediate zone). Attachment to the cortical plate (laminae ‘2-6’) can be seen throughout the rostrocaudal extent of the slice; however, it is limited to layers 4-6, i.e. the granular (‘4’) and infragranular (‘5/6’) layers. Attachment to the supragranular layers (‘2/3’) is relatively low. Dotted white lines indicate the edges of the cortex (pia at the top) and reference the same points of the slice in both photos. Scale bar, 1 mm. Fig. 9. Densities of thalamic cells attached to postnatal (P7) cortical layers. The densities of cells attached to layer 1, the cortical plate (CP, laminae 2-6) and the white matter (WM) were determined from 7 sagittal slices (light bars). Black bars represent the mean (± standard error) from the 7 samples. Data were collected from the presumed somatosensory cortex, the extent of which is indicated by the dotted lines in Fig. 8, because the laminae could be unambiguously distinguished within this region (total number of cells counted from the 7 slices: n1=891, n2-3=330, n4=1379, n5-6=7042, nwm=2400, noff slice=1874). ‘Off Slice’ data were collected from the culture substratum adjacent to the pial surface. INSET shows the density of cells attached to the laminae within the cortical plate. Cell density on supragranular layers (2/3) was much lower than on granular (4) and infragranular (5/6) layers (analysis of variance test: P<0.0005). A Newman-Keuls analysis veriﬁed the mean density on laminae 2/3 as an outlier (µ5/6 ≠µ4 ≠µ2/3). the central myelin-derived inhibitor of neurite outgrowth (Caroni and Schwab, 1988). The correlations between in vitro behavior of thalamic neurons and in vivo development Data were collected from the presumed somatosensory cortex, the extent of which is indicated by the dotted lines in Fig. 8, because the laminae could be unambiguously distinguished within this region (total number of cells counted from the 7 slices: n1=891, n2-3=330, n4=1379, n5-6=7042, nwm=2400, noff slice=1874). ‘Off Slice’ data were collected from the culture substratum adjacent to the pial surface. INSET shows the density of cells attached to the laminae within the cortical plate. Cell density on supragranular layers (2/3) was much 2820 The correlations between in vitro behavior of thalamic neurons and in vivo development Examples include the fact that most classes of neurons extend neurites in response to laminin, even though laminin is present in only a few parts of the developing brain (reviewed by Lander and Calof, 1993); that retinal axons will regenerate through peripheral nerve grafts and terminate speciﬁcally within structures that are not normal targets (Zwimpfer et al., 1992; Carter et al., 1994); and that even ﬁbroblasts – which are not found in the central nervous system – respond to the motility-inhibiting effects of the in vitro growth of thalamic axons on substrata of isolated cortical membranes: Götz et al. (1992) found that embryonic (E16), rat cortical membranes were relatively non-permissive for axon outgrowth, but postnatal (P7) membranes were not. More recently, Tuttle et al. (1993) showed that the change in the outgrowth-promoting properties of cortical membranes – from non-permissive at embryonic stages, to permissive at postnatal stages – could also be observed with membranes prepared from only the superﬁcial cortex (i.e. the cortical plate and marginal zone – of which the cortical plate is the major component). Thus, both these and the present study point to the cortical plate as the site of transient non-permissiveness, both for neuronal attachment and axon growth. It is not unreasonable to suppose that the cause is the same for both the lack of adhesion and the lack of axon growth, especially given that many of the molecules that promote axon growth also mediate cell adhesion (cf. Lander, 1989; Hynes and Lander, 1992). Indeed, while there are certainly cases in which the guidance of neurons by their substratum is not controlled by differential adhesion (cf. Gundersen, 1987; Calof and Lander, 1991; Lemmon et al., 1992), the development of the thalamocortical system may be a case where adhesion does play a dominant role. One issue not discussed above is whether the non-permis- 820 e central myelin-derived inhibitor of neurite outgrowth Caroni and Schwab, 1988). What molecules are thalamic neurons detecting in tro? he general non-permissiveness of the embryonic cortical ate and postnatal layers 2/3 for cell attachment and neurite utgrowth in vitro suggests that these regions either lack olecules that promote neuron adhesion and outgrowth, or ossess molecules that inhibit adhesion and outgrowth. The correlations between in vitro behavior of thalamic neurons and in vivo development lthough the data presented here cannot distinguish between ese possibilities, at least one major mediator of neural cell dhesion, NCAM, is clearly expressed in the embryonic ortical plate and postnatal supragranular layers (Fushiki and chachner, 1986; Chung et al., 1991; and unpublished obser- ations). In addition, no dramatic differences have been seen the relative isoform composition or polysialic acid content the NCAM in these, versus other layers, of the develop- g cortex (Chung et al., 1991; and unpublished observa- ons). Considering that NCAM can act as a homophilic adhesion olecule and that the majority of thalamic cells plated in the esent study were NCAM-immunoreactive, the possibility at the embryonic cortical plate and postnatal layers 2/3 ntain molecules that inhibit cell adhesion and axon growth worth considering seriously. Recently, several molecules, articularly certain components of the extracellular matrix, ave been claimed to have ‘anti-adhesive’ (reviewed by Sage d i d i l i l D. E. Emerling and A. D. Lander g. 8. Thalamic cell attachment to P7 rtex. (A) Laminae of a P7 cortex can seen under UV ﬂuorescence after a gittal slice is cultured with thalamic lls (3 hours), ﬁxed and stained with sbenzamide. The cell-dense, barrel alls of the somatosensory cortex can seen and used to identify the anular layer 4 (Woolsey and Van Der oos, 1970; Rice and Van Der Loos, 77). Rostral is to the left, caudal to e right. (B) The same slice viewed der rhodamine optics shows the stribution of ﬂuorescently labeled and ached thalamic cells. As in the E15 d P1 experiments, cells attached well layer 1 (‘1’, formerly the marginal ne) and the white matter (WM, rmerly the intermediate zone). tachment to the cortical plate aminae ‘2-6’) can be seen throughout e rostrocaudal extent of the slice; wever, it is limited to layers 4-6, i.e. e granular (‘4’) and infragranular 5/6’) layers. Attachment to the pragranular layers (‘2/3’) is relatively w. Dotted white lines indicate the ges of the cortex (pia at the top) and ference the same points of the slice in th photos. Scale bar, 1 mm. Fig. 9. Densities of thalamic cells attached to postnatal (P7) cortical layers. The densities of cells attached to layer 1, the cortical plate (CP, laminae 2-6) and the white matter (WM) were determined from 7 sagittal slices (light bars). Black bars represent the mean (± standard error) from the 7 samples. 2821 Thalamic cell behavior on cortical slices g De Carlos, J. A. and O’Leary, D. D. M. (1992). Growth and targeting of subplate axons and the establishment of major cortical pathways J. Neurosci. 12, 1194-1211. Dodd, J. and Jessell, T. M. (1988). Axon guidance and the patterning of neuronal projections in vertebrates. Science 242, 692-699. Easter, S. S., Jr., Ross, L. S. and Frankfurter, A. (1993). Initial tract formation in the mouse brain. J. Neurosci. 13, 285-299. Erzurumlu, R. S. and Jhaveri, S. (1992). Emergence of connectivity in the embryonic rat parietal cortex. Cereb. Cortex 2, 336-352. Faissner, A. and Kruse, J. (1990). J1/Tenascin is a repulsive substrate for central nervous system neurons. Neuron 5, 627-637. Overall, the results presented in this paper suggest that molecules that inﬂuence neuronal cell adhesion may, through their patterns and timing of expression, provide important cues for the growth of thalamic axons along the radial axis of the cortex. The continued use of rapid in vitro assays, such as the one described here, should aid in determining whether and how known adhesion molecules participate in this process. Fushiki, S. and Schachner, M. (1986). Immunocytological localization of cell adhesion molecules L1 and N-CAM and the shared carbohydrate epitope L2 during development of the mouse neocortex. Dev. Brain Res. 24, 153-167. Geisert, E. E. (1991). Characterization of the growth of cultured cortical neurons on sections of adult cortex. Cereb. Cortex 1, 134-142. Gennarini, G., Rougon, G., Deagostini-Bazin, H., Hirn, M. and Goridis, C. (1984). Studies on the transmembrane disposition of the neural cell adhesion molecule N-CAM. Eur. J. Biochem. 142, 57-64. Ghosh, A., Antonella, A., McConnell, S. K. and Shatz, C. J. (1990). Requirement for subplate neurons in the formation of thalamocortical connections. Nature 347, 179-181. We would like to thank Mary Herndon, Jon Ivins and Chris Stipp for helpful comments on the manuscript; Todd Grinnel, John Shavel and Dave Smith for technical assistance; Reha Erzurumlu and Sonal Jhaveri for assistance with anatomy; Anthony Frankfurter for the TUJ1 antibody; and Rebecca Tuttle and Dennis O’Leary for sharing unpublished data. This work was supported by a fellowship to A. D. L. from the David and Lucile Packard Foundation. Gottlieb, D. I., Rock, K. and Glaser, L. (1976). A gradient of adhesive speciﬁcity in developing avian retina. Proc. Natl. Acad. Sci. USA 73, 410- 414. Götz, M., Novak, N., Bastmeyer, M. and Bolz, J. (1992). Membrane-bound molecules in rat cerebral cortex regulate thalamic innervation. 2821 Thalamic cell behavior on cortical slices such molecule has been suggested to play a crucial role in guiding axons towards the optic ﬁssure (Brittis et al., 1992). Carter, D. A., Bray, G. M. and Aguayo, A. J. (1994). Long-term growth and remodeling of regenerated retino-collicular connections in adult hamsters. J. Neurosci. 14, 590-598. Whatever the molecules are that account for the different adhesive and neurite outgrowth-promoting properties of different cortical layers, it should not be assumed that their source is the intrinsic cells of the cortex. Thalamic axons them- selves, growing within certain cortical layers, could contribute some of the cues that thalamic cells detect when they are plated onto cortical slices (present study) or cortical membranes (Götz et al., 1992; Tuttle et al., 1993). For example, some or all of the attachment of thalamic cells to the embryonic subplate and intermediate zone (Figs 1, 2), the tangential orientation of neurites growing on those zones (Figs 3, 4B, 5), and the thalamic cell attachment to postnatal layers 4-6 (Figs 8, 9), could reﬂect the binding of thalamic neurons to thalamic axon membranes. Such homotypic interactions could play just as important a role in guiding the majority of thalamic axons in vivo as interactions of thalamic axons with intrinsic cortical cells. The assay described in the present study, performed following the selective ablation of appropriate cells or ﬁbers in vivo (cf. Ghosh et al., 1990), should prove useful in determin- ing the sources of the cues that are responsible for the layer- speciﬁc behavior of thalamic cells and their axons. , Catalano, S. M., Robertson, R. T. and Killackey, H. P. (1991). Early ingrowth of thalamocortical afferents to the neocortex of the prenatal rat. Proc. Natl. Acad. Sci. USA 88, 2999-3003. Chung, W. W., Lagenaur, C. F., Yan, Y. and Lund, J. S. (1991). Developmental expression of neural cell adhesion molecules in the mouse neocortex and olfactory bulb. J. Comp. Neurol. 314, 290-305. y Covault J., Cunningham, J. M. and Sanes, J. R. (1987). Neurite outgrowth on cryostat sections of innervated and denervated skeletal muscle. J. Cell Biol. 105, 2479-2488. Crandall, J. E. and Caviness, V. S. (1984). Axon strata of the cerebral wall in embryonic mice. Dev. Brain Res. 14, 185-195. Crutcher, K. A. (1989). Tissue sections from mature rat brain and spinal cord as substrates for neurite outgrowth in vitro: extensive growth on fray matter but little growth on white matter. Exp. Neurol. 104, 39-54. REFERENCES Hynes, R. O. and Lander, A. D. (1992). Contact and adhesive speciﬁcities in the associations, migrations, and targeting of cells and axons. Cell 68, 303- 322. Agmon, A., Yang, L. T., O’Dowd, D. K. and Jones, E. G. (1993). Organized growth of thalamocortical axons from the deep tier of terminations into layer IV of developing mouse barrel cortex. J. Neurosci. 13, 5365-5382. Kageyama, G. H. and Robertson, R. T. (1993). Development of geniculocortical projections to visual cortex in rat: evidence for early ingrowth and synaptogenesis. J. Comp. Neurol. 335, 123-148. Barbera, A. J., Marchase, R. B. and Roth, S. (1973). Adhesive recognition and retinotectal speciﬁcity. Proc. Natl. Acad. Sci. USA 70, 2482-2486. Kostovic, I. and Molliver, M. E. (1974). A new interpretation of the laminar development of the cerebral cortex: synaptogenesis in different layers of neopallium in the human fetus. Anat. Rec. 178, 395. Bayer, S. A. and Altman, J. (1990). Development of layer I and the subplate in the rat neocortex. J. Comp. Neurol. 107, 48-62. p Lander, A. D. (1989). Understanding the molecules of neural cell contacts: emerging patterns of structure and function. TINS 12, 189-195. Bayer, S. A. and Altman, J. (1991). Neocortical Development, pp. 33-36 and 65-82. New York: Raven Press. Lander, A. D. and Calof, A. L. (1993). Extracellular matrix in the developing nervous system. In Molecular Genetics of Nervous System Tumors, (eds. A. J. Levine and H. H. Schmidek), pp. 341-355. New York: Wiley-Liss. Bolz, J., Novak, N. and Staiger, V. (1992). Formation of speciﬁc afferent connections in organotypic slice cultures from rat visual cortex cocultured with lateral geniculate nucleus. J. Neurosci. 12, 3054-3070. Brittis, P. A., Canning, D. R. and Silver, J. (1992). Chondroitin sulfate as a regulator of neuronal patterning in the retina. Science 255, 733-736. Lemmon, V., Burden, S. M., Payne, H. R., Elmslie, G. J. and Hlavin, M. L. (1992). Neurite growth on different substrates: permissive versus instructive inﬂuences and the role of adhesive strength. J. Neurosci. 12, 818-826. Calof, A. L. and Lander, A. D. (1991). Relationship between neuronal migration and cell-substratum adhesion: laminin and merosin promote olfactory neuronal migration but are anti-adhesive. J. Cell Biol. 115, 779- 794. Lund, R. D. and Mustari, M. J. (1977). Development of the geniculocortical pathway in rats. J. Comp. Neurol. 173, 289-306. Luskin, M. L. and Shatz, C. J. (1985). 2821 Thalamic cell behavior on cortical slices Development 116, 507-519. Gundersen, R. W. (1987). Response of sensory neurites and growth cones to patterned substrata of laminin and ﬁbronectin in vitro. Dev. Biol. 121, 423- 431. What molecules are thalamic neurons detecting in vitro? The general non-permissiveness of the embryonic cortical plate and postnatal layers 2/3 for cell attachment and neurite outgrowth in vitro suggests that these regions either lack molecules that promote neuron adhesion and outgrowth, or possess molecules that inhibit adhesion and outgrowth. Although the data presented here cannot distinguish between these possibilities, at least one major mediator of neural cell adhesion, NCAM, is clearly expressed in the embryonic cortical plate and postnatal supragranular layers (Fushiki and Schachner, 1986; Chung et al., 1991; and unpublished obser- vations). In addition, no dramatic differences have been seen in the relative isoform composition or polysialic acid content of the NCAM in these, versus other layers, of the develop- ing cortex (Chung et al., 1991; and unpublished observa- tions). Fig. 9. Densities of thalamic cells attached to postnatal (P7) cortical layers. The densities of cells attached to layer 1, the cortical plate (CP, laminae 2-6) and the white matter (WM) were determined from 7 sagittal slices (light bars). Black bars represent the mean (± standard error) from the 7 samples. Data were collected from the presumed somatosensory cortex, the extent of which is indicated by the dotted lines in Fig. 8, because the laminae could be unambiguously distinguished within this region (total number of cells counted from the 7 slices: n1=891, n2-3=330, n4=1379, n5-6=7042, nwm=2400, noff slice=1874). ‘Off Slice’ data were collected from the culture substratum adjacent to the pial surface. INSET shows the density of cells attached to the laminae within the cortical plate. Cell density on supragranular layers (2/3) was much lower than on granular (4) and infragranular (5/6) layers (analysis of variance test: P<0.0005). A Newman-Keuls analysis veriﬁed the mean density on laminae 2/3 as an outlier (µ5/6 ≠µ4 ≠µ2/3). Considering that NCAM can act as a homophilic adhesion molecule and that the majority of thalamic cells plated in the present study were NCAM-immunoreactive, the possibility that the embryonic cortical plate and postnatal layers 2/3 contain molecules that inhibit cell adhesion and axon growth is worth considering seriously. Recently, several molecules, particularly certain components of the extracellular matrix, have been claimed to have ‘anti-adhesive’ (reviewed by Sage and Bornstein, 1991) and ‘neurite-repulsive’ (e.g. Snow et al., 1990; Faissner and Kruse, 1990) properties. Indeed, in the retina, the gradual central-to-peripheral disappearance of one 2822 D. E. Emerling and A. D. Lander connections formed between thalamus and cortex in coculture. Nature 351, 475-477. outgrowth using cryostat sections of nervous tissues as a substratum. J. Neurosci. Meth. 39, 193-201. connections formed between thalamus and cortex in coculture. Nature 351, 475-477. Naegele, J. R., Jhaveri, S. and Schneider, G. E. (1988). Sharpening of topographical projections and maturation of geniculocortical axon arbors in the hamster. J. Comp. Neurol. 277, 593-607. Tuttle, R., Schlaggar, B. L. and O’Leary, D. D. M. (1993). Maturational changes in membrane associated molecules in the cortical plate may control development of thalamocortical and intracortical connections. Soc. Neurosci. Abstr. 19, 1088. O’Leary, D. D. M. and Koester, S. E. (1993). Development of projection neuron types, axon pathways, and patterned connections of the mammalian cortex. Neuron 10, 991-1006. Valverde, F., Facal-Valverde, V., Santacana, M. and Heredia, M. (1989). Development and differentiation of early generated cells of sublayer VIb in the somatosensory cortex of the rat: a correlated golgi and autoradiographic study. J. Comp. Neurol. 290, 118-140. Reinoso, B. S. and O’Leary, D. D. M. (1988). Development of visual thalamocortical projections in the fetal rat. Soc. Neurosci. Abstr. 14, 1113. study. J. Comp. Neurol. 290, 118-140. y p Watanabe, E. and Murakami, F. (1989). Preferential adhesion of chick central neurons to the grey matter of the central nervous system. Neurosci. Lett. 97, 69-74. Rice, F. L. and Van Der Loos, H. (1977). Development of the barrels and barrel ﬁeld in the somatosensory cortex of the mouse. J. Comp. Neurol. 171, 545-560. Sage, E. H. and Bornstein, P. (1991). Extracellular proteins that modulate cell-matrix interactions. J. Biol. Chem. 266, 14831-14834. Woolsey, T. A. and Van Der Loos, H. (1970). The structural organization of Layer IV in the somatosensory region (SI) of mouse cerebral cortex. Brain Res. 17, 205-242. Sandrock, A. W. and Matthew, W. D. (1987). Identiﬁcation of a peripheral nerve neurite growth-promoting activity by development and use of an in vitro bioassay. Proc. Natl. Acad. Sci. USA 84, 6934-6938. Wood, J. N. and Anderton, B. H. (1981). Monoclonal antibodies to mammalian neuroﬁlaments. Biosci. Rep. 1, 263-268. Savio, T. and Schwab, M. E. (1989). Rat CNS white matter, but not grey matter, is nonpermissive for neuronal cell adhesion and ﬁber outgrowth. J. Neurosci. 9, 1126-1133. Yamamoto, N., Kurotani, T. and Toyama, K. (1989). Neural connections between the lateral geniculate nucleus and visual cortex in vitro. Science 245, 192-194. Schambra, U. B., Lauder, J. M. REFERENCES Studies of the earliest generated cells of the cat’s visual cortex: cogeneration of subplate and marginal zones. J. Neurosci. 5, 1062-1075. Carbonetto, S., Evans, D. and Cochard, P. (1987). Nerve ﬁber growth in culture on tissue substrata from central and peripheral nervous systems. J. Neurosci. 7, 610-620. Miller, B., Chou, L. and Finlay, B. L. (1993). The early development of thalamocortical and corticothalamic projections J. Comp. Neurol. 335, 16- 41. Caroni, P. and Schwab, M. E. (1988). Two membrane protein fractions from rat central myelin with inhibitory properties for neurite outgrowth and ﬁbroblast spreading. J. Cell Biol. 106, 1281-1288. Molnár, Z. and Blakemore, C. (1991). Lack of regional speciﬁcity for 2822 D. E. Emerling and A. D. Lander and Silver, J. (1992). Atlas of the Prenatal Mouse Brain. San Diego: Academic Press, Inc. Yamamoto, N., Yamada, K., Kurotani, T. and Toyama, K. (1992). Laminar speciﬁcity of extrinsic cortical connections studied in coculture preparations. Neuron 9, 217-228. Shepard, A. M., Hamilton, S. K. and Pearlman, A. L. (1991). Changes in the distribution of extracellular matrix components accompany early morphogenic events of mammalian cortical development. J. Neurosci. 11, 3928-3942. Zar, J. H. (1974). Biostatistical Analysis, pp. 133-139, pp. 151-155 and pp. 316-317. Englewood Cliffs, NJ: Prentice-Hall Inc. Zwimpfer, T. J., Aguayo, A. J. and Bray, G. M. (1992). Synapse formation and preferential distribution in the granule cell layer by regenerating retinal ganglion cell axons guided to the cerebellum of adult hamsters. J. Neurosci. 12, 1144-1159. Smart, I. H. M. (1983). Three dimensional growth of the mouse isocortex. J. Anat. 137, 683-694. Snow, D. M., Lemmon, V., Carrino, D. A., Caplan, A. I. and Silver, J. (1990). Sulfated proteoglycans in astroglial barriers inhibit neurite outgrowth in vitro. Exp. Neurol. 109, 111-130. (Accepted 29 June1994) (Accepted 29 June1994) g p , Tuttle, R. and Matthew, W. P. (1991). An in vitro bioassay for neurite

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